A processing technology for enhancing the probiotic feed of antibodies

Abstract

This invention discloses a processing technology for antibody-enhancing probiotic feed, which includes raw material pretreatment, substrate preparation, sterilization, cooling, strain activation, aseptic inoculation, constant-temperature anaerobic fermentation, immune-enhancing compounding, filtration and homogenization, and aseptic filling and storage. The fermentation substrate liquid includes purified water, molasses, yeast extract, compound trace elements, and buffer salts. A compound microbial community composed of *Lactobacillus plantarum*, *Bacillus subtilis*, *Bifidobacterium*, and yeast is used for fermentation. The temperature is controlled at 35–39°C during fermentation, and intermittent stirring is employed. After fermentation, astragalus polysaccharide, echinacea extract, and trehalose are added for compounding. This invention reduces the loss of active ingredients by adding immune-enhancing components after fermentation, and improves the survival rate of probiotics and fermentation stability through intermittent stirring. The resulting liquid feed has a high live bacteria content and immune-enhancing effect.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation feed technology, and in particular to a processing technology for a feed containing antibody-enhancing beneficial bacteria. Background Technology

[0002] As the scale and intensification of livestock and poultry farming continue to increase, farmed animals are kept in high-density environments for extended periods. This makes them susceptible to factors such as increased stocking density, environmental changes, the spread of pathogens, and changes in feed nutrition. Consequently, the animals' immune function declines, the balance of the intestinal microecology is disrupted, and problems such as diarrhea, reduced growth rate, decreased feed utilization, and increased susceptibility to diseases occur.

[0003] In traditional animal husbandry, antibiotics are commonly used for disease prevention and control to reduce disease incidence and improve production performance. However, long-term use of antibiotics can easily lead to intestinal flora imbalance and promote the development of drug-resistant bacteria. Furthermore, antibiotic residues pose potential risks to food safety and the ecological environment. Therefore, developing safe, efficient, and environmentally friendly functional feed additives to improve the intestinal environment and enhance the immune system of animals has become an important development direction in the feed industry.

[0004] In recent years, the use of probiotic fermentation to prepare liquid feed has attracted widespread attention. During fermentation, probiotics can produce lactic acid, organic acids, antimicrobial peptides, and various metabolites, which can regulate the intestinal flora structure of animals, inhibit the growth of harmful microorganisms, and promote animal digestion and absorption. However, current processing techniques for probiotic liquid feed still have certain shortcomings.

[0005] On the one hand, existing liquid probiotic feeds typically employ continuous stirring during fermentation to improve the uniformity of the fermentation system. However, prolonged mechanical stirring can easily generate significant shear forces, leading to damage to the cell structure of some probiotics. Simultaneously, continuous stirring increases the contact between the fermentation liquid and air, raising the dissolved oxygen level in the system. This is not conducive to the reproduction of anaerobic or facultative anaerobic bacteria such as Bifidobacteria, resulting in a decrease in bacterial count, an imbalance in bacterial ratio, and insufficient product stability after fermentation.

[0006] On the other hand, most existing probiotic feeds focus on improving the gut microbiota environment of animals, with fewer designs aimed at enhancing animal immune function. Some products improve animal disease resistance by adding natural immune-enhancing ingredients such as astragalus polysaccharide and echinacea extract. However, these plant-based active ingredients are usually heat-sensitive and often require sterilization, high-temperature treatment, and long-term fermentation during traditional processing. This can easily lead to degradation or microbial metabolism, reducing the content of effective ingredients in the final product and resulting in unstable immune-enhancing effects.

[0007] In addition, during the fermentation process of compound probiotics, due to the differences in growth requirements between different strains, if there is a lack of effective nutrient regulation and pH control measures, it is easy for the dominant strains to over-proliferate and some strains to be inhibited, resulting in large differences in the number of live bacteria between product batches, making it difficult to achieve stable industrial production.

