Plant protein stock fermentation composition, method of making and use thereof

Abstract

The present application belongs to the technical field of microbial fermentation and bioactive preparation, and particularly relates to a plant protein stock solution fermentation composition, a preparation method thereof and application. The plant protein stock solution fermentation composition is prepared by fermentation of the following raw materials in parts by mass: plant protein stock solution: 85-90 parts; sugar: 6-8 parts; superoxide dismutase: 0.2-0.3 parts; and complex probiotic flora: 0.2-3.0 parts. The complex probiotic flora comprises one or more of Lactobacillus plantarum, Lactobacillus casei or functional yeast. The sugar comprises one or both of brown sugar and glucose. The present application provides a fermentation composition which can be applied to human body and various animals for oral health care, and is helpful to integrate research and development resources, simplify product lines and reduce production cost.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology and bioactive preparation technology, and particularly relates to a plant protein stock solution fermentation composition, its preparation method and application. Background Technology

[0002] Currently, most fermented or bioactive products on the market have limited functions and applications, namely, they are developed separately as health supplements for human consumption and feed additives for livestock. For example, existing technology (Chinese invention patent publication number CN117256730A, publication date 2023-12-22) discloses a probiotic-containing feed additive for regulating animal intestines, which can only be used on animals and does not show any indication of its use on humans. This separation between human and animal systems leads to dispersed R&D resources, redundant product lines, high production costs, and an inability to realize the maximum synergistic value of product formulations based on the concept of human-animal homology.

[0003] In addition, conventional microbial fermentation processes usually require relatively high fermentation temperatures, typically above 35°C, to accelerate the reaction process. However, such conditions can easily lead to the denaturation and inactivation of protein-based bioactive substances such as superoxide dismutase, as well as the decline in the activity of some probiotics. Summary of the Invention

[0004] One of the objectives of this invention is to provide a plant protein stock solution fermentation composition that can achieve the technical effects for human use and all animal use.

[0005] The second objective of this invention is to provide a method for preparing the above-mentioned plant protein stock solution fermentation composition, which maximizes the preservation of SOD activity and the number of probiotic live bacteria through a low-temperature fermentation process.

[0006] The third objective of this invention is to provide specific applications of the aforementioned plant protein stock solution, including specific application scenarios for animals and humans.

[0007] The technical solution adopted by the present invention to solve the above problems is: a plant protein stock solution fermentation composition, which is prepared by fermentation from the following raw materials in parts by weight: plant protein stock solution: 85-90 parts; sugars: 6-8 parts; superoxide dismutase: 0.2-0.3 parts; compound probiotic flora: 0.2-3.0 parts; the compound probiotic flora includes one or more of Lactobacillus plantarum, Lactobacillus casei, or functional yeast; the sugars include one or more of brown sugar or glucose.

[0008] A further preferred technical solution is that the compound probiotic flora consists of Lactobacillus plantarum, Lactobacillus casei, and functional yeast, and the mass ratio of Lactobacillus plantarum, Lactobacillus casei, and functional yeast is (2~3):(1~2):(1~2).

[0009] A further preferred technical solution is that the compound probiotic flora consists of Lactobacillus plantarum, Lactobacillus casei, and functional yeast, and the mass ratio of Lactobacillus plantarum, Lactobacillus casei, and functional yeast is 3:1:1.

[0010] A further preferred technical solution is that the sugars are brown sugar and glucose, and the mass ratio of brown sugar to sucrose is (1~2):(1~2).

[0011] The method for preparing the plant protein stock solution fermentation composition according to any one of the above-mentioned methods includes the following steps: S1. Extract the plant protein stock solution from pasture grass; S2. Under sterile conditions, the plant protein stock solution is mixed with the sugars to obtain a fermentation base solution; S3. Place the fermentation base liquid in a closed anaerobic environment at a temperature of 22~32℃, inoculate with a compound probiotic group, and carry out constant temperature fermentation for 48h~72h to obtain the primary fermentation liquid; S4. Under conditions of 22℃~32℃, superoxide dismutase is added to the primary fermentation broth and stirred to obtain secondary fermentation broth; S5. The secondary fermentation broth is subjected to static stabilization treatment, and then centrifuged to obtain the plant protein stock solution fermentation composition.

[0012] The application of any of the above-mentioned plant protein stock solution fermentation compositions is specifically applied to the internal health care of terrestrial or aquatic animals.

[0013] A further preferred technical solution is that the terrestrial animal is a piglet.

