A method for preparing a compound post-biotic to improve the stress resistance of aquatic animals and its application in feed.

Abstract

This invention belongs to the field of aquaculture technology and discloses a method for preparing a compound metabiotic to improve the stress resistance of aquatic animals and its application in feed. The invention discloses a method for preparing a compound metabiotic that employs a spatiotemporal sequential fermentation strategy of "first propagation, then induction, and finally compensation": First, Bacillus subtilis aerobic fermentation degrades the substrate to produce specific signal peptides; then, Lactobacillus plantarum and Saccharomyces cerevisiae are introduced, utilizing the previous metabolites to induce Lactobacillus plantarum to efficiently synthesize antimicrobial peptides and organic acids, and combined with the metabolic compensation effect of Saccharomyces cerevisiae, significantly enhancing the functional component gradient of the metabiotic. The compound metabiotic prepared by this invention has high concentrations of organic acids, active enzyme systems, and high release rates of β-glucan, which can significantly improve the stress resistance and survival rate of aquatic animals such as shrimp under extreme environments such as low salinity, high ammonia nitrogen, and low oxygen, solving the industry pain point of probiotic activity loss during high-temperature feed pelleting.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically relating to a method for preparing a compound post-biotic to improve the stress resistance of aquatic animals and its application in feed. Background Technology

[0002] In recent years, with the increasing level of intensive aquaculture, various stress factors such as high density, low dissolved oxygen, temperature and salinity fluctuations, transportation, and transfer between ponds often occur in the aquaculture environment, which can easily lead to a decline in the growth performance, impaired immune function, and increased mortality of farmed animals (such as whiteleg shrimp, tilapia, and sea bass).

[0003] Probiotics and their metabolites are widely used as biological agents in traditional disease management, improving production efficiency, animal health, stress resistance, and survival rates. However, limitations exist in their use, including decreased probiotic viability and the need for low-temperature storage, leading to unstable effects and transportation difficulties. In aquatic animal pellet feed production, pelleting temperatures must be maintained above 80°C, at which many probiotics are killed, reducing their effectiveness. These limitations have spurred research into alternative probiotic formulations. Further research has revealed that specific inactive bacterial byproducts and cellular components contribute to probiotic-like physiological effects; these substances are designated as "metasitrophs."

[0004] Epibiotics, as a novel type of inactive probiotic feed additive, can improve the quality and efficiency of aquaculture. Epibiotics are derived from aquatic probiotic microorganisms and consist of a complex mixture of inactivated probiotic cells, bacterial cell components, and microbial metabolites, such as organic acids, peptidoglycans, polysaccharides, and lipids. Compared to live probiotics, epibiotics offer enhanced safety, a defined chemical structure, higher stability, a longer shelf life, simpler storage and transportation, and resistance to digestive enzyme activity. The active ingredients, such as metabolites, present in epibiotics can improve the growth performance of aquatic animals, reduce stress responses, enhance immune responses, improve gut health, and enhance disease resistance. Furthermore, the structure of epibiotics (fermentation metabolites) is not destroyed during the pelleting process of aquatic animal feed. However, the effect of a single epibiotic on regulating the immune response pathways of farmed aquatic animals is relatively limited. Additionally, aquatic animals are poikilothermic invertebrates and are extremely sensitive to environmental fluctuations. In current technologies, single epibiotics often only improve digestion and are insufficient to address extreme conditions with multiple stressors (such as hypoxia + high ammonia nitrogen). This invention generates a group of metabolites with a "cross-protective effect" through a multi-microbial symbiotic system.

[0005] The preparation process of compound postbiotics has several limitations: 1. Optimizing probiotic culture conditions to obtain high-quality probiotic cultures; 2. Optimizing the preparation process of postbiotics to obtain high-quality postbiotics; 3. Optimizing production scale, shifting from laboratory-scale to large-scale industrial production; 4. Optimizing the diversity and effectiveness of postbiotic composition. These limitations, to some extent, restrict the widespread application and development of probiotic postbiotics. Therefore, it is urgent to develop a novel compound postbiotic preparation process to solve the above problems, which will help improve breeding efficiency, reduce disease incidence, and promote the sustainable growth of aquaculture. Summary of the Invention

[0006] This invention provides a compound post-biotic preparation for aquaculture, its preparation process, and its application. Addressing the problems of intense dissolved oxygen competition, mutual inhibition of metabolites (such as premature lactic acid accumulation inhibiting non-acid-tolerant bacteria), and limited functional components caused by simultaneous fermentation of multiple microbial strains in existing technologies, this technical solution constructs an "in-situ induction and metabolic compensation" system based on a spatiotemporal sequence fermentation strategy. The core logic of this technical solution lies in achieving a qualitative change from "interspecific competition" to "metabolic synergy" through dynamic control of the transition from aerobic to microaerobic conditions. In the initial propagation and in-situ induction stage: Firstly, Bacillus subtilis is induced into the logarithmic growth phase through a high dissolved oxygen environment. Its highly expressed neutral protease precisely degrades large molecular weight proteins in the substrate into bioactive oligopeptides of specific molecular weights. These oligopeptides not only serve as a high-quality nitrogen source for subsequent strains but also act as key signaling molecules, pre-triggering the regulation of metabolic pathways in subsequent inoculated strains. Multi-microbial symbiosis and metabolic compensation stage: Introducing *Lactobacillus plantarum* and *Saccharomyces cerevisiae* into a microaerobic environment, the metabolic components produced in the early stages "reprogrammed" the synthetic metabolism of *Lactobacillus plantarum*, inducing higher titers of antimicrobial peptides and organic acids. Simultaneously, the endogenous nutrients such as B vitamins released by *Saccharomyces cerevisiae* in the later stages of fermentation provide physiological compensation to the lactobacillus under low pH pressure, maintaining the continuous output of metabolic products. This "chemical plant-style" continuous biotransformation achieves the scientific compounding and synergistic effect of multiple active ingredients. The specific metabolic profile produced (including high concentrations of total organic acids, active enzyme systems, and high-release rates of β-glucan, etc.) cannot be achieved by simple physical mixing processes. Experiments have shown that this model increases the total organic acid content by more than 60%, significantly enhancing the tolerance of active ingredients to high-temperature pelleting of feed, thereby endowing aquaculture animals with stronger immune defenses and antioxidant stress resistance.