[0008] Therefore, there is an urgent need to provide a new processing technology for antibody-enhancing probiotic feed that can improve the stability of immune-enhancing active ingredients while ensuring the high activity and high density of compound probiotics, and improve the survival rate of bacteria during product storage, thereby obtaining a functional liquid feed product with good immunomodulatory effects and stability. Summary of the Invention

[0009] This invention provides a processing technology for antibody-enhancing probiotic feed, which solves the problems of low number of live probiotics, poor flora stability, easy degradation and inactivation of immune-enhancing components during fermentation, and insufficient storage stability of finished products in the current probiotic feed processing.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: A processing method for antibody-enhancing probiotic feed includes the following steps: (1) Preparation of fermentation substrate: Molasses, yeast extract, complex trace elements and buffer salts are added to purified water and mixed to prepare fermentation substrate liquid; The fermentation substrate liquid comprises the following components by mass percentage: molasses 3-6%; yeast extract 1-3%; complex trace elements 0.1-1%; buffer salts 0.1-1%; and the remainder is purified water. (2) Sterilization of the substrate: The fermentation substrate liquid is heated to 90-100℃ and kept at 10-20 min for sterilization, and then cooled to 35-38℃; (3) Inoculation with compound bacteria: Inoculate the compound bacterial solution composed of Lactobacillus plantarum, Bacillus subtilis, Bifidobacterium and yeast into the sterilized fermentation substrate solution; (4) Anaerobic fermentation: The fermentation system after inoculation is sealed and anaerobic fermentation is carried out at 35-39℃ for 48-72h. During the fermentation process, intermittent stirring is adopted, and the pH value of the fermentation liquid is controlled to be 3.5-4.5 by means of buffer salt system and online pH monitoring. (5) Immunostimulating compound: After fermentation, the fermentation liquid is cooled to 20-30℃, and Astragalus polysaccharide, Echinacea extract and trehalose are added and mixed. (6) Post-processing: The compounded fermentation liquid is filtered and homogenized, and then aseptically filled to obtain antibody-enhanced beneficial bacteria feed.

[0011] Furthermore, the mass ratio of Lactobacillus plantarum, Bacillus subtilis, Bifidobacterium and yeast is 8:5:4:3.

[0012] Furthermore, the intermittent stirring method involves stirring for 3–8 minutes and then letting it stand for 20–40 minutes, and the stirring speed is 20–35 r / min.

[0013] Furthermore, the inoculation amount of the compound bacterial solution is 2-10% of the total volume of the fermentation substrate solution.

[0014] Furthermore, based on the total mass of the fermentation broth, the amount of Astragalus polysaccharide added during the immune-enhancing compounding process is 0.3-1.5%, the amount of Echinacea extract added is 0.05-0.5%, and the amount of trehalose added is 0.2-1%.

[0015] Furthermore, the composite trace elements include one or more of magnesium, manganese, iron, and zinc.

[0016] Furthermore, the buffer salt is selected from one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and calcium carbonate.

[0017] Furthermore, the filtration process uses a 100-300 mesh filter medium to remove undissolved particles and large bacterial clusters.

[0018] Furthermore, the homogenization process pressure is 5–10 MPa.

[0019] Compared with the prior art, the beneficial effects of the present invention are: This invention employs a complex microbial community composed of *Lactobacillus plantarum*, *Bacillus subtilis*, *Bifidobacterium*, and yeast. By controlling fermentation temperature, pH, and nutrient conditions, a good growth balance is maintained among different microbial species within the same fermentation system, increasing the quantity and stability of the complex probiotics. Intermittent stirring is used instead of continuous stirring during fermentation, ensuring uniform mixing of the fermentation broth while reducing damage to the microbial cells from mechanical shearing and minimizing the possibility of air entering the fermentation system. This helps maintain the high activity of anaerobic probiotics such as *Bifidobacterium*. By adding immune-enhancing components such as astragalus polysaccharide and echinacea extract after fermentation, the active ingredients are protected from prolonged fermentation and high-temperature treatment, reducing functional component degradation and improving the immune-enhancing effect. The addition of trehalose as a cell-protecting component improves the stability of the probiotics during storage and slows the rate of decline in cell activity during low-temperature storage, resulting in a product with good storage performance. This invention features clear process steps and controllable parameters, making it suitable for large-scale production. The resulting liquid feed combines probiotic regulation of intestinal function and immune enhancement, and can be used to improve the disease resistance of farmed animals, reducing the need for antibiotics. Detailed Implementation

[0020] To make the technical solution, technical effects, and implementation process of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be noted that the following embodiments are only used to illustrate the technical solution of the present invention and are not intended to limit the scope of protection of the present invention. Equivalent substitutions or adjustments made by those skilled in the art to process parameters, raw material ratios, and equipment forms without departing from the technical concept of the present invention should all fall within the scope of protection of the present invention.

