Small molecule active protein peptide

Abstract

Disclosed in the present invention is a method for preparing a small molecule active protein peptide from animal tissues and organs, the method comprising the following steps: taking animal tissues and organs and subjecting same to steps such as degreasing, washing, cutting, grinding and / or homogenization, enzymolysis, centrifugation, filtration, vacuumizing, ultrafiltration, nanofiltration, sterilization, and vacuum ultra-low temperature lyophilization. The steps described above are appropriately adjusted according to the characteristics of tissues and organs of different organisms. Moreover, the present invention provides an advanced technical solution to solve problems arising in the production process, thereby establishing a standardized process for preparing the small molecule active protein peptide from animal tissues and organs. The main objective is to improve the activity value, content, yield, amino acid composition, etc., of the protein peptide, while reducing industrial costs and improving the industrial production efficiency.

Description

Small molecule active protein peptides I. Technical Field

[0001] This invention relates to a method for preparing protein peptides based on animal tissues and organs, the prepared protein peptides and their uses, and belongs to the fields of meat protein physiology and biochemistry, protein biochemistry and biomanufacturing engineering. II. Background Technology

[0002] The cell is the basic unit of life; all known organisms are composed of cells (although viruses are not cells, they rely on cells for replication). Cells have complex structures and functions and are the foundation of life activities. Tissues are composed of cells with similar morphology, structure, and function linked together.

[0003] Animals mainly consist of seven major tissues: nervous tissue, epithelial tissue, connective tissue, muscle tissue, bone tissue, lymphatic tissue, and adipose tissue. Each of these tissues has a unique structure and function. Tissues enable cells to work together to complete specific physiological functions and cooperate with each other to maintain normal physiological activities and life processes of the human body. Specifically: (1) Muscle tissue: divided into skeletal muscle, smooth muscle, and cardiac muscle. Skeletal muscle can contract at will to realize the movement of the body, such as the activity of leg muscles when running; smooth muscle is mainly in the internal organs and participates in the peristalsis of organs, such as the peristalsis of the gastrointestinal tract; cardiac muscle can automatically and rhythmically contract to maintain the heartbeat; (2) Bone tissue: hard in texture, in addition to supporting the body, it is also a storage depot for minerals such as calcium and phosphorus, and bone marrow has hematopoietic function, continuously providing blood cells to the body; (3) Connective tissue: including bone tissue, blood, adipose tissue, etc. Bone tissue forms the skeleton, supporting the body and protecting internal organs; blood is responsible for transporting nutrients, oxygen, metabolic waste, etc., and maintaining the stability of the body's internal environment; (4) Adipose tissue: In addition to storing energy, it can also insulate and protect internal organs from mechanical damage. It is distributed under the skin and around the internal organs. For example, abdominal fat has a certain buffering and protective effect on organs; (5) Lymphatic tissue: It is an important part of the immune system, such as lymph nodes and spleen. It can filter lymph fluid, remove pathogens, play a key role in immune defense, and help the body resist disease invasion; (6) Epithelial tissue: It covers the surface of the body and the inner surface of various cavities in the body. For example, the epidermis of the skin can protect the body from external damage and also participate in absorption, secretion and other functions. For example, the villi epithelium of the small intestine can absorb nutrients; (7) Nervous tissue: It is composed of neurons and glial cells. Neurons can receive stimuli, integrate information and transmit nerve impulses, enabling the human body to perceive the outside world and control its own activities. For example, neurons in the brain process various sensory and cognitive information.

[0004] An organ is composed of various tissues combined in a specific order, possessing a definite form and function. For example, the heart is an organ composed of cardiac muscle, connective tissue, nerve tissue, and vascular tissue, responsible for pumping blood. A system is composed of multiple organs that work together to perform one or more physiological functions, combined in a specific order. For example, the digestive system includes the mouth, esophagus, stomach, and small intestine, which work together to digest food and absorb nutrients. An individual organism is a living organism whose different organs or systems coordinate to complete complex life activities. The individual is the basic unit of reproduction and heredity in organisms, maintaining the continuation and development of life with its unique structure and function. This demonstrates the complexity and diversity of the biological world.

[0005] Throughout the long history of biological evolution, the structure and function of some proteins have evolved alongside protein folding, development, and advancement, enabling individuals to adapt to ever-changing environments. For example, the protein structures and functions of deep-sea fish and birds in flight, as well as the function of the compound eyes of insects, are far beyond the reach of humans, who consider themselves the most advanced animals. This is a disadvantage that comes with the loss or deficiency of protein function during biological evolution.

[0006] Small molecule peptides are low-molecular-weight bioactive substances composed of two or more amino acids linked by peptide bonds. Numerous studies have shown that small molecule peptides possess a variety of physiological functions, such as regulating cellular aging, regeneration, controlling apoptosis, modulating immunity, improving cardiovascular health, and promoting cell repair. Due to their unique bioavailability and bioselectivity, small molecule peptides exhibit great potential in multiple fields, including nutritional supplements, healthcare, cosmetics, and medicine.

[0007] On the other hand, the human body's digestibility and utilization rate of food is extremely low, with some foods requiring less than 5% digestion and averaging around 20%. This doesn't even account for unfavorable factors such as individual differences in digestive system function, health status, and stomach capacity. A large amount of food resources are excreted as waste without being digested and absorbed, causing environmental pollution, energy waste, and ecological damage. This is particularly evident in protein utilization efficiency. The human digestive system contains a limited number of enzymes, thus there is an urgent need for peptides that can be directly digested, absorbed, and utilized to meet human nutritional and health needs.

[0008] With societal development, the accelerated pace and increased pressure of human life and work have led to a gradual increase in the risks of health problems, sub-health conditions, and diseases, which are becoming increasingly complex. It is common knowledge that the human body is composed of multiple proteins; however, whether this is due to a single key protein or the synergistic effect of multiple proteins remains a highly worthy subject of investigation in the field of protein science. Therefore, nutritional supplements and the consumption of polypeptides are beneficial to the human body, at least providing a form of immune protection.

[0009] Numerous methods for preparing peptides from animal tissues are disclosed in both patent literature and scientific research papers. Common methods include aqueous extraction, acid extraction, high-temperature low-pressure extraction, drying, alkali extraction, and enzymatic extraction. However, the bioactive peptides obtained by these methods are mostly denatured peptides. Furthermore, the diversity of animals and their tissue and organ structures must be considered, meaning a one-size-fits-all approach cannot be used. Simultaneously, various problems affecting product quality arise during production, such as column blockage, product yellowing, and low yield. Therefore, a novel and entirely new method for preparing bioactive peptides is urgently needed. III. Summary of the Invention

[0010] To achieve the objectives of the present invention as described above, in one aspect of this invention, a method for preparing small molecule active protein peptide lyophilized powder from animal tissues and organs is provided, wherein the method includes the following steps: taking animal tissues and organs, removing oil, washing, chopping, grinding and / or homogenizing, adjusting pH, enzymatic hydrolysis, filtration, vacuuming, ultrafiltration, nanofiltration, sterilization, and vacuum ultra-low temperature freeze drying.

[0011] The animals used in this invention are selected from one or more of the group consisting of annelids, mollusks, arthropods, fish, echinoderms, amphibians, reptiles, birds, and mammals.

[0012] Specifically, the animals are selected from one or more of the following groups: earthworms, sea cucumbers, pigs, wild boars, black pigs, suckling pigs, cattle, calves, yellow cattle, water buffalo, yaks, sheep (e.g., lambs, sheep, goats), alpacas, deer, farmed sika deer, horses, donkeys, mules, camels, free-range camels, rabbits, kangaroos, fish, shrimp, lobsters, Australian lobsters, crayfish, crabs, king crabs, mitten crabs, shellfish, scallops, oysters, snails, snails, sea urchins, abalone, clams, soft-shelled turtles, octopuses, squid, penfish, bullfrogs, chickens, ducks, geese, turkeys, pheasants, pigeons, partridges, quails, etc. Preferably, the animal is selected from one or more of the group consisting of earthworms, sea cucumbers, cattle, calves, yaks, sheep (e.g., lambs, sheep, goats), farmed sika deer, donkeys, free-range camels, fish, shrimp, lobsters, Australian lobsters, crabs, king crabs, shellfish, scallops, oysters, sea urchins, abalone, clams, soft-shelled turtles, octopuses, squid, pheasants, partridges, and quails. More preferably, the animal is selected from one or more of the group consisting of earthworms, sea cucumbers, cattle, calves, yaks, sheep, farmed sika deer, donkeys, free-range camels, fish, shrimp, crabs, shellfish, abalone, clams, soft-shelled turtles, and octopuses.

[0013] Animal tissues and organs used in this invention include, but are not limited to, carcass, meat, brain, heart, spleen, pancreas, kidney, lung, liver, eyes, ears, nose, tongue, lips, oral cavity, head, glands, chest, thymus, skin, swim bladder, fish swim bladder, muscle, skeletal muscle, smooth muscle, striated muscle, interosseous muscle, abdomen, buttock, back, marrow, bone marrow, spinal cord, tendon, flank, tendon, hoof tendon, tendon tip, whole tendon, interrupted tendon, tendon tendon, spur tendon, forehoof tendon, hindhoof tendon, tendon tendon, diaphragm, flank tendon, sirloin tendon, joint, ligament, esophagus, digestive tract, respiratory tract, membrane, periosteum, pleura, intestinal membrane, diaphragm, visceral membrane, stomach, intestine, testis, penis (whip). The vagina, uterus, gallbladder, appendix, placenta, bladder, neck, gills, gills, fish gills, miscellaneous organs, chest, tubes, blood vessels, aorta, larynx, esophagus, trachea, sperm, testes (sperm white), blood, egg, ovary, nerve, nerve fiber, limb, extremities, bone, hard bone, cartilage, rib, vertebrae, club bone, fingers, nails, feet, toes, tibia, fibula, palm, bear paw, diaphragm, intermuscular septum, tendon septum, bone septum, fleshy septum, compartment septum, claws, hump, camel hump, antlers, horns, spines, shark spines, thorns, spines (e.g., deer antlers), protrusions (e.g., camel hump, lion's nipple), scales, fins, spongy tissue, turtle skirt (water fish skirt), and tail, etc.

[0014] Another aspect of the present invention addresses problems arising in the production process by providing an oil removal process for animal tissues to solve the problem of product deterioration, which also causes pollution of production pipelines and increases operating costs. The ultimate goal is to improve the activity, yield, and content of bioactive peptides, while reducing industrial costs and improving industrial production efficiency.

[0015] Another aspect of the present invention is to improve the production process steps by performing vacuuming after filtering the protein peptide enzymatic hydrolysate, which avoids the frequent clogging of expensive imported ultrafiltration or nanofiltration columns, saves production costs and improves production efficiency. The ultimate goal is to improve the activity, yield and content of active peptides.

[0016] Specifically, the method mainly includes the following steps: (1) taking animal tissues and organs; (2) removing oil; (3) chopping; (4) adding 5 to 10 times the volume of water or salt-containing buffer solution after chopping and stirring evenly; (5) grinding and / or homogenizing; (6) adjusting pH and enzymatic hydrolysis; (7) centrifuging; (8) coarsely filtering the enzymatic hydrolysate with a filter membrane and collecting the filtrate; (9) vacuuming; (10) ultrafiltration of the filtrate with an ultrafiltration membrane of a predetermined molecular weight cutoff; (11) nanofiltration concentration of the filtrate with a nanofiltration membrane of a predetermined molecular weight cutoff, and then adding water to the original volume for nanofiltration desalting 3 to 5 times; (12) finally concentrating to a predetermined protein concentration; (13) sterilizing; (14) preparing lyophilized powder of small molecule active protein peptides of tissues and organs by ultra-low temperature vacuum freeze drying.

[0017] In another aspect of the invention, a method for preparing small molecule active protein peptides from animal tissues and organs is provided, wherein an extraction step is included before the grinding and / or homogenization step, the method comprising the following steps: taking animal tissues and organs, removing oil, washing, chopping, extracting, cleaning, grinding and / or homogenizing, enzymatic hydrolysis, centrifugation, filtration, vacuuming, ultrafiltration, nanofiltration, sterilization, and vacuum cryogenic freeze drying.

[0018] Preferably, the method mainly includes the following specific steps: (1) taking animal tissues and organs; (2) removing oil; (3) chopping; (4) extraction; (5) grinding and / or homogenizing; (6) adjusting pH and enzymatic hydrolysis; (7) centrifugation; (8) coarsely filtering the enzymatic hydrolysate with a filter membrane and collecting the filtrate; (9) vacuuming; (10) ultrafiltration of the filtrate with an ultrafiltration membrane of a predetermined molecular weight cutoff; (11) nanofiltration concentration of the filtrate with a nanofiltration membrane of a predetermined molecular weight cutoff, and then adding water to the original volume for nanofiltration desalting 3 to 5 times; (12) finally concentrating to a predetermined protein concentration; (13) sterilization; (14) preparing lyophilized powder of small molecule active protein peptides from tissues and organs by ultra-low temperature vacuum freeze drying.

[0019] In another aspect of the invention, a method for preparing small molecule active protein peptides from animal tissues and organs is provided, wherein an extraction step is further included after the grinding and / or homogenization step, the method comprising the following specific steps: taking animal tissues and organs, removing oil, washing, chopping, grinding and / or homogenizing, extraction, centrifugation, enzymatic hydrolysis, filtration, vacuuming, ultrafiltration, nanofiltration, sterilization, and vacuum cryogenic freeze drying.

[0020] Preferably, the method mainly includes the following steps: (1) taking animal tissues and organs; (2) removing oil; (3) chopping; (4) grinding and / or homogenizing; (5) extraction; (6) adjusting pH and enzymatic hydrolysis; (7) centrifugation; (8) coarsely filtering the enzymatic hydrolysate with a filter membrane and collecting the filtrate; (9) vacuuming; (10) ultrafiltration of the filtrate with an ultrafiltration membrane of a predetermined molecular weight cutoff; (11) nanofiltration concentration of the filtrate with a nanofiltration membrane of a predetermined molecular weight cutoff, and then adding water to the original volume for nanofiltration desalting 3 to 5 times; (12) finally concentrating to a predetermined protein concentration; (13) sterilization; (14) preparing lyophilized powder of small molecule active protein peptides from tissues and organs by ultra-low temperature vacuum freeze drying.

[0021] The advantage of this invention is that it provides three advanced process solutions for different animal tissues and organs, which helps those skilled in the art to make reasonable choices of technical process solutions when faced with the complex and varied composition and structural characteristics of animal tissues and organs.

[0022] A significant contribution of this invention to the prior art is the use of a step-by-step enzymatic hydrolysis method, namely a graded enzymatic hydrolysis method. By selecting the type of enzyme, the order of enzymes, and changing the appropriate temperature and pH, more tissues and organs are hydrolyzed into small molecule active peptides. This allows for control over the production of more target protein peptides through the manufacturing process, thereby achieving the objective of this invention.

[0023] Another advantage of this invention is the increased yield of small molecule bioactive protein peptides, achieving a yield (as a percentage of the body weight of the tissue or organ used) of approximately or more than 5%. In terms of protein content, this means that at least one-third of the total protein in the tissue or organ used is hydrolyzed into small molecule peptides.

[0024] Therefore, one aspect of the present invention is to provide a small molecule active protein peptide, wherein the active protein peptide prepared according to the method of the present invention contains 1 to 200 amino acids; said active protein peptide contains one or more amino acids in the group consisting of isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine, etc.

[0025] One advantage of this invention is that the protein peptide yield is expressed as the weight ratio of the obtained protein peptides to the total weight of the tissues and organs used, and the protein peptide yield is above 10 g / kg. Simultaneously, the activity of the protein peptides was tested; that is, when the protein concentration was 2.0 mg / ml, the active protein peptides exhibited an ACE inhibition rate of over 10% and an IC50 of less than 1.50 mg / mL. 50 .

[0026] The invention method and advanced manufacturing process steps are also applicable to the preparation of small molecule protein active peptides from other biological resources, such as plant resources (natural or artificially cultivated plants).

[0027] The advantages of this invention lie in the high activity, high purity, high yield, and high amino acid composition of the small molecule active protein peptides prepared by the production process of this invention. Simultaneously, the improved production process reduces industrial production costs and increases industrial production efficiency. The goal is to establish a necessary standardized process, summarize certain rules, and ensure the full and rational utilization of biological resources. The method of this invention is simple to operate, easy to implement, and suitable for large-scale industrial production. IV. Detailed Implementation Methods:

[0028] To provide a substantial understanding of the invention, certain aspects, modes, embodiments, variations, and features of the invention are described below with varying degrees of detail.

[0029] In this invention, the terms "living organism," "organism," "animal," "animal tissue," "animal organ," "tissue," "organ," "animal tissue organ," "animal organ tissue," "tissue organ," "organ tissue," "animal cell," "animal body," "part of an animal body," "its primary preparation," "intermediate product," or "final product" may have the same meaning in some contexts and may be used interchangeably.

[0030] In one embodiment of this aspect, a method for preparing small molecule active protein peptide lyophilized powder from animal tissues and organs is provided, wherein the method includes the following steps: taking animal tissues and organs, removing oil, washing, chopping, grinding and / or homogenizing, enzymatic hydrolysis, filtration, vacuuming, ultrafiltration, nanofiltration, sterilization, and vacuum ultra-low temperature freeze drying.

[0031] In one specific embodiment of the present invention, a method for preparing lyophilized protein peptide powder from animal tissues and organs is provided, which mainly includes the following steps: (1) taking animal tissues and organs; (2) removing oil; (3) chopping; (4) adding 5 to 10 times the volume of water or a salt-containing buffer solution after chopping and stirring evenly; (5) grinding and / or homogenizing; (6) adjusting pH and enzymatic hydrolysis; (7) centrifuging; (8) coarsely filtering the enzymatic hydrolysate with a filter membrane and collecting the filtrate; (9) vacuuming; (10) ultrafiltration of the filtrate with an ultrafiltration membrane of a predetermined molecular weight cutoff; (11) nanofiltration concentration of the filtrate with a nanofiltration membrane of a predetermined molecular weight cutoff, and then adding water to the original volume for nanofiltration desalting 3 to 5 times; (12) finally concentrating to a predetermined protein concentration; (13) sterilizing; (14) preparing lyophilized small molecule active protein peptide powder from tissues and organs by ultra-low temperature vacuum freeze drying.

[0032] The animals used in this invention can be derived from any kind of animal, including terrestrial or aquatic animals. The animals are generally edible, including livestock and poultry, and aquatic animals, which further include fish and general aquatic products. Therefore, the term "animal" in this document refers to all animals with physiological tissues and organs, and these tissues and organs can be used to prepare small molecule bioactive protein peptides.

[0033] Preferably, the animals used in this invention are selected from one or more of the group consisting of annelids, arthropods, mollusks, echinoderms, fish, amphibians, reptiles, birds, and mammals. Specifically, the animals are selected from one or more of the group consisting of earthworms, sea cucumbers, pigs, wild boars, black pigs, suckling pigs, cattle, calves, yellow cattle, water buffalo, yaks, sheep (e.g., lambs, sheep, goats), alpacas, deer, farmed sika deer, horses, donkeys, mules, camels, free-range camels, rabbits, kangaroos, fish, shrimp, lobsters, Australian lobsters, crayfish, crabs, king crabs, mitten crabs, shellfish, scallops, oysters, snails, snails, sea urchins, abalone, clams, soft-shelled turtles, octopuses, squid, penfish, bullfrogs, chickens, ducks, geese, turkeys, pheasants, pigeons, partridges, and quails. Preferably, the animal is selected from one or more of the group consisting of earthworms, sea cucumbers, cattle, calves, yaks, sheep (e.g., lambs, sheep, goats), farmed sika deer, donkeys, free-range camels, fish, shrimp, lobsters, Australian lobsters, crabs, king crabs, shellfish, scallops, oysters, sea urchins, abalone, clams, soft-shelled turtles, octopuses, squid, pheasants, partridges, and quails. More preferably, the animal is selected from one or more of the group consisting of earthworms, sea cucumbers, cattle, calves, yaks, sheep, farmed sika deer, donkeys, free-range camels, fish, shrimp, crabs, shellfish, abalone, clams, soft-shelled turtles, and octopuses.

[0034] All animals used in this invention are natural, domesticated, free-range, raised, and farmed, and are edible species permitted by laws and regulations, with appropriate adjustments made according to the dietary habits of various countries around the world.

[0035] The animal tissues and organs used in this invention can be derived from any kind of animal tissues and organs, including but not limited to carcass, meat, brain, heart, spleen, pancreas, kidney, lung, liver, eye, ear, nose, tongue, lips, oral cavity, head, glands, chest, thymus, skin, swim bladder, fish swim bladder, muscle, skeletal muscle, smooth muscle, striated muscle, interosseous muscle, abdomen, buttock, back, marrow, bone marrow, spinal cord, tendon, belly, tendon, hoof tendon, tendon tip, whole tendon, interrupted tendon, tendon tendon, spit tendon, fore hoof tendon, hind hoof tendon, tendon tendon, diaphragm, belly tendon, sirloin tendon, ham tendon, joint, ligament, esophagus, digestive tract, respiratory tract, membrane, periosteum, pleura, intestinal membrane, diaphragm, visceral membrane, stomach, intestine, testis, penis (whip), vagina, uterus, gallbladder, appendix, placenta, bladder, neck, gills, fish gills, miscellaneous, chest, One or more of the following tissues and organs are used in the method of the present invention: tubes, blood vessels, aorta, larynx, esophagus, trachea, sperm, testes (sperm white), blood, eggs, ovaries, nerves, nerve fibers, limbs, bones, hard bones, cartilage, ribs, vertebrae, club bones, fingers, nails, feet, toes, tibia, fibula, palms, bear paws, diaphragms, muscle septa, tendon septa, bone septa, flesh septa, compartment septa, claws, humps, camel humps, antlers, horns, spines, shark spines, thorns, spines (e.g., deer antlers), protrusions (e.g., camel humps, lion nipples), scales, fins, spongy tissue, turtle skirt (water fish skirt), and tail; preferably, the animal tissues and organs used in the method of the present invention include, but are not limited to, one or more of the following tissues and organs: flesh, brain, marrow, bone marrow, spinal cord, muscle, skeletal muscle, smooth muscle, striated muscle, abdomen, buttocks, back, eggs, sperm, and blood.

[0036] All animal tissues and organs used in this invention have undergone strict food hygiene and safety inspections and are qualified, edible animal tissues or organs, with appropriate adjustments made according to the dietary habits of various countries around the world.

[0037] External contamination: Food products are contaminated by microorganisms other than their raw materials and semi-finished products during processing, such as bacterial contamination in water, secondary bacterial contamination in the air, secondary cross-infection from employee hands, equipment, containers, tools, and turnover boxes, and contaminated packaging materials. Water sterilization generally uses ultraviolet light or ozone disinfection; airborne bacterial elimination uses plasma diffusion technology, dynamic food sterilizers, or air purification systems; hand disinfection generally involves washing hands with detergent, drying them with hot air, and then adding 75% acetic acid to an automatic sensor hand sanitizer dispenser, which automatically sprays out the disinfectant to disinfect the hands, allowing them to enter the workshop directly.

[0038] Internal contamination: This refers to bacteria naturally present in raw materials and semi-finished products. It can be categorized into different types, such as baked goods, beverages, seafood, snack foods, convenience foods, beer, soy products, and nutritional supplements, each requiring different sterilization equipment and technologies.

[0039] In some embodiments of the present invention, the workshop or equipment used for product production is disinfected. The equipment includes pipes, water sources, etc. The disinfection equipment used includes, but is not limited to, ultraviolet disinfection, ozone disinfection, and disinfection treatment of the product or intermediate product. The sterilization includes, but is not limited to, microwave sterilization, gene sterilization, electron beam sterilization, magnetic sterilization, resistance heating sterilization, pasteurization, ultra-high temperature (UHT) sterilization, superheated steam sterilization, irradiation sterilization, ultra-high pressure sterilization, ultrasonic sterilization, and other sterilization techniques. Preferably, the sterilization means and methods do not damage the activity or spatial structure of proteins, such as ultra-high pressure sterilization.

[0040] The term "fresh" refers to the immediate removal of the target organ or tissue from a live animal after slaughter, for direct use in production. The shorter the time from slaughter to use in the production process, the better. Materials used include various pure, natural, organic, and pollution-free animals, as examples only, but also including various animals. Specifically, it refers to edible parts of animals favored by consumers, including but not limited to various tissues and organs. Under low-temperature production conditions, utilizing advanced technologies and processes to maintain the full activity of proteins, various fully active protein peptide ultra-low temperature vacuum freeze-dried powders are manufactured. These powders contain multiple functional substances, such as immune-enhancing factors, hematopoietic factors, regenerative factors, small molecule active protein peptides, repair peptides, fibrin, collagen, elastin, cell signaling regulators, and other active proteins or enzymes. These are all indispensable factors for the growth, development, and repair of cells, tissues, or organs. From material selection to vacuum ultra-low temperature freeze-drying for the preparation of crystalline freeze-dried protein active peptides, every step of the production process utilizes cutting-edge protein biochemical engineering techniques to maintain the protein peptides in a fully active state. The resulting product retains the protein tree's three-dimensional structure (3D), preserving its original biological activity and ensuring that the protein peptides' function is not damaged or lost due to the manufacturing process. This also allows for direct absorption through mucous membranes. Extracting animal protein peptide essence using ultra-low temperature freeze-drying technology can effectively enhance the human body's absorption of animal protein peptides, achieving the goal of preventing and / or alleviating and / or treating and / or curing pathological symptoms or diseases caused by the loss and degeneration of human tissues and organs.

[0041] The connective tissue used in this invention can be fresh, freshly refrigerated, quick-frozen or frozen, coarsely processed, finely processed, dried or salted, preferably fresh, freshly refrigerated, quick-frozen or frozen; more preferably fresh or freshly refrigerated or express-frozen; and most preferably fresh.

[0042] In another embodiment of the present invention, a method for cryopreserving animal tissues and organs without denaturation is provided. The animal tissues and organs can be cryopreserved whole, or they can be prepared into several large pieces or even chopped for preservation. No additives may be added during preservation, or additives may be added to prevent cryopreservation denaturation.

[0043] This invention can use fresh animal tissues and organs directly or animal tissues and organs that have been cryopreserved.

[0044] The term "low-temperature preservation" includes both "refrigerated preservation" and "frozen preservation".

[0045] In another embodiment, refrigeration can preserve the animal tissues and organs at temperatures below about 10°C, for example, between about 0°C and about 9°C; between about 1°C and about 5°C; between about 2°C and about 6°C; or between about 2°C and about 4°C.