[0007] The first objective of this invention is to provide a method for preparing a composite postbiotic.

[0008] The second objective of this invention is to provide a composite post-genetic agent.

[0009] The third aspect of this invention aims to provide the application of the composite post-biotic of the second aspect of this invention in product preparation or animal husbandry.

[0010] The fourth aspect of this invention is to provide a product.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing a composite postbiotic, comprising the following steps: Bacillus subtilis was inoculated into the substrate solution and aerobic fermentation was carried out under dissolved oxygen conditions of ≥30% to obtain fermentation broth 1 containing active small peptides; Inoculate Lactobacillus plantarum and Saccharomyces cerevisiae into fermentation broth 1, adjust dissolved oxygen to 1%-15% for micro-anaerobic fermentation; obtain a compound fermentation broth; wherein, the product generated in step (1); The compound fermentation broth was inactivated to obtain the compound post-biotic.

[0012] In the aerobic process, neutral proteases produced by Bacillus subtilis are used to degrade the substrate in situ, yielding fermentation broth 1 containing bioactive peptides. During the microanaerobic fermentation, these bioactive peptides act as signaling molecules to induce *Lactobacillus plantarum* to synthesize antimicrobial peptides. Furthermore, *Saccharomyces cerevisiae* releases B vitamins in the later stages of fermentation, providing metabolic compensation for *Lactobacillus plantarum* operating under low pH conditions.

[0013] In some embodiments of the present invention, after aerobic fermentation to the late logarithmic growth phase of Bacillus subtilis, Lactobacillus plantarum and Saccharomyces cerevisiae are inoculated into the fermentation broth 1.

[0014] In some embodiments of the present invention, the dissolved oxygen content of the fermentation system during microanaerobic fermentation is 1%-15%, such as any value or a range formed by any combination of 1%, 2%, 5%, 7%, 9%, 10%, 12%, 14%, or 15%. The dissolved oxygen content of the fermentation system can be reduced by methods such as reducing aeration, stopping aeration, or intermittent aeration.

[0015] In some embodiments of the present invention, the aerobic fermentation time is 10-20 h, more specifically 12-18 h, such as any value of 12, 13, 14, 15, 16, 17 or 18 h or a range formed by any two of them.

[0016] In some embodiments of the present invention, the microanaerobic fermentation time is 30-55 h; further, it is 36-48 h, such as any value or a range formed by any two of 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 56, 47 or 48 h.

[0017] In some embodiments of the present invention, the inoculation ratio of Bacillus subtilis, Lactobacillus plantarum and Saccharomyces cerevisiae is (2-3):(2-4):1 (calculated in CFU).

[0018] In some embodiments of the present invention, the fermentation temperature of the aerobic fermentation and the micro-anaerobic fermentation is 30-38°C; further, it is 30-35°C, such as any value of 30, 31, 32, 33, 34 or 35°C or a range formed by any two of them.

[0019] In some embodiments of the present invention, the fermentation speed of the aerobic fermentation and micro-anaerobic fermentation is 200-600 rpm, such as any value of 200, 250, 300, 350, 400, 450, 500, 550 or 600 rpm or a range formed by any two of them.

[0020] In some embodiments of the present invention, the pH value of the aerobic fermentation system is controlled at 6.0-6.5.

[0021] In some embodiments of the present invention, the pH value of the aerobic fermentation system is controlled at 4.0-6.5; more specifically, at 4.5-5.8.

[0022] In some embodiments of the present invention, the inactivation conditions are: 70-85°C for 10-40 min; further, 78-84°C for 20-35 min; and even further, 80-82°C for 20-30 min. This temperature gradient is precisely at the critical point between moderate protein denaturation and protection of active small peptides, and can induce animal immunity through residual heat shock proteins.

[0023] In some embodiments of the present invention, before inactivation, the compound fermentation broth is subjected to solid-liquid separation (e.g., centrifugation at 3000-5000 rpm for 10-30 min), the liquid is collected, and inactivation is performed.

[0024] Heat treatment (inactivation) activates the denaturation of certain heat-stable small peptides and heat-sensitive antigens, thereby inducing a stronger immune response. Simultaneously, the inactivation process makes postbiotic products unrestricted by storage conditions, facilitating preservation and transportation.

[0025] In some embodiments of the present invention, the Bacillus subtilis (1×10⁻⁶) 6 -9×10 7 The inoculation amount (CFU / mL) is 1%-5% (v / v), such as any value of 1%, 2%, 3%, 4% or 5% or any range formed by both.

[0026] In some embodiments of the present invention, the *Lactobacillus plantarum* (1×10⁻⁶) 6 -9×107 The inoculation amount (CFU / mL) is 1%-5% (v / v), such as any value of 1%, 2%, 3%, 4% or 5% or any range formed by both.

[0027] In some embodiments of the present invention, the brewing yeast (1×10⁻⁶) 6 -1×10 7 The inoculation amount (CFU / mL) is 1%-5% (v / v), such as any value of 1%, 2%, 3%, 4% or 5% or any range formed by both.

[0028] In some embodiments of the present invention, the Bacillus subtilis is activated before inoculation, and the activation process is as follows: Bacillus subtilis strain is inoculated into MR liquid medium and cultured at 35-39°C for 20-50 hours.

[0029] In some embodiments of the present invention, the Bacillus subtilis is activated before inoculation. The activation process is as follows: Lactobacillus plantarum is inoculated into MRS liquid culture medium and cultured at 35-39°C for 20-50 hours.