[0021] Example 1: This embodiment provides a processing technology for antibody-enhancing beneficial bacteria feed, using a 500L stainless steel fermenter as the fermentation equipment. The fermenter is equipped with a jacketed heat exchange structure, a mechanical stirring device, a temperature detection device, an online pH detection device, and a sterile inoculation interface. Before production, the inside of the fermenter, the stirring shaft, the conveying pipeline, and the filling pipeline are cleaned. After cleaning, sterilization is performed by steam sterilization to ensure that the production equipment meets the requirements for sterile inoculation.

[0022] The raw materials used in this embodiment include: purified water; molasses; yeast extract; compound trace elements; buffer salts; *Lactobacillus plantarum*; *Bacillus subtilis*; *Bifidobacterium*; yeast; astragalus polysaccharide; echinacea extract; and trehalose. *Lactobacillus plantarum*, *Bacillus subtilis*, *Bifidobacterium*, and yeast are added in a compound culture. The mass ratio of the four cultures is: *Lactobacillus plantarum*: *Bacillus subtilis*: *Bifidobacterium*: yeast = 8:5:4:3. This ratio balances acid production capacity, tolerance, and metabolite formation. Specifically: *Lactobacillus plantarum* can rapidly produce lactic acid, lowering the pH of the fermentation system while inhibiting the growth of harmful bacteria; *Bacillus subtilis* has strong environmental adaptability and can produce various enzymes; *Bifidobacterium* regulates the intestinal flora but is sensitive to oxygen, therefore requiring cultivation in a low-oxygen environment; yeast provides various nutritional metabolites, improving the overall stability of the fermentation system.

[0023] Then, the molasses, yeast extract, and compound trace elements were sieved separately. In this embodiment, an 80-mesh sieve was used for sieving. During the sieving process, clumps, impurities, and foreign matter generated during the transportation and storage of the raw materials were removed. The sieved raw materials were sealed and stored separately for later use. Astragalus polysaccharide, echinacea extract, and trehalose were individually sieved before being added to the compound to ensure there were no clumps and to avoid prolonged exposure to high temperature and high humidity environments.

[0024] The fermentation substrate solution, prepared by weight percentage, in this embodiment consists of: 75% purified water; 4.5% molasses; 2.0% yeast extract; 0.1% complex trace elements; and 0.4% buffer salt. Molasses serves as the primary carbon source, providing usable sugars for probiotic growth; yeast extract provides amino acids, vitamins, and growth factors; the complex trace elements provide trace minerals such as magnesium, manganese, and iron; and the buffer salt reduces acidity changes during fermentation, improving the stability of the microbial community. Specifically, the calculated amount of purified water is first added to the sterilized fermenter. The stirring device is started, and the stirring speed is controlled at 45 rpm. Then, molasses, yeast extract, buffer salt, and complex trace elements are added sequentially. Continuous stirring is maintained during the addition of each component to rapidly disperse the solid materials. Stirring continues for 20 minutes to ensure complete dissolution of all components, forming a uniform and transparent fermentation substrate solution.

[0025] After preparing the substrate solution, it is sterilized. This embodiment employs a mild sterilization method: steam is introduced through the fermenter jacket to raise the temperature of the substrate solution to 95°C. Once the temperature reaches 95°C, it is maintained for 15 minutes. Heating is then stopped after sterilization. This sterilization condition effectively reduces the number of miscellaneous bacteria in the substrate while avoiding prolonged high-temperature degradation of sugars. Compared to traditional 121°C autoclaving, this method reduces nutrient loss and is more suitable for probiotic fermentation substrate treatment.

[0026] After sterilization, the fermentation substrate liquid is cooled using a jacketed circulating cooling water system to lower the temperature to 37°C. During the cooling process, the fermenter is kept sealed to prevent outside air from entering. Once the temperature stabilizes, the next inoculation step is performed. Lactobacillus plantarum, Bacillus subtilis, Bifidobacterium, and yeast are mixed in a ratio of 8:5:4:3. The mixed inoculum is then added to the sterilized sugar water activation solution.