[0046] In another embodiment, the animal tissues and organs can be cryopreserved at temperatures below about 0°C (e.g., liquid nitrogen), for example, below 0°C to about -180°C; at about -20°C to about -180°C; at about -40°C to about -180°C; at about -80°C to about -180°C; at about -20°C to about -80°C; at about -25°C to about -80°C; or at about -25°C to about -40°C.

[0047] The term "rapid cryogenic freezing" refers to the process of instantly cooling animal tissues and organs by spraying or directly immersing them in ultra-low-temperature liquids such as liquid nitrogen at -180°C. The treated tissues and organs are then frozen and preserved at the desired temperature; this method can also be called "cooling shock."

[0048] The degreasing process of animal tissues and organs is often overlooked and neglected. Peptide products often suffer from mechanical or pipeline contamination due to inadequate initial treatment of the grease adhering to the materials, resulting in yellow or dark-colored products that not only affect their appearance and color but also produce unpleasant odors. Therefore, the initial raw material treatment, especially the removal of the surface grease layer, is crucial. Many methods exist for degreasing animal tissues and organs, such as boiling and chemical treatment with strong alkalis. However, these methods can cause irreversible denaturation of the animal tissues and organs, rendering the prepared animal tissue protein peptides inactive. The degreasing method used in this invention employs physical tools or machinery to remove the grease present on the animal tissues and organs; or a freezing method, preferably rapid low-temperature freezing, is used to freeze and solidify the oil film or grease layer on the surface of the animal tissues and organs, followed by removal using physical tools, mechanical slicers, or cutting machines. The pretreatment of animal tissues and organs is a very important technical issue, entirely determined by the characteristics of the animal tissues and organs. There is no fixed model, and it is often neglected or uniformly applied, resulting in reduced activity, reduced yield, and low efficiency. Therefore, the present invention provides a practical and feasible pretreatment method for animal tissues and organs.

[0049] In another specific embodiment of the invention, various meat slicers, meat grinders, choppers, and tumblers are used in combination to cut animal tissues and organs into slices, chunks, shreds, strips, mince, and / or mechanically pulverize them into a paste, preferably powder or paste. This shape makes the tissues and organs more suitable for the following processing steps. Multiple machines can be used simultaneously. Whether to continue homogenization or grinding depends primarily on the characteristics and particle size (also referred to as "target particle size") of the selected animal tissues and organs. For example, relatively soft brain tissue is homogenized; hard animal bones or cartilage can be initially crushed using a bone crusher, then acid extracted, and finally further finely ground into powder to facilitate enzymatic hydrolysis. The target particle size of various bone powders is usually expressed in mesh (International Meters).

[0050] In another specific embodiment of the present invention, the tissues and organs are cleaned using a liquid, which can be 1 to 10 times or more than 10 times the volume of the cleaning solution. The ambient temperature can be controlled between 0 and 25°C, or at any temperature, preferably below 18°C, below 16°C, or even in a cold room below 6°C. Those skilled in the art can select the appropriate cleaning method based on the observed state of the tissues and organs. Cleaning can further remove floating fat and blood from connective tissue, usually two or more times, such as three, four, or five times. The cleaning solution can be water or a buffer solution, using the buffer to stabilize the pH value of the tissue or organ fluid. The buffer solution used is any buffer solution with a pH range between 6.0 and 8.2.

[0051] The cleaning solution can be either clean water or a buffer solution. The term "clean water" refers to water that is free of any bacteria, viruses, and impurities. It can be "distilled water," "mineral water," "purified water," "filtered water," "natural water," or "deionized water." In some cases, these terms have the same meaning and can be used interchangeably.

[0052] In another embodiment of the invention, a buffer solution is used to stabilize the pH of a tissue or organ. The buffer solution used is any buffer solution within a predetermined buffer range. Examples include MES (2-morpholinoethanesulfonic acid) buffer, Bis-Tris buffer, HEPES buffer, PIPES buffer, MOPS buffer, Tricine buffer, TEA (triethanolamine) buffer, glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer, disodium hydrogen phosphate-citric acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, malic acid-sodium citrate buffer, malic acid-sodium malate buffer, citric acid-sodium malate buffer, acetic acid-sodium acetate buffer, potassium hydrogen phthalate-sodium hydroxide buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, sodium barbital-hydrochloric acid buffer, Tris-hydrochloric acid buffer, and any saline solution capable of adjusting physiological saline. It is preferable to use a broad-spectrum, pH-adjustable, and edible buffer solution, such as glycine-hydrochloric acid buffer, disodium hydrogen phosphate-citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, malic acid-sodium citrate buffer, malic acid-sodium malate buffer, citric acid-sodium malate buffer, acetic acid-sodium acetate buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, or any physiologically adjustable saline solution.

[0053] In one specific embodiment of the invention, the buffer solution used is a phosphate buffer, typically with a concentration of 1–100 mM, sometimes exceeding this range. For example, phosphate concentrations are 1–40 mM, 1–30 mM, 1–25 mM, 2–25 mM, or 5–20 mM. Suitablely, it contains 0.1 M–2 M NaCl, for example: 0.01 M–2 M, 0.01 M–1.5 M, 0.01 M–1 M, 0.01 M–0.5 M, or 0.01 M–0.3 M. In one specific aspect of the invention, the buffer solution is a 1-20 mM phosphate buffer solution containing 50 mM to 0.5 M NaCl, with a pH adjustable range of 2-11, such as pH 3-10, pH 4-9.5, pH 5-9, pH 6-8.5, pH 6.5-8.5, pH 6.5-8, pH 6.5-7.5, pH 6.8-7.5, and pH 6.8-7.0.

[0054] The term "pulping" refers to the process of refining mechanically pulverized animal tissues and organs into smaller particles in a liquid, including grinding and / or homogenizing.

[0055] The term "mesh count" refers to the number of pores per inch. A higher mesh count indicates a smaller pore size. Generally, mesh count × pore size (in micrometers) = 15000. For example, a 400-mesh sieve has an pore size of approximately 38 micrometers; a 500-mesh sieve has an pore size of approximately 30 micrometers. The relationship between pore size and mesh count, calculated using the aforementioned formula, is shown in the table below:

[0056] The above-mentioned pulverization steps, through the selection of mechanical combinations, should aim to achieve a sufficiently small target particle size. For example, less than 10 mesh, less than 50 mesh, less than 100 mesh, less than 200 mesh, less than 500 mesh, or less than 1000 mesh. If the target particles are small enough, further processing with pulping can be performed. The selection of processing steps for various animal organ tissues is mainly based on the performance parameters of the machinery and the observation of the particle size in the desired organ tissue.

[0057] In another specific embodiment of the invention, the powdered or granulated particles are pulped. The pulping machine is a novel wet grinding device. Domestically produced equipment includes grinding wheel mills and stainless steel mills; considering hygiene and food safety factors, stainless steel mills are more commonly used. The grinding fineness can reach 80–200 mesh, with a molecular particle size of approximately 80–200 μm. The mill speed is generally below 4500 rpm.

[0058] The grinding time can be 30 minutes, 25 minutes, 20 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 3 minutes, 2 minutes, 1 minute, or less than 1 minute, such as 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds.

[0059] In another specific embodiment of the present invention, in order to improve the extraction of proteins from animal tissues or organs, the proteins are homogenized into powder or granules. A homogenizer is a commonly used device for extracting proteins from animal tissues and organs. Commonly used homogenizers include the PRO Multi-Prep multi-sample homogenizer (USA), the SB-1 brushless silent homogenizer, the PR025D digital homogenizer; the IKEA homogenizer series (Switzerland); and the Japanese homogenizer series, etc. These machines all share common characteristics: the homogenizer speed is generally higher than 5000 rpm, and through sharp and sturdy metal blades, and with a certain rotation angle and arc design, the tissue is broken down to a degree that can penetrate a filter membrane of 200 mesh or less in an instant.

[0060] The homogenization time can be less than 20 minutes, for example, 20 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 3 minutes, 2 minutes, 1 minute, or even less than 1 minute, for example, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds.

[0061] In one embodiment of this aspect, a method for preparing small molecule active protein peptide lyophilized powder from animal tissues and organs is provided, wherein an extraction step is further included before the grinding and / or homogenization step, the method comprising the following steps: taking animal tissues and organs, degreasing, washing, chopping, extraction, cleaning, grinding and / or homogenizing, enzymatic hydrolysis, centrifugation, filtration, vacuuming, ultrafiltration, nanofiltration, sterilization, and vacuum ultra-low temperature freeze drying.

[0062] In another specific embodiment of the present invention, a method for preparing lyophilized protein peptide powder from animal tissues and organs is provided, which mainly includes the following steps: (1) taking animal tissues and organs; (2) removing oil; (3) chopping; (4) extraction; (5) grinding and / or homogenizing; (6) adjusting pH and enzymatic hydrolysis; (7) centrifugation; (8) coarsely filtering the enzymatic hydrolysate with a filter membrane and collecting the filtrate; (9) vacuuming; (10) ultrafiltration of the filtrate with an ultrafiltration membrane of a predetermined molecular weight cutoff; (11) nanofiltration concentration of the filtrate with a nanofiltration membrane of a predetermined molecular weight cutoff, and then adding water to the original volume for nanofiltration desalting 3 to 5 times; (12) finally concentrating to a predetermined protein concentration; (13) sterilization; (14) preparing lyophilized small molecule active protein peptide powder from tissues and organs by ultra-low temperature vacuum freeze drying.

[0063] The animal tissues and organs used in this invention can be derived from any kind of animal tissues and organs, including but not limited to carcass, meat, brain, heart, spleen, pancreas, kidney, lung, liver, eye, ear, nose, tongue, lips, oral cavity, head, glands, chest, thymus, skin, swim bladder, fish swim bladder, muscle, skeletal muscle, smooth muscle, striated muscle, interosseous muscle, abdomen, buttock, back, marrow, bone marrow, spinal cord, tendon, flank, tendon, hoof tendon, tendon shank, tendon tip, whole tendon, interrupted tendon, tendon tendon, spur tendon, forehoof tendon, hindhoof tendon, tendon tendon, diaphragm, flank tendon, etc. Cold tendons, tendons, joints, ligaments, esophagus, digestive tract, respiratory tract, membranes, periosteum, pleura, intestinal membrane, diaphragm, visceral membranes, stomach, intestines, testes, penis (whip), vagina, uterus, gallbladder, appendix, placenta, bladder, neck, gills, gills, fish gills, miscellaneous, chest, canal, blood vessels, aorta, larynx, esophagus, trachea, sperm, testes (serum), blood, egg, ovary, nerve, nerve fiber, limbs, extremities, bones, hard bone, cartilage, ribs, vertebrae, club bones, fingers, nails, feet, toes, tibia, fibula, palms, bear paws One or more of the following tissues and organs: septum, muscle septum, tendon septum, bone septum, flesh septum, compartment septum, claws, humps, camel humps, antlers, horns, spines, shark spines, thorns (e.g., deer antlers), protrusions (e.g., camel humps, lion's milk), scales, fins, spongy tissue, turtle skirt (soft-shelled turtle skirt), and tail; preferably, including but not limited to, the carcass, heart, spleen, pancreas, kidneys, lungs, liver, eyes, ears, nose, tongue, lips, oral cavity, head, glands, thorax, thymus, esophagus, digestive tract, respiratory tract, membranes, periosteum, pleura, intestinal membrane, septum. Membranes, visceral membranes, testes, vagina, uterus, gallbladder, appendix, placenta, bladder, neck, gills, gills, fish gills, miscellaneous organs, chest, ovaries, nerves, nerve fibers, limbs, bones, hard bones, cartilage, ribs, vertebrae, club bones, fingers, nails, feet, toes, tibia, fibula, claws, hump, camel hump, antlers, horns, spines, shark spines, thorns, spines (e.g., deer antlers), protrusions (e.g., camel hump, lion's nipple), scales, fins, spongy tissue, turtle skirt (water fish skirt), and tail, etc.

[0064] In some embodiments of the present invention, tissue and organ serous fluid is extracted. The extraction is an acid extraction, and the acid used in the present invention can be any organic or inorganic acid, such as one or more selected from the group consisting of hydrochloric acid, boric acid, nitric acid, nitrous acid, carbonic acid, sulfuric acid, sulfurous acid, chloric acid, hypochlorous acid, dichromic acid, bromic acid, hypobromic acid, iodic acid, hypoiodic acid, lactic acid, citric acid, formic acid, acetic acid (also called acetic acid or glacial acetic acid), propionic acid, butyric acid, valeric acid, hexanoic acid, caprylic acid, capric acid, lauric acid, cinnamic acid, succinic acid, tartaric acid, maleic acid, malic acid, oxalic acid, malonic acid, glutaric acid, adipic acid, pimelic acid, succinic acid, azelaic acid, etc. Preferably, the acid used in the extraction process of the present invention is one or more selected from the group consisting of hydrochloric acid, lactic acid, malic acid, citric acid, formic acid, acetic acid (also called acetic acid or glacial acetic acid), lauric acid, cinnamic acid, succinic acid, tartaric acid, maleic acid, oxalic acid, etc. More preferably, the acid used in the extraction process of this invention is one or more selected from the group consisting of hydrochloric acid, lactic acid, malic acid, boric acid, nitric acid, citric acid, acetic acid (also called acetic acid or glacial acetic acid), oxalic acid, etc. Citric acid, malic acid, and glacial acetic acid are preferred; glacial acetic acid is most preferred.

[0065] The concentration of the acid depends on the specific properties of the acid. When using acetic acid, the final concentration range can be 0.01% to 99.9%, for example, 0.01% to 90%, 0.01% to 75%, 0.01% to 60%, 0.01% to 50%, 0.01% to 30%, 0.1% to 30%, 0.5% to 30%, 1% to 30%, 1% to 20%, 1% to 10%, 2% to 10%, and 3% to 10%. The extraction pH range is 1 to 6.8, for example, pH 6.7, 6.5, 6.2, 6.0, 5.8, 5.5, 5.0, 4.5, 4.0, and 3.5. Sometimes it may even be lower than 3.0, for example, pH below 3.0, 2.5, 2.0, or even below 2.0, for example, below 1.0. The extraction temperature can be selected to be at room temperature or below room temperature, below 20°C, for example below 10°C, below 6°C, below 4°C, or close to 0°C. The extraction time depends on the extraction temperature, extraction effect, and the properties of the small molecule active protein peptides. For example, extraction can be performed for 1, 2, 3, 4, 5, or 6 days within 7 days. In some embodiments, the extraction time is controlled within 24 hours, for example 0.5–24 hours, 1–20 hours, or 2–12 hours.

[0066] In another embodiment of this aspect, a method for preparing small molecule active protein peptide lyophilized powder from animal tissues and organs is provided, wherein the extraction process step is placed after the grinding and / or homogenization step. The method includes the following steps: taking animal tissues and organs, removing oil, washing, chopping, grinding and / or homogenizing, extraction, centrifugation, enzymatic hydrolysis, filtration, vacuuming, ultrafiltration, nanofiltration, sterilization, and vacuum ultra-low temperature freeze drying.

[0067] In another specific embodiment of the present invention, a method for preparing lyophilized protein peptide powder from animal connective tissue is provided, which mainly includes the following steps: (1) taking animal tissues and organs; (2) removing oil; (3) chopping; (4) grinding and / or homogenizing; (5) extraction; (6) adjusting pH and enzymatic hydrolysis; (7) centrifugation; (8) coarsely filtering the enzymatic hydrolysate with a filter membrane and collecting the filtrate; (9) vacuuming; (10) ultrafiltration of the filtrate with an ultrafiltration membrane of a predetermined molecular weight cutoff; (11) nanofiltration concentration of the filtrate with a nanofiltration membrane of a predetermined molecular weight cutoff, and then adding water to the original volume for nanofiltration desalting 3 to 5 times; (12) finally concentrating to a predetermined protein concentration; (13) sterilization; (14) preparing lyophilized small molecule active protein peptide powder from tissues and organs by ultra-low temperature vacuum freeze drying.

[0068] The animal tissues and organs used in this invention can be derived from any kind of animal tissues and organs, including but not limited to carcass, meat, brain, heart, spleen, pancreas, kidney, lung, liver, eye, ear, nose, tongue, lips, oral cavity, head, glands, chest, thymus, skin, swim bladder, fish swim bladder, muscle, skeletal muscle, smooth muscle, striated muscle, interosseous muscle, abdomen, buttock, back, marrow, bone marrow, spinal cord, tendon, belly, tendon, hoof tendon, tendon tip, whole tendon, interrupted tendon, tendon Tendons, shin tendons, foreleg tendons, hind leg tendons, ligaments, diaphragm, brisket tendons, sirloin tendons, braised tendons, joints, ligaments, esophagus, digestive tract, respiratory tract, membranes, periosteum, pleura, intestinal membrane, diaphragm, visceral membranes, stomach, intestines, testes, penis (whip), vagina, uterus, gallbladder, appendix, placenta, bladder, neck, gills, fish gills, miscellaneous, chest, canal, blood vessels, aorta, larynx, esophagus, trachea, sperm, testes (sperm white), blood, eggs, ovaries, nerves The tissues and organs include, but are not limited to, nerve fibers, limbs, bones, hard bones, cartilage, ribs, vertebrae, club bones, fingers, nails, feet, toes, tibia, fibula, palms, bear paws, diaphragms, muscle septa, tendon septa, bone septa, flesh septa, compartment septa, claws, humps, camel humps, antlers, horns, spines, shark spines, thorns, spines (e.g., deer antlers), protrusions (e.g., camel humps, lion nipples), scales, fins, spongy tissue, turtle skirt (soft-shelled turtle skirt), and tail; preferably, Including but not limited to one or more of the following tissues and organs: skin, swim bladder, fish swim bladder, interosseous muscles, tendons, brisket, tendons, hoof tendons, slab tendons, slab tendon tips, whole slab tendons, interrupted tendons, tendon tendons, spit tendons, forehoof tendons, hindhoof tendons, tendon tendons, diaphragm, brisket tendons, sirloin tendons, hamstring tendons, joints, ligaments, stomach, intestines, penis (whip), canals, blood vessels, aorta, larynx, esophagus, trachea, palm, bear paw, diaphragm, muscle septum, tendon septum, bone septum, flesh septum, and compartment septum.

[0069] The term "separation" refers to the process of separating proteins from a solution, either dissolved or suspended. This can be achieved through centrifugation, filtration, or salting out. A common method is neutral salt precipitation, where the neutral salt can be ammonium sulfate, sodium sulfate, sodium chloride, potassium chloride, or other neutral salts. The typical method involves adding powdered neutral salt to the liquid while stirring, then allowing all the protein to precipitate. After precipitation, the mixture is filtered or centrifuged to collect more protein. A salting-out curve can be plotted, with the horizontal axis representing the amount of salt added (in grams or kilograms) and the vertical axis representing the amount of protein precipitated. The final concentration of the added salt is typically 0.01%–99.9%, for example, 0.1%–90%, 1%–75%, 2%–60%, 5%–60%, 10%–60%, 20%–60%, 30%–60%, etc.

[0070] The pH of crude small-molecule bioactive protein peptides can be adjusted to a range of acidic, neutral, or alkaline, specifically 1–11, such as 2–11, 2–10, 2–9, 3–9, 4–9, 5–9, 6–9, 6.5–9, 6.5–8.5, 6.8–8.5, 6.8–8, or 6.8–7.5. Choosing a suitable pH is not the primary factor in enzymatic hydrolysis; the key is selecting a suitable combination of enzymes to improve the hydrolysis efficiency and yield more of the desired small-molecule bioactive protein peptides.

[0071] The terms “a,” “an,” and “the”: Unless otherwise specified, the singular forms “a,” “an,” and “the” used in this description and the appended claims include the plural references. For example, “an animal brain” refers to a single animal brain, such as a pig brain; two different animal brains, such as a pig brain and a cow brain; or a combination of more than one animal brain, such as a pig brain, a cow brain, and a deer brain, etc.

[0072] In one specific embodiment of the present invention, the process includes an enzymatic hydrolysis step of the protein liquid. The enzyme used for hydrolysis is selected from one or more combinations of the group consisting of pepsin, chymotrypsin, trypsin, cathepsin, carboxypeptidase, aminopeptidase, thiol protease, serine protease, aspartic protease, glutamine protease, serine protease, cysteine ​​protease, metalloproteinase, basic protease, neutral protease, acidic protease, proteinase K, fig protease, papain, bromelain, flavor protease, complex protease, and subtilisin. Preferably, the enzyme used for hydrolysis is selected from one or more combinations of the group consisting of pepsin, chymotrypsin, trypsin, cathepsin, thiol protease, serine protease, aspartic protease, glutamine protease, serine protease, cysteine ​​protease, neutral protease, proteinase K, papain, bromelain, flavor protease, complex protease, and subtilisin. More preferably, the enzyme used for enzymatic hydrolysis is selected from one or more combinations of pepsin, chymotrypsin, trypsin, cathepsin, serine protease, neutral protease, proteinase K, papain, bromelain, flavor protease, complex protease, and subtilisin. For certain specific tissues and organs, a single protease may be most effective. Determining which protease to use for unfamiliar tissues and organs requires inventive effort similar to that of this invention. Therefore, one inventive point of this invention is to solve the enzymatic hydrolysis problem for all tissues and organs by finding suitable hydrolysis times and temperatures.

[0073] When using two or more proteases for enzymatic hydrolysis, adding the enzymes in different proportions, simultaneously, or at different times (i.e., sequentially) is a preferred method to improve hydrolysis efficiency. The weight ratio of the different enzymes can be selected from 1:1 to 100, for example, 1:1 to 90, 1:1 to 75, 1:1 to 50, 1:1 to 25, 1:1 to 10, 1:1 to 5, 1:1 to 2, or added in equal amounts. The time intervals for adding various enzymes can be selected from 0 to 48 hours, 0 to 24 hours, 0 to 12 hours, 0 to 6 hours, 0 to 4 hours, 0 to 2 hours, 0 to 1.5 hours, 0 to 1 hour, 0 to 0.5 hours, 0 to 0.25 hours, etc.

[0074] The total amount of enzymes used can be any value relative to the weight of the tissues and organs, but it is generally not less than 1‰, for example, not less than 2‰, 3‰, 4‰, 5‰, 6‰, 7‰, 8‰, 9‰, 1%, 2%, 3%, 5%, 7%, 10%, etc.

[0075] In the enzymatic hydrolysis process, the temperature of the animal tissue and organ slurry is first adjusted to 0–65°C, and the total hydrolysis time is 0.1–48 hours. For example, in the enzymatic hydrolysis process, the temperature of the animal tissue and organ slurry is first adjusted to 0–55°C, and the hydrolysis time is 1–24 hours. Suitablely, in the enzymatic hydrolysis process, the temperature of the animal tissue and organ slurry is first adjusted to 10–55°C, and the hydrolysis time is 2–18 hours. More preferably, in the enzymatic hydrolysis process, the temperature of the animal tissue and organ slurry is first adjusted to 20–55°C, and the hydrolysis time is 1–16 hours. Preferably, in the enzymatic hydrolysis process, the temperature of the animal tissue and organ slurry is first adjusted to 30–50°C, and the hydrolysis time is 4–12 hours.

[0076] The enzyme hydrolysate can be separated by centrifugation or filtration.

[0077] Another specific embodiment of the present invention employs centrifugation for sample separation. Centrifugation can be performed using various commonly used laboratory centrifugation equipment, such as vacuum centrifuges or non-vacuum centrifuges. Based on rotational speed, centrifuges can be categorized as low-speed, high-speed, and ultra-speed centrifuges. Based on whether refrigeration is used, centrifuges can be classified as refrigerated centrifuges or ambient-temperature centrifuges. Centrifuges commonly used in industrial production include, for example, high-speed continuous centrifuges.

[0078] Centrifugal force is usually calculated in grams to set the rotational speed. Centrifugal force can be above 100,000g, or between 1,000g and 20,000g, for example, between 1,000g and 10,000g, specifically 1,500g, 2,000g, 3,000g, 5,000g, 6,000g, 7,000g, 8,000g, and 9,000g.

[0079] Centrifugation can also be expressed in revolutions per minute (rpm). Depending on the nature of the sample being centrifuged, speeds of 1000, 2000, 3000, 5000, 10000 or more, 15000 or more, 20000 or even 100000 or more are possible.

[0080] The centrifugation temperature can be selected to be at room temperature or below room temperature, for example below 20°C or below 15°C, preferably below 10°C, more preferably 6°C or below 4°C.

[0081] Centrifugation time can be any time, but it should not be too long, as this will cause excessive precipitation of impurities and reduce yield. Also, excessively long centrifugation times can easily lead to collagen denaturation. Generally, a time within 1 hour is chosen, such as 45 minutes, 30 minutes, 15 minutes, 10 minutes, or 5 minutes.

[0082] Centrifugation can effectively separate the product into two parts: supernatant and precipitate, which can then be used for their respective purposes.

[0083] A key technical measure in this invention is the improvement of the technical process steps, namely, vacuuming the filtered sample. The vacuum pump used in this invention is a device that uses mechanical, physical, chemical, or physicochemical methods to extract gas from a container to achieve a vacuum effect. Common types of vacuum pumps include dry screw vacuum pumps, water ring pumps, reciprocating pumps, slide valve pumps, rotary vane pumps, Roots pumps, and diffusion pumps. Vacuuming the sample can be done by performing small-volume, multiple vacuuming operations, or by performing large-volume, small-volume vacuuming operations, starting with a weak vacuum and gradually increasing the intensity. Simultaneously, careful observation and adjustment of the vacuuming time, ambient temperature, and vacuum intensity are crucial to maintain appropriate vacuuming conditions and prevent liquid spillage and loss. Existing technologies involve vacuuming the sample during ultrafiltration or nanofiltration within ultrafiltration or nanofiltration columns, resulting in column blockage. Furthermore, these columns are often professionally manufactured, with long ordering and delivery cycles, and are mostly imported, making them relatively expensive. Blockage causes production stoppages, leading to losses. Therefore, it is urgent to find the root cause of the problem and a key technical solution.

