A method and system for separating and purifying bacterial protease hydrolysate and the application of its products in broiler chickens.

Abstract

This invention discloses a method and system for separating and purifying bacterial protease hydrolysate, and the application of its products in broiler chickens. The invention employs a two-stage hollow fiber membrane coupled with equal-volume washing filtration, enabling the targeted enrichment of small peptides up to 3 kDa with high separation efficiency and a small peptide recovery rate of 77.52%. This invention combines activated carbon deodorization, titanium rod filtration, ultra-high temperature instantaneous sterilization, and low-temperature freeze-drying to effectively remove odors and impurities while maximizing the retention of small peptide activity, resulting in stable product quality and good water solubility. The invention is equipped with a dedicated processing system, ensuring a continuous process flow and controllable parameters, making it suitable for industrial-scale production. When applied at a dosage of 0.04% to high-density stress-prone broilers, this invention significantly improves growth performance, enhances antioxidant capacity, improves meat quality, and demonstrates good anti-stress effects. This invention achieves high-value utilization of bacterial protein, reducing dependence on imported protein raw materials such as soybean meal, and exhibits outstanding economic and application value.

Description

Technical Field

[0001] This invention relates to the field of separation and purification technology, specifically to a method and system for separating and purifying bacterial protease hydrolysate and the application of its products in broiler chickens. Background Technology

[0002] Since 1999, my country has shifted from a major soybean meal exporter to a major soybean meal importer, with its dependence on imported soybean meal reaching no less than 80% in recent years. This heavy reliance on imported protein sources presents a significant challenge. The development of microbial protein offers an effective way to alleviate this dependence. Microbial protein boasts advantages such as high protein levels, excellent amino acid composition, and rich functional active ingredients, resulting in high nutritional value and functional properties. For Clostridium ethanolica protein, the crude protein content is ≥80%, far exceeding that of conventional protein sources (fishmeal has approximately 65% ​​crude protein, and soybean meal approximately 45%). Simultaneously, the content of essential amino acids (amino acids that the body cannot synthesize or cannot synthesize sufficiently and must obtain from food) is also significantly higher than that of conventional protein sources, with an essential amino acid content ≥40%, including lysine ≥7.5% and methionine ≥2.3%. Furthermore, Clostridium ethanolica protein has been found to possess functional activity, containing high levels of functional branched-chain amino acids (leucine, valine, and isoleucine) and nucleotides, playing a crucial role in improving energy metabolism and immune function. This type of protein, with its high nutritional value and functional activity, fails to fully realize its potential when used solely in animal feed. Enzymatic hydrolysis is one of the most effective ways to diversify protein functions and increase product added value. It involves using enzymes to break peptide bonds between amino acids, degrading large protein molecules in raw materials into smaller, bioactive peptides. Bioactive peptides are small molecular fragments composed of 2-20 amino acids obtained from proteins through enzymatic hydrolysis or fermentation. They are characterized by their small molecular weight, high activity, easy absorption, and safe origin. They are widely found in animals, plants, microorganisms, and the human body, possessing various biological activities such as antioxidant, blood pressure-lowering, immune-enhancing, anti-aging, and lipid-lowering effects. They have been applied in cosmetics, functional foods, animal feed, and biopesticides. Therefore, using enzyme engineering technology to enzymatically hydrolyze microbial proteins is an effective way to promote the high-value utilization of microbial proteins.

[0003] The applicant's prior patent, "A Method and System for Processing Bacterial Protein Mash and Its Application" (Application No.: CN119823913A), discloses a method and system for processing bacterial protein mash, which prepares a bacterial protein treatment solution through concentration, cell disruption, enzymatic hydrolysis, filtration, desalination, and sterilization. However, this patent only yields a crude enzymatic hydrolysate, containing various components including insoluble components, large molecular weight proteins, large molecular weight peptides, and small molecular weight peptides. Previous studies have shown that smaller polypeptide molecular weights exhibit stronger antioxidant properties, a conclusion consistent with multiple literature reports. Therefore, to obtain polypeptide products with stronger biological activity, this patent further separates and purifies the crude enzymatic hydrolysate and employs a freeze-drying process to maintain the biological activity of the polypeptide sample, ultimately obtaining a pure, dried polypeptide powder. Simultaneously, the crude enzymatic hydrolysate has been found to have a culture medium odor, limiting its use in feed, cosmetics, and pharmaceuticals, requiring the removal of this odor through physical, chemical, or microbial deodorization methods. Ultimately, relying on this patented technology and system, a small peptide powder product with high biological activity, stable physicochemical properties, and no odor can be obtained. Furthermore, the antioxidant stress effect of this small peptide powder product was verified at the organismal level using a high-density broiler chicken farming model. Summary of the Invention

[0004] Therefore, embodiments of the present invention provide a method and system for separating and purifying bacterial protease hydrolysate and the application of the product in broiler chickens.