[0027] In this embodiment, a 3% glucose solution was used as the sugar water activation solution. The activation temperature was controlled at 37°C, and the activation time was 2 hours. The process was kept sealed. This pre-activation treatment restored the metabolic capacity of the dormant microorganisms, improving their adaptation speed after inoculation. After activation, the resulting compound bacterial solution was added to the fermenter at 5% of the total volume of the fermentation substrate solution (i.e., 5% inoculation amount) through a sterile inoculation pipeline. The fermenter was kept sealed during inoculation. After inoculation, the inoculation valve was closed, allowing the fermentation system to enter an anaerobic culture state.

[0028] After inoculation, the fermentation system was subjected to isothermal anaerobic culture. In this embodiment, the fermentation temperature was controlled at 37℃, and the total fermentation time was controlled at 60 hours. During fermentation, the inlet and outlet valves of the fermenter were closed to keep the fermentation system in a relatively sealed state. To avoid oxygen entry and mechanical shear damage caused by traditional continuous stirring, this embodiment adopted an intermittent stirring mode. The specific control method was: stirring for 5 minutes; settling for 30 minutes; and circulating. The stirring speed was controlled at 25 r / min. Through the above stirring method, the uniform distribution of nutrients in the fermentation broth was ensured, avoiding local nutrient deficiency, while reducing cell damage caused by prolonged mechanical stirring. Especially for Bifidobacteria, since they are strict anaerobic or semi-anaerobic bacteria, they are quite sensitive to the oxygen environment. Continuous stirring can easily lead to an increase in dissolved oxygen in the fermentation broth, thereby affecting their proliferation. This embodiment uses a combination of short-time stirring and long-time settling to gradually create a low-oxygen environment in the fermentation system, which is conducive to maintaining high activity of Bifidobacteria. During fermentation, the pH of the fermentation broth was monitored in real time by a pH detection device installed inside the fermenter. As lactic acid bacteria and other microorganisms continuously metabolize and produce organic acids, the pH value of the fermentation broth gradually decreases. In this embodiment, a buffer salt system in the fermentation substrate, along with the addition of food-grade acid-base regulators as needed, maintains the pH value within the range of 3.8–4.2 during fermentation. Specifically: when the pH value decreases too rapidly, an appropriate amount of calcium carbonate buffer system is added; when the pH value is too high, a small amount of lactic acid solution is added for adjustment. By controlling the fermentation environment, different microorganisms can grow together under suitable conditions. After fermentation, samples of the fermentation broth are taken for testing. The test results show that the total viable count of the compound probiotics reaches 2.3 × 10^9 CFU / mL.

[0029] After the primary fermentation is complete, the temperature of the fermentation broth is lowered. In this embodiment, the temperature of the fermentation broth is lowered to 25°C using a jacketed cooling method. After cooling, the immune-enhancing components are added under low-speed stirring. Specifically, based on the total mass of the fermentation broth, the following are added: Astragalus polysaccharide 0.8%; Echinacea extract 0.2%; Trehalose 0.5%. The order of addition is: first, add Astragalus polysaccharide; after it is fully dispersed, add Echinacea extract; and finally, add Trehalose. After addition, control the stirring speed at 15 r / min and continue stirring for 15 min to ensure thorough mixing of all components. This step differs from the traditional direct fermentation method. In this invention, Astragalus polysaccharide and Echinacea extract are not added to the fermentation system in advance, but rather supplemented after the probiotics have completed proliferation. This avoids: loss of immune-active substances during high-temperature sterilization; consumption by bacterial metabolism during long-term fermentation; and reduction of effective components due to an acidic environment. Simultaneously, trehalose can form a protective structure on the bacterial surface, improving the stability of the probiotics during subsequent storage.

[0030] After the immune-enhancing components were formulated, the fermentation broth was filtered. A 200-mesh stainless steel filter was used to remove undissolved particles and larger bacterial clusters. After filtration, the resulting liquid was fed into a high-pressure homogenizer. In this embodiment, the homogenization pressure was set to 8 MPa. Low-pressure homogenization further dispersed the components in the fermentation broth evenly, reducing sedimentation and stratification during storage, and no significant decrease in viable cell count was observed before and after homogenization.