[0084] In another embodiment of the invention, a method for filtering and nanofiltration desalting crude highly active proteins or protein peptides is provided. The filtration employs a plate and frame filter press and a gauze filter. Commonly used plate and frame filter presses include manual, mechanical, and hydraulic types. The filter press is typically equipped with a filter membrane, the size of which can be adjusted arbitrarily, for example, 10 micrometers, 5 micrometers, 2 micrometers, 1 micrometer, 0.45 micrometers, 0.2 micrometers, 0.1 micrometers, 0.05 micrometers, etc. Ultrafiltration and nanofiltration are defined based on the molecular weight of the protein or protein peptide. Ultrafiltration machines are typically equipped with ultrafiltration membranes, the size of which is set according to the desired protein molecule size. They can retain large proteins of any molecular weight, typically between 2,000 and 1,500,000. Examples of suitable molecular weight ranges include 2,000 to 1,000,000, 2,000 to 750,000, 2,000 to 500,000, 2,000 to 300,000, 2,000 to 200,000, 2,000 to 150,000, and 2,000 to 10,000. The concentrations range from 0 to 3000,000, 5000 to 100000, 8000 to 100000, 10000 to 100000, 15000 to 100000, 20000 to 100000, 20000 to 80000, 20000 to 60000, 20000 to 50000, 20000 to 40000, 20000 to 30000, and 20000 to 25000. Generally, nanofiltration is used simultaneously for concentration and desalination. Commonly used nanofiltration machines include hollow fiber column nanofiltration machines and reverse osmosis nanofiltration machines. Nanofiltration machines are typically equipped with nanofiltration membranes, the size of which is set according to the size of the protein molecules to be retained. Typically, the molecular weight is between 50 and 3000, for example, between 100 and 3000, between 100 and 2500, between 200 and 2500, between 200 and 2000, between 200 and 1500, between 200 and 1200, between 200 and 1000, between 200 and 800, between 200 and 600, between 200 and 400, etc.

[0085] In one embodiment of the present invention, the obtained protein peptides are detected, quantified, and qualitatively analyzed. Detection methods include: Van Gieson staining, picric acid-Sirius red-polarized light method, and composite staining methods (including a composite staining method consisting of ponceau S-picric acid and resorcinol-fuchsine). Quantitative analyses include: Kjeldahl method, biuret method, ultraviolet absorption method, Coomassie brilliant blue method, Sirius red method, FPLC, and HPLC. Qualitative analyses include: amino acid sequencing and activity detection methods. Commonly used methods for protein amino acid determination include the Edman assay and mass spectrometry. Mass spectrometers include the British Biochrom 30+ fully automated amino acid analyzer, the (HITACHI) Hitachi L-8900 fully automated amino acid analyzer, the British AA4300 amino acid analyzer, the German Manmerbohr A300 automated amino acid analyzer, the German Sykam fully automated and semi-automated amino acid analyzers, the German Sykam+S433D, the American ABI Procise™ 491 protein sequencer, circular dichroism and parallax scanning calorimeters, protein dynamics analyzers, electron microscopes, NMR, protein three-dimensional structure diffractometers, and the Japanese Shimadzu PPSQ-31A automated protein sequencer, etc.

[0086] In some embodiments of the present invention, the protein content of the final product or intermediate active peptides is determined, and the content is usually expressed in mg / ml. For example, it is between 0.001 and 100,000 mg / ml, 0.01 and 100,000 mg / ml, 0.1 and 10,000 mg / ml, 1 and 1,000 mg / ml, 1 and 500 mg / ml, 1 and 100 mg / ml, 10 and 100 mg / ml, 20 and 100 mg / ml, and 20 and 50 mg / ml.

[0087] Those skilled in the art will understand that the advancement of a manufacturing method is expressed by the yield of protein peptides, as different tissues and organs of an animal contain varying amounts of protein. This is typically expressed as the yield of small molecule active protein peptides, that is, the weight ratio of the obtained protein peptides to the total weight of the tissue or organ used. The protein peptide yield is usually between 50 and 350 g / kg. In one embodiment of the present invention, the total amount of protein peptides is weighed to determine the protein peptide yield, and then the protein peptide yield is calculated. Suitablely, the yield of protein peptides is above 8g / Kg, for example, above 10g / Kg, and above 15g / Kg, 20g / Kg, 30g / Kg, 40g / Kg, 50g / Kg, 60g / Kg, 70g / Kg, 80g / Kg, 90g / Kg, 100g / Kg, 110g / Kg, 125g / Kg, 150g / Kg, 175g / Kg, 200g / Kg, 250g / Kg, 300g / Kg, 350g / Kg, etc.

[0088] One embodiment of this invention calculates the utilization rate of protein peptides in an organism by measuring the protein content in animal tissues and organs and the obtained protein peptides. Protein content varies depending on the species; for example, the protein content in camel and yak meat typically exceeds 20%. Protein content also varies within the same organism's tissues and organs, typically containing approximately 5-25% protein, with some tissues and organs containing extremely high levels, such as approximately 5-95%, 6-65%, 8-35%, 8-25%, 10-25%, 10-23%, 10-21%, 10-19%, 11-19%, 12-18%, and 12-17% protein. These values ​​are subject to frequent fluctuations; that is, it is impossible to know the precise protein content of a specific organ or tissue for every organism, but approximate values ​​can be obtained. One of the objectives of this invention is to effectively utilize as much protein as possible by converting it into small-molecule bioactive peptides to meet human needs for improved nutrition and health. Most importantly, the method of this invention produces more target small-molecule bioactive peptides.

[0089] Typically, the yield of small molecule bioactive peptides is expressed as the percentage of the total amount of bioactive peptides obtained relative to the total protein content of the tissue or organ used. Using the method of this invention, the yield of small molecule bioactive peptides is above 1%, for example, above 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75%. This value is actually related to the bioavailability of the organism. This value can never reach the ideal 100% because organisms contain tens of thousands, hundreds of thousands, millions, or even tens of millions of proteins, which are difficult to decompose using enzymatic methods.

[0090] In one embodiment of the present invention, the obtained protein peptides are subjected to activity determination, wherein the activity can be total activity or specific activity. A commonly used method, such as that used in the protein products prepared according to the present invention, is to calculate the activity of the protein peptides using the percentage of inhibition against ACE (angiotensin-converting enzyme, ACE) as the activity parameter. The specific operation is as follows:

[0091] I. Reagents and Solutions

[0092] (I) Reagents

[0093] 1. Hippuric acid (Hip), a product of Sigma-Aldrich, USA;

[0094] 2. Hippuryl-His-Leu tetrahydrate (HHL), Sigma-Aldrich, USA;

[0095] 3. Angiotensin-converting enzyme (ACE), 0.1U, Sigma-Aldrich, USA;

[0096] 4. Acetonitrile, HPLC grade;

[0097] 5. Boric acid (H3BO3), borax, and sodium chloride were all of analytical grade;

[0098] (II) Preparation of test solution

[0099] 1. 0.1 mol / L boric acid-borax buffer solution (pH 8.3, containing 0.3 mol / L NaCl): Weigh 12.37 g boric acid, dissolve by heating, and then bring the volume to 1 L. Weigh 19.07 g borax (Na₂B₄O₇·10H₂O), dissolve by heating, and then bring the volume to 1 L. Mix 175 mL of borax solution and 325 mL of boric acid solution together, adjust the pH to 8.3 with HCl or NaOH, then add 17.532 g NaCl and bring the volume to 1 L.

[0100] 2. The protein peptide solution of the present invention: using 0.1 mol·L⁻¹ -1 Borate buffer (pH 8.3, containing 0.3 mol·L⁻¹) -1 A blood pressure-lowering peptide solution of appropriate concentration was prepared using NaCl. This solution is abbreviated as "ACEI".

[0101] 3. ACE solution: Dissolve 0.1 U of ACE in 1 mL of 0.1 mol·L⁻¹ solution. -1 Borate buffer (pH 8.3, containing 0.3 mol·L⁻¹) -1 NaCl).

[0102] 4. HCl solution: Take an appropriate amount of HCl and use 0.1 mol·L⁻¹ solution. -1 Borate buffer (pH 8.3, containing 0.3 mol·L⁻¹) -1 Prepare a solution of NaCl at 6.5 mmol·L⁻¹ -1 HHL solution.

[0103] 5.1 mg / mL hippuric acid standard solution: Take an appropriate amount of hippuric acid standard and prepare a hippuric acid standard solution with double-distilled water.

[0104] II. Analytical Methods

[0105] (I) Preparation of reaction solution

[0106] Add 5 μL of ACE solution to sample tube (B) and blank tube (A), 10 μL of ACEI to sample tube, and 10 μL of buffer to blank tube. Incubate at 37°C for 5 min, then add 50 μL of HCl solution and react at 37°C for 30 min. Finally, add 85 mL of 1.0 mol / L HCl to stop the reaction, obtaining the reaction solution. Filter through 0.45 μm and use for HPLC analysis.

[0107] (II) Chromatographic conditions

[0108] 1. Chromatographic column: VYDAC 238EV54 C18 (250mm×4.6mm, 5μm).

[0109] 2. Mobile phase and chromatographic conditions: Acetonitrile:ultrapure water = 25:75 (containing 0.05% (v / v) TFA and 0.1% (v / v) triethylamine), flow rate: 0.5 mL / min -1 Detection wavelength: 228nm; Column temperature: 30℃; Injection volume: 20μL.

[0110] (III) Calculation of Results

[0111] HHL rapidly decomposes under the catalysis of ACE to produce hippuric acid (Hip) and a dipeptide (His-Leu, HL), with hippuric acid showing maximum absorption at 228 nm. When an ACEI sample is added, the activity of the ACE enzyme is inhibited, and the amount of hippuric acid produced decreases. Therefore, the inhibition rate of ACEI on ACE activity can be evaluated by determining the amount of hippuric acid produced using high-performance liquid chromatography.

[0112] The calculation formula is: Inhibition rate R = (AB) / A × 100%

[0113] R: Inhibition rate of ACE by ACEI sample (%)

[0114] A: Peak area of ​​hippuric acid in the blank control group

[0115] B: Peak area of ​​hippuric acid in the ACEI-added group

[0116] IC 50 IC50 is defined as the inhibitor concentration required to inhibit ACE enzyme activity by half under certain conditions. Since the inhibition rate and the formulation concentration are not linearly related, a curve showing the relationship between inhibitor concentration and inhibition rate must first be plotted, and then the IC50 concentration can be determined from the curve. 50 Or IC 50 Obtain it using the following method:

[0117] (1) Plot log[ACEI] against log(R / (1-R)) (it should be a straight line) to obtain the equation Y=aX+b, where Y is log(R / (1-R)).

[0118] (2) Calculate the value of X when Y = 0. Taking the antilogarithm of X gives IC. 50 .

[0119] In another embodiment of the present invention, a small molecule active protein peptide is provided, which exhibits a percentage inhibition of ACE (angiotensin-converting enzyme) and an IC50 concentration. 50 Simultaneous characterization was performed. Using the percentage inhibition of ACE (angiotensin-converting enzyme) as the activity parameter, a comparative experiment was first conducted on the ACE effects of various preparation methods and peptides derived from different tissues and organs. It was found that when using different peptides with the same protein concentration, the protein peptide products prepared according to this invention inhibited more than 10% of ACE, for example, more than 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, and 95% of ACE. IC 50 It is an indicator for measuring the efficacy of ACE inhibitors and also an indicator for measuring protein activity. The IC50 of various proteins prepared using the method of this invention... 50 It is possible for levels below 20.0 mg / mL, such as below 15.0 mg / mL, 10.0 mg / mL, 5.0 mg / mL, 2.0 mg / mL, 1.5 mg / mL, 1.0 mg / mL, 0.8 mg / mL, 0.6 mg / mL, 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.08 mg / mL, 0.06 mg / mL, 0.05 mg / mL, 0.01 mg / mL, 0.005 mg / mL, and 0.001 mg / mL.

[0120] In some preferred embodiments of the present invention, when the protein concentration is 2.0 mg / ml, the small molecule active protein peptide has an ACE inhibition rate of more than 30% and an IC50 of less than 1.29 mg / mL. 50When the protein concentration is 1.5 mg / mL, the small molecule active protein peptide exhibits an ACE inhibition rate exceeding 35% and an IC50 concentration below 0.95 mg / mL. 50 When the protein concentration is 1.0 mg / mL, the small molecule active protein peptide exhibits an ACE inhibition rate of over 40% and an IC50 concentration of less than 0.68 mg / mL. 50 When the protein concentration is 0.75 mg / mL, the small molecule active protein peptide exhibits an ACE inhibition rate exceeding 45% and an IC50 concentration below 0.47 mg / mL. 50 When the protein concentration is 0.5 mg / mL, the small molecule active protein peptide exhibits an ACE inhibition rate exceeding 48% and an IC50 concentration below 0.37 mg / mL. 50 When the protein concentration is 0.25 mg / ml, the small molecule active protein peptide exhibits an ACE inhibition rate exceeding 50% and a concentration below 0.16 mg / ml. 50 When the protein concentration is 0.1 mg / mL, the small molecule active protein peptide exhibits an ACE inhibition rate of over 55% and an IC50 concentration of less than 0.07 mg / mL. 50 When the protein concentration is 0.05 mg / mL, the small molecule active protein peptide exhibits an ACE inhibition rate exceeding 60% and an IC50 concentration below 0.039 mg / mL. 50 When the protein concentration is 0.01 mg / mL, the small molecule active protein peptide exhibits an ACE inhibition rate exceeding 65% and an IC50 concentration below 0.0076 mg / mL. 50 When the protein concentration is 0.005 mg / mL, the small molecule active protein peptide exhibits an ACE inhibition rate exceeding 70% and an IC50 concentration below 0.0037 mg / mL. 50 .

[0121] Simultaneously, the purity of the prepared active peptides was determined. Purity can be measured using common methods in existing technologies such as protein band staining intensity, FPLC, HPLC, and mass spectrometry. In some embodiments, the purity was above 60%, for example, all above 80%. In still other embodiments of the present invention, high-purity active peptides were prepared using the method of the present invention, for example, with purities above 85%, 86%, 87%, 88%, 89%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, and 99.9%.

[0122] The terms “peptide” or “protein peptide” or “active peptide” or “active protein peptide” or “active protein peptide” or “small molecule peptide” or “small molecule protein peptide” or “small molecule active peptide” or “small molecule active protein peptide” or “active protein” or “small molecule active protein peptide” may have the same meaning in some contexts and may be used interchangeably.

[0123] The peptides prepared by the method of this invention generally contain 1 to 200 amino acids, for example, 1 to 180, 1 to 150, 1 to 100, 1 to 80, 2 to 80, 3 to 80, 3 to 60, 5 to 60, 7 to 60, 10 to 30, 10 to 20, or 1 to 5 amino acids. The prepared bioactive peptides are small molecule protein bioactive peptides, containing one or more amino acids from a group consisting of isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine, etc. The content of the above-mentioned amino acids, i.e., the weight percentage of the total amino acids, can be above 1%, for example, above 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0124] In some embodiments of the present invention, the small molecule active protein peptides prepared by the method of the present invention contain at least 1% leucine, for example, at least 1% leucine and lysine; at least 2% leucine, lysine, and phenylalanine; at least 3% leucine, lysine, phenylalanine, and tryptophan; at least 4% leucine, lysine, phenylalanine, tryptophan, and valine; at least 5% leucine, lysine, phenylalanine, tryptophan, valine, lysine, and methionine; at least 6% leucine, lysine, phenylalanine, tryptophan, valine, lysine, methionine, and threonine; at least 7% leucine, lysine, phenylalanine, tryptophan, valine, lysine, methionine, and histidine; and at least 8% leucine, lysine, phenylalanine, tryptophan, valine, lysine, methionine, threonine, and histidine.

[0125] In some other embodiments of the present invention, the small molecule active protein peptides prepared by the method of the present invention contain one or more amino acids in the group consisting of glutamic acid, cystine, hydroxyproline, arginine, serine, and aspartic acid, and the content of the above amino acids, i.e., the weight ratio of the total amino acids, can be more than 1%, for example, more than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, or 30%.

[0126] The technical solution of this invention enables the targeted and large-scale production of high-quality natural target protein peptides. The term "target protein peptide" refers to small molecule peptides that have been proven in scientific research fields such as food, medicine, nutrition, and cosmetics to prevent and / or alleviate and / or treat and / or cure pathological symptoms or diseases caused by the loss and degeneration of human tissues and organs. Therefore, the products of this invention may contain one or more target protein peptides, exhibiting broader applicability.

[0127] One specific embodiment of the present invention is to use the active peptides of the present invention in the preparation of food, medicine, nutritional health products or cosmetics for the prevention and / or relief and / or treatment of pathological symptoms or diseases caused by decreased immune function, wherein the pathological symptoms or diseases caused by decreased immune function include, but are not limited to, hay fever, allergies, etc.

[0128] One specific embodiment of the present invention is the use of the active peptides of the present invention in the preparation of food, medicine, nutritional supplements or cosmetics for the prevention and / or reduction and / or treatment of aging and dysfunction of tissues and organs.

[0129] The protein-active peptides of the present invention can be administered as a single formulation or in combination in a unit dosage form composition, or sequentially to the subject.

[0130] The present invention can be better understood through the following embodiments, but is not limited to these embodiments.

[0131] Referring to Figure 1, the present invention provides a further explanation of the technical solution for preparing high-molecular-weight active protein peptides from animal tendons, achieving the goal of industrial production.

[0132] Example

[0133] Example 1: Preparation method of small molecule protein bioactive peptides from annelids

[0134] Example 1. Preparation process of earthworm small molecule protein bioactive peptides (1)

[0135] Take 500g of fresh earthworms (also known as soil worms), wash them, remove any surface impurities, and pat dry with kitchen paper towels. Add approximately 5 times the volume of 50mM NaCl-phosphate buffer (pH 6.8), and grind them using a grinder for about 8 minutes, with 45-second intervals. Then homogenize them using a homogenizer for 5 minutes, with 30-second intervals. Stir to mix well, adjust the pH to 6.8, add 1g of neutral protease, and incubate at 45℃ with stirring for 2 hours; then adjust the pH to 9.0, add 1g of serine protease, and incubate at 50℃ with stirring for about 4 hours. Centrifuge the enzymatic hydrolysate at 12500rpm for 45 minutes, and filter the centrifuged solution using a filter equipped with a 0.45-micron filter membrane. Vacuum the solution until no air bubbles remain. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 15000–100000. The liquid was nanofiltered four times using a nanofiltration machine equipped with a predetermined molecular weight cutoff of 115-3000, and finally concentrated to about 290 ml. After sterilization and freeze-drying, 34.8 g of white, small-molecule active protein peptide lyophilized powder from earthworms with a purity of approximately 99.1% was obtained.

[0136] Example 2. Preparation process of earthworm small molecule protein active peptides (2)

[0137] Take 500g of fresh earthworms (also known as soil worms), wash them, remove any surface impurities, and pat dry with kitchen paper towels. Add approximately 5 times the volume of 50mM NaCl-phosphate buffer (pH 6.5), and grind them using a grinder for about 8 minutes, with 45-second intervals. Then homogenize them using a homogenizer for 5 minutes, with 30-second intervals. Stir and mix well, adjust the pH to 6.5, add 1g of papain, and incubate at 55°C with stirring for 2 hours; adjust the pH to 7.5, add 1g of subtilisin, and incubate at 50°C with stirring for 3 hours. Centrifuge the enzymatic hydrolysate at 12500rpm for 45 minutes, and filter the centrifuged solution using a filter equipped with a 0.45-micron filter membrane. Vacuum the solution until no air bubbles remain. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 15000–100000. The liquid was nanofiltered four times using a nanofiltration unit equipped with a predetermined molecular weight cutoff of 200-3000, and finally concentrated to approximately 322 ml. After sterilization and freeze-drying, approximately 37.3 g of white, small-molecule active protein peptide lyophilized powder from earthworm was obtained, with a purity of approximately 99.3%.

[0138] Example 2: Preparation method of small molecule bioactive protein peptides from mollusks

[0139] Example 3. Preparation of lyophilized oyster small molecule active protein peptide powder (1)

[0140] Take approximately 5 kg of fresh oysters, open them with a shell opener, and separate them into two edible parts: the mantle and the foot. Rinse the oysters with about 5-10 times their volume of water to remove dirt and inedible parts. Pat dry with kitchen paper towels and take 400 g of the edible part. Add about 5 times their volume of 50 mM NaCl-phosphate buffer (pH 9.0) and grind the oysters using a grinder for about 8 minutes, with 45-second intervals. Homogenize the oysters using a homogenizer for 5 minutes, with 30-second intervals. Adjust the pH to 9.0, add 2 g of serine protease, and incubate at 50°C with stirring for about 5 hours. Centrifuge the enzymatic hydrolysate at 16,000 rpm for 30 minutes, and filter the centrifuged solution using a filter fitted with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. Perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10,000-100,000. The solution was filtered four times using a nanofiltration unit equipped with a predetermined molecular weight cutoff of 200-3000, and then concentrated to 325 ml using the nanofiltration unit. The nanofiltrate was diluted to its original volume, and the nanofiltration was repeated 3-5 times. Finally, it was sterilized and freeze-dried to obtain 26.7 g of oyster small molecule active protein peptide freeze-dried powder.

[0141] Example 4. Preparation of lyophilized oyster small molecule active protein peptide powder (2)

[0142] Take approximately 5 kg of fresh oysters, open them with a shell opener, and separate them into two edible parts: the mantle and the foot. Rinse with approximately 5-10 times their volume of water to remove dirt and inedible parts. Pat dry with kitchen paper towels. Take 400 g of the edible part. Add approximately 5 times their volume of 50 mM NaCl-phosphate buffer (pH 6.5), and grind using a grinder for approximately 8 minutes, with 45-second intervals. Homogenize using a homogenizer for 5 minutes, with 30-second intervals. Adjust the pH to 6.5, add 2 g of papain, and incubate at 55°C with stirring for approximately 5 hours. Centrifuge the enzymatic hydrolysate at 16,000 rpm for 45 minutes, and filter the centrifuged liquid through a filter fitted with a 0.25-micron filter membrane. Vacuum the liquid until no air bubbles remain. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10,000-100,000. The permeate was concentrated to 315 ml using a nanofiltration unit equipped with a predetermined molecular weight cutoff of 200-3000. The nanofiltrate was diluted to its original volume, and the nanofiltration was repeated four times. Finally, it was sterilized and freeze-dried to obtain 25.3 g of oyster small molecule active protein peptide freeze-dried powder.

[0143] Example 5. Preparation of lyophilized oyster small molecule active protein peptide powder (3)

[0144] Take approximately 5 kg of fresh oysters, open them with a shell opener, and separate them into the edible mantle and foot parts. Rinse with approximately 5-10 times their volume of water to remove dirt and non-edible parts. Pat dry with kitchen paper towels, and take 400 g of the edible part. Add approximately 5 times their volume of 50 mM NaCl-phosphate buffer (pH 6.8), and grind using a grinder for approximately 8 minutes, with 45-second intervals. Homogenize using a homogenizer for 5 minutes, with 30-second intervals. Adjust the pH to 6.8, add 2 g of neutral protease, and incubate at 45°C with stirring for approximately 6 hours. Centrifuge the enzymatic hydrolysate at 16,000 rpm for 60 minutes, and filter the centrifuged liquid through a filter equipped with a 0.25-micron filter membrane. Vacuum the liquid until no air bubbles remain. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10,000-100,000. The permeate was concentrated to 326 ml using a nanofiltration unit equipped with a predetermined molecular weight cutoff of 200–3000. The nanofiltrate was diluted to its original volume, and the nanofiltration was repeated four times. Finally, it was sterilized and freeze-dried to obtain 27.1 g of oyster small molecule active protein peptide freeze-dried powder.

[0145] Example 6. Preparation of lyophilized oyster small molecule active protein peptide powder (4)

[0146] Take approximately 5 kg of fresh oysters, open them with a shell opener, and separate them into two edible parts: the mantle and the foot. Rinse the oysters with about 5-10 times their volume of water to remove dirt and inedible parts. Pat dry with kitchen paper towels and take 400 g of the edible part. Add about 5 times their volume of 50 mM NaCl-phosphate buffer (pH 8.0) and grind the oysters using a grinder for about 8 minutes, with 45-second intervals. Homogenize the oysters using a homogenizer for 5 minutes, with 30-second intervals. Adjust the pH to 8.0, add 2 g of trypsin, and incubate at 37°C with stirring for about 8 hours. Centrifuge the enzymatic hydrolysate at 16,000 rpm for 60 minutes, and filter the centrifuged solution using a filter equipped with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. Perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10,000-100,000. The permeate was concentrated to 368 ml using a nanofiltration unit equipped with a predetermined molecular weight cutoff of 200–3000. The nanofiltrate was diluted to its original volume, and the nanofiltration was repeated four times. Finally, it was sterilized and freeze-dried to obtain 28.3 g of oyster small molecule active protein peptide freeze-dried powder.

[0147] Example 7. Preparation of freeze-dried squid small molecule active protein peptide powder (1)

[0148] Take a squid, scrape and remove oil with a knife, remove dirt and non-edible parts (squid bones, cuttlebone), rinse with clean water, and pat dry with kitchen paper towels. Weigh 500g of squid. Cut into thin strips, grind into a paste for 20 minutes, with 45-second intervals. Add 3% acetic acid to the paste and stir to extract overnight. Centrifuge at 14000 rpm and collect the precipitate. Add phosphate buffer (50mM NaCl-20mM phosphate buffer) to adjust the pH to 8.0, add 1g of trypsin, and incubate at 37°C with stirring for about 2 hours; adjust the pH to 9.0, then add 1g of serine protease, and incubate at 50°C with stirring for about 4 hours. Centrifuge the enzymatic hydrolysate at 16000 rpm for 60 minutes, and filter the centrifuged liquid through a filter equipped with a 0.22-micron filter membrane. Vacuum until no air bubbles remain in the liquid. First, ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000. The permeate was then concentrated to 373 ml using a nanofiltration unit equipped with a molecular weight cutoff of 200–3000. The nanofiltrate was diluted to its original volume, and the nanofiltration was repeated four times. Finally, the solution was sterilized and freeze-dried to obtain 32.1 g of lyophilized squid small molecule active protein peptide powder.

[0149] Example 8. Preparation of freeze-dried squid small molecule active protein peptide powder (2)

[0150] Take a squid, scrape and remove oil with a knife, remove dirt and non-edible parts (squid bones, cuttlebone), rinse with clean water, and pat dry with kitchen paper towels. Weigh 500g of squid. Cut into thin strips, grind into a paste for 20 minutes, with 45-second intervals. Add 3% acetic acid to the paste and stir to extract overnight. Centrifuge at 14000 rpm and collect the precipitate. Add phosphate buffer (50mM NaCl-20mM phosphate buffer) to adjust the pH to 6.8, add 1g of neutral protease, and incubate at 45°C with stirring for about 2 hours; raise the temperature to 55°C, then add 1g of papain, and incubate at 55°C with stirring for about 4 hours. Centrifuge the enzymatic hydrolysate at 16000 rpm for 60 minutes, and filter the centrifuged liquid through a filter equipped with a 0.22-micron filter membrane. Vacuum until no air bubbles remain in the liquid. First, ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000. The permeate was then concentrated to 347 ml using a nanofiltration unit equipped with a molecular weight cutoff of 200–3000. The nanofiltrate was diluted to its original volume, and the nanofiltration was repeated four times. Finally, the solution was sterilized and freeze-dried to obtain 30.7 g of lyophilized squid small molecule active protein peptide powder.