[0030] In some embodiments of the present invention, the yeast is activated before inoculation, and the activation process is as follows: Lactobacillus plantarum is inoculated into YPD liquid medium and cultured at 35-39°C for 20-50 hours.

[0031] In some embodiments of the present invention, the substrate solution includes a carbon source, a protein source, vitamins, inorganic salts, and trace elements.

[0032] In some embodiments of the present invention, the carbon source includes at least one selected from glucose, fructose, sucrose, lactose, maltose, starch, and cellulose.

[0033] In some embodiments of the present invention, the protein source includes at least one of fish meal, meat and bone meal, plasma protein powder, egg powder, whey protein, soybean meal, rapeseed meal, peanut meal, cottonseed meal, corn gluten powder, and yeast powder.

[0034] In some embodiments of the present invention, the vitamins include at least one of vitamin B (such as B1, B2, B3, B5, B6, etc.), vitamin A, vitamin D, vitamin E, vitamin K, and vitamin C.

[0035] In some embodiments of the present invention, KH2PO4, (NH4)2SO4 and MgSO4 are used.

[0036] In some embodiments of the present invention, the trace elements include at least one of Fe, Mn, Zn, Cu and Se.

[0037] In some embodiments of the present invention, the substrate solution further includes a pH adjuster, such as CaCO3.

[0038] In some embodiments of the present invention, the concentration of the carbon source in the substrate solution is 10-40 g / L; more specifically, 20-30 g / L, such as any value or a range formed by any two of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 g / L.

[0039] In some embodiments of the present invention, the concentration of the protein source in the substrate solution is 20-60 g / L; further, it is 30-50 g / L, any value of 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50 g / L or a range formed by any two of them.

[0040] In some embodiments of the present invention, the concentration of inorganic salt in the substrate solution is 1-5 g / L; more specifically, 1-3 g / L, such as any value of 1, 1.5, 2, 2.5 or 3 g / L or a range formed by any two of them.

[0041] In some embodiments of the present invention, the concentration of the pH adjuster in the substrate solution is 4-15 g / L; more specifically, 5-10 g / L, such as any value of 5, 6, 7, 8, 9 or 10 g / L or a range formed by any two of them.

[0042] In some embodiments of the present invention, the pH value of the substrate solution is 6.0-6.5.

[0043] This invention relates to a method for preparing a complex postbiotic to enhance stress resistance. The method utilizes a combination of Bacillus subtilis strains, Lactobacillus plantarum strains, and Saccharomyces cerevisiae strains to form a special complex postbiotic. This complex postbiotic exhibits excellent proliferative effects on beneficial bacteria in the intestines of various aquatic animals and strong inhibitory effects on pathogenic bacteria in the intestines of various aquatic animals, effectively improving the composition and structure of the intestinal flora of aquatic animals. Furthermore, after large-scale preparation, the product has shown a significant effect in enhancing the stress resistance of aquatic animals in aquaculture trials.

[0044] When using the same dosage of compound metabiotics, compared with compounding metabiotics obtained by fermenting single Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae strains, the preparation method provided by this invention can significantly increase the content of beneficial metabiotics (short-chain fatty acids, low-molecular-weight peptides, enzymes, intracellular / extracellular components, etc.) in the compound metabiotics. This suggests that there is a potential interaction between Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae strains, which can cooperate to exert a synergistic effect in promoting the balance of intestinal microecology in aquatic animals. In a second aspect, the present invention provides a composite post-genetic agent prepared by the method of the first aspect of the present invention.

[0045] In some embodiments of the present invention, the total organic acid content in the composite post-biotic is 8-10 g / L; more specifically, it is 9-9.5 g / L.

[0046] In some embodiments of the present invention, the lactic acid content in the composite postbiotic is 4-8 g / L; more specifically, it is 5-6 g / L.

[0047] In some embodiments of the present invention, the total content of acetic acid and propionic acid in the composite post-generic is 1.5-3 g / L; more specifically, it is 2-3 g / L.

[0048] In some embodiments of the present invention, the content of protease in the composite postbiotic is 1800-2300 U / mL; more specifically, it is 2000-2100 U / mL.

[0049] In some embodiments of the present invention, the content of amylase in the composite post-biotic is 2400-2600 U / mL; more specifically, it is 2400-2500 U / mL.

[0050] In some embodiments of the present invention, the content of lipase in the composite postbiotic is 400-450 U / mL; more specifically, it is 410-430 U / mL.

[0051] In some embodiments of the present invention, the content of cellulase in the composite post-biotic is 150-180 U / mL; more specifically, it is 160-170 U / mL.

[0052] The digestive enzymes and other bioactive substances in the compound metabiotics of this invention can promote the digestion and absorption of nutrients by aquatic animals, improve feed utilization, thereby promoting the growth of aquatic animals, shortening the breeding cycle, and increasing breeding output.

[0053] In some embodiments of the present invention, the content of antimicrobial peptide activity in the composite postbiotic is 1000-1200 AU / mL; more specifically, it is 1050-1100 AU / mL.

[0054] In some embodiments of the present invention, the content of B vitamins in the compound postbiotic is 40-50 mg / L; more specifically, it is 40-45 mg / L.

[0055] In some embodiments of the present invention, the β-glucan release rate in the composite postbiotic is 60%-70%; more specifically, it is 60%-70%.

[0056] In some embodiments of the present invention, the content of mannan oligosaccharides in the composite post-biotic is 3-5 g / L; more specifically, it is 3.5-4 g / L.

[0057] A third aspect of the present invention provides the application of the composite post-biotic of the second aspect of the present invention in the preparation of products or in animal husbandry.

[0058] In some embodiments of the present invention, the product has the following functions: improving the animal's stress resistance (such as oxidative stress, low salt stress, ammonia nitrogen stress, dew stress, hypoxia stress, etc.), regulating the animal's intestinal flora, and / or improving the animal's growth performance (improving the animal's weight gain rate, body length gain rate, and survival rate, etc.).