[0005] The technical problem to be solved by this invention:

[0006] (1) In view of the shortcomings of existing technologies that can only obtain crude enzymatic hydrolysis products, the crude enzymatic hydrolysis products are further separated and purified. Hollow fiber membrane technology is used to prepare bioactive peptide products with a molecular weight of less than 3 kDa.

[0007] (2) To address the issue of culture medium odor in bioactive peptide products, activated carbon and its supporting process system are used to remove the culture medium odor from the bioactive peptide products.

[0008] (3) To address the issues of unstable functional properties and darkening color of bioactive peptide products, a sterilization device is used to remove the bacteria that cause unstable quality of bioactive peptide products, thereby ensuring the stability of the functional properties and color of bioactive peptide products.

[0009] (4) To address the problem that high-temperature drying methods in the existing technology can easily damage the structure of bioactive peptide products, the drying method is optimized to freeze drying.

[0010] (5) The antioxidant stress effect of the small peptide powder product was verified by using an animal stress model.

[0011] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0012] According to a first aspect of the present invention, the present invention provides a method for separating and purifying bacterial protease hydrolysate, the method comprising:

[0013] The bacterial protein hydrolysate was clarified through a hollow fiber membrane to obtain the first part of the first permeate and the first concentrate.

[0014] After washing and filtering an equal volume of the primary concentrate, further clarification is carried out using a hollow fiber membrane to obtain a second part of the primary permeate and primary residual liquid.

[0015] The first part of the primary permeate and the second part of the primary permeate are combined and mixed evenly to form a primary permeate. The primary permeate is then clarified in a secondary stage to obtain a secondary permeate and a secondary residual liquid.

[0016] The secondary permeate was passed into a reaction vessel, activated carbon was added and stirred, and then the activated carbon was removed through a titanium rod filter to obtain a small peptide solution with the culture medium odor removed.

[0017] The small peptide solution, from which the culture medium odor was removed, was sterilized using an ultra-high temperature instantaneous sterilizer to obtain a small peptide treated solution.

[0018] The small peptide treatment solution was concentrated by electric heating to obtain a small peptide concentrate.

[0019] The small peptide concentrate was freeze-dried using a freeze dryer to obtain lyophilized small peptide flakes.

[0020] The lyophilized flake peptides were sieved to obtain a small peptide powder product.

[0021] Furthermore, the hollow fiber membrane of the primary clarification has a precision of 0.2 μm, the feed pressure of the primary clarification is 0.6~1.5 Bar, the volume of the bacterial protease hydrolysate is V1, the volume of the first part of the primary permeate is 0.5V1~0.6V1, and the volume of the primary concentrate is 0.4V1~0.5V1.

[0022] Furthermore, the primary concentrate has an equal-volume washing filtration ratio of 3.5, and the volume of the concentrate obtained after washing and filtration is 1.4V1 to 1.75V1; and / or

[0023] The total volume V2 of the primary permeate and the volume V3 of the primary residual liquid satisfy the following relationship: V2:V3 ≥ 5.

[0024] Furthermore, the hollow fiber membrane of the secondary clarification has a resolution of 3 kDa, the feed pressure of the secondary clarification is 0.7~0.8 Bar, and the volume V4 of the secondary permeate and the volume V5 of the secondary residual liquid satisfy the relationship: V4:V5 ≥ 25.

[0025] Furthermore, the activated carbon addition amount is 0.5~1.0% w / v, the stirring speed of the reaction vessel is 400~500 rpm, the stirring time is 20~30 min, and the titanium rod filter has a size of Φ 60~80 mm × 400~500 mm and a filtration accuracy of 20~30 μm.

[0026] Furthermore, the sterilization temperature of the ultra-high temperature instantaneous sterilizer is 120℃~140℃, the high-temperature treatment time of the ultra-high temperature instantaneous sterilizer is 2s~6s, and the processing capacity of the ultra-high temperature instantaneous sterilizer is 0.8 tons / hour~1 ton / hour.

[0027] Further, the temperature of the electrothermal concentration is 70℃~72℃, the electrothermal concentration time is 1.5 h~2.0 h, and the temperature of the small peptide concentrate is 43℃~50℃ during the electrothermal concentration; and / or

[0028] The small peptide treatment liquid-solid content is 40 g / L~60 g / L; and / or

[0029] The concentrated peptide solution has a solid content of 400 g / L to 600 g / L; and / or

[0030] The freeze-drying temperature is -80.5℃ to -80℃, and the vacuum degree is 1 Pa to 10 Pa.