[0031] After homogenization, the product is transported to the filling equipment via sterile piping. Filling and sealing are performed in a clean environment. Immediately after filling, the product is transferred to a low-temperature storage environment. In this embodiment, the storage temperature is 4°C. Testing showed that the antibody-enhancing probiotic liquid feed obtained in this embodiment has good stability, with a final pH value of 4.1.

[0032] Example 2: This embodiment provides another processing technology for antibody-enhancing probiotic feed. This embodiment is basically the same as Example 1, except for the fermentation parameters and the proportion of immune-enhancing components. In this embodiment: the fermentation temperature is controlled at 35℃; the fermentation time is controlled at 72h. The intermittent stirring method is adjusted to: stirring for 3min; standing for 40min. The stirring speed is still controlled at 25r / min. The inoculum amount is also 5%. By lowering the fermentation temperature and extending the fermentation time, the probiotics have more time to proliferate. Simultaneously, this embodiment adjusts the amount of immune-enhancing components added: based on the total mass of the fermentation broth, Astragalus polysaccharide 1.2%; Echinacea extract 0.3%; Trehalose 1.0%. The remaining steps include: raw material pretreatment; substrate sterilization; strain activation; aseptic inoculation; low-temperature compounding after fermentation; filtration using a 200-mesh filter; homogenization at 8MPa; filling and storage; all are the same as in Example 1. Testing shows that the total viable count of the compound probiotics in this embodiment reaches 2.6×10^9 CFU / mL. The obtained product was stored at 4°C for 90 days and then tested, and the viable bacteria retention rate reached 87%. This indicates that a high level of viable bacteria can be obtained under lower temperature and longer storage conditions, and that the product has good storage stability.

[0033] Example 3: This embodiment provides another processing technology for antibody-enhancing probiotic feed. The difference between this embodiment and Example 1 lies in adjusting the composition of the fermentation substrate. This embodiment uses the following mass percentages: purified water 70%; molasses 6%; yeast extract 3%; compound trace elements 0.5%; buffer salts 0.5%. After using the above substrate formula, inoculation was performed according to the strain ratio and 5% inoculation amount in Example 1. During fermentation: temperature controlled at 37℃; fermentation time 60h; stirring method: stirring for 5min, standing for 30min, and circulation; pH controlled at 3.8–4.2. After fermentation, astragalus polysaccharide, echinacea extract, and trehalose were added according to the method in Example 1. Subsequently, the product was filtered through a 200-mesh screen, homogenized at 8MPa, and then filled and stored. Testing showed that the total viable count of the compound probiotics in the product obtained in this embodiment reached 2.1 × 10^9 CFU / mL, and the product pH value was 3.9. This indicates that even after adjusting the nutrient substrate ratio, a high level of bacterial proliferation can still be maintained.

[0034] Comparative Example 1: This comparative example provides a processing technology for antibody-enhancing beneficial bacteria feed. The overall processing procedure is the same as in Example 1, except that trehalose is not added in the immune-enhancing compounding step, while the amounts of Astragalus polysaccharide and Echinacea extract remain unchanged. The specific steps are as follows: First, the fermentation substrate liquid is prepared according to the method in Example 1. The composition of the fermentation substrate liquid is as follows: purified water 75%; molasses 4.5%; yeast extract 2.0%; complex trace elements 0.1%; buffer salts 0.4%. Then, sterilization is performed according to the method in Example 1. The sterilization temperature is 95℃, and the holding time is 15 min. After cooling, the activated complex bacterial solution is added to the fermentation substrate liquid at a 5% inoculum. The ratio of the complex bacterial strains is: Lactobacillus plantarum: Bacillus subtilis: Bifidobacterium: yeast = 8:5:4:3. The fermentation temperature is controlled at 37℃. The fermentation time is 60 h. During fermentation, intermittent stirring is used: stirring for 5 min; standing for 30 min. The pH value is maintained at 3.8–4.2 by adjusting the buffer salts and acid-base balance. After fermentation, the temperature was lowered to 25°C. Then, based on the total mass of the fermentation broth, 0.8% Astragalus polysaccharide and 0.2% Echinacea extract were added. Unlike Example 1, trehalose was not added in this comparative example. After thorough mixing, the mixture was sequentially filtered through a 200-mesh filtration system, homogenized at 8 MPa, and aseptically filled. The resulting product was stored at 4°C. Viability testing was conducted after 30, 60, and 90 days of storage. The results showed that, with storage times of 30, 60, and 90 days, the viability retention rates in Example 1 were 96%, 91%, and 87%, respectively, while those in Comparative Example 1 were 82%, 52%, and 29%, respectively. The results indicate that without the addition of trehalose, the rate of decline in probiotics in the product during storage was significantly accelerated. This is because, during the storage of liquid feed, the cell membranes of probiotics are easily affected by changes in osmotic pressure, low temperatures, and metabolic products, causing the bacteria to gradually lose their activity. After the addition of trehalose, it can form a protective effect with the phospholipid structure on the bacterial cell surface, improving the stability of the bacterial cell membrane structure and thus increasing the survival rate of probiotics during storage. Therefore, trehalose, as a post-addition protective agent, has a significant effect on improving the stability of liquid probiotic feed.