[0151] Example 3: Preparation method of small molecule bioactive protein peptides from arthropods

[0152] Example 9. Preparation process of grasshopper small molecule active protein peptides (1)

[0153] Take grasshoppers, wash them with about 5 liters of water, and weigh 1 kg. Grind them in a grinder for about 10 minutes, with 45-second intervals; homogenize for 5 minutes, with 30-second intervals. Adjust the pH to 6.80, add 3 g of neutral protease, and incubate at 45°C with stirring for about 8 hours. Centrifuge the enzymatic hydrolysate at 12500 rpm for 45 minutes, and filter the centrifuged liquid through a filter equipped with a 0.20-micron filter membrane. Vacuum the liquid until no air bubbles remain. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000. Collect the centrifugation, filtration, and ultrafiltration residues for analysis and future use. Concentrate the permeate to 766 ml using a nanofiltration unit equipped with a molecular weight cutoff of 200–3000. Dilute the nanofiltration liquid to the original volume, and repeat nanofiltration 3–5 times before sterilization and freeze-drying to obtain 44.1 g of grasshopper small molecule active protein peptide lyophilized powder.

[0154] Example 10. Preparation process of grasshopper small molecule active protein peptides (2)

[0155] Take grasshoppers, wash them with about 5 liters of water, and weigh 1 kg. Grind them in a grinder for about 10 minutes, with 45-second intervals; homogenize for 5 minutes, with 30-second intervals. Adjust the pH to 6.5, add 3 g of papain, and incubate at 55°C with stirring for about 4 hours. Centrifuge the enzymatic hydrolysate at 15000 rpm for 30–45 minutes, and filter the centrifuged liquid through a filter equipped with a 0.20-micron filter membrane. Vacuum the liquid until no air bubbles remain. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000. Collect the centrifugation, filtration, and ultrafiltration residues for analysis and future use. Concentrate the permeate to 689 ml using a nanofiltration unit equipped with a molecular weight cutoff of 200–3000. Dilute the nanofiltration liquid to the original volume, and repeat nanofiltration 3–5 times before sterilization and freeze-drying to obtain 40.3 g of grasshopper small molecule active protein peptide lyophilized powder.

[0156] Example 11. Preparation process of grasshopper small molecule active protein peptides (3)

[0157] Take grasshoppers, wash them with about 5 liters of water, and weigh 1 kg. Grind them in a grinder for about 10 minutes, with 45-second intervals; homogenize for 5 minutes, with 30-second intervals. Adjust the pH to pH 6.8, add 1.5 g of neutral protease, and incubate at 45°C with stirring for about 2 hours; raise the temperature to 55°C, then add 1.5 g of papain, and incubate at 55°C with stirring for about 4 hours. Centrifuge the enzymatic hydrolysate at 12000–16000 rpm for 30–60 minutes, and filter the centrifuged liquid through a filter equipped with a 0.20-micron filter membrane. Vacuum the solution until no air bubbles remain. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000. Collect the centrifugation, filtration, and ultrafiltration residues for analysis and future use. Concentrate the permeate to 765 ml using a nanofiltration unit equipped with a molecular weight cutoff of 200–3000. The nanofiltration solution was diluted to its original volume, and the nanofiltration solution was repeatedly nanofiltered 3 to 5 times before sterilization and freeze-drying to obtain 45.8g of grasshopper small molecule active protein peptide freeze-dried powder.

[0158] Example 12. Preparation process of grasshopper small molecule active protein peptides (4)

[0159] Take grasshoppers, wash them with about 5 liters of water, and weigh 1 kg. Grind them in a grinder for about 10 minutes, with 45-second intervals; homogenize for 5 minutes, with 30-second intervals. Adjust the pH to pH 8.0, add 1.5 g of trypsin, and incubate at 45°C with stirring for about 2 hours; adjust the pH to 9.0, then add 1.5 g of serine protease, and incubate at 50°C with stirring for about 4 hours. Centrifuge the enzymatic hydrolysate at 12000–16000 rpm for 30–60 minutes, and filter the centrifuged liquid through a filter equipped with a 0.20-micron filter membrane. Vacuum the solution until no air bubbles remain. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000. Collect the centrifugation, filtration, and ultrafiltration residues for analysis and future use. Concentrate the permeate to 729 ml using a nanofiltration unit equipped with a molecular weight cutoff of 200–3000. The nanofiltration solution was diluted to its original volume, and the nanofiltration solution was repeatedly nanofiltered 3 to 5 times before sterilization and freeze-drying to obtain 46.5g of grasshopper small molecule active protein peptide freeze-dried powder.

[0160] Example 13. Manufacturing process of shrimp small molecule protein bioactive peptides (1)

[0161] Take 1 kg of fresh, live shrimp, remove the shells, remove the shrimp vein and internal organs with a needle, rinse quickly, and pat dry with kitchen paper towels. Weigh out 450 g of pure shrimp meat. Add 2 liters of 50 mM NaCl-phosphate buffer (pH 6.8). Homogenize for 5 minutes with 30-second intervals. Adjust the pH to 6.80, add 3.0 g of neutral protease, and incubate at 45°C for approximately 8 hours while stirring. Centrifuge the enzymatic hydrolysate at 13500 rpm for 45 minutes, and filter the centrifuged solution using a filter equipped with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000. Then, desalinate the permeate using a nanofiltration unit equipped with a molecular weight cutoff of 200–3000. Dilute the nanofiltrate to its original volume and repeat nanofiltration 5 times. Sterilization and ultra-low temperature vacuum freeze drying yielded 32.8g of shrimp small molecule active protein peptide freeze-dried powder.

[0162] Example 14. Manufacturing process of shrimp small molecule protein bioactive peptides (2)

[0163] Take 1 kg of fresh, live shrimp, remove the shells, remove the shrimp vein and internal organs with a needle, rinse quickly, and pat dry with kitchen paper towels. Weigh out 450 g of pure shrimp meat. Add 2 liters of 50 mM NaCl-phosphate buffer (pH 6.5). Homogenize for 5 minutes with 30-second intervals. Adjust the pH to 6.5, add 3.0 g of papain, and incubate at 55°C for approximately 4 hours while stirring. Centrifuge the enzymatic hydrolysate at 13500 rpm for 45 minutes, and filter the centrifuged solution using a filter equipped with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000. Then, desalinate the permeate using a nanofiltration unit equipped with a molecular weight cutoff of 200–3000. Dilute the nanofiltrate to its original volume and repeat nanofiltration 5 times. Sterilization and ultra-low temperature vacuum freeze drying yielded 34.4g of shrimp small molecule active protein peptide freeze-dried powder.

[0164] Example 15. Manufacturing process of shrimp small molecule protein bioactive peptides (3)

[0165] Take 1 kg of fresh, live shrimp, remove the shells, remove the shrimp vein and internal organs with a needle, rinse quickly, and pat dry with kitchen paper towels. Weigh out 450 g of pure shrimp meat. Add 2 liters of 50 mM NaCl-phosphate buffer (pH 8.0). Homogenize for 5 minutes with 30-second intervals. Adjust the pH to 8.0, add 3.0 g of trypsin, and incubate at 37°C for approximately 8 hours while stirring. Centrifuge the enzymatic hydrolysate at 13500 rpm for 45 minutes, and filter the centrifuged solution using a filter equipped with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000. Then, desalinate the permeate using a nanofiltration unit equipped with a molecular weight cutoff of 200–3000. Dilute the nanofiltrate to its original volume and repeat nanofiltration 5 times. Sterilization and ultra-low temperature vacuum freeze drying yielded 31.6g of shrimp small molecule active protein peptide freeze-dried powder.

[0166] Example 16. Manufacturing process of shrimp small molecule protein bioactive peptides (4)

[0167] Take 1 kg of fresh, live shrimp, remove the shells, remove the shrimp vein and internal organs with a needle, rinse quickly, and pat dry with kitchen paper towels. Weigh out 450 g of pure shrimp meat. Add 2 liters of 50 mM NaCl-phosphate buffer (pH 6.8). Homogenize for 5 minutes with 30-second intervals. Adjust the pH to 6.8, add 1 g of neutral protease, and incubate at 37°C for about 2 hours while stirring. Then raise the temperature to 53°C, add 1 g of flavor protease, and incubate at 50°C for about 2 hours while stirring. Adjust the pH to 7.5, add 1 g of subtilisin, and incubate at 50°C for about 2 hours while stirring. Centrifuge the enzymatic hydrolysate at 13500 rpm for 45 minutes, and filter the centrifuged solution using a filter equipped with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. Perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000. The solution was desalted by nanofiltration using a nanofiltration unit equipped with a molecular weight cutoff of 200–3000. The nanofiltrate was diluted to its original volume and then repeatedly nanofiltered five times. The solution was then sterilized and freeze-dried at ultra-low temperature under vacuum to obtain 33.8 g of shrimp small molecule active protein peptide freeze-dried powder.

[0168] Example 17. Manufacturing process of crab small molecule protein bioactive peptides (1)

[0169] Take 2 kg of fresh, live crabs, remove the shells and internal organs, rinse quickly, and pat dry with kitchen paper towels. Weigh out 435 g of pure crab meat. Add 2 liters of 50 mM NaCl-phosphate buffer (pH 6.8) and homogenize using a homogenizer with 30-second intervals. Adjust the pH to 6.80, add 2 g of neutral protease, and incubate at 45°C for approximately 8 hours while stirring. Centrifuge the enzymatic hydrolysate at 15,000 rpm for 45 minutes, and filter the centrifuged liquid using a filter equipped with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 2,000–150,000. Then, desalinate the permeate using a nanofiltration unit equipped with a molecular weight cutoff of 200–2,000. Dilute the nanofiltrate to its original volume and repeat nanofiltration 5 times. Sterilize and freeze-dry at ultra-low temperature under vacuum to obtain 35.1 g of lyophilized crab small molecule active protein peptide powder.

[0170] Example 18. Manufacturing process of crab small molecule protein bioactive peptides (2)

[0171] Take 2 kg of fresh, live crabs, remove the shells and internal organs, rinse quickly, and pat dry with kitchen paper towels. Weigh out 435 g of pure crab meat. Add 2 liters of 50 mM NaCl-phosphate buffer (pH 6.5) and homogenize using a homogenizer with 30-second intervals. Adjust the pH to 6.5, add 2 g of papain, and incubate at 55°C for approximately 4 hours while stirring. Centrifuge the enzymatic hydrolysate at 15,000 rpm for 45 minutes, and filter the centrifuged liquid using a filter equipped with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 2,000–150,000. Then, desalinate the permeate using a nanofiltration unit equipped with a molecular weight cutoff of 200–2,000. Dilute the nanofiltrate to its original volume and repeat nanofiltration 5 times. Sterilize and freeze-dry at ultra-low temperature under vacuum to obtain 34.3 g of lyophilized crab small molecule active protein peptide powder.

[0172] Example 19. Manufacturing process of crab small molecule protein bioactive peptides (3)

[0173] Take 2 kg of fresh, live crabs, remove the shells and internal organs, rinse quickly, and pat dry with kitchen paper towels. Weigh out 435 g of pure crab meat. Add 2 liters of 50 mM NaCl-phosphate buffer (pH 6.8) and homogenize using a homogenizer at 30-second intervals. Adjust the pH to 8.0, add 2 g of trypsin, and incubate at 45°C for approximately 8 hours while stirring. Centrifuge the enzymatic hydrolysate at 15,000 rpm for 45 minutes, and filter the centrifuged liquid using a filter equipped with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 2,000–150,000. Then, desalinate the permeate using a nanofiltration unit equipped with a molecular weight cutoff of 200–2,000. Dilute the nanofiltrate to its original volume and repeat nanofiltration 5 times. Sterilize and freeze-dry at ultra-low temperature under vacuum to obtain 31.4 g of lyophilized crab small molecule active protein peptide powder.

[0174] Example 20. Manufacturing process of crab small molecule protein bioactive peptides (4)

[0175] Take 2 kg of fresh, live crabs, remove the shells and internal organs, rinse quickly, and pat dry with kitchen paper towels. Weigh out 435 g of pure crab meat. Add 2 liters of 50 mM NaCl-phosphate buffer (pH 6.8) and homogenize using a homogenizer, with 30-second intervals. Adjust the pH to 6.8, add 1 gram of neutral protease, and incubate at 45°C for approximately 2 hours while stirring. Then, raise the temperature to 53°C, add 1 gram of flavor protease, and incubate at 50°C for approximately 2 hours while stirring. Adjust the pH to 7.5, add 1 gram of subtilisin, and incubate at 50°C for approximately 4 hours while stirring. Centrifuge the enzymatic hydrolysate at 15,000 rpm for 45 minutes, and filter the centrifuged solution using a filter equipped with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. Perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 2,000–150,000. The solution was desalted by nanofiltration using a nanofiltration unit equipped with a molecular weight cutoff of 200–2000. The nanofiltrate was diluted to its original volume and then repeatedly nanofiltered five times. The solution was then sterilized and freeze-dried at ultra-low temperature under vacuum to obtain 35.5 g of lyophilized crab small molecule active protein peptide powder.

[0176] Example 4: Preparation process of small molecule active peptides from echinoderms

[0177] Example 21. Method for manufacturing small molecule protein bioactive peptides from sea cucumber (1)

[0178] Take fresh sea cucumbers, remove the internal organs and foreign objects, rinse quickly, and pat dry with kitchen paper towels. Weigh out 500g of pure sea cucumber. Add 2 liters of 50mM NaCl-phosphate buffer (pH 6.8), and grind using a grinder for about 8 minutes, with 45-second intervals; then homogenize using a homogenizer, with 30-second intervals. Adjust the pH to 6.8, add 2g of neutral protease, and incubate at 45℃ with stirring for about 6 hours. Centrifuge the enzymatic hydrolysate at 12500rpm for 45 minutes, and filter the centrifuged liquid through a filter equipped with a 0.22-micron filter membrane. Vacuum until no air bubbles remain in the liquid. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000. Then, nanofilter the permeate four times using a nanofiltration machine equipped with a molecular weight cutoff of 200–3000, and finally concentrate the solution to about 350ml. Sterilization and freeze-drying yielded approximately 29.3g of sea cucumber small molecule active protein peptide freeze-dried powder with a purity of approximately 99.8%.

[0179] Example 22. Method for manufacturing small molecule protein bioactive peptides from sea cucumber (2)

[0180] Take fresh sea cucumbers, remove the internal organs and foreign objects, rinse quickly, and pat dry with kitchen paper towels. Weigh out 500g of pure sea cucumbers. Add 50mM NaCl-phosphate buffer (pH 6.5), and grind using a grinder for about 8 minutes, with an interval of 45 seconds; then homogenize using a homogenizer, with an interval of 30 seconds. Adjust the pH to 6.8, add 1g of neutral protease, and incubate at 45℃ with stirring for 2 hours; then adjust the pH to 9.0, add 1g of serine protease, and incubate at 50℃ with stirring for about 4 hours. Centrifuge the enzymatic hydrolysate at 20,000 rpm for 45 minutes, and filter the centrifuged solution using a filter equipped with a 0.22-micron filter membrane. Vacuum the solution until no air bubbles remain. First, perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3,000–100,000. The liquid was filtered four times using a nanofiltration machine equipped with a molecular weight cutoff of 200-3000, and finally concentrated to about 400 ml. After sterilization and freeze-drying, approximately 32.7 g of white sea cucumber small molecule active protein peptide freeze-dried powder with a purity of about 99.5% was obtained.

[0181] Example 23. Method for manufacturing small molecule protein bioactive peptides from sea cucumber (3)

[0182] Take fresh sea cucumbers, remove the internal organs and foreign objects, rinse quickly, and pat dry with kitchen paper towels. Weigh out 500g of pure sea cucumbers. Add 50mM NaCl-phosphate buffer (pH 8.0), grind using a grinder for about 8 minutes, with an interval of 45 seconds; then homogenize using a homogenizer, with an interval of 30 seconds. Adjust the pH to 6.8, add 1g of neutral protease, and incubate at 37℃ for about 2 hours while stirring; then raise the temperature to 53℃, add 1g of flavor protease, and incubate at 50℃ for about 2 hours while stirring; adjust the pH to 7.5, add 1g of subtilisin, and incubate at 50℃ for about 2 hours while stirring. Centrifuge the enzymatic hydrolysate at 20,000 rpm for 30–45 minutes, and filter the centrifuged liquid through a filter equipped with a 0.22-micron filter membrane. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 3,000–100,000. The liquid was filtered four times using a nanofiltration machine equipped with a molecular weight cutoff of 200-3000, and finally concentrated to about 400 ml. After sterilization and freeze-drying, approximately 33.4 g of white sea cucumber small molecule active protein peptide freeze-dried powder with a purity of about 99.5% was obtained.

[0183] Example 24. Method for manufacturing small molecule protein bioactive peptides from sea cucumber (4)

[0184] Take fresh sea cucumbers, remove the internal organs and foreign objects, rinse quickly, and pat dry with kitchen paper towels. Weigh out 500g of pure sea cucumbers. Add 50mM NaCl-phosphate buffer (pH 9.0), grind using a grinder for about 8 minutes, with an interval of 45 seconds; then homogenize using a homogenizer, with an interval of 30 seconds. Stir well, adjust the pH to 6.5, add 1g of papain, and incubate at 55℃ with stirring for 2 hours. Adjust the pH to 7.5, add 1g of subtilisin, and incubate at 50℃ with stirring for 3 hours. Centrifuge the enzymatic hydrolysate at 20,000 rpm for 45 minutes, and filter the centrifuged liquid through a filter equipped with a 0.25-micron filter membrane. Vacuum until no air bubbles remain in the liquid. Filter the filtrate first using an ultrafiltration membrane with a molecular weight cutoff of 3,000–100,000. The liquid was nanofiltered four times using a nanofiltration unit equipped with a molecular weight cutoff of 200-3000, and finally concentrated to approximately 416 ml. The solution was then sterilized and freeze-dried to obtain approximately 32.9 grams of white sea cucumber small molecule active protein peptide freeze-dried powder with a purity of approximately 99.8%.

[0185] Example 5: Preparation method of small molecule bioactive protein peptides from fish

[0186] Example 25. Effects of various process conditions on the production of small molecule bioactive protein peptides in fish.

[0187] Take fresh fish tissues and organs, remove small bones, manually remove blood and fat, rinse quickly, and pat dry with kitchen paper towels. Weigh out 50–300g. Chop finely, add phosphate buffer (1–200mM NaCl-1–200mM phosphate buffer), and grind using a grinder for approximately 1–60 minutes, with intervals of 5–120 seconds. Then homogenize using a homogenizer for 1–60 minutes, with intervals of 5–120 seconds. Adjust the pH, add 1‰–100‰ neutral protease and / or papain and / or flavor protease and / or pepsin and / or trypsin and / or complex protease and / or serine protease and / or cathepsin and / or proteinase K, etc., and perform enzymatic hydrolysis for 0.1–24 hours, maintaining the hydrolysis temperature at 20–60℃. Centrifuge at 3000–25000 rpm for 5–120 minutes. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter packed with a 0.01–10 micrometer filter membrane. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 2000–1,000,000, then dilute with water to the original volume. Next, desalt and concentrate the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, then dilute with water to the original volume. Perform nanofiltration desalting 1–5 times. Sterilize. Prepare lyophilized powder of small-molecule active protein peptides from fish tissues and organs by freeze-drying.

[0188] Example 26. Preparation method of small molecule bioactive protein peptides from fish meat

[0189] Take fresh carp back fillet (Skeletal Muscle), remove small bones, manually remove blood and fat, rinse quickly, and pat dry with kitchen paper towels. Weigh 1000g. Chop finely, add phosphate buffer (50mM NaCl-20mM phosphate buffer), and blend using a blender for about 8 minutes, with 45-second intervals; then homogenize using a homogenizer, with 30-second intervals. Add phosphate buffer (50mM NaCl-20mM phosphate buffer, pH 6.8). Add 1g of neutral protease and incubate at 37°C with stirring for 1 hour; raise the temperature to 50°C, add 1g of subtilisin, and incubate at 50°C with stirring for 1 hour; add 1g of papain and incubate at 55°C with stirring for 1 hour; adjust the pH to 7.5, add 1g of flavor protease, and continue incubating at 50°C with stirring for 3 hours. Centrifuge at 18500rpm for 45 minutes. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.20-micron filter membrane. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 59.2g of freeze-dried fish meat small-molecule active protein peptide powder by freeze-drying.

[0190] Example 27. Preparation method of small molecule active protein peptides from fish vertebrae

[0191] Take the carp spine, scrape off the surface fat, meat residue, and blood with a knife, rinse, drain, and weigh 1000 grams. Crush to a certain size using a bone crusher, extract with 5% acetic acid for 4-8 hours, rinse, drain with kitchen paper towels, and grind into powder using a bone meal machine. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust the pH to 2.0, add 1g of pepsin, and stir at 37℃ for 2 hours for enzymatic hydrolysis; adjust the pH to 6.5, add 1g of neutral protease, and incubate at 37℃ with stirring for 2 hours; raise the temperature to 55℃, add 1g of papain, and incubate at 55℃ with stirring for 1 hour; adjust the pH to 7.5, add 1g of subtilisin, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 2g of serine protease, and incubate with stirring for 2 hours. Centrifuge at 18500 rpm for 45 minutes. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 43.2 g of freeze-dried fish vertebral bone small molecule active protein peptide powder by freeze drying.

[0192] Example 28. Preparation method of small molecule active protein peptides from shark fin

[0193] Take fresh frozen shark fins, use scissors to cut off any remaining meat, fat, impurities, and blood from the surface, rinse, drain, and weigh 1000 grams. Crush it to a certain size using a bone crusher, extract with 5% acetic acid for 4-8 hours, rinse, drain with kitchen paper towels, and grind into powder using a bone meal machine. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust the pH to 2.0, add 1g of pepsin, and stir at 37℃ for 2 hours for enzymatic hydrolysis; adjust the pH to 6.5, add 1g of neutral protease, and incubate at 37℃ with stirring for 2 hours; raise the temperature to 55℃, add 1g of papain, and incubate at 55℃ with stirring for 1 hour; adjust the pH to 7.5, add 1g of subtilisin, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 2g of serine protease, and incubate with stirring for 2 hours. Centrifuge at 22500rpm for 60 minutes. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 44g of freeze-dried shark fin small molecule active protein peptide powder by freeze drying.

[0194] Example 29. Preparation method of small molecule active protein peptides from fish scales

[0195] Fresh, frozen carp scales were rinsed to remove impurities, drained, and 1000g were weighed. Extraction was performed with 3% acetic acid for 4 hours, followed by powdering using a bone meal mill. Phosphate buffer (50mM NaCl-20mM phosphate buffer) was added, pH adjusted to 2.0, and 1g of pepsin was added. Enzymatic hydrolysis was initiated at 37°C with stirring for 2 hours. pH was adjusted to 6.5, and 1g of neutral protease was added. The mixture was incubated at 37°C with stirring for 2 hours. The temperature was raised to 55°C, and 1g of papain was added. The mixture was incubated at 55°C with stirring for 1 hour. pH was adjusted to 7.5, and 1g of subtilisin was added. The mixture was incubated at 50°C with stirring for 1 hour. pH was adjusted to 9.0, and 2g of serine protease was added. The mixture was centrifuged at 18500 rpm for 45 min. The supernatant was collected, and the crude protein peptide supernatant was filtered through a 0.20-micron filter plate. Vacuum was applied until no air bubbles remained in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 38.7 g of fish scale small molecule active protein peptide lyophilized powder was prepared by freeze-drying.

[0196] Example 30. Preparation method of small molecule active peptides from fish skin

[0197] Take fresh frozen catfish skin, scrape off the scales, internal fat, meat residue, and remove surface blood and dirt with a knife, rinse with clean water, drain, and weigh 1 kg. Cut into appropriate sizes, grind for 10 minutes with 45-second intervals. Add 3% acetic acid to the slurry and extract overnight with stirring. Centrifuge at 14000 rpm and collect the precipitate. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer), grind with a grinder for about 8 minutes with 45-second intervals; then homogenize with a homogenizer with 30-second intervals. Adjust pH to 6.5, add 1 g of neutral protease, and incubate at 37°C with stirring for 2 hours; raise temperature to 55°C, add 1 g of papain, and incubate at 55°C with stirring for 1 hour; adjust pH to 7.5, add 1 g of subtilisin, and incubate with stirring for 1 hour; adjust pH to 9.0, add 2 g of serine protease, and incubate with stirring for 2 hours. Centrifuge at 18500 rpm for 45 min. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.25 μm filter membrane. Vacuum the liquid until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 66.7 g of freeze-dried fish skin small molecule protein active peptide powder by freeze drying.

[0198] Example 31. Preparation method of small molecule active peptides from fish maw

[0199] Weigh 500g of fresh frozen fish maw. Cut into appropriate sizes and grind for 20 minutes with 45-second intervals. Add 3% acetic acid to the mixture and extract overnight with stirring. Centrifuge at 14000 rpm and collect the precipitate. Add phosphate buffer (50mM NaCl-20mM phosphate buffer) and grind for about 8 minutes with 45-second intervals; then homogenize for 5 minutes with 30-second intervals. Adjust pH to 6.5, add 1g of neutral protease, and incubate at 37°C with stirring for 1 hour; raise the temperature to 50°C, add 1g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1g of papain, and incubate with stirring for 1 hour; adjust pH to 9.0, add 1g of serine protease, and incubate with stirring for 3 hours. Centrifuge at 18500 rpm for 45 minutes. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.25-micron filter membrane. Vacuum the liquid until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 70.5g of freeze-dried fish maw small molecule protein active peptide powder by freeze-drying.

[0200] Example 6: Preparation method of small molecule bioactive protein peptides from amphibians

[0201] Example 32. Preparation method of small molecule active protein peptides from bullfrog meat

[0202] Scrape fresh bullfrog meat, manually remove blood and fat, rinse with water, and pat dry with kitchen paper towels. Weigh 1 kg of fresh bullfrog meat. Chop to appropriate size, then use a meat chopper to prepare minced meat. Homogenize using a homogenizer for 5 minutes, with 30-second intervals. Add phosphate buffer (50mM NaCl-20mM phosphate buffer, pH 6.8), adjust pH to 6.8, add 1.5g of neutral protease, and incubate at 37°C with stirring for 2 hours. Increase temperature to 55°C, add 1.5g of bromelain, and incubate for 1 hour. Add 1.5g of flavor protease, and incubate with stirring for 1 hour. Adjust pH to 9.0, add 1.5g of serine protease, and incubate with stirring for 2 hours. Centrifuge at 16000 rpm for 30 minutes. Collect the supernatant. Filter the crude protein peptide supernatant through a 0.25-micron filter plate. Vacuum until no air bubbles remain in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 8000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–2500, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 57.1 g of small-molecule active protein peptide lyophilized powder from bullfrog meat was prepared by freeze-drying.