[0031] According to a second aspect of the present invention, the present invention provides a processing system for bacterial protein mash enzymatic hydrolysate, the system being adapted to the processing method described in any of the preceding claims, the processing system comprising:

[0032] The hollow fiber membrane clarification section includes a protease hydrolysate buffer tank, a primary clarification hollow fiber membrane, a primary permeate buffer tank, a secondary clarification hollow fiber membrane, and a secondary permeate buffer tank. The outlet of the protease hydrolysate buffer tank is connected to the inlet of the primary clarification hollow fiber membrane. The outlet 1 of the primary clarification hollow fiber membrane is connected to the inlet of the primary permeate buffer tank. The outlet 2 of the primary clarification hollow fiber membrane returns to the inlet of the protease hydrolysate buffer tank. The raw material in the protease hydrolysate buffer tank circulates through the primary clarification hollow fiber membrane, ultimately forming a first portion of primary permeate and a first portion of concentrate. The primary concentrate is washed 3.5 times its volume with pure water in the protease hydrolysate buffer tank and thoroughly stirred before being discharged again through the protease hydrolysate buffer tank. The first and second portions of the primary clarification hollow fiber membrane are circulated and clarified at the inlet and outlet of the primary clarification hollow fiber membrane to obtain a second portion of primary permeate and a primary residual liquid. The second portion of the primary permeate enters the primary permeate buffer tank through the outlet 1 of the primary clarification hollow fiber membrane. The first and second portions of the primary permeate are stirred evenly in the tank to obtain the primary permeate. The outlet of the primary permeate buffer tank is connected to the inlet of the secondary clarification hollow fiber membrane, the outlet 1 of the secondary clarification hollow fiber membrane is connected to the secondary permeate buffer tank, and the outlet 2 of the secondary clarification hollow fiber membrane is connected to the primary permeate buffer tank. The liquid in the primary permeate buffer tank circulates through the secondary clarification hollow fiber membrane to obtain a bacterial protease hydrolysate solution with a molecular weight of less than 3 kDa.

[0033] The deodorization section of the reactor includes a reactor and a titanium rod filter. The outlet of the secondary permeate buffer tank is connected to the inlet of the reactor. Activated carbon is added to the reactor and stirred thoroughly. The outlet of the reactor is connected to the inlet of the titanium rod filter to obtain a small peptide treatment solution that removes the odor of the culture medium and activated carbon particles.

[0034] The sterilization section includes an ultra-high temperature instantaneous sterilizer. The outlet of the titanium rod filter is connected to the inlet of the ultra-high temperature instantaneous sterilizer to achieve sterilization of the small peptide treatment solution and obtain a sterilized small peptide treatment solution.

[0035] The concentrated freeze-dried product is prepared by connecting the outlet of the ultra-high temperature instantaneous sterilizer to the inlet of the electro-concentrating device to concentrate the small peptide treatment solution to obtain a small peptide concentrate. The outlet of the electro-concentrating device is connected to the freeze dryer to freeze-dry the small peptide concentrate to obtain a small peptide powder product.

[0036] According to a third aspect of the present invention, the present invention provides the application of a small peptide powder product obtained by the method described in any of the preceding claims in the high-density stress-induced broiler chicken rearing.

[0037] Furthermore, the application includes:

[0038] a. Broiler stocking density is 10 birds / m²2 High-density stress conditions;

[0039] b. Add the small peptide powder product to the complete feed at a mass ratio of 0.04%;

[0040] c. The small peptide powder product is used throughout the entire broiler chicken rearing process.

[0041] The embodiments of the present invention have the following advantages:

[0042] 1. This invention uses a two-stage hollow fiber membrane coupled with equal volume washing filtration, which can directionally enrich small molecule peptides within 3 kDa, with high separation efficiency and small peptide recovery rate of 77.52%.

[0043] 2. This invention combines activated carbon deodorization, titanium rod filtration, ultra-high temperature instantaneous sterilization and low temperature freeze drying to effectively remove odors and impurities, retain the activity of small peptides to the greatest extent, and produce products with stable quality and good water solubility.

[0044] 3. The present invention is equipped with a dedicated processing system, with a continuous process flow and controllable parameters, making it suitable for industrial-scale production.

[0045] 4. When this invention is applied to high-density stress-affected broilers at a dosage of 0.04%, it can significantly improve growth performance, enhance antioxidant capacity, improve meat quality, and has a good anti-stress effect.

[0046] 5. This invention enables the high-value utilization of microbial protein, reduces dependence on imported protein raw materials such as soybean meal, and has outstanding economic and application value. Attached Figure Description

[0047] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0048] Figure 1 This is a schematic diagram of a method for separating and purifying bacterial protease hydrolysate provided in an embodiment of the present invention. Detailed Implementation

[0049] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] Example 1

[0051] See Figure 1 This invention provides a method for separating and purifying bacterial protein mash, the method comprising:

[0052] S1, obtain the bacterial protein hydrolysate (this product to be separated and purified is the bacterial protein treatment solution obtained from CN119823913A), and perform primary clarification using a hollow fiber membrane. The principle of hollow fiber concentration is mainly based on membrane separation technology, utilizing the pore size of the hollow fiber to achieve interception according to molecular weight: when the feed liquid passes through the hollow fiber membrane under pressure, small molecules can permeate through the membrane, while large molecules are retained because their size is larger than the membrane pore size, thus achieving separation. The hollow fiber membrane for primary clarification has a precision of 0.2 μm, is made of polyvinylidene fluoride, has a fiber inner diameter of 1 mm, and a membrane loading of 20 L / m³. 2 Up to 30L / m 2 The bacterial protease hydrolysate enters a 0.2 μm hollow fiber membrane under pressure, flowing within the membrane channels. Some solvents and soluble small molecules with particle sizes smaller than 0.2 μm permeate through the membrane, forming the first-stage permeate. Other molecules with particle sizes smaller than 0.2 μm, soluble macromolecules, and suspended solids are retained by the hollow fiber membrane and returned to the feed tank. The feed then passes through a rotary pump into a first-stage clarification hollow fiber membrane, continuously circulating and permeating to form the first-stage permeate. The concentration of the retained concentrate gradually increases. When the volume of the concentrate (V6) and the volume of the original solution (V1) satisfy V1:V6≥2.5, the first-stage clarification is paused, yielding the first-stage permeate and the first-stage concentrate.

[0053] S2, the primary concentrate is washed with an equal volume of ultrapure water (3.5 times) to obtain a washing filtrate. This washing filtrate is then passed through a 0.2 μm hollow fiber membrane under the pressure of a rotary pump, and circulated again through the primary clarifying hollow fiber membrane. Some solvent particles smaller than 0.2 μm and soluble small molecules in the washing filtrate permeate through the membrane to form a secondary permeate. Other components are circulated by the rotary pump, continuously permeating through the membrane to form a clarified liquid. The unpermeated portion is returned to the raw material tank to form residual liquid. Clarification is paused when the total volume V2 of the primary permeate and the volume V3 of the primary residual liquid satisfy the relationship: V2:V3 ≥ 5, yielding a second portion of the primary permeate and a primary residual liquid. The first and second portions of the primary permeate are then mixed thoroughly to obtain the primary permeate.

[0054] S3, the primary permeate is obtained, and secondary clarification is performed using hollow fiber membranes. The secondary clarification hollow fiber membrane has a resolution of 3 kDa, is made of modified polyethersulfone, has a fiber inner diameter of 0.5 mm, and a membrane loading of 20 L / m³. 2 Up to 30L / m 2The bacterial protein hydrolysate enters a 3 kDa hollow fiber membrane under pressure. Some solvents and soluble small molecules with a particle size smaller than 3 kDa permeate through the membrane to form a secondary permeate. Other molecules with a particle size smaller than 3 kDa, soluble large molecules, and suspended solids are retained and refluxed by the hollow fiber membrane. The solution then passes through a rotary pump into a secondary clarification hollow fiber membrane, continuously circulating and permeating through the membrane to form a secondary permeate. The concentration of the retained concentrate gradually increases, eventually forming a secondary permeate that has permeated through the membrane and a secondary residual liquid that has not. When the volume of the secondary permeate V4 and the volume of the secondary residual liquid V5 satisfy the relationship V4:V5≥25, the secondary clarification is paused, yielding the secondary permeate and the secondary concentrate.

[0055] S4. The obtained secondary permeate is passed into a reaction vessel, and 1% activated carbon is added. After stirring thoroughly for 30 minutes, it is passed into a titanium rod filter. The titanium rod filter has a filtration accuracy of 30 μm, a filter element size of Φ60 mm × 500 mm, and 11 filter elements. The hourly processing capacity can reach 3 to 6 tons. The feed solution with activated carbon added in the reaction vessel passes through the titanium rod filter to obtain a preliminary peptide treatment solution with removed culture medium odor and activated carbon. The preliminary peptide treatment solution is then subjected to an ultra-high temperature instantaneous sterilizer, where it is treated at a high temperature of approximately 130°C for 4 seconds before flowing out to obtain a sterilized peptide treatment solution. After the above treatment, a deodorized and sterilized peptide treatment solution is obtained.

[0056] S5. An SDR electro-concentrator is used to concentrate the small peptide solution. The SDR electro-concentrator operates on a principle similar to refrigeration, utilizing the phase changes of the energy-carrying medium to achieve exothermic and endothermic processes. The released heat is used to heat the liquid in the tank for continuous evaporation, while the absorbed heat comes from the condensation heat of the secondary steam. During concentration, the heating temperature is 72℃, the heating time is 1.5 hours, and the temperature of the concentrated material, i.e., the small peptide concentrate temperature, is 45℃. Throughout the concentration process, the material maintains a suitable temperature, avoiding a high-temperature unstable state, which is beneficial for preserving the functional activity and value attributes of the small peptide solution. Through SDR electro-concentration, the material concentration is concentrated 10 times from 50g / L to 500g / L. The small peptide concentrate is then fed into a freeze dryer with a daily processing capacity of up to 180L. The freeze-drying temperature is -80℃, and the vacuum degree is 1.2pa. After sieving, the flaky freeze-dried small peptides are pulverized and passed through a 60-mesh sieve, finally obtaining a small peptide powder product with a moisture content of less than 8%.