[0014] A further preferred technical solution is that the aquatic animal is the Pacific white shrimp.

[0015] The application of the plant protein stock solution fermentation composition described in any of the above-mentioned claims is specifically applied to human oral health products. A further preferred technical solution is that the oral health product is used to regulate the gastrointestinal tract.

[0016] In summary, the present invention has the following advantages: 1. This invention provides a fermented composition that can be applied to both humans and various animals for oral health care, which helps to integrate R&D resources, simplify product lines, and reduce production costs.

[0017] 2. This invention employs a low-temperature fermentation process, which effectively preserves biological activity and minimizes the denaturation and inactivation of protein-based bioactive substances and the decline in the activity of some probiotics caused by high temperatures, thereby retaining high SOD activity and high live bacteria count in the final product.

[0018] 3. This invention uses plant protein stock solution extracted from pasture grass. Compared with using alternative raw materials such as soy protein isolate, it can obtain higher SOD activity, total number of live bacteria, proportion of small molecule peptides and antioxidant capacity after fermentation.

[0019] 4. This invention uses a specific ratio of Lactobacillus plantarum, Lactobacillus casei, and functional yeast to ferment a compound probiotic group. Compared with single-strain fermentation or natural fermentation, it shows significant advantages in terms of acid production capacity, total number of viable bacteria, yield of small molecule peptides, antioxidant activity, vitamin synthesis, and acid resistance, and can effectively inhibit miscellaneous bacteria. Detailed Implementation

[0020] Composition of plant protein stock solution: In this embodiment, the plant protein stock solution is extracted from Pennisetum genus forage grasses. Preferably, the plant protein stock solution is extracted from Gramineae family forage grasses. Preferably, the plant protein stock solution is extracted from one or a mixture of several of the following grasses: Napier grass, sweet elephant grass, giant Napier grass, red elephant grass, hybrid Pennisetum genus, Mutali grass, ryegrass, orchardgrass, sheepgrass, awnless bromegrass, ice grass, crested wheatgrass, timothy grass, sedge, bermudagrass, and June grass. Preferably, the plant protein stock solution is extracted from Zhonghua Liangmu forage grass (Latin name: Pennisetum hydridum Z. yundan), which was purchased from Zhonghua Shengshi Ecological Agriculture Co., Ltd.

[0021] Extraction methods for plant protein stock solution: In this embodiment, the extraction method of plant protein stock solution specifically includes the following steps: S1. Extracting the plant protein stock solution from the pasture. Step S1 includes: S11. Remove impurities such as dead leaves, weeds, and mud from the raw materials, wash the hay with running water, drain the surface water, and obtain clean hay. S12. Cut the washed hay into 1-3cm pieces and pre-cool the chopped hay in a low temperature environment of 4-10℃ for 30-60 minutes; S13. The chopped forage is put into a grinding device, and water pre-cooled to 4~10℃ is added at the same time. The mixture is ground until the cell wall breakage rate is greater than 85% to form a forage slurry, wherein the mass ratio of forage to water is 1:1~3; preferably, the mass ratio of forage to water is 1:1. S14. The forage slurry is fed into a pressing device through the feed inlet, with a pressure range of 5~20MPa, to obtain primary pressed liquor; S15. Sieve the primary pressed liquid through a sieve with a mesh size of 100-200 mesh, then put it into a centrifuge and centrifuge it at 4-10℃, with a centrifugation speed of 8000-15000 rpm and a centrifugation time of 15-30 min. After centrifugation, take the supernatant to obtain the plant protein stock solution.

[0022] The relevant equipment models are as follows: Forage chopping equipment: chaff crusher, Qufu Muyuan Machinery Co., Ltd.

[0023] Low-temperature environment equipment: Walk-in high and low temperature test chamber, Shanghai Shangqi Group Test Equipment Co., Ltd.

[0024] Grinding equipment: Vertical stainless steel colloid mill, Zhengzhou Yongchuang Machinery Equipment Co., Ltd.

[0025] Pressing equipment: Single screw press dewatering machine, Jiangsu Hanlong Machinery Manufacturing Co., Ltd.

[0026] Screen: Basic multi-layer vibrating screen filter, Shanghai Laosong Machinery Co., Ltd.

[0027] Centrifuge: GF105 liquid-liquid tubular centrifuge, Shanghai Tianben Machinery Technology Co., Ltd.