[0059] In some embodiments of the present invention, the product includes at least one of feed, feed additives, and immunomodulators.

[0060] In some embodiments of the present invention, the animals include aquatic animals, such as fish and shrimp.

[0061] A fourth aspect of the present invention provides a product comprising the composite post-genetic agent of the second aspect of the present invention.

[0062] In some embodiments of the present invention, the product has the following functions: improving the animal's stress resistance (such as oxidative stress, low salt stress, ammonia nitrogen stress, dew stress, hypoxia stress, etc.), regulating the animal's intestinal flora, and / or improving the animal's growth performance (improving the animal's weight gain rate, body length gain rate, and survival rate, etc.).

[0063] In some embodiments of the present invention, the product has the following functions: improving the stress resistance of animals, regulating the intestinal flora of animals, and / or improving the growth performance of animals.

[0064] In some embodiments of the present invention, the animals include aquatic animals, such as fish and shrimp.

[0065] In some embodiments of the present invention, the product is feed.

[0066] In some embodiments of the present invention, the feed further includes a basic feed.

[0067] In some embodiments of the present invention, the basic feed includes fish meal, soybean meal, peanut meal, flour, corn gluten meal, fish oil, phospholipid oil, and shrimp premix.

[0068] In some embodiments of the present invention, the feed comprises, by weight, 18-25 parts fish meal, 18-25 parts soybean meal, 10-20 parts peanut meal, 15-25 parts wheat flour, 5-15 parts corn gluten meal, 1-5 parts fish oil, 1-5 parts phospholipid oil, 1-5 parts compound postbiotic, 1-4 parts shrimp premix, and 0.1-0.5 parts antioxidant.

[0069] In some embodiments of the present invention, the feed comprises, by weight, 20-23 parts fish meal, 21-24 parts soybean meal, 14-16 parts peanut meal, 20-23 parts wheat flour, 8-12 parts corn gluten meal, 1-3 parts fish oil, 1-3 parts phospholipid oil, 1-3 parts compound postbiotics, 1-4 parts shrimp premix, and 0.1-0.5 parts antioxidant.

[0070] In some embodiments of the present invention, the shrimp premix includes vitamin A ≥ 9000 IU / kg, vitamin D3 ≥ 1900-360000 IU / kg, vitamin E ≥ 62 mg / kg, vitamin K3 ≥ 7 mg / kg, vitamin B1 ≥ 8.5 mg / kg, vitamin B2 ≥ 8.5 mg / kg, D-calcium pantothenate ≥ 41 mg / kg, nicotinamide ≥ 61 mg / kg, manganese 41-420 mg / kg, zinc 70-600 mg / kg, copper 37-310 mg / kg, and iron 415-4000 mg / kg.

[0071] In some embodiments of the present invention, the antioxidant includes BHT.

[0072] In some embodiments of the present invention, the feed is prepared by the following method: mixing fish meal, soybean meal, peanut meal, corn gluten meal, fish oil, phospholipid oil, shrimp premix and compound post-biotic, adjusting the moisture content of the materials to 10%-20%, granulating, and drying to obtain the feed.

[0073] In some embodiments of the present invention, the granulation temperature is 85-95°C.

[0074] In the feed, the main active ingredients of the compound postbiotic are preserved through micro Maillard reaction at a pelleting temperature of 90℃, which solves the industry pain point that "probiotics are not heat-resistant in feed".

[0075] The beneficial effects of this invention are: This invention provides a novel method for preparing a composite metabiotic, employing a spatiotemporal fermentation strategy of "first propagation, then induction, and finally compensation." First, *Bacillus subtilis* aerobic fermentation degrades the substrate to produce specific signal peptides. Then, *Lactobacillus plantarum* and *Saccharomyces cerevisiae* are inoculated, utilizing the metabolites from the initial fermentation to induce *Lactobacillus plantarum* to efficiently synthesize antimicrobial peptides and organic acids. Combined with the metabolic compensation effect of *Saccharomyces cerevisiae*, the functional component gradient of the metabiotic is significantly enhanced. The composite metabiotic prepared by this invention possesses high concentrations of organic acids, active enzyme systems, and high-release-rate β-glucan, significantly improving the stress resistance and survival rate of shrimp and other aquatic animals under extreme environments such as low salinity, high ammonia nitrogen, and low oxygen. This addresses the industry pain point of probiotic activity loss during high-temperature feed pelleting.

[0076] The composite metabiotic provided by this invention can enhance the stress resistance of aquatic animals and improve their intestinal microecological balance. This composite metabiotic has a good proliferative effect on various beneficial bacteria in the aquatic animal gut and a strong inhibitory effect on various pathogenic bacteria. It can precisely regulate and restore a healthy microbial community structure. Moreover, the functional feed products further prepared using it have shown a significant effect on improving the intestinal microecological balance in aquaculture trials, meeting the need for stress mitigation in aquaculture and providing a long-term, natural microecological management strategy for aquaculture and water quality conditions.

[0077] When using the same dosage, compared with a single Bacillus subtilis strain, a single Lactobacillus plantarum strain, or a single Saccharomyces cerevisiae strain, the metabiotic obtained by inactivation after mixed fermentation of the three strains showed a significant improvement in the above-mentioned efficacy. They can work together to achieve synergistic effects in promoting the balance of intestinal microecology in aquatic animals.

[0078] When fermented alone, *Lactobacillus plantarum* only produces conventional lactic acid. However, in the preparation method provided by this invention, during fermentation, the neutral protease produced by *Bacillus subtilis* consuming oxygen first degrades the substrate into specific oligopeptides. These oligopeptides act as signaling molecules, inducing *Lactobacillus plantarum* to synthesize higher concentrations of antimicrobial peptides and organic acids. During fermentation, the B vitamins released by *Saccharomyces cerevisiae* in the later stages of fermentation compensate for the metabolic losses of *Lactobacillus plantarum* under low pH conditions, enabling it to maintain a high level of metabolic product output even under extreme environments (such as pH 3.9).