[0057] Test Example 1

[0058] I. Model Construction and Grouping

[0059] Establishment of a high-density stress broiler model: 720 one-day-old male white-feathered broiler chicks with an initial weight of (45.2±0.3) g were used in the experiment. After conventional brooding until 7 days of age, they were reared in a single layer on the floor.

[0060] High-density rearing is adopted, 10 birds / m² 2 (Slaughter density 32 kg / m³) 2 No additional ventilation was provided throughout the process. The temperature was maintained at 32°C for 4 hours daily starting from the third week, and the ammonia concentration was controlled at 20-25 ppm to establish a triple stress model of "high temperature + high density + high ammonia".

[0061] The small peptide powder prepared in Example 1 was mixed evenly with the basal diet at a ratio of 0.04% (w / w) in a mixer, granulated (75°C, 30 s), and then cooled to obtain the experimental diet.

[0062] The constructed stress model was divided into experimental groups:

[0063] T1 (negative control) = stress model + basal diet;

[0064] T2 (experimental group) = stress model + diet containing 0.04% small peptide powder;

[0065] T3 (positive control) = stress model + basal diet + 200 mg / kg (0.02%) vitamin C.

[0066] Each group had 6 replicates, with 40 animals per replicate. Production performance, serum antioxidant-related indicators, and meat quality were recorded. A free-range feeding and watering system was adopted. The experiment lasted 42 days, divided into two phases: 0–21 days and 22–42 days. A corn-soybean meal basal diet was used, formulated with reference to the NRC (1994) nutrient levels.

[0067] II. Experimental Research

[0068] 1. Detection Method

[0069] (1) Detection of crude protein and moisture: according to the method provided by AOAC (2006). Among them, the moisture content was detected in an oven at 105℃; the crude protein was detected by the Kjeldahl method, and the crude protein content was calculated by multiplying the nitrogen content determined by the Kjeldahl method by 6.25.

[0070] (2) Antioxidant performance: Total antioxidant capacity (T-AOC) was tested using the Total Antioxidant Capacity (T-AOC) kit (ABTS method) (Nanjing Jiancheng Bioengineering Institute). The kit instructions were followed. The T-AOC value of the samples was calculated based on the Trolox standard curve.

[0071] (3) Solubility: Accurately weigh 2.5g of sample, add 30 mL of water to the sample, shake for 30 min, then centrifuge at 3500 rpm for 10 min, pour out the supernatant and dry it at 135 ℃ for 2 hours, and calculate the ratio of the solid content of the supernatant to the mass of the sample.

[0072] (4) Peptide molecular weight determination: Weigh 0.125 g of small peptide powder using weighing paper, transfer it to a 25 mL volumetric flask, and dilute to the mark using the mobile phase (a mixed solution of acetonitrile, water, and trifluoroacetic acid with a volume ratio of 40:60:0.05). Then, use ultrasonic oscillation to fully dissolve the Clostridium ethanolate enzymatic hydrolysis product, and filter it through a 0.2 μm filter membrane. The filtrate is then loaded into the liquid chromatography vial to complete the sample preparation for the liquid chromatography. Then, according to the detection method described in GB 31645-2018, prepare the mobile phase, set the chromatographic parameters, and determine the weight content of peptides and the weight content of peptides with a relative molecular mass ≤10000 Da.

[0073] (5) Amino acid composition: The amino acid composition of the sample was determined using an automatic amino acid analyzer, referring to the method provided in GB / T 18246-2019.

[0074] (6) Broiler test indicators:

[0075] a. Production performance: Body weight and feed intake were recorded on days 21 and 42 of the experiment. The average daily gain (ADG), average daily feed intake (ADFI), and feed to gain ratio (F:G) of broilers were calculated based on the above values ​​for 0–21 days, 22–42 days, and 0–42 days.

[0076] b. Antioxidant indicators: Blood was collected from the wing veins of 42-day-old chicks, and serum was obtained by centrifugation. The activity of catalase (CAT), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) in the serum were determined using an A6 semi-automatic biochemical analyzer (Beijing Songshang Technology Co., Ltd., Beijing). The reagent kits were manufactured by Beijing Huaying Biotechnology Research Institute.

[0077] c. Meat quality: On day 42 of the experiment, six chickens from each treatment (one chicken per replicate) were randomly selected for slaughter, and the breast muscle was collected for meat quality index determination. The determination method is as follows:

[0078] Drip loss: During slaughter, the middle portion of the left pectoralis major muscle is taken, trimmed into a cuboid with dimensions of 30 mm × 15 mm × 5 mm, and weighed (m1). One end of the meat sample is then hooked with a wire, ensuring the muscle fibers are vertically upward. The sample is placed in an air-filled plastic bag, ensuring it does not contact the bag wall. The bag is sealed and hung in a 4°C refrigerator for 24 hours. After 24 hours, the sample is removed, dried with filter paper, and weighed again (m2). The drip loss is calculated using the formula: Drip loss = (m2 - m1) / m1 × 100%.