[0203] Example 33. Preparation method of bullfrog small molecule bioactive protein peptides

[0204] Take a fresh, whole bullfrog, skinned and gutted, scrape off the surface oil and blood with a knife, rinse, drain, and weigh 1 kg. Chop it into small pieces, extract with 3% acetic acid for 4 hours, and grind into powder using a bone meal machine. Add phosphate buffer (50 mM NaCl-20 mM phosphate buffer), adjust the pH to 2.0, add 2 g of pepsin, and stir at 37°C for 2 hours for enzymatic hydrolysis; adjust the pH to 6.8, add 1.5 g of neutral protease, and incubate at 37°C with stirring for 2 hours; raise the temperature to 55°C, add 1.5 g of bromelain, and incubate for 1 hour; add 1.5 g of flavor protease, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1.5 g of serine protease, and incubate with stirring for 2 hours. Centrifuge at 15000 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.25 μm filter plate and frame. Vacuum until no air bubbles remain in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 8000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–2500, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalination was performed 3–5 times. The solution was then sterilized. 48.6 g of lyophilized bullfrog small molecule active protein peptide powder was prepared by freeze-drying.

[0205] Example 7: Preparation method of reptile small molecule bioactive protein peptides

[0206] Example 34. Preparation method of small molecule active protein peptides from turtle meat

[0207] Take fresh turtle meat, remove blood and fat manually, rinse with water, and pat dry with kitchen paper towels. Weigh 1 kg of fresh turtle meat. Chop to an appropriate size, then use a meat chopper to make minced meat. Homogenize using a homogenizer for 5 minutes, with 30-second intervals. Add phosphate buffer (50mM NaCl-20mM phosphate buffer, pH 6.8), adjust pH to 6.8, add 1.5g of neutral protease, and incubate at 37℃ with stirring for 2 hours. Increase the temperature to 55℃, add 1.5g of bromelain, and incubate for 1 hour. Add 1.5g of flavor protease, and incubate with stirring for 1 hour. Adjust pH to 9.0, add 1.5g of serine protease, and incubate with stirring for 2 hours. Centrifuge at 12500 rpm for 60 minutes. Collect the supernatant. Filter the crude protein peptide supernatant through a 0.25-micron filter plate. Vacuum until no air bubbles remain in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 8,000–100,000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3,000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 55.3 g of freeze-dried small-molecule active protein peptide powder from turtle meat was prepared by freeze-drying.

[0208] Example 35. Preparation method of small molecule active protein peptides from turtle shell

[0209] Take the turtle shell (also called tortoise shell or turtle plate, with the outer skin attached), scrape off the surface oil and blood with a knife, rinse, dry, and weigh about 1 kg. Crush it to a certain size using a bone crusher, extract with 5% acetic acid for 4 hours, and grind it into powder using a bone meal machine. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust the pH to 2.0, add 1.5g of pepsin, and stir at 37℃ for 2 hours for enzymatic hydrolysis; adjust the pH to 6.8, add 1.5g of neutral protease, and incubate at 37℃ with stirring for 2 hours; raise the temperature to 55℃, add 1.5g of bromelain, and incubate for 1 hour; add 1.5g of flavor protease, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1.5g of serine protease, and incubate with stirring for 2 hours. Centrifuge at 15000 rpm for 45 min. Take the supernatant, and filter the crude protein peptide supernatant through a filter plate frame fitted with a 0.20 micrometer filter membrane. Vacuum the liquid until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 8000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 29.8g of freeze-dried turtle shell small molecule active protein peptide powder.

[0210] Example 36. Preparation method of small molecule active peptides from turtle skirt

[0211] Take the tender, skin-like tissue (skirt) around a fresh, frozen turtle, scrape off impurities, grease, residue, and blood with a knife, rinse, drain, and weigh 1 kg. Cut into shreds to a certain size, add 3% acetic acid and extract for 4 hours, then use a chopper to prepare a paste; grind with a grinder for 8 minutes with 45-second intervals; homogenize with a homogenizer for 5 minutes with 30-second intervals. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust pH to 2.0, add 1.5g pepsin, and stir at 37℃ for 2 hours for enzymatic hydrolysis; adjust pH to 6.5, add 1.5g neutral protease, and incubate at 37℃ with stirring for 1 hour; raise temperature to 50℃, add 1.5g subtilisin, and incubate with stirring for 1 hour; raise temperature to 55℃, add 1.5g papain, and incubate with stirring for 1 hour; adjust pH to 8.5, add 1.5g serine protease, and incubate with stirring for 3 hours. Centrifuge at 18500rpm for 45min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.25μm filter plate and frame. Vacuum until no air bubbles remain in the liquid. Filter the filtrate first through an ultrafiltration membrane with a molecular weight cutoff of 8000-100000, then dilute with water to the original volume. The ultrafiltrate was first nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume. Then, water was added to dilute it to its original volume, and the solution was desalted by nanofiltration 3–5 times. The solution was then sterilized. 69.1 g of freeze-dried small-molecule protein active peptide powder from turtle skirt was prepared by freeze-drying.

[0212] Example 8: Preparation method of small molecule bioactive protein peptides for birds

[0213] (I) Chicken small molecule protein active peptides

[0214] Example 37. Preparation method of chicken small molecule protein bioactive peptides

[0215] Take freshly slaughtered free-range chicken meat, manually remove fat and small blood vessels, and weigh 1 kg. Chop to an appropriate size, then use a meat chopper to prepare minced meat. Homogenize using a homogenizer for 5 minutes, with 30-second intervals. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), add 1g of neutral protease, and incubate at 37°C with stirring for 2 hours; raise the temperature to 50°C, add 1g of subtilisin, and incubate with stirring for 1 hour; add 1g of papain, and incubate with stirring for 1 hour; adjust the pH to 7.5, add 1g of flavor protease, and continue incubating with stirring for 3 hours. Centrifuge at 12000 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.45-micron filter plate and frame. Vacuum until no air bubbles remain in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 58.3 g of lyophilized chicken small molecule protein active peptide powder was prepared by freeze-drying.

[0216] Example 38. Preparation method of chicken small molecule active protein peptides

[0217] Take 1.98 kg of freshly slaughtered free-range chicken, slaughter it, remove blood and pluck feathers, and weigh 1 kg. Cut it to a suitable size using a machine, add 4% acetic acid, and stir for 8 hours. Grind it into powder using a grinder. Add phosphate buffer (50 mM NaCl-20 mM phosphate buffer), adjust the pH to 2.0, add 2 g of pepsin, and stir at 37°C for 2 hours for enzymatic hydrolysis; adjust the pH to 6.5, add 1.5 g of neutral protease, and incubate at 37°C with stirring for 2 hours; adjust the pH to 7.5, raise the temperature to 50°C, add 1.5 g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1.5 g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1.5 g of serine protease, and incubate with stirring for 2 hours. Take the supernatant, and filter the crude protein peptide supernatant through a filter plate and frame fitted with a 0.25 micron filter membrane. Vacuum the liquid until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 49.7g of freeze-dried chicken small molecule active protein peptide powder.

[0218] Example 39. Preparation method of small molecule protein bioactive peptides from chicken skeleton

[0219] Take 1 kg of chicken carcass (with meat thoroughly removed). Chop it to a suitable size, add 5% acetic acid, and stir for 8 hours. Grind it into powder using a grinder. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer), adjust the pH to 2.0, add 2 g of pepsin, and stir at 37°C for 2 hours for enzymatic hydrolysis; adjust the pH to 6.5, add 1.5 g of neutral protease, and incubate at 37°C with stirring for 2 hours; adjust the pH to 7.5, raise the temperature to 50°C, add 1.5 g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1.5 g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1.5 g of serine protease, and incubate with stirring for 2 hours. Centrifuge at 18500 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.25 μm filter plate. Vacuum the liquid until no air bubbles remain. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 42.6 g of lyophilized chicken skeleton small molecule protein active peptide powder was prepared by freeze-drying.

[0220] Example 40. Preparation method of small molecule protein bioactive peptides from chicken claw tendons

[0221] Remove the boneless chicken feet tendons. Scrape off the surface grease and dirt with a knife, rinse, drain, and weigh 1 kg. Chop to a suitable size, add 5% acetic acid, and stir to extract for 8 hours. Add water, then use a chopper to prepare the tendon paste; grind with a grinder for 8 minutes with 45-second intervals; homogenize with a homogenizer for 5 minutes with 30-second intervals. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust pH to 2.0, add 2g pepsin, and incubate at 37℃ for 2 hours with stirring for enzymatic hydrolysis; adjust pH to 6.5, add 1.5g neutral protease, and incubate at 37℃ with stirring for 2 hours; adjust pH to 7.5, heat to 50℃, add 1.5g subtilisin, and incubate with stirring for 1 hour; heat to 55℃, add 1.5g papain, and incubate with stirring for 1 hour; adjust pH to 9.0, add 1.5g serine protease, and incubate with stirring for 2 hours. Centrifuge at 18500 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a plate and frame filter fitted with a 0.25-micron filter membrane. Vacuum until no air bubbles remain in the liquid. Filter the filtrate first using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, then dilute with water to the original volume. The ultrafiltrate was first nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume. Then, water was added to dilute it to its original volume, and the solution was desalted by nanofiltration 3–5 times. The solution was then sterilized. 61.4 g of lyophilized chicken tendon small molecule protein active peptide powder was prepared by freeze-drying.

[0222] (II) Partridge Small Molecule Protein Active Peptides

[0223] Example 41. Preparation method of small molecule protein bioactive peptides from partridge meat

[0224] Take 1 kg of slaughtered free-range partridge meat, manually remove fat and small blood vessels. Chop to an appropriate size, then use a meat chopper to prepare minced meat. Homogenize using a homogenizer for 5 minutes, with 30-second intervals. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer), add 1 g of neutral protease, and incubate at 37°C with stirring for 2 hours. Increase the temperature to 50°C, add 1 g of subtilisin, and incubate with stirring for 1 hour. Add 1 g of papain, and incubate with stirring for 1 hour. Adjust the pH to 7.5, add 1 g of flavor protease, and continue incubating with stirring for 3 hours. Centrifuge at 12000 rpm for 45 minutes. Collect the supernatant. Filter the crude protein peptide supernatant through a 0.25-micron filter plate. Vacuum until no air bubbles remain in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 60.6 g of partridge meat small molecule protein peptide lyophilized powder was prepared by freeze-drying.

[0225] Example 42. Preparation method of partridge small molecule bioactive protein peptides

[0226] Take 1.98 kg of slaughtered free-range partridges, slaughter them, remove blood and pluck feathers, and weigh 1 kg. Cut them to the appropriate size using a machine, add 4% acetic acid, and stir for 8 hours. Grind them into powder using a grinder. Add phosphate buffer (50 mM NaCl-20 mM phosphate buffer), adjust the pH to 2.0, add 2 g of pepsin, and stir at 37°C for 2 hours for enzymatic hydrolysis; adjust the pH to 6.5, add 1.5 g of neutral protease, and incubate at 37°C with stirring for 2 hours; adjust the pH to 7.5, raise the temperature to 50°C, add 1.5 g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1.5 g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1.5 g of serine protease, and incubate with stirring for 2 hours. Take the supernatant, and filter the crude protein peptide supernatant through a filter plate and frame fitted with a 0.25 micron filter membrane. Vacuum the liquid until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 51.6g of partridge small molecule active protein peptide lyophilized powder by freeze-drying.

[0227] Example 43. Preparation method of small molecule protein bioactive peptides from the partridge skeleton

[0228] Take 1 kg of free-range partridge skeleton (with meat thoroughly removed). Chop to a suitable size, add 5% acetic acid, and stir for 8 hours for extraction. Grind into powder using a grinder. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer), adjust the pH to 2.0, add 2 g of pepsin, and stir at 37°C for 2 hours for enzymatic hydrolysis; adjust the pH to 6.5, add 1.5 g of neutral protease, and incubate at 37°C with stirring for 2 hours; adjust the pH to 7.5, raise the temperature to 50°C, add 1.5 g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1.5 g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1.5 g of serine protease, and incubate with stirring for 2 hours. Centrifuge at 18500 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.25-micron filter plate and frame. Vacuum the liquid until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalination 3–5 times. Sterilize. Prepare 44.3g of partridge skeleton small molecule protein peptide lyophilized powder by freeze drying.

[0229] Example 44. Preparation method of small molecule protein bioactive peptides from partridge claw tendons

[0230] Collect the claw tendons (deboned) of free-range partridges. Scrape off the surface grease and dirt with a knife, rinse, drain, and weigh 1 kg. Chop to a suitable size, add 5% acetic acid, and stir for 8 hours. Add water, then use a chopper to prepare claw tendon paste; grind with a grinder for 8 minutes with 45-second intervals; homogenize with a homogenizer for 5 minutes with 30-second intervals. Adjust the pH to 2.0, add 2g of pepsin, and stir at 37℃ for 2 hours for enzymatic hydrolysis; adjust the pH to 6.5, add 1.5g of neutral protease, and incubate at 37℃ with stirring for 2 hours; adjust the pH to 7.5, raise the temperature to 50℃, add 1.5g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55℃, add 1.5g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1.5g of serine protease, and incubate with stirring for 2 hours. Centrifuge at 18500 rpm for 45 min. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.25 μm filter membrane. Vacuum until no air bubbles remain in the liquid. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 62.7 g of freeze-dried partridge claw tendon small molecule protein active peptide powder by freeze drying.

[0231] Example 9: Preparation method of mammalian small molecule bioactive protein peptides

[0232] (I) Preparation method of whole sheep small molecule bioactive peptides

[0233] Example 45. Effects of various process steps on the preparation of sheep small molecule bioactive peptides

[0234] Take fresh sheep tissues and organs, manually remove blood and fat, and weigh 50–300g. Chop finely, add phosphate buffer (1–200mM NaCl-1–200mM phosphate buffer), and grind using a grinder for approximately 1–60 minutes, with intervals of 5–120 seconds; then homogenize using a homogenizer for 1–60 minutes, with intervals of 5–120 seconds. Adjust the pH, add 1‰–100‰ neutral protease and / or papain and / or flavor protease and / or pepsin and / or trypsin and / or complex protease and / or serine protease and / or cathepsin and / or proteinase K, etc., and perform enzymatic hydrolysis for 0.1–24 hours, maintaining the hydrolysis temperature at 20–60℃. Centrifuge at 3000–25000 rpm for 5–120 minutes. Collect the supernatant; filter the crude protein peptide supernatant through a filter plate and frame fitted with a filter membrane of 0.01–10 micrometers. Vacuum until no air bubbles remain in the liquid. The filtrate is first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10,000–100,000, and then diluted with water to its original volume. The ultrafiltrate is then nanofiltered and desalted using a nanofiltration membrane with a molecular weight cutoff of 200–2000, and then diluted with water to its original volume. This nanofiltration desalting process is repeated 1–5 times. The solution is then sterilized. Finally, it is freeze-dried to prepare fish bone protein peptide lyophilized powder.

[0235] Example 46. Preparation method of small molecule bioactive protein peptides from mutton.

[0236] Take 1 kg of fresh lean lamb meat, remove blood and fat by hand. Chop it to an appropriate size, then use a meat chopper to make minced meat. Homogenize the minced meat using a homogenizer at 30-second intervals. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer) to adjust the pH to 6.8, add 1.0 g of neutral protease, and incubate at 37°C with stirring for 2 hours. Increase the temperature to 50°C, add 1.0 g of subtilisin, and incubate with stirring for 1 hour. Add 1.0 g of papain, and incubate with stirring for 1 hour. Adjust the pH to 7.5, add 1.0 g of flavor protease, and continue incubating for 1 hour. Add 2 g of proteinase K, and incubate at 55°C with stirring for 2 hours. Centrifuge at 18500 rpm for 45 min. Collect the supernatant. Filter the crude protein peptide supernatant through a 0.20 μm filter plate. Vacuum the liquid until no air bubbles remain. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10,000–100,000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–2000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 57g of freeze-dried mutton small-molecule active protein peptide powder was obtained.

[0237] Example 47. Preparation method of small molecule active protein peptides from sheep vertebrae

[0238] Take sheep vertebrae (or sheep spine bones), scrape off the surface fat and blood with a knife, rinse, drain the water, and weigh 1 kg. Crush the bones with a bone crusher, extract with 8% acetic acid for 4 hours, and grind into powder using a bone meal machine. Add phosphate buffer (50 mM NaCl-20 mM phosphate buffer), adjust the pH to 2.0, add 1 g of pepsin, and stir at 37°C for 2 hours for enzymatic hydrolysis; adjust the pH to 5.0, add 1 g of cathepsin (a mixture of D+B+K in equal proportions), and incubate at 40°C with stirring for 1.5 hours; adjust the pH to 6.5, add 1 g of neutral protease, and incubate at 37°C with stirring for 2 hours; adjust the pH to 7.5, raise the temperature to 50°C, add 1 g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1 g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1 g of proteinase K, and incubate with stirring for 2 hours. Centrifuge at 18500 rpm for 45 min. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.20 μm filter membrane. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 10,000–100,000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–2000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 38.3 g of small molecule active protein peptides from sheep vertebrae by freeze-drying.

[0239] Example 48. Preparation method of small molecule active protein peptides from sheep lungs

[0240] Take fresh sheep lungs, trim off the surface fat and blood with scissors, rinse quickly, and pat dry with kitchen paper towels. Weigh 1 kg. Chop and extract with 5% acetic acid for 8 hours. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer), grind for 8 minutes with 45-second intervals. Adjust the pH to 2.0, add 1 g of pepsin, and stir at 37°C for 1 hour for enzymatic hydrolysis; adjust the pH to 5.0, add 1 g of cathepsin (a mixture of D+B+L+H in equal proportions), and incubate at 40°C with stirring for 1 hour; adjust the pH to 6.5, add 1 g of neutral protease, and incubate at 37°C with stirring for 1 hour; adjust the pH to 7.5, raise the temperature to 50°C, add 1 g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1 g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1 g of serine protease, and incubate with stirring for 2 hours. The enzymatic hydrolysate was centrifuged at 12500 rpm, and the supernatant was collected. The crude protein peptide supernatant was filtered through a plate and frame filter fitted with a 0.25-micron filter membrane. Vacuum was applied until no air bubbles were present in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10,000–100,000, and then diluted with water to the original volume. The ultrafiltrate was first nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–2000, concentrated to 1 / 2–1 / 10 of the original volume, and then diluted with water to the original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 44.7 g of lyophilized sheep lung small molecule active protein peptide powder was prepared by freeze drying.

[0241] Example 49. Preparation method of small molecule bioactive protein peptides from sheep kidney

[0242] Take fresh sheep kidneys, trim off the surface fat (which is very thick) and remove blood vessels and blood with scissors, rinse quickly, and pat dry with kitchen paper towels. Weigh out 1 kg. Chop and extract with 3% acetic acid for 8 hours. Grind into a paste for 8 minutes, with 45-second intervals between grindings. Add phosphate buffer (50mM NaCl-20mM phosphate buffer, pH 7.0), adjust the pH to 2.0, add 1g of pepsin, and incubate at 37℃ for 1 hour with stirring. Adjust the pH to 5.0, add 1g of cathepsin (a mixture of D+B+L+H in equal proportions), and incubate at 40℃ with stirring for 1 hour. Adjust the pH to 6.5, add 1g of neutral protease, and incubate at 37℃ with stirring for 1 hour. Adjust the pH to 7.5, raise the temperature to 50℃, add 1g of subtilisin, and incubate with stirring for 1 hour. Raise the temperature to 55℃, add 1g of papain, and incubate with stirring for 1 hour. Adjust the pH to 9.0, add 1g of serine protease, and incubate with stirring for 2 hours. Centrifuge the enzymatic hydrolysate at 12500rpm for 45min, collect the supernatant, and filter the crude protein peptide supernatant through a 0.25-micron filter plate. Vacuum the solution until no air bubbles remain. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10,000–100,000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–2000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 44.5 g of lyophilized sheep kidney small molecule active protein peptide powder was prepared by freeze-drying.

[0243] Example 50. Preparation method of small molecule active protein peptides from sheep liver

[0244] Weigh out 1 kg of fresh sheep liver, and use scissors to remove surface fat, blood vessels, and blood. Chop the liver and extract with 3% acetic acid for 8 hours. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer, pH 7.0), and homogenize for 8 minutes with 45-second intervals. Homogenize for 5 minutes with 30-second intervals. Adjust the pH to 2.0, add 1g of pepsin, and incubate at 37°C with stirring for 1 hour for enzymatic hydrolysis; adjust the pH to 5.0, add 1g of cathepsin (a mixture of D+B+L+H in equal proportions), and incubate at 40°C with stirring for 1 hour; adjust the pH to 6.5, add 1g of neutral protease, and incubate at 37°C with stirring for 1 hour; adjust the pH to 7.5, raise the temperature to 50°C, add 1g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1g of serine protease, and incubate with stirring for 2 hours. Centrifuge at 12500 rpm for 45 min (centrifugal force RCF above 10000g). Collect the supernatant, and filter the crude protein peptide supernatant through a plate and frame filter fitted with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10,000–100,000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–2000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 46.5 g of lyophilized sheep liver small molecule active protein peptide powder was prepared by freeze-drying.

[0245] Example 51. Preparation method of small molecule protein bioactive peptides from sheep stomach

[0246] Weigh 1 kg of fresh sheep tripe, scrape off the surface grease (very much), impurities, and fat with a knife, rinse quickly, chop, and grind into a paste using a colloid mill. Add 3% glacial acetic acid, stir and extract overnight. Centrifuge at 14000 rpm and collect the precipitate. Add phosphate buffer (50 mM NaCl-20 mM phosphate buffer, pH 7.0), stir well, adjust the pH to 2.0, add 10 g of pepsin, and stir at 37°C for 4 hours for enzymatic hydrolysis; adjust the pH to 6.5, add 5.0 g of neutral protease, and incubate at 37°C for 1 hour; raise the temperature to 50°C, add 7.0 g of subtilisin, and incubate for 1 hour; raise the temperature to 55°C, add 8.0 g of papain, and incubate for 1 hour; adjust the pH to 9.0, add 5.0 g of serine protease, and incubate while stirring for 3 hours. Centrifuge at 12500 rpm for 45 min. Collect the supernatant. Filter the crude protein peptide supernatant using a 0.25-micron filter plate and frame. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 10,000–100,000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–2000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 40.3g of freeze-dried sheep stomach small molecule protein active peptide powder by freeze-drying.

[0247] Example 52. Preparation method of small molecule protein active peptides from sheep spleen

[0248] Take fresh sheep spleen, manually cut or use a knife to remove attached tissues such as oil and blood vessels, weigh 1 kg. Chop into fine shreds, add 3% hydrochloric acid, and extract for 4.5 hours. Grind into a paste for 10 minutes, with an interval of 45 seconds. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer), adjust pH to 6.8, add 1.0 g neutral protease, and incubate at 37°C with stirring for 2 hours; raise the temperature to 50°C, add 1.0 g subtilis protease, and incubate with stirring for 1 hour; add 1.0 g papain, and incubate with stirring for 1 hour; adjust pH to 7.5, add 1.0 g flavor protease, and continue incubation for 1 hour; add 2 g proteinase K, and incubate at 55°C with stirring for 2 hours. Centrifuge at 15000 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a filter plate and frame fitted with a 0.45 μm filter membrane. Vacuum until no air bubbles remain in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10,000–100,000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–2000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 45.2 g of lyophilized sheep spleen small molecule protein active peptide powder was prepared by freeze-drying.

[0249] Example 53. Preparation method of small molecule protein bioactive peptides from sheep pancreas

[0250] Take 1 kg of fresh sheep pancreas, manually cut or with a knife to remove oil, blood vessels, and other attached tissues. Chop into fine shreds and grind for 10 minutes, with 45-second intervals; homogenize for 5 minutes, with 30-second intervals. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer), add 1 g of neutral protease, and incubate at 37°C with stirring for 2 hours; raise the temperature to 50°C, add 1 g of subtilisin, and incubate with stirring for 1 hour; add 1 g of papain, and incubate with stirring for 1 hour; adjust the pH to 7.5, add 1 g of flavor protease, and continue incubating with stirring for 3 hours. Centrifuge at 15000 rpm for 45 minutes. Collect the supernatant; filter the crude protein peptide supernatant through a 0.45-micron filter plate. Vacuum until no air bubbles remain in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10,000–100,000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–2000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 41.9 g of lyophilized sheep pancreas small molecule protein active peptide powder was prepared by freeze-drying.

[0251] Example 54. Preparation method of small molecule active protein peptides from sheep heart

[0252] Take fresh sheep heart, trim off the surface fat and remove blood with scissors, and weigh 1 kg. Chop it and extract with 10% acetic acid for 6 hours. Grind into a paste for 15 minutes, with 45-second intervals. Adjust the pH to 2.0, add 1 g of pepsin, and stir at 37°C for 2 hours for enzymatic hydrolysis; adjust the pH to 5.0, add 1 g of cathepsin (a mixture of D+B+L+K in equal proportions), and incubate at 40°C with stirring for 1.5 hours; adjust the pH to 6.5, add 1 g of neutral protease, and incubate at 37°C with stirring for 2 hours; adjust the pH to 7.5, raise the temperature to 50°C, add 1 g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1 g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1 g of proteinase K, and incubate with stirring for 2 hours. Centrifuge at 10000 rpm for 45 minutes. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.22-micron filter membrane. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 10,000–100,000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–2000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 42.2 g of lyophilized sheep heart small molecule active protein peptide powder by freeze drying.

[0253] Example 55. Preparation method of small molecule active protein peptides from ram horn

[0254] Take 1 kg of sheep horn and scrape off any dirt or impurities with a knife. Crush the horn using a bone crusher, extract with 3% acetic acid for 4 hours, and then grind it into powder using a bone meal machine. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer), adjust the pH to 2.0, add 1 g of pepsin, and stir at 37°C for 2 hours for enzymatic hydrolysis. Adjust the pH to 5.0, add 1 g of cathepsin (a mixture of D, B, L, and K in equal proportions), and incubate at 40°C with stirring for 1.5 hours. Adjust the pH to 6.5, add 1 g of neutral protease, and incubate at 37°C with stirring for 2 hours. Adjust the pH to 7.5, raise the temperature to 50°C, add 1 g of subtilisin, and incubate with stirring for 1 hour. Raise the temperature to 55°C, add 1 g of papain, and incubate with stirring for 1 hour. Adjust the pH to 9.0, add 1 g of proteinase K, and incubate with stirring for 2 hours. Filter the crude protein peptide supernatant using a 0.25-micron filter plate and frame. Vacuum the liquid until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 10,000–100,000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–2000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 36.3g of lyophilized horn-shaped small molecule active protein peptide powder by freeze-drying.