[0079] The compound postbiotic provided by this invention contains inactivated Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae strains, which will not multiply in the body during use, making it suitable for long-term use and unlikely to cause infection risks. Furthermore, this compound postbiotic exhibits high stability; the inactivation treatment eliminates storage limitations, facilitating preservation and transportation. When added to feed, it retains a high rate of its main active ingredients. Detailed Implementation The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0080] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0081] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0082] Example 1 A method for preparing a composite post-biotic to enhance stress resistance includes the following steps: (1) The strain of Bacillus subtilis (preservation number CCTCC AB 130168, its classification name is Bacillus subtilis) was identified. Bacillus subtilis The strain (CCTCC DB 20081542, classified as *Lactobacillus plantarum*) was inoculated into MR liquid medium on July 23, 2013. Lactobacillus plantarum The *Saccharomyces cerevisiae* strain (preservation number CCTCC KY 2008635, taxonomically named *Saccharomyces cerevisiae*, preservation date June 24, 2006) was inoculated into MRS liquid medium. The *Saccharomyces cerevisiae* strain was inoculated into YPD liquid medium and cultured at 37°C for 24 hours for activation. This activation was repeated twice to obtain the activated solution, which was activated to 1×10⁻⁶ *Bacillus subtilis*. 7 -1×10 8 CFU / mL, Lactobacillus plantarum 1×10 7 -1×10 8 CFU / mL; Saccharomyces cerevisiae 1×10 6 -1×10 7 CFU / mL.

[0083] (2) Prepare substrate according to a volume of 3L: 3L of sterile water / deionized water; 75g of carbon source (glucose or sucrose); 130g of protein source (soybean meal hydrolysate or fish meal hydrolysate); 6g, 2g, and 1g of inorganic salts (KH2PO4, (NH4)2SO4, MgSO2·7H2O); 3mL of vitamins and trace elements (compound trace element solution, Shanghai Lianmai Biotechnology Co., Ltd., catalog number SL-6); and 20g of pH adjuster (CaCO3).

[0084] (3) Fermentation: Phase 1 (Aerobic phase, dissolved oxygen maintained at ≥30%): Bacillus subtilis is introduced at a concentration of 1×10⁻⁶. 7 The CFU / mL concentration was inoculated into the substrate of step (2) at an inoculation rate of 3% (v / v), and fermented for 16 h. The fermentation temperature was 34±2℃, the initial pH was 6.3, and the mixture was stirred at 450 rpm to ensure material mixing and oxygen mass transfer during the aerobic stage. Phase 2 (microanaerobic phase, dissolved oxygen decreases to 5%-15% or continuous ventilation is stopped): Add *Lactobacillus plantarum* (1×10⁻⁶) at the end of Phase 1. 7 CFU / mL) and Saccharomyces cerevisiae (1×10⁻⁶ CFU / mL) 6 The inoculum concentration (CFU / mL) was 3% (v / v). Fermentation was carried out for 48 h at a temperature of 34±2℃, with an initial pH of 6.5 (the pH is allowed to drop to 5.2 at this stage due to lactic acid production; CaCO3 buffering can prevent the pH from dropping too quickly), and stirring at 400 rpm.

[0085] During the fermentation process described above, the pH, temperature, dissolved oxygen (DO), and colony count (qPCR or OD) of the fermentation system were monitored every 12 hours. 600 ), residual sugar, short-chain fatty acids, etc.

[0086] (4) Centrifuge the cultured compound fermentation broth at 4000 r / min for 15 min and collect the supernatant. The supernatant contains organic acids (such as lactic acid and acetic acid), bacteriocins, vitamins, enzymes and other bioactive substances produced by the three probiotics. Then inactivate the cells at 80℃ for 20-30 min, inactivating the cells and retaining the metabolic products to form a compound postbiotic.

[0087] The colony counts and metabolite contents of each bacterium in the compound fermentation broth were detected and counted using a spectrophotometer. The results are shown in Tables 1 and 2. Using the method of this embodiment for segmented compound fermentation, the total viable cell count can reach 5.0 × 10⁻⁶. 9CFU / L; total organic acids up to 9.2 g / L; protease, amylase, lipase and cellulase contents of 2,050 U / mL, 2,480 U / mL, 420 U / mL and 165 U / mL respectively; antimicrobial peptide activity up to 1,050 AU / mL; β-glucan release rate up to 68%; mannan oligosaccharide content up to 3.6 g / L.

[0088] Table 1. Results of viable cell count in the compound bacterial solution after fermentation

[0089] Table 2 Results of metabolite detection

[0090] Example 2 A method for preparing a composite postbiotic includes the following steps: (1) The strain of Bacillus subtilis (preservation number CCTCC AB 130168, its classification name is Bacillus subtilis) was identified. Bacillus subtilis The strain (CCTCC DB 20081542, classified as *Lactobacillus plantarum*) was inoculated into MR liquid medium on July 23, 2013. Lactobacillus plantarum The *Saccharomyces cerevisiae* strain (preservation number CCTCC KY 2008635, taxonomically named *Saccharomyces cerevisiae*, preservation date June 24, 2006) was inoculated into MRS liquid medium. The *Saccharomyces cerevisiae* strain was inoculated into YPD liquid medium and cultured at 37°C for 24 hours for activation. This activation was repeated twice to obtain the activated solution, which was activated to 1×10⁻⁶ *Bacillus subtilis*. 7 -1×10 8 CFU / mL, Lactobacillus plantarum 1×10 7 -1×10 8 CFU / mL; Saccharomyces cerevisiae 1×10 6 -1×10 7 CFU / mL.

[0091] (2) Prepare substrate according to a volume of 3L: 3L of sterile water / deionized water; 75g of carbon source (glucose or sucrose); 130g of protein source (soybean meal hydrolysate or fish meal hydrolysate); 6g, 2g, and 1g of inorganic salts (KH2PO4, (NH4)2SO4, MgSO2·7H2O); 3mL of vitamins and trace elements (compound trace element solution, Shanghai Lianmai Biotechnology Co., Ltd., catalog number SL-6); and 20g of pH adjuster (CaCO3).