[0079] Shear force: Measured using a muscle tenderness meter with a load of 15 kg and a shearing speed of 200 mm / min. Cut a piece of meat with a length × width × thickness of 3 cm × 1 cm × 1 cm along the direction parallel to the muscle fibers, place it into the blade, and cut in the direction perpendicular to the direction of the blade.

[0080] pH: Right sternum muscle was harvested at slaughter. A calibrated portable pH meter (Testo 105, Testo, Germany) was used. The pH probe was inserted into the meat for measurement. Three different locations were measured for each piece of meat, and the average value was taken as the final result, recorded as pH. 45min After measuring the pH value, the chicken breast on the right side was placed in a 4°C refrigerator. After 24 hours, it was removed, and the pH value was measured at three different locations on each piece of meat. The average value was taken as the final result and recorded as pH. 24h .

[0081] Meat color: Lightness (L) of the right muscle was measured within 2 hours after slaughter using a Metan colorimeter (CR-410, Konica Metan Holdings, Japan). * Redness (a) * ) and Yellowness (b * ).

[0082] 2. Test Results

[0083] (1) Calculation of hollow fiber membrane recovery rate in existing process

[0084] The parameters for the extraction of bioactive peptides from the crude enzymatically hydrolyzed bacterial protein using hollow fiber membrane separation and purification are shown in Table 1 below.

[0085] Table 1

[0086]

[0087] The crude enzymatically hydrolyzed bacterial protein product was microfiltered through a 0.2 μm hollow fiber membrane. The primary concentrate was concentrated 2.5 times and then washed and filtered 3.5 times. The washed and filtered liquid was microfiltered again to obtain all the primary permeate. The primary permeate was then ultrafiltered through a 3 kDa hollow fiber membrane to obtain the desired small peptide solution with a molecular weight within 3 kDa, as shown in Table 1. The primary and secondary residues are non-recoverable portions, while the primary and secondary permeates are recoverable portions. Based on the protein concentration and the volume of each portion, the recovery rate of this separation and purification process can reach 77.52%.

[0088] (2) Analysis of product indicators of small peptide powder

[0089] The physicochemical properties are shown in Table 2 below.

[0090] Table 2

[0091]

[0092] The enzymatic hydrolysis of bacterial protein is processed by the separation and purification process of this patent to obtain small peptide powder. The color is white to light yellow, the particles are uniform, and it is odorless. Physicochemical analysis shows that its crude protein content is ≥80%, the moisture content is ≤7%, the antioxidant capacity is ≥1.0 mM, and the water solubility of the small peptide powder is 100%.

[0093] The molecular weight distribution is shown in Table 3 below.

[0094] Table 3

[0095]

[0096] The amino acid composition is shown in Table 4 below.

[0097] Table 4

[0098]

[0099] To further explore the relationship between the structure and activity of purified small peptides, this study determined their molecular weight and amino acid composition. The purified small peptides were predominantly small molecules, with 100% below 3 kDa. Among these, peptides with molecular weights less than 1 kDa accounted for 89.84%, and low-molecular-weight peptides often exhibited stronger antioxidant capacity. The amino acid composition of the purified small peptides was then analyzed, revealing that hydrophobic amino acids accounted for approximately 40.57%. Generally, the more hydrophobic amino acids in a peptide, the stronger its antioxidant activity. This structure-activity relationship explains the possible reason why the purified peptides possess antioxidant activity.

[0100] (3) Effects of dietary supplementation with small peptide powder on high-density stress broilers

[0101] a. Composition of the basic diet, see Table 5 below.

[0102] Table 5. Composition of the basal diet (%, feeding basal level)

[0103]

[0104] Note: 1 The premix provides the following per kilogram of diet: Vitamin A, 9,000 IU; Vitamin D3, 3,000 IU; Vitamin E, 24 mg; Vitamin K3, 1.8 mg; Vitamin B1, 2.0 mg; Riboflavin, 5.0 mg; Vitamin B6, 3.0 mg; Vitamin B2... 12 0.1 mg; niacin, 40 mg; pantothenic acid, 15 mg; folic acid, 1.0 mg; biotin, 0.05 mg; choline chloride, 500 mg; iron (ferrous sulfate monohydrate), 80 mg; copper (copper sulfate pentahydrate), 20 mg; zinc (zinc sulfate monohydrate), 90 mg; iodine (potassium iodide), 0.35 mg; selenium (sodium selenite), 0.30 mg.

[0105] b. Nutritional levels of the experimental diets are shown in Table 6 below.

[0106] Table 6. Nutritional levels of the experimental diets (%, basal feeding)

[0107]

[0108] Note: All nutritional levels, except for metabolizable energy, are measured values.

[0109] c. Growth performance, see Table 7 below.