[0255] (II) Preparation methods of small molecule protein bioactive peptides from animal brain

[0256] Example 56. Preparation method of porcine brain small molecule protein bioactive peptides

[0257] Take fresh pig brains, manually remove fat, blood vessels, and blood, and weigh 1 kg. Chop to an appropriate consistency, add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer, pH 6.8), and then prepare brain paste using a chopper, grinding for 5 minutes with 45-second intervals; homogenize for 5 minutes with 30-second intervals. Adjust pH to 6.8, add 1.0 g neutral protease, and incubate at 37°C with stirring for 2 hours; raise temperature to 50°C, add 1.0 g subtilis protease, and incubate with stirring for 1 hour; add 1.0 g papain, and incubate with stirring for 1 hour; adjust pH to 7.5, add 1.0 g flavor protease, and continue incubation for 1 hour; add 2 g proteinase K, and incubate at 55°C with stirring for 2 hours. Centrifuge at 12500 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a filter plate and frame fitted with a 0.45-micron filter membrane. Vacuum the liquid until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 15,000–100,000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–1500 or higher, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 36.7g of freeze-dried porcine brain small molecule protein active peptide powder.

[0258] Example 57. Preparation method of bovine brain small molecule protein bioactive peptides

[0259] Take fresh bovine brain, manually remove fat, blood vessels, and blood, and weigh 1 kg. Chop to an appropriate consistency, add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer, pH 7.0), and then prepare brain paste using a chopper, grinding for 5 minutes with 45-second intervals; homogenize for 5 minutes with 30-second intervals. Adjust pH to 6.8, add 1.0 g neutral protease, and incubate at 37°C with stirring for 2 hours; raise temperature to 50°C, add 1.0 g subtilis protease, and incubate with stirring for 1 hour; add 1.0 g papain, and incubate with stirring for 1 hour; adjust pH to 7.5, add 1.0 g flavor protease, and continue incubation for 1 hour; add 2 g proteinase K, and incubate at 55°C with stirring for 2 hours. Centrifuge at 12500 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.45-micron filter plate and frame. Vacuum the liquid until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 15,000–100,000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–1500, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 38.6 g of lyophilized bovine brain small molecule protein active peptide powder by freeze-drying.

[0260] Example 58. Preparation method of deer brain small molecule protein active peptides

[0261] Take fresh deer brains, manually remove fat, blood vessels, and blood, and weigh 1 kg. Chop to an appropriate consistency, add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer, pH 7.0), and then prepare brain paste using a chopper, grinding for 5 minutes with 45-second intervals; homogenize for 5 minutes with 30-second intervals. Adjust pH to 6.8, add 1.0 g neutral protease, and incubate at 37°C with stirring for 2 hours; raise temperature to 50°C, add 1.0 g subtilis protease, and incubate with stirring for 1 hour; add 1.0 g papain, and incubate with stirring for 1 hour; adjust pH to 7.5, add 1.0 g flavor protease, and continue incubation for 1 hour; add 2 g proteinase K, and incubate at 55°C with stirring for 2 hours. Centrifuge at 12500 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a filter plate and frame fitted with a 0.45-micron filter membrane. Vacuum the liquid until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 15,000–100,000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–1500, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 37.1 g of freeze-dried deer brain small molecule protein active peptide powder by freeze drying.

[0262] (III) Preparation method of camel small molecule bioactive peptides

[0263] Example 59. Effects of various process steps on the preparation of camel small molecule bioactive peptides

[0264] Camel tissues and organs were harvested, and blood and fat were removed by hand. 50–300 g of the harvested tissues and organs were weighed. The tissues were chopped, and phosphate buffer (1–200 mM NaCl-1–200 mM phosphate buffer) was added. The tissues were then homogenized using a grinder for approximately 1–60 minutes, with intervals of 5–120 seconds. The homogenate was then homogenized for 1–60 minutes, with intervals of 5–120 seconds. The pH was adjusted, and 1‰–100‰ neutral protease and / or papain and / or flavor protease and / or pepsin and / or trypsin and / or complex protease and / or serine protease and / or cathepsin and / or proteinase K were added. Enzymatic hydrolysis was performed for 0.1–24 hours at a temperature maintained at 20–60°C. The mixture was centrifuged at 3000–25000 rpm for 5–120 minutes. The supernatant was collected, and the crude protein peptide supernatant was filtered through a plate and frame filter fitted with a 0.01–10 micrometer filter membrane. Vacuum was applied until no air bubbles remained in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 2000–1,000,000, and then diluted with water to its original volume. The ultrafiltrate was then concentrated by nanofiltration using a nanofiltration membrane with a molecular weight cutoff of 200–3000, and then diluted with water to its original volume. This nanofiltration desalting process was repeated 1–5 times. The solution was then sterilized. Finally, freeze-drying was used to prepare lyophilized powder of small molecule active peptides from camel tissues and organs.

[0265] Example 60. Preparation method of small molecule bioactive protein peptides from camel meat

[0266] Take camel meat, manually remove blood and fat, and weigh 1 kg. Chop to the appropriate consistency, then use a meat chopper to prepare a paste, grinding for 8 minutes with 45-second intervals; homogenize for 5 minutes with 30-second intervals. Add phosphate buffer (50 mM NaCl-20 mM phosphate buffer), adjust pH to 6.8, add 1.0 g neutral protease, and incubate at 37°C with stirring for 2 hours; raise temperature to 50°C, add 1.0 g subtilis protease, and incubate with stirring for 1 hour; add 1.0 g papain, and incubate with stirring for 1 hour; adjust pH to 7.5, add 1.0 g flavor protease, and continue incubation for 1 hour; add 2 g proteinase K, and incubate at 55°C with stirring for 2 hours. Centrifuge the enzymatic hydrolysate at 18500 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.20-micron filter plate and frame. Vacuum until no air bubbles remain in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–2500, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 61.4 g of camel meat small-molecule active protein peptide lyophilized powder was prepared by freeze-drying.

[0267] Example 61. Preparation method of small molecule active protein peptides from camel rib cage

[0268] Take camel carcass bones, scrape off the surface fat and blood with a knife, and weigh 1 kg. Crush the bones using a bone crusher, extract with 3% acetic acid for 4 hours, and then grind into powder using a bone meal machine. Add phosphate buffer (50 mM NaCl-20 mM phosphate buffer), adjust the pH to 2.0, add 1.5 g of pepsin, and stir at 37°C for 4 hours for enzymatic hydrolysis; adjust the pH to 6.5, add 1.5 g of neutral protease, and incubate at 37°C for 1 hour; raise the temperature to 50°C, add 1.0 g of subtilisin, and incubate for 1 hour; raise the temperature to 55°C, add 1.0 g of papain, and incubate for 1 hour; raise the temperature to 55°C and adjust the pH to 9.0, add 2.0 g of serine protease, and continue incubating for 3 hours. Centrifuge at 13500 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.25 μm filter plate and frame. Vacuum until no air bubbles remain in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–2500, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 36.7 g of camel rib cartilage small molecule active protein peptide lyophilized powder was prepared by freeze-drying.

[0269] Example 62. Preparation method of small molecule active protein peptides from camel lungs

[0270] Take camel lungs, trim off the surface grease and remove blood with scissors, rinse, filter, and weigh 1 kg. Chop and extract with 6% acetic acid for 8 hours. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer), grind for 15 minutes with 45-second intervals. Adjust pH to 2.0, add 1 g pepsin, and stir at 37°C for 2 hours for enzymatic hydrolysis; adjust pH to 5.0, add 1 g cathepsin (a mixture of D+B+L+K in equal proportions), and incubate at 40°C with stirring for 1.5 hours; adjust pH to 6.5, add 1 g neutral protease, and incubate at 37°C with stirring for 2 hours; adjust pH to 7.5, raise temperature to 50°C, add 1 g subtilisin, and incubate with stirring for 1 hour; raise temperature to 55°C, add 1 g papain, and incubate with stirring for 1 hour; adjust pH to 9.0, add 1 g proteinase K, and incubate with stirring for 2 hours. Centrifuge at 12500 rpm for 45 min. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.25 μm filter membrane. Vacuum the liquid until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–2500, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 43.0 g of lyophilized camel lung small molecule active protein peptide powder by freeze drying.

[0271] Example 63. Preparation method of camel kidney small molecule active protein peptides

[0272] Weigh out 1 kg of camel kidneys (kidneys), and use scissors to remove the excess surface fat, blood vessels, and blood. Chop the kidneys and extract with 3% acetic acid for 5 hours. Grind the mixture for 8 minutes, with 45-second intervals between grindings. Adjust the pH to 2.0, add 1 g of pepsin, and incubate at 37°C for 2 hours with stirring for enzymatic hydrolysis. Adjust the pH to 5.0, add 1 g of cathepsin (a mixture of D, B, L, and K in equal proportions), and incubate at 40°C with stirring for 1.5 hours. Adjust the pH to 6.5, add 1 g of neutral protease, and incubate at 37°C with stirring for 2 hours. Adjust the pH to 7.5, raise the temperature to 50°C, add 1 g of subtilisin, and incubate with stirring for 1 hour. Raise the temperature to 55°C, add 1 g of papain, and incubate with stirring for 1 hour. Adjust the pH to 9.0, add 1 g of serine protease, and incubate with stirring for 2 hours. Centrifuge at 12500 rpm for 45 minutes. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–2500, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 45.2 g of camel kidney small molecule active protein peptide lyophilized powder by freeze drying.

[0273] Example 64. Preparation method of small molecule active protein peptides from camel liver

[0274] Weigh 1 kg of camel liver, and use scissors to remove the surface fat, blood vessels, and blood. Chop the liver and extract with 5% acetic acid for 6 hours. Grind the liver into a paste for 12 minutes, with 45-second intervals. Adjust the pH to 2.0, add 1 g of pepsin, and incubate at 37°C for 2 hours with stirring for enzymatic hydrolysis. Adjust the pH to 5.0, add 1 g of cathepsin (a mixture of D, B, L, and K in equal proportions), and incubate at 40°C with stirring for 1.5 hours. Adjust the pH to 6.5, add 1 g of neutral protease, and incubate at 37°C with stirring for 2 hours. Adjust the pH to 7.5, raise the temperature to 50°C, add 1 g of subtilisin, and incubate with stirring for 1 hour. Raise the temperature to 55°C, add 1 g of papain, and incubate with stirring for 1 hour. Adjust the pH to 9.0, add 1 g of serine protease, and incubate with stirring for 2 hours. Centrifuge at 12500 rpm for 45 minutes (centrifugal force RCF above 10000g). Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–2500, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 45.7g of lyophilized camel liver small molecule active protein peptide powder by freeze-drying.

[0275] Example 65. Preparation method of small molecule protein bioactive peptides from camel stomach

[0276] Weigh out 1 kg of camel stomach, scrape off the surface grease (very much), impurities, and fat with a knife, rinse, dry, and weigh out. Chop and grind into a paste for 20 minutes, with 45-second intervals between grindings. Add 3% acetic acid to the paste and extract overnight by stirring. Centrifuge at 14,000 rpm and collect the precipitate. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust pH to 2.0, add 1g pepsin, and incubate at 37℃ for 2 hours with stirring for enzymatic hydrolysis; adjust pH to 5.0, add 1g cathepsin (D+B+L+K mixture), and incubate at 40℃ with stirring for 1.5 hours; adjust pH to 6.5, add 1g neutral protease, and incubate at 37℃ with stirring for 2 hours; adjust pH to 7.5, raise temperature to 50℃, add 1g subtilisin, and incubate with stirring for 1 hour; raise temperature to 55℃, add 1g papain, and incubate with stirring for 1 hour; adjust pH to 9.0, add 1g serine protease, and incubate with stirring for 2 hours. Centrifuge at 12500rpm for 45min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.25μm filter plate. Vacuum the solution until no air bubbles remain. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–2500, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 41.1 g of camel stomach small molecule protein active peptide lyophilized powder was prepared by freeze-drying.

[0277] Example 66. Preparation method of camel paw small molecule active protein peptide

[0278] Take camel paws (foot paws), scrape off surface dirt and grease with a knife, rinse, dry, and weigh 1 kg. Cut into pieces, grind into a pulp for 20 minutes, with 45-second intervals. Add 3% acetic acid to the pulp, stir and extract overnight. Centrifuge at 14,000 rpm and collect the precipitate. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust pH to 2.0, add 1g pepsin, and incubate at 37℃ for 2 hours with stirring for enzymatic hydrolysis; adjust pH to 5.0, add 1g cathepsin (a mixture of D+B+L+K in equal proportions), and incubate at 40℃ with stirring for 1.5 hours; adjust pH to 6.5, add 1g neutral protease, and incubate at 37℃ with stirring for 2 hours; adjust pH to 7.5, raise temperature to 50℃, add 1g subtilisin, and incubate with stirring for 1 hour; raise temperature to 55℃, add 1g papain, and incubate with stirring for 1 hour; add 2g proteinase K, and incubate at 55℃ with stirring for 2 hours. Centrifuge at 18500rpm for 45min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.20μm filter plate. Vacuum the solution until no air bubbles remain. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–2500, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 86.7 g of camel's foot small molecule active protein peptide lyophilized powder was prepared by freeze-drying.

[0279] (iv) Preparation method of small molecule bioactive peptides from animal skin

[0280] Example 67. Preparation method of donkey skin small molecule active protein peptides

[0281] Take donkey hide, scrape off the surface hair and remove grease and other dirt from the inner surface with a knife, and weigh 1 kg. Chop and grind the hide into a pulp using a chopper and a grinder, with an interval of 45 seconds. Add 3% acetic acid and extract for 4 hours, stirring and extracting overnight. Centrifuge at 14,000 rpm and collect the precipitate. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust pH to 2.0, add 1g pepsin, and incubate at 37℃ for 2 hours with stirring for enzymatic hydrolysis; adjust pH to 5.0, add 1g cathepsin (D+B+L+K mixture), and incubate at 40℃ with stirring for 1.5 hours; adjust pH to 6.5, add 1g neutral protease, and incubate at 37℃ with stirring for 2 hours; adjust pH to 7.5, raise temperature to 50℃, add 1g subtilisin, and incubate with stirring for 1 hour; raise temperature to 55℃, add 1g papain, and incubate with stirring for 1 hour; adjust pH to 9.0, add 1g serine protease, and incubate with stirring for 2 hours. Centrifuge at 18500rpm for 45min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.45μm filter plate. Vacuum the solution until no air bubbles remain. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 52.1 g of donkey skin small molecule active protein peptide lyophilized powder was prepared by freeze-drying.

[0282] Example 68. Preparation method of small molecule active protein peptides from bovine hide

[0283] Take a cowhide, scrape off the surface hair and remove grease and other dirt from the inner surface with a knife, and weigh 1 kg. Chop and grind the hide with a chopper for 20 minutes, with 45-second intervals. Add 3% acetic acid to the pulp, stir and extract overnight. Centrifuge at 14,000 rpm and collect the precipitate. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust pH to 2.0, add 1g pepsin, and incubate at 37℃ for 2 hours with stirring for enzymatic hydrolysis; adjust pH to 5.0, add 1g cathepsin (D+B+L+K mixture), and incubate at 40℃ with stirring for 1.5 hours; adjust pH to 6.5, add 1g neutral protease, and incubate at 37℃ with stirring for 2 hours; adjust pH to 7.5, raise temperature to 50℃, add 1g subtilisin, and incubate with stirring for 1 hour; raise temperature to 55℃, add 1g papain, and incubate with stirring for 1 hour; adjust pH to 9.0, add 1g serine protease, and incubate with stirring for 2 hours. Centrifuge at 18500rpm for 45min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.45μm filter plate. Vacuum the solution until no air bubbles remain. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 53.9 g of lyophilized bovine skin small molecule active protein peptide powder was prepared by freeze-drying.

[0284] Example 69. Preparation method of small molecule active protein peptides from sheepskin

[0285] Take a sheepskin, scrape off the surface hair and remove grease and other dirt from the inner surface with a knife, and weigh 1 kg. Chop and mix the hide with a chopper, then grind it into a pulp using a grinder for 20 minutes with 45-second intervals. Add 3% acetic acid to the pulp and extract overnight by stirring. Centrifuge at 14,000 rpm and collect the precipitate. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust the pH to 2.0, add 1g of pepsin, and incubate at 37°C with stirring for 2 hours for enzymatic hydrolysis; adjust the pH to 5.0, add 1g of cathepsin (a mixture of D+B+L+K in equal proportions), and incubate at 40°C with stirring for 1.5 hours; adjust the pH to 6.5, add 1g of neutral protease, and incubate at 37°C with stirring for 2 hours; adjust the pH to 7.5, raise the temperature to 50°C, add 1g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1g of serine protease, and incubate with stirring for 2 hours. Collect the supernatant, and filter the crude protein peptide supernatant through a filter plate and frame fitted with a 0.45-micron filter membrane. Vacuum the solution until no air bubbles remain. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 45.3 g of lyophilized sheepskin small molecule active protein peptide powder was prepared by freeze-drying.

[0286] Example 70. Preparation method of small molecule active protein peptides from porcine skin

[0287] Take pigskin, scrape off the surface hair and remove grease and other dirt from the inner surface with a knife, and weigh 1 kg. Chop and mix the pigskin with a chopper, then grind it into a pulp using a grinder for 20 minutes with 45-second intervals. Add 5% acetic acid to the pulp and extract it overnight by stirring. Centrifuge at 14,000 rpm and collect the precipitate. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust pH to 2.0, add 1g pepsin, and incubate at 37℃ for 2 hours with stirring for enzymatic hydrolysis; adjust pH to 5.0, add 1g cathepsin (D+B+L+K mixture), and incubate at 40℃ with stirring for 1.5 hours; adjust pH to 6.5, add 1g neutral protease, and incubate at 37℃ with stirring for 2 hours; adjust pH to 7.5, raise temperature to 50℃, add 1g subtilisin, and incubate with stirring for 1 hour; raise temperature to 55℃, add 1g papain, and incubate with stirring for 1 hour; adjust pH to 9.0, add 1g serine protease, and incubate with stirring for 2 hours. Centrifuge at 18500rpm for 45min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.45μm filter plate. Vacuum the solution until no air bubbles remain. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 49.4 g of lyophilized pigskin small molecule active protein peptide powder was prepared by freeze-drying.

[0288] (V) Preparation method of small molecule active peptides from animal tendons

[0289] Example 71. Effects of various improvements in preparation process steps on the preparation effect of animal tendon active peptides

[0290] Weigh 100-300g of fresh animal tendons and manually remove blood and fat. Chop and grind into a paste. Grind for 20 minutes, with 45-second intervals. Add 3% acetic acid to the paste and extract overnight with stirring. Centrifuge at 14000 rpm and collect the precipitate. Add phosphate buffer (50mM NaCl-20mM phosphate buffer) and 3% acetic acid, and extract for 6 hours. Adjust the pH and add pepsin and / or bromelain and / or serine protease and / or neutral protease and / or papain and / or flavor protease and / or trypsin and / or complex protease, etc., for enzymatic hydrolysis for 4-12 hours, maintaining the hydrolysis temperature at 35-60℃. Centrifuge at 8000-20000 rpm for 45 minutes. Collect the supernatant. Filter the crude protein peptide supernatant through a plate and frame filter fitted with a 0.20-0.45 micrometer filter membrane. Vacuum until no air bubbles remain in the liquid. The filtrate is first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 2000–1,000,000, and then diluted with water to its original volume. The ultrafiltrate is then nanofiltered and desalted using a nanofiltration membrane with a molecular weight cutoff of 200–2000, and then diluted with water to its original volume. This nanofiltration desalting process is repeated 3–5 times. The solution is then sterilized. Finally, it is freeze-dried to prepare animal fascia peptide lyophilized powder.

[0291] Example 72. Preparation method of small molecule active protein peptides from bovine tendon

[0292] Take fresh beef tendon, remove oil and impurities by hand or with a knife, rinse, drain, and weigh 1 kg. Chop, chop with a chopper, and grind into a tendon slurry using a grinder. Grind for 20 minutes with 45-second intervals. Add 3% acetic acid to the slurry, stir, and extract overnight. Centrifuge at 14,000 rpm and collect the precipitate. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust pH to 2.0, add 1g pepsin, and incubate at 37℃ for 2 hours with stirring for enzymatic hydrolysis; adjust pH to 5.0, add 1g cathepsin (a mixture of D+B+L+K in equal proportions), and incubate at 40℃ with stirring for 1.5 hours; adjust pH to 6.5, add 1g neutral protease, and incubate at 37℃ with stirring for 2 hours; adjust pH to 7.5, raise temperature to 50℃, add 1g subtilisin, and incubate with stirring for 1 hour; raise temperature to 55℃, add 1g papain, and incubate with stirring for 1 hour; adjust pH to 9.0, add 1g proteinase K, and incubate with stirring for 2 hours. Centrifuge at 18500rpm for 45min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.20μm filter plate. Vacuum the solution until no air bubbles remain. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 2000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–2000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 55.6 g of lyophilized small-molecule active protein peptide powder from beef tendon was prepared by freeze-drying.

[0293] Example 73. Preparation method of small molecule protein bioactive peptides from pig tendons

[0294] Take fresh, frozen pig tendons, remove excess oil and impurities by hand or with a knife, rinse, drain, and weigh 1 kg. Chop, mince using a chopper, and grind into a tendon slurry using a grinder. Grind for 20 minutes with 45-second intervals. Add 3% acetic acid to the slurry, stir, and extract overnight. Centrifuge at 14,000 rpm and collect the precipitate. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust pH to 2.0, add 1g pepsin, and incubate at 37℃ for 2 hours with stirring for enzymatic hydrolysis; adjust pH to 5.0, add 1g cathepsin (D+B+L+K mixture), and incubate at 40℃ with stirring for 1.5 hours; adjust pH to 6.5, add 1g neutral protease, and incubate at 37℃ with stirring for 2 hours; adjust pH to 7.5, raise temperature to 50℃, add 1g subtilisin, and incubate with stirring for 1 hour; raise temperature to 55℃, add 1g papain, and incubate with stirring for 1 hour; adjust pH to 9.0, add 1g serine protease, and incubate with stirring for 2 hours. Centrifuge at 18500rpm for 45min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.20μm filter plate. Vacuum until no air bubbles remain in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 2000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–2000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 52.9 g of lyophilized pig tendon small molecule protein active peptide powder was prepared by freeze-drying.

[0295] Example 74. Preparation method of deer tendon small molecule active protein peptides

[0296] Take fresh, frozen, farmed sika deer tendons (limb tendons), manually cut or use a knife to remove oil and attached tissue, rinse, and weigh 1 kg. Chop, mince with a chopper, and grind into a tendon slurry using a grinder for 20 minutes with 45-second intervals. Add 3% acetic acid to the slurry and extract overnight with stirring. Centrifuge at 14,000 rpm and collect the precipitate. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust pH to 2.0, add 1g pepsin, and incubate at 37°C with stirring for 2 hours for enzymatic hydrolysis; adjust pH to 5.0, add 1g cathepsin (a mixture of D+B+L+K in equal proportions), and incubate at 40°C with stirring for 1.5 hours; adjust pH to 6.5, add 1g neutral protease, and incubate at 37°C with stirring for 2 hours; adjust pH to 7.5, raise temperature to 50°C, add 1g subtilisin, and incubate with stirring for 1 hour; raise temperature to 55°C, add 1g papain, and incubate with stirring for 1 hour; adjust pH to 9.0, add 1g proteinase K, and incubate with stirring for 2 hours. Centrifuge at 18500 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.20 μm filter plate. Vacuum the solution until no air bubbles remain. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 2000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–2000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalination was performed 3–5 times. The solution was then sterilized. 50.4 g of deer tendon small molecule active protein peptide lyophilized powder was prepared by freeze-drying.

[0297] (vi) Preparation method of bovine small molecule protein bioactive peptides

[0298] Example 75. Effects of various preparation processes on the production of bovine small molecule bioactive protein peptides

[0299] Take fresh bovine tissues and organs, manually remove blood and fat, and weigh 100–300 g. Chop finely, add phosphate buffer (50 mM NaCl-20 mM phosphate buffer), and grind into a paste. Adjust the pH, add pepsin and / or bromelain and / or serine protease and / or neutral protease and / or papain and / or flavor protease and / or trypsin and / or complex protease, etc., and perform enzymatic hydrolysis for 0.5–12 hours, maintaining the hydrolysis temperature at 35–60℃. Centrifuge at 2000–25000 rpm for 5–90 min. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.20–1 micrometer filter membrane. Vacuum until no air bubbles remain in the liquid. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 2000–1000000, then dilute with water to the original volume. The ultrafiltrate was first concentrated by nanofiltration using a nanofiltration membrane with a molecular weight cutoff of 200–3000, then diluted with water to its original volume, and the nanofiltration process was repeated 3–5 times. The solution was then sterilized. Finally, it was freeze-dried to prepare bovine small molecule protein active peptide lyophilized powder.

[0300] Example 76. Preparation method of small molecule bioactive peptides from beef

[0301] Take fresh yak meat, manually remove blood and fat, and weigh 1 kg. Chop to an appropriate size, then use a meat chopper to prepare minced meat, grinding for 8 minutes with 45-second intervals. Homogenize for 6 minutes with 30-second intervals. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust pH to 6.8, add 1g of neutral protease, and incubate at 37°C with stirring for 2 hours; adjust pH to 7.5, raise temperature to 50°C, add 1g of subtilisin, and incubate with stirring for 1 hour; raise temperature to 55°C, add 1g of papain, and incubate with stirring for 1 hour; adjust pH to 9.0, add 1g of proteinase K, and incubate with stirring for 2 hours. Centrifuge at 15000 rpm for 45 minutes. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.25-micron filter plate. Vacuum until no air bubbles remain in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. This process was repeated 3–5 times for nanofiltration desalting. The solution was then sterilized. 60.8 g of yak meat small molecule protein active peptide freeze-dried powder was prepared by freeze-drying.