[0028] Composition of the plant protein stock solution fermentation composition: In this embodiment, the composition of the plant protein stock solution fermentation composition is as follows: Plant protein stock solution: 85-90 parts; Carbohydrates: 6-8 servings; Superoxide dismutase: 0.2~0.3 parts; Compound probiotic flora: 0.2~3.0 parts; The compound probiotic flora includes one or more of Lactobacillus plantarum, Lactobacillus casei, or functional yeasts; The sugars include one or both of brown sugar and glucose.

[0029] Lactobacillus plantarum breaks down carbohydrates through glycolysis and phosphoketose pathways, producing a mixture of organic acids, primarily lactic acid. It also metabolizes various bacteriocins, such as plant lactobacillusin, to inhibit bacterial growth. This rapid acid production lowers the pH of the fermentation system to 4.0-5.0, effectively inhibiting the growth of unwanted bacteria and spoilage microorganisms, ensuring the safety of the fermentation process. Furthermore, Lactobacillus plantarum secretes extracellular proteases and peptidases, degrading large protein molecules in the plant protein concentrate into smaller peptides and free amino acids, significantly improving the product's bioavailability and absorbability. Lactobacillus plantarum is a recognized safe strain both domestically and internationally, listed in my country's "List of Microbial Strains that Can Be Used in Food," and is safe for human consumption.

[0030] *Lactobacillus casei* and *Lactobacillus plantarum* both belong to the family Lactobacillus and are facultative heterofermentative bacteria. They produce acid through glycolysis and possess strong amino acid conversion capabilities. In plant protein stock solution fermentation systems, *Lactobacillus casei* possesses a rich extracellular proteolytic enzyme system, including cell wall-anchored proteases and peptide transport systems, enabling it to deeply degrade complex polypeptide structures in plant proteins and efficiently generate bioactive short peptides. *Lactobacillus casei* exhibits high tolerance thresholds to osmotic pressure fluctuations, low temperatures, and low pH, maintaining good metabolic activity in the later stages of fermentation, ensuring the stability of the fermentation process and the consistency of the final product quality. Its cell wall components can be recognized by pattern recognition receptors on the surface of intestinal immune cells, activating innate immune responses, regulating cytokine secretion profiles, and enhancing the body's immune function. *Lactobacillus casei* can regulate Th1 / Th2 immune balance and enhance intestinal secretory immunoglobulin A levels. *Lactobacillus casei* and *Lactobacillus plantarum* are complementary in substrate utilization profiles and enzyme types; co-inoculation can significantly improve the degradation efficiency of plant proteins and the abundance of functional metabolites. Lactobacillus casei is also included in my country's "List of Microbial Strains that Can Be Used in Food" and is safe for human consumption.

[0031] The brown sugar composition conforms to the Chinese national standard GB / T 35885-2018 "Brown Sugar," with a sucrose content ≥90wt%. Sucrose in brown sugar is the most readily assimilated disaccharide carbon source for lactic acid bacteria. After being hydrolyzed into glucose and fructose by extracellular sucrase, it enters the sugar metabolism pathway, providing the carbon skeleton and energy for bacterial growth and acid production. Glucose, without extracellular hydrolysis, can directly enter the microbial cell through the glucose-specific transport system on the cell membrane. After phosphorylation, it enters the glycolysis pathway, making it the most preferentially utilized fast-acting carbon source for all heterotrophic microorganisms. Supplementing with glucose in the early stages of fermentation can significantly shorten the lag phase of the strain, allowing it to quickly enter the logarithmic growth phase. Brown sugar primarily provides minerals and flavor, while glucose primarily provides fast-acting carbon; the two exert a synergistic and complementary effect.

[0032] Among them, the functional yeast is *Saccharomyces cerevisiae*, a natural and highly efficient producer of vitamins B1, B2, B6, pantothenic acid, and biotin, which can significantly enhance the nutritional value of the final fermentation product. During its metabolism, *Saccharomyces cerevisiae* releases alkaline phosphatase, esterase, and various coenzyme factors extracellularly, providing growth-promoting factors such as amino acids, polypeptides, and trace metal conjugates for the growth of lactobacilli in the system, thus promoting the proliferation and activity of lactic acid bacteria.

[0033] In a preferred embodiment, the mass ratio of *Lactobacillus plantarum*, *Lactobacillus casei*, and functional yeast is (2-3):(1-2):(1-2). Preferably, the mass ratio of *Lactobacillus plantarum*, *Lactobacillus casei*, and functional yeast is 3:1:1.

[0034] In a preferred embodiment, the mass ratio of brown sugar to sucrose is (1~2):(1~2). Preferably, the mass ratio of brown sugar to sucrose is 2:1.