[0092] (3) Fermentation: The bacterial culture (Bacillus subtilis strain, Lactobacillus plantarum strain and Saccharomyces cerevisiae strain in an inoculation ratio of 2:3:1 (calculated based on CFU)) was inoculated into the substrate in step (2) at an inoculation rate of 3% (v / v). Fermentation was carried out for 36-48 h at a fermentation temperature of 34±2℃, an initial pH of 6.0-6.5, and stirring at 400 rpm to ensure material mixing and oxygen mass transfer during the aerobic stage. Dissolved oxygen was controlled at 15-20%.

[0093] During the fermentation process described above, the pH, temperature, dissolved oxygen (DO), and colony count (qPCR or OD) of the fermentation system were monitored every 6-12 hours. 600 ), residual sugar, short-chain fatty acids, etc.

[0094] (4) Centrifuge the cultured compound fermentation broth at 4000 r / min for 15 min and collect the supernatant. The supernatant contains organic acids (such as lactic acid and acetic acid), bacteriocins, vitamins, enzymes and other bioactive substances produced by the three probiotics. Then inactivate the cells at 80℃ for 20-30 min, inactivating the cells and retaining the metabolic products to form a compound postbiotic.

[0095] The colony counts and metabolite contents of each bacterium in the composite fermentation broth were detected and statistically analyzed using a spectrophotometer. The results are shown in Tables 1 and 2. The method used in this example was employed for mixed fermentation, while Example 2 used simultaneous fermentation. This resulted in oxidative stress in Bacillus subtilis and Saccharomyces cerevisiae under oxygen-limited conditions, and a lack of induction signals from early oligopeptides. Consequently, their core indicators (such as antimicrobial peptide activity and β-glucan release rate) were significantly lower than in Example 1. This demonstrates the necessity of spatiotemporal sequential fermentation for metabolic pathway reconstruction.

[0096] Example 3 A method for preparing a composite postbiotic includes the following steps: (1) The strain of Bacillus subtilis (preservation number CCTCC AB 130168, its classification name is Bacillus subtilis) was identified. Bacillus subtilis The strain (CCTCC DB 20081542, classified as *Lactobacillus plantarum*) was inoculated into MR liquid medium on July 23, 2013. Lactobacillus plantarum The *Saccharomyces cerevisiae* strain (preservation number CCTCC KY 2008635, taxonomically named *Saccharomyces cerevisiae*, preservation date June 24, 2006) was inoculated into MRS liquid medium. The *Saccharomyces cerevisiae* strain was inoculated into YPD liquid medium and cultured at 37°C for 24 hours for activation. This activation was repeated twice to obtain the activated solution, which was activated to 1×10⁻⁶ *Bacillus subtilis*. 7 -1×10 8CFU / mL, Lactobacillus plantarum 1×10 7 -1×10 8 CFU / mL; Saccharomyces cerevisiae 1×10 6 -1×10 7 CFU / mL.

[0097] (2) Prepare substrate according to a volume of 3L: 3L of sterile water / deionized water; 75g of carbon source (glucose or sucrose); 130g of protein source (soybean meal hydrolysate or fish meal hydrolysate); 6g, 2g, and 1g of inorganic salts (KH2PO4, (NH4)2SO4, MgSO2·7H2O); 3mL of vitamins and trace elements (compound trace element solution, Shanghai Lianmai Biotechnology Co., Ltd., catalog number SL-6); and 20g of pH adjuster (CaCO3).

[0098] (3) Fermentation: Bacillus subtilis, Lactobacillus plantarum and Saccharomyces cerevisiae were inoculated into the substrate of step (2) at an inoculation rate of 3% (v / v). Fermentation was carried out for 36-48 h at a fermentation temperature of 34±2℃ and an initial pH of 6.0-6.5. The mixture was stirred at 400 rpm to ensure material mixing and oxygen mass transfer during the aerobic stage, and dissolved oxygen was controlled at 15-20%.

[0099] During the fermentation process described above, the pH, temperature, dissolved oxygen (DO), and colony count (qPCR or OD) of the fermentation system were monitored every 6-12 hours. 600 ), residual sugar, short-chain fatty acids, etc.

[0100] (4) Centrifuge each cultured fermentation broth at 4000 r / min for 15 min and collect the supernatant. Mix the supernatants of the three broths and then inactivate them at 80℃ for 20-30 min to inactivate the cells and retain the metabolites, forming a complex post-biotic.

[0101] The colony counts and metabolite contents of each bacterium in the compound fermentation broth were detected and statistically analyzed. The results are shown in Tables 1 and 2.

[0102] Example 4 A shrimp feed containing a compound metabiotic, by weight, comprises 22 parts fish meal, 23 parts soybean meal, 15 parts peanut meal, 21 parts wheat flour, 10 parts corn gluten meal, 2 parts fish oil, 2 parts phospholipid oil, 2 parts compound metabiotic from Example 1, 3 parts shrimp premix (Qingyuan Branch of Guangdong Haid Group Co., Ltd., Q / GDHDQY 002-2025), and 0.1 parts antioxidant (BHT; Aladdin, item number: B431698).

[0103] The above-mentioned shrimp feed is prepared by the following steps: Weigh each ingredient by weight, crush the fish meal, soybean meal, peanut meal, and corn gluten powder, pass them through a 60-mesh sieve, and mix them evenly to obtain a mixture. The mixture is thoroughly mixed with fish oil, phospholipid oil, shrimp premix and other ingredients; compound post-biotics and appropriate amount of water are added to adjust the moisture content to 15%; then it is extruded through a twin-screw pellet mill (CD4-1TS extruder, Guangzhou Huagong Opto-Mechatronics Technology Co., Ltd.) to produce pellet feed with a particle size of 2mm. The prepared pelleted feed is dried at 60℃ until the moisture content is less than 10%, then crushed and sieved (20 mesh), and finally packaged to obtain shrimp feed.