[0110] Table 7 Effects of small peptide powder on growth performance of broiler chickens

[0111]

[0112] Note: n = 6.

[0113] d. Antioxidant function, see Table 8 below.

[0114] Table 8 Effects of small peptide powder on antioxidant indicators in broiler chicken serum

[0115]

[0116] Note: n = 6.

[0117] e. Meat quality, see Table 9 below.

[0118] Table 9. Effects of small peptide powder on the quality of broiler chicken.

[0119]

[0120] Note: n = 6.

[0121] Among them, T1 was the negative control, stress model + basal diet; T2 was the experimental group, stress model + diet containing 0.04% small peptide powder; T3 was the positive control, stress model + basal diet + 200 mg / kg (0.02%) vitamin C.

[0122] Effects of small peptide powder on the growth performance of broiler chickens: Compared with group T1, groups T2 and T3 significantly increased the average daily weight gain of broiler chickens from 0 to 42 days (P < 0.05). Among them, group T2 also significantly increased the average daily weight gain in the later stage of the experiment and significantly reduced the feed conversion ratio in the later stage and throughout the entire experiment (P < 0.05).

[0123] Effects of small peptide powder on antioxidant indicators in broiler serum: Compared with group T1, groups T2 and T3 significantly increased catalase activity at 21 days of age, total antioxidant capacity at 42 days of age, and significantly reduced malondialdehyde (MDA) content (P < 0.05). It is understood that CAT and T-AOC are important parameters for assessing antioxidant capacity, with higher values ​​indicating stronger antioxidant capacity; while MDA is a key indicator of oxidative stress, with higher values ​​indicating weaker antioxidant capacity.

[0124] Effects of small peptide powder on the quality of broiler chicken: Compared with group T1, groups T2 and T3 significantly reduced drip loss in chicken breast and significantly increased brightness value (P < 0.05).

[0125] Meat quality is a comprehensive evaluation index involving physicochemical properties related to the appearance, nutritional value, and palatability of meat. Meat color, as a direct evaluation indicator, directly influences consumers' meat choices. Evaluation indicators for meat color include L... * value, a * value and b * Value, where b * The value refers to the yellowness index, which reflects the antioxidant capacity of meat. The lower the value, the stronger the antioxidant capacity. * The values ​​and drip loss indicate the water-holding capacity of the meat, where L * The value represents the brightness of the meat. Within a certain range, this value is positively correlated with the water-holding capacity of the meat, while drip loss is negatively correlated with the water-holding capacity of the meat.

[0126] Therefore, as can be seen from the above data, the small peptide powder prepared by this invention can improve the growth performance of broiler chickens, improve the body's antioxidant capacity and meat quality when added at a concentration of 0.04%, thanks to its own good antioxidant capacity. At the same time, its effect is similar to that of vitamin C, a typical antioxidant.

[0127] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for separating and purifying bacterial protease hydrolysate, characterized in that, The method includes: The bacterial protein hydrolysate was clarified through a hollow fiber membrane to obtain the first part of the first permeate and the first concentrate. After the primary concentrate is washed and filtered with an equal volume of pure water, the resulting filtrate is further clarified by a hollow fiber membrane to obtain a second part of the primary permeate and a primary residual liquid. The first part of the primary permeate and the second part of the primary permeate are combined and mixed evenly to form a primary permeate. The primary permeate is then clarified in a secondary stage to obtain a secondary permeate and a secondary residual liquid. The secondary permeate was passed into a reaction vessel, activated carbon was added and stirred, and then the activated carbon was removed through a titanium rod filter to obtain a small peptide solution with the culture medium odor removed. The small peptide solution, from which the culture medium odor was removed, was sterilized using an ultra-high temperature instantaneous sterilizer to obtain a small peptide treated solution. The small peptide treatment solution was concentrated by electric heating to obtain a small peptide concentrate. The small peptide concentrate was freeze-dried using a freeze dryer to obtain lyophilized small peptide flakes. The lyophilized flake peptides were sieved to obtain a small peptide powder product.

2. The method for separating and purifying bacterial protease hydrolysate according to claim 1, characterized in that, The hollow fiber membrane used for primary clarification has a precision of 0.2 μm, the feed pressure for primary clarification is 0.6~1.5 Bar, the volume of the bacterial protease hydrolysate is V1, the volume of the first portion of the primary permeate is 0.5V1~0.6V1, and the volume of the primary concentrate is 0.4V1~0.5V1.

3. The method for separating and purifying bacterial protease hydrolysate according to claim 1, characterized in that, The primary concentrate has an equal-volume washing filtration ratio of 3.5, and the volume of the concentrate obtained after washing and filtration is 1.4V1 to 1.75V1; and / or The total volume V2 of the primary permeate and the volume V3 of the primary residual liquid satisfy the following relationship: V2:V3 ≥ 5.