[0035] Preparation method of plant protein stock solution fermentation composition: S2. Under sterile conditions, the plant protein stock solution is mixed with the sugars to obtain a fermentation base solution; S3. Place the fermentation base liquid in a closed anaerobic environment at a temperature of 22~32℃, inoculate with a compound probiotic group, and carry out constant temperature fermentation for 48h~72h to obtain the primary fermentation liquid; S4. Under conditions of 22℃~32℃, superoxide dismutase is added to the primary fermentation broth and stirred to obtain secondary fermentation broth; S5. The secondary fermentation broth is subjected to static stabilization treatment, and then centrifuged to obtain the plant protein stock solution fermentation composition.

[0036] The specific steps of the static stabilization treatment are as follows: the secondary fermentation broth is transferred to a clean, covered stabilization tank and allowed to stand at 22°C to 32°C for 12 to 48 hours, preferably 24 hours. After standing, the supernatant is taken for further processing, and any sediment at the bottom is discarded.

[0037] The centrifugation process involves centrifuging at 4-10°C, with a centrifugation speed of 8000-15000 rpm and a centrifugation time of 15-30 min. After centrifugation, the supernatant is collected to obtain the plant protein stock solution fermentation composition.

[0038] Preferably, after centrifugation, aseptic filling is also included to dispense the treated high-purity, high-activity plant protein stock solution fermentation composition into the final packaging container in a sterile environment to prevent secondary contamination and ensure the safety of the product's microbiological indicators during its shelf life.

[0039] The relevant equipment models are as follows: Aseptic mixing equipment: Aseptic stainless steel mixing tank, Wenzhou Maitron Machinery Co., Ltd.

[0040] Fermentation equipment: Stainless steel anaerobic fermenter, Wenzhou Lihong Machinery Technology Co., Ltd.

[0041] Anaerobic equipment: High-purity nitrogen cylinders, purity ≥99.99%.

[0042] Aseptic filling equipment: Portable strip packaging machine, Dongguan Chengyi Intelligent Equipment Co., Ltd. Bag dimensions: Length: 50~180mm, Width: 17~105mm.

[0043] The present invention will be specifically illustrated below with reference to embodiments: The following description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention.

[0044] Example 1: In this embodiment, the preparation method of the plant protein stock solution fermentation composition is as follows: S1. Extract the plant protein stock solution from Zhonghua Liangmu; wherein, step S1 includes: S11. Remove dead leaves, weeds and mud, wash with running water and drain the surface water; S12. Cut into 1cm pieces and pre-cool at 8℃ for 45 minutes; S13. Put the mixture into a colloid mill, add purified water pre-cooled to 8°C at a mass ratio of 1:1 forage to water, and wet grind until the cell wall breakage rate is >85% to obtain forage slurry; S14. The forage slurry is fed into a single screw press dewatering machine at a pressure of 15MPa to obtain primary press liquor; S15. Pass the primary pressed liquid through a 150-mesh vibrating screen, centrifuge the filtrate at 8℃ and 12000rpm for 20min, and take the supernatant to obtain the plant protein stock solution.

[0045] S2. Take 90 parts of plant protein stock solution and 6 parts of sugar (of which the mass ratio of brown sugar to glucose is 2:1), mix them evenly in a sterile mixing tank to obtain the fermentation base liquid; S3. Transfer the fermentation base liquid to an anaerobic fermenter, purge with nitrogen for 5 minutes to replace the air, seal the tank, control the temperature at 28℃, and inoculate with 1.5 parts of compound probiotics (of which Lactobacillus plantarum: Lactobacillus casei: Saccharomyces cerevisiae mass ratio 3:1:1). Perform constant temperature anaerobic fermentation for 60 hours to obtain the primary fermentation liquid. S4. At 25℃, add 0.25 parts of superoxide dismutase to the primary fermentation broth and mix at low speed for 10 min to obtain the secondary fermentation broth. S5. The secondary fermentation broth is transferred to a covered stabilizer and allowed to stand at 28°C for 24 hours. The supernatant is then centrifuged at 8°C and 12,000 rpm for 20 minutes. The supernatant is then aseptically filled to obtain the plant protein stock solution fermentation composition.

[0046] Example 2 This embodiment is basically the same as the steps in Embodiment 1, except that: S1. Extract the plant protein stock solution from the timothy forage grass.

[0047] Example 3 This embodiment is basically the same as the steps in Embodiment 1, except that: S1. Extract the plant protein stock solution from ryegrass.