[0104] Example 5 A shrimp feed containing a compound post-biotic is different from Example 4 only in that the compound post-biotic is the same as that in Example 3.

[0105] Example 6: Evaluation of the efficacy of compound post-biotics in shrimp feed 1. The effects of compound post-biotics on shrimp farming Prior to the feeding trial, shrimp underwent a four-week acclimatization period in temporary holding tanks. All shrimp were fed a commercially formulated feed (48% protein content, Shenzhen, China). After the temporary holding period, 1000 shrimp of similar size (0.64 ± 0.04 g) were randomly assigned to 10 culture cages, with a stocking density of 100 shrimp per cage. They were randomly divided into two groups: an experimental group (fed the shrimp feed of Example 4) and a control group (fed the shrimp feed of Example 5), with each group assigned to five replicate cages. Shrimp were fed four times daily (7:00, 12:00, 18:00, and 23:00) at a saturation rate of 6%–8% of their total body weight for eight weeks. The weight of all shrimp was measured weekly to determine the feeding amount. During the experiment, water quality was maintained at a temperature of 28 ± 2℃, a pH of 8.0 ± 0.3, dissolved oxygen ≥ 5.6 mg / L, and ammonia nitrogen ≤ 0.2 mg / L.

[0106] After the aquaculture trial, the shrimp in the experimental and control groups were comprehensively tested from multiple dimensions, including growth performance, immune capacity, gut health, and antioxidant capacity. The specific indicators are as follows: (1) Growth performance indicators Weight and body length: Accurately measure the weight and body length of each shrimp, and calculate the average weight, body length, weight growth rate, and body length growth rate. Compare the two sets of data to assess the impact of feed on shrimp growth rate.

[0107] Weight gain rate (%) = (Final weight - Initial weight) / Initial weight × 100%; Body length growth rate (%) = (final body length - initial body length) / initial body length × 100%.

[0108] Feed conversion ratio (FCR): The total feed intake and total weight gain of shrimp in each group are counted to calculate the feed conversion ratio. The formula is: FCR = feed intake / weight gain. The lower the feed conversion ratio, the higher the feed utilization rate.

[0109] Survival rate: Record the number of surviving shrimp in each group at the end of the experiment, calculate the survival rate, and reflect the impact of feed on the survival of shrimp.

[0110] (2) Digestive enzyme activity indicators Hepatopancreatic digestive enzyme activities: The activities of protease (catalog number A080-2-2), lipase (catalog number A054-1-1), and amylase (catalog number C016-1-1) were detected. These enzymes play a crucial role in the digestion and absorption of nutrients in shrimp, and their activity levels reflect the shrimp's ability to digest and absorb basic nutrients. Biochemical parameters were analyzed using commercial kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions.

[0111] (3) Content of serum biochemical components Serum biochemical content: The levels of total protein (TP; catalog number A045-2), triglycerides (TG; catalog number A110-1-1), glucose (GLU; catalog number A154-2-1), and cholesterol (TC; catalog number A111-1-1) in serum were measured. These biochemical components in shrimp serum are indicators of shrimp physiology and energy metabolism; their levels reflect changes in shrimp nutrition, health, and environmental conditions. Biochemical parameters were analyzed using commercial kits according to the manufacturer's instructions (Nanjing Jiancheng Biotechnology Institute, China).

[0112] (4) Immune capacity indicators Immune-related enzyme activities: The activities of immune enzymes such as acid phosphatase (ACP; catalog number A060-2-1), alkaline phosphatase (AKP; catalog number A059-2-2), and lysozyme (LZM; catalog number A050-1-1) in shrimp serum were detected. These enzymes play an important role in the immune defense of shrimp, and their activities directly reflect the shrimp's immune capacity. Biochemical parameters were analyzed using commercial kits according to the manufacturer's instructions (Nanjing Jiancheng Biotechnology Institute, China).

[0113] (5) Antioxidant capacity index Antioxidant enzyme activity: The activities of antioxidant enzymes such as superoxide dismutase (SOD; catalog number A001-3-2), catalase (CAT; catalog number A007-1-1), and glutathione peroxidase (GPx; catalog number A005-1-2) in the hepatopancreas or muscle tissue of shrimp were measured. These enzymes can scavenge free radicals in the body and protect cells from oxidative damage. Malondialdehyde (MDA; catalog number A003-1-2) content: The content of MDA in tissues was detected, and its level reflects the degree of oxidative damage to cells. Biochemical parameters were analyzed using commercial kits according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, China).

[0114] The experimental results are shown in Tables 3 and 4. The addition of the compound post-biotic feed from Example 1 (i.e., the shrimp feed from Example 4) significantly improved the growth performance, immunity, and antioxidant levels of Litopenaeus vannamei. During the 8-week rearing period, the average weight and body length of the shrimp in the experimental group were significantly higher than those in the control group. P The weight gain rate increased by approximately 12.7%, and the feed conversion ratio (FCR) decreased significantly, indicating that the compound metabiotic in Example 1 effectively promoted feed utilization efficiency and growth rate. The survival rate of shrimp in the experimental group was approximately 8.3% higher than that in the control group, showing that the compound metabiotic in Example 1 has a positive effect on improving health status and reducing aquaculture stress. From a physiological and biochemical perspective, the activities of protease, lipase, and amylase in the hepatopancreas of the experimental group were significantly increased, indicating that the compound metabiotic in Example 1 can improve the intestinal microecological environment and promote nutrient absorption and utilization. Serum total protein (TP) and glucose (GLU) levels increased, while triglyceride (TG) and cholesterol (TC) levels decreased slightly, reflecting a more balanced energy metabolism in shrimp. Regarding the activity of immune-related enzymes, the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM) in the experimental group were significantly increased, indicating that the compound metabiotic in Example 1 can enhance the innate immune defense function of shrimp. Regarding antioxidant capacity, the activities of antioxidant enzymes such as SOD, CAT, and GPx were significantly increased in the hepatopancreatic tissue of the experimental group, while the content of MDA was significantly decreased. P <0.05), indicating that the composite postbiotic of Example 1 can effectively reduce oxidative stress damage and enhance cell defense capabilities.