[0035] Comparative Example 2: This comparative example illustrates the importance of adding the immune-enhancing components after fermentation. The difference between this comparative example and Example 1 is that Astragalus polysaccharide and Echinacea extract are added to the fermentation substrate solution beforehand to participate in sterilization and subsequent fermentation. The specific steps are as follows: The fermentation substrate solution was prepared according to the method in Example 1. The difference is that, along with molasses, yeast extract, trace elements, and buffer salts, the following were added by weight of the total fermentation liquid: 0.8% Astragalus polysaccharide and 0.2% Echinacea extract. These were then sterilized together. The sterilization conditions were: temperature 95℃; time 15 min. After sterilization, the temperature was lowered to 37℃. A 5% inoculum of the compound bacterial solution was added for fermentation. The fermentation conditions were: temperature 37℃; time 60 h; stirring speed 25 r / min; stirring for 5 min, followed by standing for 30 min and cycling. After fermentation, only 0.5% trehalose was added. Subsequent steps, including 200-mesh filtration, 8 MPa homogenization, and filling, were the same as in Example 1. Testing revealed that the actual retention rate of Astragalus polysaccharides in the product obtained in this comparative example was approximately 58%, and the retention rate of active ingredients in Echinacea extract was approximately 62%. However, in Example 1, the retention rate of Astragalus polysaccharides reached over 94%, and the retention rate of active ingredients in Echinacea extract reached over 90%. The reasons for this are as follows: Astragalus polysaccharides are natural polysaccharide active substances, which are easily affected by microbial metabolism during long-term fermentation. Some active ingredients in Echinacea extract are also prone to degradation during high-temperature sterilization and long-term fermentation. Adding them prematurely would result in a significant loss of immune-functional components. This invention employs a low-temperature addition method after fermentation, allowing immune-enhancing components to bypass the fermentation process, thereby ensuring the content of active ingredients in the final product. Therefore, adding immune-enhancing components later is an important technical measure to improve the immune function of the product.

[0036] Comparative Example 3: Compared with Example 1, this comparative example only changed the stirring method during fermentation. Specifically, Example 1 involved stirring for 5 minutes, letting it stand for 30 minutes, and then circulating the mixture. This comparative example used continuous stirring, meaning the stirring mechanism was continuously turned on during fermentation, the stirring speed was maintained at 25 r / min, and it ran continuously for 60 hours. All other steps (including 5% inoculum, post-fermentation compounding, 200-mesh filtration, and 8 MPa homogenization) were the same as in Example 1. After fermentation, the resulting product was tested. The results are as follows: Example 1 had a total viable count of 2.3 × 10^9 CFU / mL, a high number of Bifidobacteria, and a viable count retention rate of 96% after 30 days of storage; Comparative Example 3 had a total viable count of 1.6 × 10^9 CFU / mL, a significantly reduced number of Bifidobacteria, and a viable count retention rate of 76% after 30 days of storage. The test results indicate that while continuous stirring can improve the mixing degree of the fermentation system, prolonged mechanical shearing can damage some of the bacterial structure. Meanwhile, continuous stirring increases the contact between the fermentation broth and air, raising the dissolved oxygen content and hindering the growth of anaerobic bacteria such as Bifidobacteria. This invention employs intermittent stirring, ensuring uniform distribution of nutrients while reducing mechanical damage and oxygen entry, thus improving the stability of the complex microbial community.