[0048] Example 4 This embodiment is basically the same as the steps in Embodiment 1, except that: S1. Extract the plant protein stock solution from sweet elephant grass.

[0049] Comparative Example 1 This comparative example is basically the same as the steps in Example 1, except that: S1. Soy protein isolate (ISOPRO910, Shandong Jiahua Biotechnology Co., Ltd.) was used directly as the plant protein stock solution.

[0050] Performance tests were conducted on Examples 1-4 and Comparative Example 1, and the results are shown in Table 1: ; Table 1. Performance test results of Examples 1-4 and Comparative Example 1 As shown in Table 1, the total number of viable bacteria in fermented soybean protein was only about 1 / 4 of that extracted from forage, and the protective effect of SOD activity decreased significantly. This fully demonstrates that the plant protein stock solution extraction method of the present invention, especially the extraction from Zhonghua Liangmu, has certain advantages.

[0051] Example 5 This embodiment is basically the same as the steps in Embodiment 1, except that: S3. Transfer the fermentation base liquid to an anaerobic fermenter, purge with nitrogen for 5 minutes to replace the air, seal the tank, control the temperature at 28℃, and inoculate with 1.5 parts of compound probiotics (of which Lactobacillus plantarum: Lactobacillus casei: Saccharomyces cerevisiae mass ratio is 2:1:1). Perform constant temperature anaerobic fermentation for 60 hours to obtain the primary fermentation liquid.

[0052] Example 6 This embodiment is basically the same as the steps in Embodiment 1, except that: S3. Transfer the fermentation base liquid to an anaerobic fermenter, purge with nitrogen for 5 minutes to replace the air, seal the tank, control the temperature at 28℃, and inoculate with 1.5 parts of compound probiotics (of which Lactobacillus plantarum: Lactobacillus casei: Saccharomyces cerevisiae mass ratio 3:2:2). Perform constant temperature anaerobic fermentation for 60 hours to obtain the primary fermentation liquid.

[0053] Example 7 This embodiment is basically the same as the steps in Embodiment 1, except that: S3. Transfer the fermentation base liquid to an anaerobic fermenter, purge with nitrogen for 5 minutes to replace the air, seal the tank, control the temperature at 28℃, and inoculate with 1.5 parts of compound probiotics (with Lactobacillus plantarum: Lactobacillus casei: Saccharomyces cerevisiae in a mass ratio of 1:1:1). Perform constant temperature anaerobic fermentation for 60 hours to obtain the primary fermentation liquid.

[0054] Example 8 This embodiment is basically the same as the steps in Embodiment 1, except that: S3. Transfer the fermentation base liquid to an anaerobic fermenter, purge with nitrogen for 5 minutes to replace the air, seal the tank, control the temperature at 28℃, add 1.5 portions of compound probiotics (containing only Lactobacillus plantarum), and perform constant temperature anaerobic fermentation for 60 hours to obtain the primary fermentation liquid; Comparative Example 2 This comparative example is basically the same as the steps in Example 1, except that: S3. Transfer the fermentation base liquid to an anaerobic fermenter, purge with nitrogen for 5 minutes to replace the air, then seal the tank, control the temperature at 28℃, and allow natural fermentation without inoculating bacteria. Perform constant-temperature anaerobic fermentation for 60 hours to obtain the primary fermentation liquid. Performance tests were conducted on Examples 1, 5, and 6, as well as Comparative Example 2. The results are shown in Table 2. ; Table 2. Performance test results of Examples 1, 5-8 and Comparative Example 2 As shown in Table 2, the single-strain fermentation in Example 8 lost the synergistic metabolic effect among multiple strains, with significant decreases in small molecule peptide yield and DPPH scavenging rate. The B vitamin synthesis capacity was only about 1 / 7 of that in the complex microbial fermentation. In Comparative Example 2, the natural fermentation, lacking a dominant strain, resulted in severe proliferation of miscellaneous bacteria, low acid production, and a viable cell count only one ten-thousandth of that in the complex microbial group. Antioxidant activity and protein degradation capacity were both extremely low, essentially indicating uncontrolled fermentation. The three-strain complex system composed of *Lactobacillus plantarum*, *Lactobacillus casei*, and functional yeast in Examples 1, 5-7 achieved synergistic effects.

[0055] In this embodiment, the application of the plant protein stock solution fermentation composition in the preparation of oral health care for terrestrial or aquatic animals is also disclosed. Oral health care includes feed additives or functional preparations for nutritional conditioning, growth promotion, immunity enhancement, or relief of breeding stress.