[0302] Example 77. Preparation method of small molecule protein bioactive peptides from calf spleen

[0303] Take fresh calf spleen (sometimes called calf liver), manually cut or use a knife to remove attached tissues such as oil and blood vessels, weigh 1 kg. Chop into fine shreds, add 3% hydrochloric acid, and extract for 4.5 hours. Grind into a paste for 10 minutes, with an interval of 45 seconds. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust the pH to 2.0, add 1g of pepsin, and incubate at 37°C with stirring for 2 hours for enzymatic hydrolysis; adjust the pH to 5.0, add 1g of cathepsin (a mixture of D+B+L+K in equal proportions), and incubate at 40°C with stirring for 1.5 hours; adjust the pH to 6.5, add 1g of neutral protease, and incubate at 37°C with stirring for 2 hours; adjust the pH to 7.5, raise the temperature to 50°C, add 1g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1g of proteinase K, and incubate with stirring for 2 hours. Centrifuge at 15000 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a plate and frame filter fitted with a 0.25-micron filter membrane. Vacuum the solution until no air bubbles remain. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 43.8 g of lyophilized calf spleen small molecule protein active peptide powder was prepared by freeze-drying.

[0304] Example 78. Preparation method of bovine pancreatic small molecule protein bioactive peptides

[0305] Take 1 kg of fresh yak pancreas, manually cut or with a knife to remove oil, blood vessels, and other attached tissues. Chop it into fine shreds and grind it into a paste for 10 minutes, with an interval of 45 seconds; homogenize for 5 minutes, with an interval of 30 seconds. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer), add 1 g of neutral protease, and incubate at 37°C with stirring for 2 hours; raise the temperature to 50°C, add 1 g of subtilisin, and incubate with stirring for 1 hour; add 1 g of papain, and incubate with stirring for 1 hour; adjust the pH to 7.5, add 1 g of flavor protease, and continue incubating with stirring for 3 hours. Centrifuge at 15000 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a filter plate and frame fitted with a 0.22-micron filter membrane. Vacuum the liquid until no air bubbles remain. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 42.4 g of lyophilized yak pancreatic small molecule protein active peptide powder was prepared by freeze-drying.

[0306] Example 79. Preparation method of small molecule bioactive protein peptides from bovine heart

[0307] Take fresh yak calf heart, remove surface fat and blood with scissors, and weigh 1 kg. Chop and extract with 6% acetic acid for 3 hours. Grind into a paste for 15 minutes, with 45-second intervals. Adjust the pH to 2.0, add 1 g of pepsin, and stir at 37°C for 2 hours for enzymatic hydrolysis; adjust the pH to 5.0, add 1 g of cathepsin (a mixture of D+B+L+K in equal proportions), and incubate at 40°C with stirring for 1.5 hours; adjust the pH to 6.5, add 1 g of neutral protease, and incubate at 37°C with stirring for 2 hours; adjust the pH to 7.5, raise the temperature to 50°C, add 1 g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1 g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1 g of proteinase K, and incubate with stirring for 2 hours. Centrifuge at 10000 rpm for 45 minutes. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.22-micron filter membrane. Vacuum the liquid until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 44.8 g of yak heart small molecule active protein peptide lyophilized powder by freeze drying.

[0308] Example 80. Preparation method of small molecule bioactive peptides from animal intestines

[0309] Take fresh yak intestines, cut off the surface grease and remove blood vessels and blood with scissors, manually scrape off surface grease and impurities, soak in salt water, rinse with clean water, filter dry, and weigh 1 kg. Grind into a paste using a colloid mill for 8 minutes with 45-second intervals; homogenize using a homogenizer for 5 minutes with 30-second intervals. Add 5% acetic acid and extract overnight. Centrifuge at 14000 rpm and collect the precipitate. Adjust the pH to 2.0, add 1g of pepsin, and incubate at 37°C for 2 hours with stirring for enzymatic hydrolysis; adjust the pH to 5.0, add 1g of cathepsin (a mixture of D+B+L+K in equal proportions), and incubate at 40°C with stirring for 1.5 hours; adjust the pH to 6.5, add 1g of neutral protease, and incubate at 37°C with stirring for 2 hours; adjust the pH to 7.5, raise the temperature to 50°C, add 1g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1g of proteinase K, and incubate with stirring for 2 hours. Centrifuge at 15000 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.45-micron filter membrane. Vacuum the solution until no air bubbles remain. Ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, and then dilute with water to the original volume. The ultrafiltrate was first nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume. Then, water was added to dilute it to its original volume, and the solution was desalted by nanofiltration 3–5 times. The solution was then sterilized. 40.3 g of yak intestine small molecule active peptide lyophilized powder was prepared by freeze-drying.

[0310] Example 81. Preparation method of bovine gastric small molecule bioactive peptides

[0311] Take tripe, trim off the surface grease and remove blood vessels and blood with scissors, manually scrape off surface grease and impurities, soak in salt water, rinse with clean water, filter dry, and weigh 1 kg. Grind into a paste using a colloid mill for 8 minutes with 45-second intervals; homogenize using a homogenizer for 5 minutes with 30-second intervals. Add 5% acetic acid and extract overnight. Centrifuge at 14000 rpm and collect the precipitate. Adjust the pH to 2.0, add 1g of pepsin, and incubate at 37°C for 2 hours with stirring for enzymatic hydrolysis; adjust the pH to 5.0, add 1g of cathepsin (a mixture of D+B+L+K in equal proportions), and incubate at 40°C with stirring for 1.5 hours; adjust the pH to 6.5, add 1g of neutral protease, and incubate at 37°C with stirring for 2 hours; adjust the pH to 7.5, raise the temperature to 50°C, add 1g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1g of proteinase K, and incubate with stirring for 2 hours. Centrifuge at 15000 rpm for 45 min. Filter the crude protein peptides through a 0.2-micron filter membrane. Vacuum the liquid until no air bubbles remain. Ultrafilter the coarse filtrate first using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, then dilute with water to the original volume. The ultrafiltrate was first concentrated to 1 / 10 to 1 / 50 of its original volume using a nanofiltration membrane with a molecular weight cutoff of 200–3000. Then, it was diluted to its original volume with buffer solution and desalted by nanofiltration 3–5 times. Finally, it was sterile filtered. 41.6 g of bovine tripe protein peptide lyophilized powder was prepared by freeze-drying.

[0312] Example 82. Preparation method of small molecule bioactive protein peptides from bovine liver

[0313] Weigh 1 kg of fresh yak liver, and use scissors to remove surface fat, blood vessels, and blood. Chop into fine shreds, add 3% hydrochloric acid, and extract for 4.5 hours. Grind into a paste for 10 minutes, with 45-second intervals. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer), adjust the pH to 2.0, add 1 g of pepsin, and stir at 37°C for 2 hours for enzymatic hydrolysis; adjust the pH to 5.0, add 1 g of cathepsin (a mixture of D+B+L+K in equal proportions), and incubate at 40°C with stirring for 1.5 hours; adjust the pH to 6.8, add 1 g of neutral protease, and incubate at 37°C with stirring for 2 hours; adjust the pH to 7.5, raise the temperature to 50°C, add 1 g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1 g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1 g of proteinase K, and incubate with stirring for 2 hours. Centrifuge at 12500 rpm for 45 min (centrifugal force RCF above 10000 g). Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.25 μm filter membrane. Vacuum until no air bubbles remain in the liquid. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000 or higher, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 47.8 g of yak liver small molecule protein peptide lyophilized powder by freeze drying.

[0314] Example 83. A novel preparation method for small molecule bioactive protein peptides from bovine heart tubes.

[0315] After slaughtering cattle, take 1 kg of freshly frozen heart tubes, chop them, add tap water, soak overnight, and rinse three times with clean water the next day to thoroughly remove blood. Grind the heart tubes into a slurry, add 3% acetic acid, and extract overnight with stirring. Centrifuge at 14,000 rpm and collect the precipitate. Add phosphate buffer (50 mM NaCl-20 mM phosphate buffer) to the precipitate, adjust the pH to 2.0, add 2.0 g of pepsin, and stir at 37°C for 4 hours for enzymatic hydrolysis; adjust the pH to 6.8, add 1.0 g of neutral protease, and incubate at 37°C with stirring for 2 hours; raise the temperature to 50°C, add 1.0 g of subtilisin, and incubate with stirring for 1 hour; add 1.0 g of papain, and incubate with stirring for 1 hour; adjust the pH to 7.5, add 1.0 g of flavor protease, and continue to incubate with stirring for 1 hour; adjust the pH to 9.0, add 2 g of serine protease, and incubate with stirring for 2 hours. Centrifuge at 18500 rpm for 45 min. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.20 μm filter membrane. Vacuum the liquid until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 54.8 g of lyophilized bovine heart tube small molecule active protein peptide powder by freeze drying.

[0316] Example 84. A novel preparation method for bovine placental small molecule bioactive protein peptides

[0317] After calving, 1 kg of frozen placenta was collected from dairy cows, chopped, soaked in tap water overnight, and rinsed three times with clean water the next day to thoroughly remove blood. The placenta was then ground into a slurry using a colloid mill, and 3% acetic acid was added. Extraction was carried out overnight with stirring. The precipitate was collected by centrifugation at 14,000 rpm for 40 minutes. The precipitate was added to phosphate buffer (50 mM NaCl - 20 mM phosphate buffer), the pH was adjusted to 2.0, 2.0 g of pepsin was added, and the mixture was stirred at 37°C for 4 hours for enzymatic hydrolysis. The pH was adjusted to 6.8, 1.0 g of neutral protease was added, and the mixture was incubated at 37°C with stirring for 2 hours. The temperature was raised to 50°C, 1.0 g of subtilisin was added, and the mixture was incubated with stirring for 1 hour. 1.0 g of papain was added, and the mixture was incubated with stirring for 1 hour. The pH was adjusted to 7.5, 1.0 g of flavor protease was added, and the mixture was incubated with stirring for 1 hour. The pH was adjusted to 9.0, 2 g of serine protease was added, and the mixture was incubated with stirring for 2 hours. Centrifuge at 18500 rpm for 45 min. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.20 μm filter membrane. Vacuum the liquid until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 62.3 g of lyophilized bovine placental small molecule active protein peptide powder by freeze drying.

[0318] Example 85. Preparation method of small molecule active protein peptides from bovine trachea

[0319] Take the trachea of ​​a yellow cattle and scrape off any dirt or debris with a knife. Crush the trachea into pieces using a bone crusher and grind it into a paste for 20 minutes, with 45-second intervals. Add 3% acetic acid to the paste and extract overnight with stirring. Centrifuge at 14,000 rpm and collect the precipitate. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust the pH to 2.0, and incubate at 37°C with stirring for 4 hours for enzymatic hydrolysis. Adjust the pH to 6.8, add 1.0g of neutral protease, and incubate at 37°C with stirring for 2 hours. Increase the temperature to 50°C, add 1.0g of subtilisin, and incubate with stirring for 1 hour. Add 1.0g of papain, and incubate with stirring for 1 hour. Adjust the pH to 7.5, add 1.0g of flavor protease, and continue incubating with stirring for 1 hour. Adjust the pH to 9.0, add 2g of serine protease, and incubate with stirring for 2 hours. Centrifuge at 18,500 rpm for 45 minutes. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.20-micron filter membrane. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 3000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 45.4 g of lyophilized small-molecule active protein peptide powder from bovine trachea using freeze-drying.

[0320] Example 86. Preparation method of small molecule active protein peptides from animal bone marrow

[0321] Weigh 500g of fresh bovine bone marrow. Chop it and add phosphate buffer (50mM NaCl-20mM phosphate buffer, pH 6.8). Homogenize for 5 minutes, then add 1g of neutral protease at 30-second intervals. Incubate at 37°C with stirring for 2 hours. Increase the temperature to 50°C, add 1g of subtilisin, and incubate with stirring for 1 hour. Add 1g of papain and incubate with stirring for 1 hour. Adjust the pH to 7.5, add 1g of flavor protease, and continue incubating with stirring for 3 hours. Centrifuge at 12500rpm for 90 minutes. Collect the supernatant. Filter the crude protein peptide supernatant using a 0.22-micron filter plate and frame. Vacuum until no air bubbles remain in the liquid. Ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, then dilute with water to the original volume. The ultrafiltrate was first nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume. Then, water was added to dilute it to its original volume, and the nanofiltration process was repeated 3–5 times for desalting. The solution was then sterilized. 12.5 g of lyophilized bovine bone marrow small molecule protein active peptide powder was prepared by freeze-drying.

[0322] 10. Preparation methods of small molecule protein bioactive peptides from animal tissues and organs

[0323] Example 87. Effects of various novel preparation processes on the production efficiency of small molecule bioactive protein peptides from animal tissues and organs.

[0324] Take fresh animal tissues and organs, remove small thorns, manually remove blood and grease, rinse quickly, and pat dry with kitchen paper towels. Weigh out 50–300g. Chop finely, add phosphate buffer (1–200mM NaCl-1–200mM phosphate buffer), adjust pH to 1–11, and grind using a grinder for approximately 1–60 minutes, with intervals of 5–120 seconds; then homogenize using a homogenizer for 1–60 minutes, with intervals of 5–120 seconds. Adjust the pH, add 1‰–100‰ neutral protease and / or papain and / or flavor protease and / or pepsin and / or trypsin and / or complex protease and / or serine protease and / or cathepsin and / or proteinase K, etc., and perform enzymatic hydrolysis for 0.1–24 hours, maintaining the hydrolysis temperature at 20–60℃. Centrifuge at 3000–25000 rpm for 5–120 minutes. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter packed with a 0.01–10 micrometer filter membrane. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 2000–1,000,000, then dilute with water to the original volume. Next, desalt and concentrate the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–2000, then dilute with water to the original volume. Perform nanofiltration desalting 1–5 times. Sterilize. Prepare lyophilized powder of small molecule active protein peptides from animal tissues and organs by freeze-drying.

[0325] Example 88. Preparation method of small molecule bioactive protein peptides from animal meat

[0326] Take fresh venison from farmed sika deer, manually remove blood and fat, and weigh 1 kg. Chop to the appropriate consistency, then use a meat grinder to prepare a paste, grinding for 8 minutes with 45-second intervals. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer), adjust pH to 7.0, add 2.0 g of neutral protease, and incubate at 37°C with stirring for 1 hour; raise the temperature to 50°C, add 1.0 g of subtilisin, and incubate with stirring for 1 hour; add 1.0 g of papain, and incubate with stirring for 1 hour; adjust pH to 7.5, add 1.0 g of flavor protease, and continue incubating for 1 hour; raise the temperature to 55°C and adjust pH to 9.0, add 2.0 g of serine protease, and continue incubating with stirring for 3 hours. Centrifuge at 15000 rpm for 45 min. Collect the supernatant; filter the crude protein peptide supernatant through a 0.45-micron filter plate and frame. Vacuum until no air bubbles remain in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 62.4 g of venison small molecule active protein peptide freeze-dried powder was prepared by freeze-drying.

[0327] Example 89. Preparation method of small molecule bioactive peptides from connective tissue between animal muscles

[0328] Collect the connective tissue between animal muscles, trim away surface fat, remove blood vessels and blood with scissors, manually scrape off surface fat and impurities, rinse with water, filter dry, and weigh 1 kg of connective tissue between animal muscles. Grind into a paste for 8 minutes with 45-second intervals; homogenize for 5 minutes with 30-second intervals. Add 3% acetic acid, stir and extract overnight. Centrifuge at 14000 rpm and collect the precipitate. The precipitate was added to phosphate buffer (50mM NaCl-20mM phosphate buffer), the pH was adjusted to 2.0, 1g of pepsin was added, and the mixture was stirred at 37°C for 2 hours for enzymatic hydrolysis. The pH was adjusted to 5.0, 1g of cathepsin (a mixture of D+B+L+K in equal proportions) was added, and the mixture was incubated at 40°C with stirring for 1.5 hours. The pH was adjusted to 6.5, 1g of neutral protease was added, and the mixture was incubated at 37°C with stirring for 2 hours. The pH was adjusted to 7.5, the temperature was raised to 50°C, 1g of subtilisin was added, and the mixture was incubated with stirring for 1 hour. The temperature was raised to 55°C, 1g of papain was added, and the mixture was incubated with stirring for 1 hour. The pH was adjusted to 9.0, 1g of proteinase K was added, and the mixture was incubated with stirring for 2 hours. The mixture was centrifuged at 15000 rpm for 45 min. The crude protein peptides were filtered through a 0.2 μm filter. Vacuum was applied until no air bubbles remained in the liquid. The coarse filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5,000–100,000, and then diluted with water to its original volume. The ultrafiltrate was then concentrated using a nanofiltration membrane with a molecular weight cutoff of 200–3,000 to 1 / 10–1 / 50 of its original volume, and then diluted with buffer solution to its original volume, undergoing nanofiltration desalting 3–5 times. After sterile filtration, the solution was freeze-dried to prepare 57 g of small molecule protein peptide lyophilized powder from connective tissue between animal muscles.

[0329] Example 90. Preparation method of small molecule active peptides from animal interosseous muscle

[0330] Collect the connective tissue of the animal's interosseous muscle, trim away the surface fat and remove blood vessels and blood with scissors, manually scrape off the surface fat and impurities, rinse with water, filter dry, and weigh 1 kg of animal interosseous muscle. Grind into a paste for 8 minutes with 45-second intervals; homogenize for 5 minutes with 30-second intervals. Add 3% acetic acid, stir and extract overnight. Centrifuge at 14000 rpm and collect the precipitate. The precipitate was added to phosphate buffer (50mM NaCl-20mM phosphate buffer), the pH was adjusted to 2.0, 2.0g of pepsin was added, and the mixture was stirred at 37℃ for 4 hours for enzymatic hydrolysis. The pH was adjusted to 6.8, 1.0g of neutral protease was added, and the mixture was incubated at 37℃ with stirring for 2 hours. The temperature was raised to 50℃, 1.0g of subtilisin was added, and the mixture was incubated with stirring for 1 hour. The pH was adjusted to 8.0, and the temperature was raised to 43℃, 1.0g of complex protease and chymotrypsin were added respectively, and the mixture was incubated with stirring for 2 hours. The pH was adjusted to 9.0, 2.0g of proteinase K was added, and the mixture was incubated at 55℃ with stirring for 2 hours. The mixture was centrifuged at 15000rpm for 45min. The crude protein peptides were filtered through a 0.2μm filter membrane. The crude filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, and then diluted with water to the original volume. The ultrafiltrate was first concentrated to 1 / 10 to 1 / 50 of its original volume using a nanofiltration membrane with a molecular weight cutoff of 200 to 3000. Then, it was diluted to its original volume with buffer solution and desalted by nanofiltration 3 to 5 times. After sterile filtration, it was freeze-dried to prepare 58.3g of animal interosseous muscle protein peptide lyophilized powder.

[0331] Example 91. Preparation method of deer lung small molecule active protein peptides

[0332] Take fresh lungs from farmed sika deer, remove surface fat and blood with scissors, and weigh 1 kg. Chop finely. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer), grind for 8 minutes, with 45-second intervals. Adjust pH to 2.0, add 2.0 g pepsin, and stir at 37°C for 4 hours for enzymatic hydrolysis; adjust pH to 6.8, add 1.0 g neutral protease, and incubate at 37°C for 1 hour; raise temperature to 50°C, add 1.0 g subtilis protease, and incubate for 1 hour; raise temperature to 55°C, add 1.0 g papain, and incubate for 1 hour; adjust pH to 9.0, add 2.0 g serine protease, and incubate while stirring for 3 hours. Centrifuge at 12500 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.25-micron filter plate and frame. Vacuum until no air bubbles remain in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 44.6 g of lyophilized deer lung small molecule active protein peptide powder was prepared by freeze-drying.

[0333] Example 92. Preparation method of deer kidney small molecule active protein peptides

[0334] Take fresh deer kidneys (kidneys) from farmed sika deer, remove the excess fat and blood vessels with scissors, and weigh 1 kg. Cut into thin strips, add 3% hydrochloric acid, and extract for 6 hours. Grind into a paste for 10 minutes, with 45-second intervals. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust the pH to 2.0, add 1g of pepsin, and stir at 37℃ for 2 hours for enzymatic hydrolysis; adjust the pH to 6.8, add 1.0g of neutral protease, and incubate at 37℃ with stirring for 2 hours; raise the temperature to 50℃, add 1.0g of subtilisin, and incubate with stirring for 1 hour; adjust the pH to 8.0 and raise the temperature to 43℃, add 1g of complex protease and chymotrypsin respectively, and incubate with stirring for 2 hours; adjust the pH to 9.0, add 2g of proteinase K, and incubate at 55℃ with stirring for 2 hours. Centrifuge at 12500 rpm for 45 minutes. Collect the supernatant. Filter the crude protein peptide supernatant using a 0.25-micron filter plate and frame. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 42.1 g of lyophilized deer kidney small molecule active protein peptide powder by freeze-drying.

[0335] Example 93. Preparation method of small molecule active protein peptides from deer liver

[0336] Take fresh deer liver, trim off the surface fat and remove blood vessels and blood with scissors, weigh 1 kg. Cut into thin strips, add 3% hydrochloric acid, and extract for 6 hours. Grind into a paste for 10 minutes, with an interval of 45 seconds. Add phosphate buffer (50 mM NaCl-20 mM phosphate buffer), adjust the pH to 2.0, add 1 g of pepsin, and stir at 37°C for 2 hours for enzymatic hydrolysis; adjust the pH to 6.8, add 1.0 g of neutral protease, and incubate at 37°C with stirring for 2 hours; raise the temperature to 50°C, add 1.0 g of subtilisin, and incubate with stirring for 1 hour; adjust the pH to 8.0 and raise the temperature to 43°C, add 1 g of complex protease and chymotrypsin respectively, and incubate with stirring for 2 hours; adjust the pH to 9.0, add 2 g of proteinase K, and incubate at 55°C with stirring for 2 hours. Centrifuge at 12500 rpm for 45 min. Collect the supernatant. Filter the crude protein peptide supernatant using a 0.25-micron filter plate and frame. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 44.3g of freeze-dried deer liver small molecule active protein peptide powder by freeze drying.

[0337] Example 94. Preparation method of deer antler small molecule active protein peptide

[0338] Take the deer penis and scrape off any unwanted impurities from its surface. Cut it into several small pieces and grind them into a paste for 20 minutes, with 45-second intervals between grinding. Add 3% acetic acid to the paste and extract overnight by stirring. Centrifuge at 14,000 rpm and collect the precipitate. Add phosphate buffer (50mM NaCl-20mM phosphate buffer), adjust pH to 2.0, add 10g pepsin, and incubate at 37℃ for 2 hours with stirring for enzymatic hydrolysis; adjust pH to 5.0, add 1g cathepsin (a mixture of D+B+L+K in equal proportions), and incubate at 40℃ with stirring for 1.5 hours; adjust pH to 6.5, add 1g neutral protease, and incubate at 37℃ with stirring for 2 hours; adjust pH to 7.5, raise temperature to 50℃, add 1g subtilisin, and incubate with stirring for 1 hour; raise temperature to 55℃, add 1g papain, and incubate with stirring for 1 hour; adjust pH to 9.0, add 1g serine protease, and incubate with stirring for 2 hours. Centrifuge at 18500rpm for 45min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.20μm filter plate and frame. Vacuum until no air bubbles remain in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalination was performed 3–5 times. The solution was then sterilized. 52.5 g of deer antler velvet small molecule active protein peptide lyophilized powder was prepared by freeze-drying.

[0339] Example 95. A novel preparation method for small molecule bioactive protein peptides from porcine heart tubes.

[0340] After slaughtering pigs, take 1 kg of fresh frozen heart tubes, chop them, add tap water, soak overnight, and rinse repeatedly with clean water 3 times the next day to thoroughly remove blood. Grind the blood into a slurry using a colloid mill, add 3% acetic acid, stir and extract overnight. Centrifuge at 14,000 rpm and collect the precipitate. The precipitate was added to phosphate buffer (50mM NaCl-20mM phosphate buffer), the pH was adjusted to 2.0, 1g of pepsin was added, and the mixture was stirred at 37°C for 4 hours for enzymatic hydrolysis. The pH was adjusted to 5.0, 1g of cathepsin (a mixture of D+B+L+K in equal proportions) was added, and the mixture was incubated at 40°C with stirring for 1.5 hours. The pH was adjusted to 6.5, 1g of neutral protease was added, and the mixture was incubated at 37°C with stirring for 2 hours. The pH was adjusted to 7.5, the temperature was raised to 50°C, 1g of subtilisin was added, and the mixture was incubated with stirring for 1 hour. The temperature was raised to 55°C, 1g of papain was added, and the mixture was incubated with stirring for 1 hour. The pH was adjusted to 9.0, 1g of serine protease was added, and the mixture was incubated with stirring for 2 hours. The mixture was centrifuged at 18500 rpm for 45 min. The supernatant was collected, and the crude protein peptide supernatant was filtered through a 0.20 μm filter plate. Vacuum was applied until no air bubbles remained in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 53g of lyophilized porcine heart tube small molecule active protein peptide powder was prepared by freeze-drying.

[0341] Example 96. Preparation method of deer spleen small molecule protein active peptides

[0342] Take fresh deer spleen, manually cut or use a knife to remove attached tissues such as oil and blood vessels, weigh 1 kg. Chop into fine shreds, add 3% hydrochloric acid, and extract for 4.5 hours. Grind into a paste for 10 minutes, with an interval of 45 seconds. Add phosphate buffer (50 mM NaCl-20 mM phosphate buffer), adjust the pH to 2.0, add 1 g of pepsin, and stir at 37°C for 2 hours for enzymatic hydrolysis; adjust the pH to 6.8, add 1.0 g of neutral protease, and incubate at 37°C with stirring for 2 hours; raise the temperature to 50°C, add 1.0 g of subtilisin, and incubate with stirring for 1 hour; add 1.0 g of papain, and incubate with stirring for 1 hour; adjust the pH to 7.5, add 1.0 g of flavor protease, and continue incubation for 1 hour; adjust the pH to 9.0, add 2 g of proteinase K, and incubate at 55°C with stirring for 2 hours. Centrifuge at 15000 rpm for 45 min. Collect the supernatant. Filter the crude protein peptide supernatant using a plate and frame filter fitted with a 0.45-micron filter membrane. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 44.2 g of freeze-dried deer spleen small molecule active protein peptide powder by freeze drying.

[0343] Example 97. Preparation method of small molecule protein bioactive peptides from deer pancreas

[0344] Take 1 kg of fresh deer pancreas, manually cut or with a knife to remove attached tissues such as oil and blood vessels. Chop into fine shreds and grind for 10 minutes with 45-second intervals; homogenize for 5 minutes with 30-second intervals. Add phosphate buffer (50 mM NaCl - 20 mM phosphate buffer), add 1 g of neutral protease, and incubate at 37°C with stirring for 2 hours; raise the temperature to 50°C, add 1 g of subtilisin, and incubate with stirring for 1 hour; add 1 g of papain, and incubate with stirring for 1 hour; adjust the pH to 7.5, add 1 g of flavor protease, and continue incubating with stirring for 3 hours. Centrifuge at 15000 rpm for 45 min. Collect the supernatant, and filter the crude protein peptide supernatant through a 0.45-micron filter plate. Vacuum until no air bubbles remain in the liquid. The filtrate was first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, and then diluted with water to its original volume. The ultrafiltrate was then nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrated to 1 / 2–1 / 10 of its original volume, and then diluted with water to its original volume. Nanofiltration desalting was performed 3–5 times. The solution was then sterilized. 38.9 g of deer pancreas small molecule active protein peptide lyophilized powder was prepared by freeze-drying.