[0115] Table 3 Results of shrimp farming experiments

[0116] Table 4 Results of Biochemical Indicators

[0117] 2. Effects of compound post-biotics on stress resistance in shrimp The shrimp farming and treatment were the same as above (1. The effect of compound post-biotics on shrimp farming). After the farming was completed, acute salinity stress test, acute ammonia nitrogen stress test, acute dew stress test, and hypoxia stress tolerance test were conducted, as follows: (1) Acute salinity stress test After the shrimp farming in each group was completed, some shrimp were placed in a low-salt (3‰) environment for 6 hours. The survival rate was calculated by recording the number of shrimp that survived in each group after the experiment was completed and placed in the low-salt environment, which reflects the effect of compound post-biotic feed on the shrimp's resistance to low salt.

[0118] (2) After the shrimp farming in each group was completed, some shrimp were placed in a high ammonia nitrogen (10 mg / L) environment for 48 hours. The survival rate was calculated and the number of shrimp surviving in each group after the experiment was completed was recorded as time went on under the high ammonia nitrogen environment. This reflected the effect of compound post-biotic feed on the shrimp's resistance to ammonia nitrogen stress.

[0119] (3) Acute dry dew stress test After the shrimp farming in each group was completed, some shrimp were placed in a humid, out-of-water environment (humidity 80%). The survival rate was calculated by recording the number of shrimp surviving in each group in the humid, out-of-water environment over time after the experiment ended, reflecting the effect of compound post-biotic feed on the shrimp's resistance to desiccation stress.

[0120] (4) Hypoxia stress test After the shrimp farming in each group was completed, some shrimp were placed in a low-oxygen environment (dissolved oxygen <2mg / L) for 24 hours. The survival rate was calculated by recording the number of shrimp surviving in each group under low-oxygen conditions over time after the experiment ended, reflecting the effect of compound post-biotic feed on the shrimp's resistance to low-oxygen stress.

[0121] The results are shown in Tables 5-8. Under low-salt, high-ammonia-nitrogen, low-oxygen, and dry-dry conditions, the survival rates of shrimp in the experimental group were significantly higher than those in the control group. Specifically, the survival rate increased by >9% under acute low-salt stress, >14% under acute ammonia-nitrogen stress, and >15% under low-oxygen stress. The average tolerance time under dry-dry stress was also prolonged. In summary, the compound postbiotic in Example 1, through the synergistic effect of multiple bacterial metabolites, significantly improved the intestinal health and immune status of shrimp, enhanced their tolerance to environmental stress, and improved their overall growth performance, providing an efficient and safe functional feed additive solution for healthy shrimp farming.

[0122] Table 5 Results of the Acute Low-Salt Stress (6h) Test

[0123] Table 6 Results of the acute ammonia nitrogen stress test (48 h, 10 mg / L)

[0124] Table 7 Results of Acute Dry Dew Stress Test

[0125] Table 8 Results of hypoxia stress (DO < 2 mg / L, 24 h) test

[0126] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for preparing a composite postbiotic, comprising the following steps: Bacillus subtilis was inoculated into the substrate solution and aerobic fermentation was carried out under dissolved oxygen conditions of ≥30% to obtain fermentation broth 1 containing active small peptides; Inoculate Lactobacillus plantarum and Saccharomyces cerevisiae into fermentation broth 1, adjust the dissolved oxygen level to 1%-15% for micro-anaerobic fermentation; to obtain a compound fermentation broth; The compound fermentation broth was inactivated to obtain the compound post-biotic.

2. The preparation method according to claim 1, characterized in that, The aerobic fermentation time is 10-20 hours.

3. The preparation method according to claim 1, characterized in that, The micro-anaerobic fermentation time is 30-55 hours.

4. The preparation method according to any one of claims 1-3, characterized in that, The inoculation ratio of Bacillus subtilis, Lactobacillus plantarum and Saccharomyces cerevisiae is (2-3):(2-4):

1.

5. The preparation method according to any one of claims 1-3, characterized in that, The substrate solution includes a carbon source, a protein source, vitamins, inorganic salts, and trace elements; and / or, the pH of the substrate solution is 6.0-6.

5.

6. The preparation method according to any one of claims 1-3, characterized in that, The aerobic fermentation and micro-anaerobic fermentation are carried out at a temperature of 30-38℃ and a fermentation speed of 200-60 rpm; and / or the inactivation conditions are inactivation at 70-85℃ for 10-40 min.

7. A composite postgenetic agent, prepared by the method of any one of claims 1-6.

8. The composite post-genetic agent according to claim 7, characterized in that, The total organic acid content in the compound postbiotic is 8-10 g / L; and / or, the protease content in the compound postbiotic is 1800-2300 U / mL; and / or, the amylase content in the compound postbiotic is 2400-2600 U / mL; and / or, the lipase content in the compound postbiotic is 400-450 U / mL; and / or, the cellulase content in the compound postbiotic is 150-180 U / mL; and / or, the antimicrobial peptide activity in the compound postbiotic is 1000-1200 AU / mL; and / or, the β-glucan release rate in the compound postbiotic is 60%-70%; and / or, the mannan oligosaccharide content in the compound postbiotic is 3-5 g / L.

9. The application of the composite postbiotic as described in claim 7 or 8 in product preparation or animal husbandry; Preferably, the product has the following functions: improving the animal's stress resistance, regulating the animal's intestinal flora, and / or improving the animal's growth performance; Preferably, the product includes at least one of feed, feed additives, and immunomodulators.

10. A product comprising the composite post-genetic agent as described in claim 7 or 8.

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