[0056] This disclosure focuses on two major aquaculture scenarios: terrestrial animals (represented by piglets) and aquatic animals (represented by whiteleg shrimp). Multiple application examples and comparative examples are designed to systematically verify the efficacy of the plant protein stock solution fermentation composition of this invention in animal oral health care.

[0057] Application Example 1 Experimental animals: 28-day-old weaned piglets, weighing approximately 7.5 ± 0.5 kg, randomly divided into groups of 30 piglets each.

[0058] Experimental content: 0.2% (mass fraction, based on the total mass of the feed) of the plant protein stock solution fermentation composition prepared in Example 1 of this invention was added to the basal diet, sprayed onto the surface of the feed in liquid form, mixed evenly, and then fed.

[0059] Experimental period: 28 consecutive days of feeding.

[0060] Feeding and management: Free access to feed and water, routine immunization program, and temperature control in the enclosure at 24-26℃. Daily records of feed intake and diarrhea occurrences.

[0061] Testing indicators: Growth performance: Average daily gain (ADG), average daily feed intake (ADFI), feed conversion ratio (F / G) Diarrhea rate: Diarrhea rate (%) = Number of piglets with diarrhea per group during the experimental period / (Number of piglets per group × Number of experimental days) × 100% Serum immune indicators: Blood was collected from the anterior vena cava at the end of the test to detect serum IgG and IgA levels (immunoturbidimetric method).

[0062] Gut microbiota: At the end of the experiment, 6 animals were randomly selected from each group for slaughter, and cecal contents were collected. The number of lactobacilli and Escherichia coli was determined by plate count method.

[0063] Serum antioxidant indicators: serum total superoxide dismutase (T-SOD) activity (hydroxylamine method), malondialdehyde (MDA) content (TBA method). Application Comparative Example 1 The plant protein stock solution fermentation composition in Application Example 1 was replaced with an equal amount of purified water, while all other indicators remained the same.

[0064] Application Comparative Example 2 The plant protein stock solution fermentation composition in Application Example 1 was replaced with a commercially available compound probiotic feed additive (Yihao, compound feed bacteria), with all other indicators remaining identical.

[0065] The test results of Application Example 1 and Application Comparative Examples 1 and 2 are shown in Table 3.

[0066] ; Table 3. Test results of Application Example 1 and Comparative Examples 1 and 2 In Application Example 1, the plant protein stock solution fermentation composition, at an addition of 0.2%, increased the average daily weight gain of weaned piglets by 37.9% compared to the control group, reduced the feed conversion ratio to 1.40, and significantly reduced the diarrhea rate from 12.56% to 1.85%, demonstrating comprehensive superiority over commercially available probiotic preparations. This indicates that the composition of the present invention can positively regulate the intestinal flora and simultaneously enhance the body's antioxidant capacity.

[0067] Application Example 2 Experimental animals: Litopenaeus vannamei, with an initial average weight of 1.25±0.15g and a body length of 3.2±0.3cm. They were randomly divided into groups of 3 replicates, with 500 shrimp per replicate.

[0068] Experimental procedure: 0.15% (mass fraction) of the plant protein stock solution fermentation composition prepared in Example 1 of this invention was added to the basic feed, and the mixture was uniformly sprayed onto the feed surface in liquid form, dried at low temperature (40°C) and then fed.

[0069] Experimental period: 42 consecutive days of feeding.

[0070] Feeding and Management: Shrimp are raised in indoor cement ponds (2m×3m×1.2m) at a water temperature of 28±1℃ and a salinity of 15±2‰. Continuous aeration is provided for 24 hours, maintaining dissolved oxygen >5.0mg / L, pH 7.8~8.2, ammonia nitrogen <0.2mg / L, and nitrite <0.05mg / L. Feeding is provided four times daily (6:00, 11:00, 16:00, and 21:00), with a daily feed amount of approximately 5-8% of the shrimp's body weight, adjusted according to feeding behavior.

[0071] Testing indicators: Growth performance: weight gain (WGR), specific growth rate (SGR), feed conversion ratio (FCR), survival rate.

[0072] Non-specific immune markers: serum phenol oxidase (PO) activity, serum lysozyme (LSZ) activity, and serum superoxide dismutase (SOD) activity.

[0073] Challenge experiment: After the experiment, 60 fish were randomly selected from each group and injected intraperitoneally at a concentration of 1×10⁻⁶. 7 0.05 mL of Vibrio parahaemolyticus suspension with CFU / mL was used to calculate the cumulative mortality rate over 7 days.