4. The method for separating and purifying bacterial protease hydrolysate according to claim 1, characterized in that, The hollow fiber membrane used for secondary clarification has a resolution of 3 kDa, the feed pressure for secondary clarification is 0.7~0.8 Bar, and the volume V4 of the secondary permeate and the volume V5 of the secondary residual liquid satisfy the relationship: V4:V5 ≥ 25.

5. The method for separating and purifying bacterial protease hydrolysate according to claim 1, characterized in that, The activated carbon addition amount is 0.5~1.0% w / v, the stirring speed of the reaction vessel is 400~500 rpm, the stirring time is 20~30 min, and the titanium rod filter has a size of Φ 60~80 mm×400~500 mm and a filtration accuracy of 20~30 μm.

6. The method for separating and purifying bacterial protease hydrolysate according to claim 1, characterized in that, The ultra-high temperature instantaneous sterilizer has a sterilization temperature of 120℃~140℃, a high-temperature treatment time of 2s~6s, and a processing capacity of 0.8 tons / hour~1 ton / hour.

7. The method for separating and purifying bacterial protease hydrolysate according to claim 1, characterized in that, The electrothermal concentration temperature is 70℃~72℃, the electrothermal concentration time is 1.5 h~2.0 h, and the temperature of the small peptide concentrate is 43℃~50℃ during the electrothermal concentration; and / or The small peptide treatment liquid-solid content is 40 g / L~60 g / L; and / or The concentrated peptide solution has a solid content of 400 g / L to 600 g / L; and / or The freeze-drying temperature is -80.5℃ to -80℃, and the vacuum degree is 1 Pa to 10 Pa.

8. A system for separating and purifying enzymatic hydrolysate of bacterial protein mash, characterized in that, The system is adapted to the method as described in any one of claims 1 to 7, and the separation and purification system comprises: The hollow fiber membrane clarification section includes a protease hydrolysate buffer tank, a primary clarification hollow fiber membrane, a primary permeate buffer tank, a secondary clarification hollow fiber membrane, and a secondary permeate buffer tank. The outlet of the protease hydrolysate buffer tank is connected to the inlet of the primary clarification hollow fiber membrane. The outlet 1 of the primary clarification hollow fiber membrane is connected to the inlet of the primary permeate buffer tank. The outlet 2 of the primary clarification hollow fiber membrane returns to the inlet of the protease hydrolysate buffer tank. The raw material in the protease hydrolysate buffer tank circulates through the primary clarification hollow fiber membrane, ultimately forming a first portion of primary permeate and a first portion of concentrate. The primary concentrate is washed 3.5 times its volume with pure water in the protease hydrolysate buffer tank and thoroughly stirred before being discharged again through the protease hydrolysate buffer tank. The first and second portions of the primary clarification hollow fiber membrane are circulated and clarified at the inlet and outlet of the primary clarification hollow fiber membrane to obtain a second portion of primary permeate and a primary residual liquid. The second portion of the primary permeate enters the primary permeate buffer tank through the outlet 1 of the primary clarification hollow fiber membrane. The first and second portions of the primary permeate are stirred evenly in the tank to obtain the primary permeate. The outlet of the primary permeate buffer tank is connected to the inlet of the secondary clarification hollow fiber membrane, the outlet 1 of the secondary clarification hollow fiber membrane is connected to the secondary permeate buffer tank, and the outlet 2 of the secondary clarification hollow fiber membrane is connected to the primary permeate buffer tank. The liquid in the primary permeate buffer tank circulates through the secondary clarification hollow fiber membrane to obtain a bacterial protease hydrolysate solution with a molecular weight of less than 3 kDa. The deodorization section of the reactor includes a reactor and a titanium rod filter. The outlet of the secondary permeate buffer tank is connected to the inlet of the reactor. Activated carbon is added to the reactor and stirred thoroughly. The outlet of the reactor is connected to the inlet of the titanium rod filter to obtain a small peptide treatment solution that removes the odor of the culture medium and activated carbon particles. The sterilization section includes an ultra-high temperature instantaneous sterilizer. The outlet of the titanium rod filter is connected to the inlet of the ultra-high temperature instantaneous sterilizer to achieve sterilization of the small peptide treatment solution and obtain a sterilized small peptide treatment solution. The concentrated freeze-dried product is prepared by connecting the outlet of the ultra-high temperature instantaneous sterilizer to the inlet of the electro-concentrating device to concentrate the small peptide treatment solution to obtain a small peptide concentrate. The outlet of the electro-concentrating device is connected to the freeze dryer to freeze-dry the small peptide concentrate to obtain a small peptide powder product.

9. The application of the small peptide powder product obtained by the method according to any one of claims 1 to 7 in the high-density stress-induced broiler chicken farming.

10. The application according to claim 9, characterized in that, The applications include: a. Broiler stocking density is 10 birds / m² 2 High-density stress conditions; b. Add the small peptide powder product to the complete feed at a mass ratio of 0.04%; c. The small peptide powder product is used throughout the entire broiler chicken rearing process.

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