[0345] Example 98. Preparation method of deer heart small molecule active protein peptides

[0346] Take fresh deer heart, trim off the surface fat and remove the blood with scissors, and weigh 1 kg. Chop it and extract with 6% acetic acid for 6 hours. Grind into a paste for 15 minutes, with 45-second intervals. Adjust the pH to 2.0, add 1 g of pepsin, and stir at 37°C for 2 hours for enzymatic hydrolysis; adjust the pH to 5.0, add 1 g of cathepsin (a mixture of D+B+L+K in equal proportions), and incubate at 40°C with stirring for 1.5 hours; adjust the pH to 6.5, add 1 g of neutral protease, and incubate at 37°C with stirring for 2 hours; adjust the pH to 7.5, raise the temperature to 50°C, add 1 g of subtilisin, and incubate with stirring for 1 hour; raise the temperature to 55°C, add 1 g of papain, and incubate with stirring for 1 hour; adjust the pH to 9.0, add 1 g of proteinase K, and incubate with stirring for 2 hours. Centrifuge at 10000 rpm for 45 minutes. Collect the supernatant. Filter the crude protein peptide supernatant using a 0.50-micron filter plate and frame. Vacuum the solution until no air bubbles remain. First, ultrafilter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 5000–100000, then dilute with water to the original volume. Next, nanofilter the ultrafiltrate using a nanofiltration membrane with a molecular weight cutoff of 200–3000, concentrating it to 1 / 2–1 / 10 of its original volume, then dilute with water to the original volume. Perform nanofiltration desalting 3–5 times. Sterilize. Prepare 40.8 g of freeze-dried deer heart small molecule active protein peptide powder.

[0347] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A method of preparing active protein peptides from animal tissue organs, characterized by: The method includes the following steps: taking animal tissues and organs, removing oil, washing, chopping, grinding and / or homogenizing, enzymatic hydrolysis, filtration, vacuuming, ultrafiltration, nanofiltration, sterilization, and vacuum ultra-low temperature freeze drying. Specifically, (1) taking animal tissues and organs; (2) removing oil; (3) chopping; (4) adding 5 to 10 times the volume of water or salt-containing buffer solution after chopping and stirring evenly; (5) grinding and / or homogenizing; (6) adjusting pH and enzymatic hydrolysis; (7) centrifuging; (8) coarsely filtering the enzymatic hydrolysate with a filter membrane and collecting the filtrate; (9) vacuuming; (10) ultrafiltration of the filtrate with an ultrafiltration membrane of a predetermined molecular weight cutoff; (11) nanofiltration concentration of the filtrate with a nanofiltration membrane of a predetermined molecular weight cutoff, and then adding water to the original volume for nanofiltration desalting 3 to 5 times; (12) finally concentrating to a predetermined protein concentration; (13) sterilization; (14) preparing small molecule active protein peptide freeze-dried powder of tissues and organs by ultra-low temperature vacuum freeze drying.

2. The method of claim 1, wherein: The animals mentioned are selected from one or more of the group consisting of annelids, arthropods, mollusks, echinoderms, fish, amphibians, reptiles, birds, and mammals.

3. The method of claim 1 or 2, wherein: The animals mentioned are selected from one or more of the following groups: earthworms, sea cucumbers, pigs, wild boars, black pigs, suckling pigs, cattle, calves, yellow cattle, water buffalo, yaks, sheep, alpacas, deer, farmed sika deer, horses, donkeys, mules, camels, free-range camels, rabbits, kangaroos, fish, shrimp, lobsters, Australian lobsters, crayfish, crabs, king crabs, mitten crabs, shellfish, scallops, oysters, snails, snails, sea urchins, abalone, clams, soft-shelled turtles, octopuses, squid, penfish, bullfrogs, chickens, ducks, geese, turkeys, pheasants, pigeons, partridges, and quails.

4. The method of claim 3, wherein: The animals mentioned are selected from one or more of the following groups: earthworms, sea cucumbers, cattle, calves, yaks, sheep, farmed sika deer, donkeys, free-range camels, fish, shrimp, lobsters, Australian lobsters, crabs, king crabs, shellfish, scallops, oysters, sea urchins, abalone, clams, soft-shelled turtles, octopuses, squid, pheasants, partridges, and quails.

5. The method of claim 4, wherein: The animals mentioned are selected from one or more of the following groups: earthworm, sea cucumber, cattle, calves, yaks, sheep, farmed sika deer, donkeys, free-range camels, fish, shrimp, crabs, shellfish, abalone, clams, soft-shelled turtles, and octopuses.

6. The method according to claims 1 to 5, characterized in that: The animal tissues and organs mentioned include, but are not limited to, carcass, meat, brain, heart, spleen, pancreas, kidney, lung, liver, eyes, ears, nose, tongue, lips, oral cavity, head, glands, chest, thymus, skin, swim bladder, fish swim bladder, muscles, skeletal muscles, smooth muscles, striated muscles, interosseous muscles, abdomen, buttocks, back, marrow, bone marrow, spinal cord, tendons, flank, tendons, hoof tendons, tendon tips, whole tendons, interrupted tendons, tendon tendons, spur tendons, forehoof tendons, hindhoof tendons, tendon tendons, diaphragm, flank tendons, sirloin tendons, ham tendons, joints, ligaments, esophagus, digestive tract, respiratory tract, membranes, periosteum, pleura, intestinal membrane, diaphragm, visceral membranes, stomach. The following are some of the following tissues and organs: intestines, testes, penis, vagina, uterus, gallbladder, appendix, placenta, bladder, neck, gills, gills, fish gills, miscellaneous organs, chest, tubes, blood vessels, aorta, larynx, esophagus, trachea, sperm, testes, blood, eggs, ovaries, nerves, nerve fibers, limbs, bones, hard bones, cartilage, ribs, vertebrae, club bones, fingers, nails, feet, toes, tibia, fibula, palms, bear paws, diaphragm, muscle septum, tendon septum, bone septum, fleshy septum, compartment septum, claws, hump, camel hump, velvet, horns, spines, shark spines, thorns, protrusions, scales, fins, spongy tissue, turtle skirt, and tail.

7. The method of claim 6, wherein: The animal tissues and organs mentioned include, but are not limited to, one or more of the following: flesh, brain, marrow, bone marrow, spinal cord, muscle, skeletal muscle, smooth muscle, striated muscle, abdomen, buttocks, back, egg, sperm, and blood.

8. The method according to claims 1 to 6, characterized in that: The method further includes an extraction step prior to the pulping and / or homogenizing steps. The process includes the following steps: taking animal tissues and organs, removing oil, washing, chopping, extracting, rinsing, grinding and / or homogenizing, enzymatic hydrolysis, centrifugation, filtration, vacuuming, ultrafiltration, nanofiltration, sterilization, and vacuum ultra-low temperature freeze drying. Specifically, (1) taking animal tissues and organs; (2) removing oil; (3) chopping; (4) extracting; (5) grinding and / or homogenizing; (6) adjusting pH and enzymatic hydrolysis; (7) centrifugation; (8) coarsely filtering the enzymatic hydrolysate with a filter membrane and collecting the filtrate; (9) vacuuming; (10) ultrafiltration of the filtrate with an ultrafiltration membrane of a predetermined molecular weight cutoff; (11) nanofiltration concentration of the filtrate with a nanofiltration membrane of a predetermined molecular weight cutoff, and then adding water to the original volume for nanofiltration desalting 3 to 5 times; (12) finally concentrating to a predetermined protein concentration; (13) sterilization; and (14) preparing lyophilized powder of small molecule active protein peptides from tissues and organs by ultra-low temperature vacuum freeze drying.

9. The method of claim 8, wherein: The animal tissues and organs mentioned include, but are not limited to, carcass, meat, brain, heart, spleen, pancreas, kidney, lung, liver, eyes, ears, nose, tongue, lips, oral cavity, head, glands, chest, thymus, skin, swim bladder, fish swim bladder, muscles, skeletal muscles, smooth muscles, striated muscles, interosseous muscles, abdomen, buttocks, back, marrow, bone marrow, spinal cord, tendons, flank, tendons, hoof tendons, tendon tips, whole tendons, interrupted tendons, tendon tendons, spur tendons, forehoof tendons, hindhoof tendons, tendon tendons, diaphragm, flank tendons, sirloin tendons, ham tendons, joints, ligaments, esophagus, digestive tract, respiratory tract, membranes, periosteum, pleura, intestinal membrane, diaphragm, visceral membranes, stomach. The following are some of the following tissues and organs: intestines, testes, penis, vagina, uterus, gallbladder, appendix, placenta, bladder, neck, gills, gills, fish gills, miscellaneous organs, chest, tubes, blood vessels, aorta, larynx, esophagus, trachea, sperm, testes, blood, eggs, ovaries, nerves, nerve fibers, limbs, bones, hard bones, cartilage, ribs, vertebrae, club bones, fingers, nails, feet, toes, tibia, fibula, palms, bear paws, diaphragm, muscle septum, tendon septum, bone septum, fleshy septum, compartment septum, claws, hump, camel hump, velvet, horns, spines, shark spines, thorns, protrusions, scales, fins, spongy tissue, turtle skirt, and tail.

10. The method of claim 9, wherein: The animal tissues and organs mentioned include, but are not limited to, one or more of the following: carcass, heart, spleen, pancreas, kidney, lung, liver, eye, ear, nose, tongue, lips, oral cavity, head, gland, chest, thymus, esophagus, digestive tract, respiratory tract, membrane, periosteum, pleura, intestinal membrane, diaphragm, visceral membrane, testis, vagina, uterus, gallbladder, appendix, placenta, bladder, neck, gills, gills, fish gills, miscellaneous organs, chest, ovary, nerve, nerve fiber, limb, four limbs, bone, hard bone, cartilage, rib, vertebrae, club bone, finger, nail, foot, toe, tibia, fibula, claw, hump, camel hump, antler, horn, spine, shark spine, thorn, spine (e.g., deer antler), protrusion (e.g., camel hump, lion's nipple), scale, fin, spongy tissue, turtle skirt (water fish skirt), and tail.

11. The method of claims 1-5, wherein: The method includes an extraction step after the grinding and / or homogenization step. The method includes the following steps: taking animal tissues and organs, removing oil, washing, chopping, grinding and / or homogenizing, extraction, centrifugation, enzymatic hydrolysis, filtration, vacuuming, ultrafiltration, nanofiltration, sterilization, and vacuum ultra-low temperature freeze drying. Specifically, (1) taking animal tissues and organs; (2) removing oil; (3) chopping; (4) grinding and / or homogenizing; (5) extraction; (6) adjusting pH and enzymatic hydrolysis; (7) centrifugation; (8) coarsely filtering the enzymatic hydrolysate with a filter membrane and collecting the filtrate; (9) vacuuming; (10) ultrafiltration of the filtrate with an ultrafiltration membrane of a predetermined molecular weight cutoff; (11) nanofiltration concentration of the filtrate with a nanofiltration membrane of a predetermined molecular weight cutoff, and then adding water to the original volume for nanofiltration desalting 3 to 5 times; (12) finally concentrating to a predetermined protein concentration; (13) sterilization; (14) preparing lyophilized powder of small molecule active protein peptides from tissues and organs by ultra-low temperature vacuum freeze drying.

12. The method of claim 11, wherein: The animal tissues and organs mentioned include, but are not limited to, carcass, meat, brain, heart, spleen, pancreas, kidney, lung, liver, eyes, ears, nose, tongue, lips, oral cavity, head, glands, chest, thymus, skin, swim bladder, fish swim bladder, muscles, skeletal muscles, smooth muscles, striated muscles, interosseous muscles, abdomen, buttocks, back, marrow, bone marrow, spinal cord, tendons, flank, tendons, hoof tendons, tendon tips, whole tendons, interrupted tendons, tendon tendons, spur tendons, forehoof tendons, hindhoof tendons, tendon tendons, diaphragm, flank tendons, sirloin tendons, ham tendons, joints, ligaments, esophagus, digestive tract, respiratory tract, membranes, periosteum, pleura, intestinal membrane, diaphragm, visceral membranes, stomach. The following are some of the following tissues and organs: intestines, testes, penis, vagina, uterus, gallbladder, appendix, placenta, bladder, neck, gills, gills, fish gills, miscellaneous organs, chest, tubes, blood vessels, aorta, larynx, esophagus, trachea, sperm, testes, blood, eggs, ovaries, nerves, nerve fibers, limbs, bones, hard bones, cartilage, ribs, vertebrae, club bones, fingers, nails, feet, toes, tibia, fibula, palms, bear paws, diaphragm, muscle septum, tendon septum, bone septum, fleshy septum, compartment septum, claws, hump, camel hump, velvet, horns, spines, shark spines, thorns, protrusions, scales, fins, spongy tissue, turtle skirt, and tail.

13. The method of claim 12, wherein: The animal tissues and organs mentioned include, but are not limited to, skin, swim bladder, fish swim bladder, interosseous muscles, tendons, brisket, tendons, hoof tendons, shin tendons, shin tendon tips, whole shin tendons, interrupted tendons, tendon tendons, spit tendons, forehoof tendons, hindhoof tendons, tendon tendons, diaphragm, brisket tendons, sirloin tendons, shin tendons, joints, ligaments, stomach, intestines, penis (whip), canals, blood vessels, aorta, larynx, esophagus, trachea, paws, bear paws, diaphragm, muscle septum, tendon septum, bone septum, flesh septum, and compartment septum, etc., one or more of the following tissues and organs.

14. The method of claims 1-13, wherein: The aforementioned degreasing process involves physically removing the fatty tissue or oil film present in animal tissues and organs using various knives or machines, thereby increasing the activity, yield, and content of bioactive peptides, while simultaneously reducing industrial costs and improving industrial production efficiency.

15. The method of claim 14, wherein: The degreasing process also includes freezing the fatty tissue or oil film present in animal tissues and organs, and then removing it cleanly using various physical tools or machinery, thereby improving the activity, yield, and content of active peptides, while reducing industrial costs and improving industrial production efficiency.

16. The method of claim 15, wherein: The degreasing process also includes rapidly freezing the fatty tissue or oil film present in animal tissues and organs at low temperatures, and then removing it cleanly using various physical tools or machinery, thereby improving the activity, yield, and content of bioactive peptides, while reducing industrial costs and increasing industrial production efficiency.

17. The method of claims 1-16, wherein: The vacuuming step involves evacuating the filtered active peptide hydrolysate to prevent it from clogging the ultrafiltration or nanofiltration column, thereby improving the activity, yield, and content of the active peptides, while simultaneously reducing industrial costs and increasing industrial production efficiency.

18. The method of claims 1-17, wherein: Pulping is a process of refining mechanically pulverized animal tissues and organs into smaller particles in a liquid, including grinding and / or homogenizing. The grinding and / or homogenizing involves reducing the target particles in the slurry to below 10 mesh.

19. The method of claim 18, wherein: The pulping process involves pulping the target particles in the slurry to a mesh size of less than 100.

20. The method of claim 19, wherein: The pulping process involves pulping the target particles in the slurry to a mesh size of less than 500.

21. The method of claim 20, wherein: The pulping process involves pulping the target particles in the slurry to a mesh size of less than 1000.

22. The method of claims 8-21, wherein: The extraction method described is acid extraction.

23. The method of claim 22, wherein: The acids mentioned are malic acid, citric acid, sulfuric acid, hydrochloric acid, boric acid, nitric acid, acetic acid, and formic acid.

24. The method of claim 23, wherein: The acids are malic acid, citric acid, and acetic acid.

25. The method of claim 24, wherein: The acid mentioned is acetic acid.

26. The method of claims 8-25, wherein: The final concentration of the acetic acid solution is 0.01% to 60%, and the standing time is 0.5 to 24 hours.

27. The method of claim 26, wherein: The final concentration of the glacial acetic acid solution is 0.01% to 30%, and the standing time is 0.5 to 20 hours.

28. The method of claim 27, wherein: The final concentration of the glacial acetic acid solution is 0.5% to 25%, and the standing time is 0.5 to 20 hours.

29. The method of claim 28, wherein: The final concentration of the glacial acetic acid solution is 1% to 25%, and the standing time is 2 to 16 hours.

30. The method of claims 1-29, wherein: The enzymes used in the enzymatic hydrolysis are selected from one or more combinations of pepsin, chymotrypsin, trypsin, cathepsin, carboxypeptidase, aminopeptidase, thiol protease, serine protease, aspartic protease, glutamine protease, serine protease, cysteine ​​protease, metalloproteinase, alkaline protease, neutral protease, acidic protease, proteinase K, fig protease, papain, bromelain, flavor protease, complex protease, and subtilisin.

31. The method of claim 30, wherein: The enzymes used in the enzymatic hydrolysis are selected from one or more combinations of pepsin, chymotrypsin, trypsin, cathepsin, thiol protease, serine protease, aspartic protease, glutamine protease, serine protease, cysteine ​​protease, neutral protease, proteinase K, papain, bromelain, flavor protease, complex protease, and subtilisin.

32. The method of claim 31, wherein: The enzymes used in the enzymatic hydrolysis are selected from one or more combinations of pepsin, chymotrypsin, trypsin, cathepsin, serine protease, neutral protease, proteinase K, papain, bromelain, flavor protease, complex protease, and subtilisin.

33. The method of claims 30-32, wherein: During the enzymatic hydrolysis process, when two or more enzymes are added, the enzymes can be added in different weight ratios.

34. The method of claim 33, wherein: The weight ratio of the various added enzymes is 1:1 to 100.

35. The method of claim 34, wherein: The weight ratio of the various added enzymes is 1:1 to 10.

36. The method of claims 33-35, wherein: During the enzymatic hydrolysis process, when two or more enzymes are added, the enzymes can be added simultaneously or sequentially in different weight ratios.

37. The method of claim 36, wherein: During the enzymatic hydrolysis process, when two or more enzymes are added, the enzymes can be added sequentially in different weight ratios, with an interval of 0.1 to 48 hours between each enzyme.

38. The method of claim 37, wherein: During the enzymatic hydrolysis process, when two or more enzymes are added, the enzymes can be added sequentially in different weight ratios, with an interval of 0.5 to 24 hours between each enzyme.

39. The method of claim 38, wherein: During the enzymatic hydrolysis process, when two or more enzymes are added, the enzymes can be added sequentially in different weight ratios, with an interval of 1 to 12 hours between each enzyme.

40. The method of claims 1-39, wherein: During the enzymatic hydrolysis process, the temperature of the slurry is first adjusted to 0–65°C, and the total enzymatic hydrolysis time is 0.1–48 hours.

41. The method of claim 40, wherein: In the enzymatic hydrolysis process, the temperature of the slurry is first adjusted to 0-55℃, and the total enzymatic hydrolysis time is 1-24 hours.

42. The method of claim 61, wherein: In the enzymatic hydrolysis process, the temperature of the slurry is first adjusted to 10-55℃, and the total enzymatic hydrolysis time is 2-18 hours.

43. The method of claim 42, wherein: In the enzymatic hydrolysis process, the temperature of the slurry is first adjusted to 20-55℃, and the total enzymatic hydrolysis time is 3-16 hours.

44. The method of claim 43, wherein: In the enzymatic hydrolysis process, the temperature of the slurry is first adjusted to 30-55℃, and the total enzymatic hydrolysis time is 4-12 hours.

45. The method of claims 1-44, wherein: The filtration, ultrafiltration, and nanofiltration concentration process involves filtering the protein peptide hydrolysate through a 0.2–0.45 micrometer filter membrane, then performing ultrafiltration through a filter membrane with a molecular weight cutoff of 2,000–100,000, and finally concentrating the ultrafiltrate to 1 / 5–1 / 50 of its original volume using a nanofiltration membrane with a molecular weight cutoff of 50–2,000. This process is repeated 3–5 times with the addition of buffer solution to dilute the solution to its original volume.

46. The method of claim 45, wherein: The ultrafiltration and nanofiltration concentration process involves filtering the protein peptide hydrolysate through a 0.2–0.45 micrometer filter membrane, then performing ultrafiltration through a filter membrane with a molecular weight cutoff of 2000–20000, and finally concentrating the ultrafiltrate to 1 / 5–1 / 50 of its original volume using a nanofiltration membrane with a molecular weight cutoff of 50–1500. This process is repeated 3–5 times with the addition of buffer solution to dilute the solution to its original volume.

47. The method of claim 46, wherein: The ultrafiltration and nanofiltration concentration process involves filtering the protein peptide hydrolysate through a 0.25–0.45 micrometer filter membrane, then performing ultrafiltration through a filter membrane with a molecular weight cutoff of 2000–10000, and finally concentrating the ultrafiltrate to 1 / 5–1 / 50 of its original volume using a nanofiltration membrane with a molecular weight cutoff of 100–1000. This process is repeated 3–5 times, and then diluted to the original volume with buffer solution.

48. The method of claim 47, wherein: The ultrafiltration and nanofiltration concentration process involves filtering the protein peptide hydrolysate through a 0.25–0.45 micrometer filter membrane, then performing ultrafiltration through a filter membrane with a molecular weight cutoff of 5000–8000, and finally concentrating the ultrafiltrate to 1 / 5–1 / 50 of its original volume using a nanofiltration membrane with a molecular weight cutoff of 200–800. This process is repeated 3–5 times, and then diluted to the original volume with buffer solution.

49. [Rule 91 Correction 26.03.2026] An active protein peptide, characterized in that: The active peptide is a synthetic active protein peptide or an active protein peptide prepared according to the methods of claims 1 to 48.

50. [R 91 amendment 26.03.2026] The active protein peptide as claimed in claim 49, characterized in that: The active peptide mentioned is collagen.

51. [Amended according to Rule 91 on 26.03.2026] The active protein peptide as claimed in claim 49 or 50, characterized in that: The active peptides are lyophilized powders.

52. [R 91 amendment 26.03.2026] The active protein peptide as claimed in claims 49 to 51, characterized in that: When the protein concentration is 2.0 mg / mL, the active protein peptide exhibits an ACE inhibition rate of over 10% and an IC50 concentration of less than 1.75 mg / mL. 50 .

53. [R 91 amended 26.03.2026] The active protein peptide as claimed in claims 49 to 52, characterized in that: When the protein concentration is 1.5 mg / mL, the active protein peptide exhibits an ACE inhibition rate of over 20% and an IC50 concentration of less than 0.95 mg / mL. 50 .

54. [R 91 amended 26.03.2026] The active protein peptide as claimed in claims 49 to 53, characterized in that: When the protein concentration is 1.0 mg / mL, the active protein peptide exhibits an ACE inhibition rate of over 30% and an IC50 concentration of less than 0.68 mg / mL. 50 .

55. [R 91 amended 26.03.2026] The active protein peptide as claimed in claims 49 to 54, characterized in that: When the protein concentration is 0.5 mg / mL, the active protein peptide exhibits an ACE inhibition rate of over 40% and an IC50 concentration of less than 0.37 mg / mL. 50 .

56. [R 91 amendment 26.03.2026] The active protein peptide as claimed in claims 49-55, characterized in that: The active protein peptide has an ACE inhibitory rate of more than 45% and an IC 50 of less than 0.07 mg / mL when the protein concentration is 0.1 mg / mL.

57. [R 91 amendment 26.03.2026] The active protein peptide as claimed in claims 49-56, characterized in that: Protein peptide yield is expressed as the weight ratio of the obtained protein peptides to the total protein in the entire tissue or organ. The protein peptide yield is above 20 g / kg.

58. [R 91 amendment 26.03.2026] The active protein peptide as claimed in claim 57, characterized in that: The yield of the protein peptides is above 35g / Kg.

59. [Amended according to Rule 91 on 26.03.2026] The active protein peptide as claimed in claim 58, characterized in that: The yield of the protein peptides is above 50 g / kg.

60. [R 91 amendment 26.03.2026] The active protein peptide according to claim 59, characterized in that: The yield of the protein peptides is above 75 g / kg.

61. [Amended according to Rule 91 on 26.03.2026] The active protein peptide as claimed in claim 60, characterized in that: The yield of the protein peptides is above 90 g / kg.

62. [Correction 26.03.2026 according to Rule 91] The active protein peptide as described in claim 61, characterized in that: The yield of the protein peptides is above 100g / Kg.

63. [Correction 26.03.2026 according to Rule 91] The active protein peptide as described in claim 62, characterized in that: The yield of the protein peptides is above 120 g / kg.

64. [Correction 26.03.2026 according to Rule 91] The active protein peptide as described in claim 63, characterized in that: The yield of the protein peptides is above 150 g / kg.

65. [Correction 26.03.2026 according to Rule 91] The active protein peptide according to claims 49-64, characterized in that: The active protein peptides contain 1 to 200 amino acids.

66. [Correction 26.03.2026 according to Rule 91] The active protein peptide as described in claim 65, characterized in that: The active protein peptides contain 1 to 150 amino acids.

67. [Correction 26.03.2026 according to Rule 91] The active protein peptide as described in claim 66, characterized in that: The active protein peptides contain 1 to 100 amino acids.

68. [Correction 26.03.2026 according to Rule 91] The active protein peptide as described in claim 67, characterized in that: The active protein peptides contain 3 to 80 amino acids.

69. [Correction 26.03.2026 according to Rule 91] The active protein peptide as described in claim 68, characterized in that: The active protein peptides contain 10 to 60 amino acids.

70. [Correction 26.03.2026 according to Rule 91] The active protein peptide according to claims 49-69, characterized in that: The active protein peptides mentioned above contain glutamic acid.

71. [Correction 26.03.2026 according to Rule 91] The active protein peptide as described in claim 70, characterized in that: The active protein peptides also contain hydroxyproline.

72. [Correction 26.03.2026 according to Rule 91] The active protein peptide as described in claim 71, characterized in that: The active protein peptides also contain arginine.

73. [Correction 26.03.2026 according to Rule 91] The active protein peptide as described in claim 72, characterized in that: The active protein peptides also contain serine.

74. [Correction 26.03.2026 according to Rule 91] The active protein peptide as described in claim 73, characterized in that: The active protein peptides also contain aspartic acid.

75. [Correction 26.03.2026 according to Rule 91] The active protein peptide according to claims 70-74, characterized in that: The active peptide contains one or more amino acids in a group consisting of isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine, etc.

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