[0074] Application Comparative Example 3: The plant protein stock solution fermentation composition in Application Example 2 was replaced with an equal amount of purified water for feed spraying, while the remaining feeding management and testing indicators were exactly the same.

[0075] Application Comparative Example 4: The plant protein stock solution fermentation composition in Application Example 2 was replaced with a commercially available aquatic compound Bacillus preparation (Nanhua Qianmu, compound Bacillus stock powder), while the remaining feeding management and testing indicators were exactly the same.

[0076] The test results of Application Example 2 and Application Comparative Examples 3 and 4 are shown in Table 4.

[0077] ; Table 4. Test results of application example 2 and application comparison examples 3 and 4 The plant protein stock solution fermentation composition of Example 2, at an addition of 0.15%, increased the weight gain rate of Litopenaeus vannamei by 77.3% (compared to the blank control), significantly reduced the feed conversion ratio to 1.18, and increased the survival rate to 94.8%. Regarding non-specific immune indicators, serum phenol oxidase activity increased by 85.9%, lysozyme activity by 106.3%, and SOD activity by 170.3%, all significantly superior to the commercially available Bacillus preparation control group. Crucially, in the Vibrio parahaemolyticus challenge test, the cumulative mortality rate of the composition group after 7 days was only 16.7%, significantly lower than the blank control group (68.3%) and the commercially available Bacillus preparation group (38.3%), with a relative immune protection rate of 75.5% (compared to the blank control), confirming the efficacy of the composition in enhancing the disease resistance of aquatic animals.

[0078] In this embodiment, the application of the plant protein stock solution fermentation composition in human oral health products is also disclosed.

[0079] This disclosure presents the application of a plant protein stock solution fermentation composition in the human gastrointestinal regulation, particularly in the adjunctive treatment of Helicobacter pylori infection.

[0080] Application Example 3: Test subjects: 13 C-urea breath test ( 13 Adult volunteers who were confirmed positive for Helicobacter pylori (Hp) with a DOB value ≥4.0 by C-UBT, had no peptic ulcers or atrophic gastritis on gastrointestinal endoscopy, and had not received Hp eradication therapy within the past 3 months were randomly assigned to groups of 40.

[0081] Oral preparation: Take the plant protein stock solution fermentation composition prepared in Example 1 of this invention, filter it through a 0.22μm microporous membrane for sterilization, and dilute it with pharmaceutical-grade purified water until the SOD activity is ≥1000U / mL and the viable count is ≥2.0×10⁻⁶. 8 The product is CFU / mL and filled into 15mL brown oral liquid bottles. The bottle surface is pasteurized (63℃, 30min). It is then stored at 4℃ for 30 days. Each 15mL vial contains SOD ≥ 15000U and a total viable count ≥ 3.0 × 10⁻⁶. 9 CFU, plant small molecule peptides ≥720mg.

[0082] Trial protocol: 15 mL orally twice daily, 30 minutes before breakfast and dinner, for 28 consecutive days. Participants were instructed to maintain their normal dietary habits and not to take any additional probiotics, antibiotics, or antacids. A repeat trial was conducted at the end of the trial. 13 C-UBT detection.

[0083] Testing indicators: Hp eradication rate: at the end of the experiment13 C-UBT DOB value negative conversion rate, DOB < 4.0 is negative; The decrease in Hp DOB value (ΔDOB); Pepsinogen I / II ratio (PGR, serum measured on days 0 and 28); Gastrin-17 levels (days 0 and 28); Gastrointestinal Symptom Rating Scale (GSRS, assessing symptoms such as upper abdominal pain, bloating, acid reflux, and belching on days 0, 14, and 28); Fecal microbiota metagenomics (16S rRNA sequencing analysis of gut microbiota α and β diversity on days 0 and 28). Other adverse event records are expressed as a percentage of abnormal events. The higher the percentage, the more abnormal records there are. For example, 1% means that 1 out of 100 subjects reported an adverse event.

[0084] Application Comparative Example 5: The oral liquid in Application Example 3 was replaced with an equal amount of placebo with the same appearance and taste, while the rest of the experimental protocol remained exactly the same.

[0085] Application Comparative Example 6: The oral liquid in Application Example 3 was replaced with commercially available compound probiotic powder (Kang En Bei Probiotics), one sachet each morning and evening, dissolved in warm water, and all other test indicators were exactly the same.

[0086] Application Comparative Example 7: The oral solution in Application Example 3 was replaced with a bismuth quadruple eradication regimen: esomeprazole 20mg bid combined with bismuth potassium citrate 220mg bid combined with amoxicillin 1.0g bid combined with clarithromycin 500mg bid, for a course of 14 days.

[0087] The test results of Application Example 3 and Application Comparative Examples 5-7 are shown in Table 5.

[0088] ; Table 5. Test results of Application Example 3 and Comparative Examples 5-7 Although the Hp eradication rate (42.5%) of the oral solution composition of this invention was lower than that of the standard quadruple antibiotic regimen (85.0%) in the adjunctive treatment of Hp-positive individuals, the proposed regimen has the following unique advantages: extremely low adverse reaction rate (2.5% vs 38.5%), with no common antibiotic side effects such as taste abnormalities and gut microbiota dysbiosis; GSRS gastrointestinal symptoms improved by 72.5%, superior to the quadruple regimen (65.2%), indicating that the oral solution composition is effective in relieving upper gastrointestinal discomfort symptoms; the Shannon index of gut microbiota increased by 0.85, while the quadruple regimen led to a sharp decrease in gut microbiota α diversity by 1.25, indicating that the composition composition of this invention can positively nourish the gut microbiota while conditioning the stomach; the increase in pepsinogen PGR and the decrease in gastrin-17 indicate that the gastric mucosal function has been improved. The oral solution composition of this invention can be an ideal choice for antibiotic-intolerant, low DOB Hp-positive individuals, or those maintaining and consolidating after eradication therapy.

[0089] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

[0090] It should be noted that the product models and manufacturer specifications mentioned in the above specific embodiments of this specification are only used to illustrate the specific implementation process of the present invention and to help understand the technical solution, and are not intended to limit the scope of protection of the present invention. Those skilled in the art can use other models of products with the same functional type to replace the implementation based on the technical concept disclosed in this invention, without departing from the scope of protection of the present invention.

Claims

1. A plant protein stock solution fermentation composition, characterized in that, It is prepared by fermentation from the following raw materials in parts by weight: Plant protein stock solution: 85-90 parts; Carbohydrates: 6-8 servings; Superoxide dismutase: 0.2~0.3 parts; Compound probiotic flora: 0.2~3.0 parts; The compound probiotic flora includes one or more of Lactobacillus plantarum, Lactobacillus casei, or functional yeasts; The sugars include one or both of brown sugar and glucose.

2. The composition according to claim 1, characterized in that, The compound probiotic flora consists of Lactobacillus plantarum, Lactobacillus casei, and functional yeast, and the mass ratio of Lactobacillus plantarum, Lactobacillus casei, and functional yeast is (2~3):(1~2):(1~2).

3. The composition according to claim 1, characterized in that, The compound probiotic flora consists of Lactobacillus plantarum, Lactobacillus casei, and functional yeast, with a mass ratio of 3:1:

1.

4. The composition according to claim 1, characterized in that, The sugars are brown sugar and glucose, and the mass ratio of brown sugar to sucrose is (1~2):(1~2).

5. The method for preparing the plant protein stock solution fermentation composition according to any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Extract the plant protein stock solution from pasture grass; S2. Under sterile conditions, the plant protein stock solution is mixed with the sugars to obtain a fermentation base solution; S3. Place the fermentation base liquid in a closed anaerobic environment at a temperature of 22~32℃, inoculate with a compound probiotic group, and carry out constant temperature fermentation for 48h~72h to obtain the primary fermentation liquid; S4. Under conditions of 22℃~32℃, superoxide dismutase is added to the primary fermentation broth and stirred to obtain secondary fermentation broth; S5. The secondary fermentation broth is subjected to static stabilization treatment, and then centrifuged to obtain the plant protein stock solution fermentation composition.

6. The application of the plant protein stock solution fermentation composition according to any one of claims 1 to 4, characterized in that, The plant protein stock solution fermentation composition is used for internal health care of terrestrial or aquatic animals.

7. The application of the plant protein stock solution fermentation composition according to claim 6, characterized in that, The terrestrial animal in question is a piglet.

8. The application of the plant protein stock solution fermentation composition according to claim 6, characterized in that, The aquatic animal in question is the whiteleg shrimp.

9. The application of the plant protein stock solution fermentation composition according to any one of claims 1 to 4, characterized in that, The plant protein stock solution fermentation composition is used in human oral health products.

10. The application of the plant protein stock solution fermentation composition according to claim 9, characterized in that, The oral health supplement is used to regulate the gastrointestinal tract.