[0037] To verify the application effect of the antibody-enhanced probiotic feed of the present invention, the performance of the products obtained in Examples 1 to 3 was tested, and commercially available ordinary probiotic liquid feed was set as a control.

[0038] The viable bacteria count was determined using the plate count method to detect the quantities of *Lactobacillus plantarum*, *Bacillus subtilis*, *Bifidobacterium*, and yeast. The results are as follows: Example 1 showed a viable bacteria count of 2.3 × 10^9 CFU / mL; Example 2 showed a viable bacteria count of 2.6 × 10^9 CFU / mL; Example 3 showed a viable bacteria count of 2.1 × 10^9 CFU / mL; and the commercially available product showed a viable bacteria count of 8.5 × 10^8 CFU / mL. The results indicate that the product prepared using the processing technology of this invention has a high concentration of probiotics.

[0039] Low-temperature storage stability tests were conducted by storing the products obtained in Examples 1-3 at 4°C. Changes in the number of viable bacteria were measured at different time points. The results are as follows: the time points correspond to 0 days, 30 days, 60 days, and 90 days respectively. Specifically, the viable bacteria retention rates for Example 1 were 100%, 96%, 91%, and 87% respectively; for Example 2, they were 100%, 97%, 93%, and 87% respectively; and for Example 3, they were 100%, 94%, 88%, and 84% respectively. The results indicate that the present invention improves the long-term stability of the product through the addition of trehalose after fermentation and low-temperature storage.

[0040] Although embodiments of the present invention have been shown and described above, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A processing technology for antibody-enhancing probiotic feed, characterized in that, Includes the following steps: (1) Preparation of fermentation substrate: Molasses, yeast extract, complex trace elements and buffer salts are added to purified water and mixed to prepare fermentation substrate liquid; The fermentation substrate liquid comprises the following components by mass percentage: molasses 3-6%; yeast extract 1-3%; complex trace elements 0.1-1%; buffer salts 0.1-1%; and the remainder is purified water. (2) Sterilization of the substrate: The fermentation substrate liquid is heated to 90-100℃ and kept at 10-20 min for sterilization, and then cooled to 35-38℃; (3) Inoculation with compound bacteria: Inoculate the compound bacterial solution composed of Lactobacillus plantarum, Bacillus subtilis, Bifidobacterium and yeast into the sterilized fermentation substrate solution; (4) Anaerobic fermentation: The fermentation system after inoculation is sealed and anaerobic fermentation is carried out at 35-39℃ for 48-72h. During the fermentation process, intermittent stirring is adopted, and the pH value of the fermentation liquid is controlled to be 3.5-4.5 by means of buffer salt system and online pH monitoring. (5) Immunostimulating compound: After fermentation, the fermentation liquid is cooled to 20-30℃, and Astragalus polysaccharide, Echinacea extract and trehalose are added and mixed. (6) Post-processing: The compounded fermentation liquid is filtered and homogenized, and then aseptically filled to obtain antibody-enhanced beneficial bacteria feed.

2. The processing technology for an antibody-enhancing beneficial bacteria feed according to claim 1, characterized in that, The mass ratio of Lactobacillus plantarum, Bacillus subtilis, Bifidobacterium and yeast is 8:5:4:

3.

3. The processing technology for an antibody-enhancing beneficial bacteria feed according to claim 1, characterized in that, The intermittent stirring method involves stirring for 3–8 minutes and then letting it stand for 20–40 minutes, repeating the cycle, with a stirring speed of 20–35 r / min.

4. The processing technology for an antibody-enhancing beneficial bacteria feed according to claim 1, characterized in that, The inoculation amount of the compound bacterial solution is 2-10% of the total volume of the fermentation substrate solution.

5. The processing method for an antibody-enhancing beneficial bacteria feed according to claim 1, characterized in that, Based on the total mass of the fermentation broth, the amount of Astragalus polysaccharide added during the immune-enhancing compounding process is 0.3-1.5%, the amount of Echinacea extract added is 0.05-0.5%, and the amount of trehalose added is 0.2-1%.

6. The processing method for an antibody-enhancing beneficial bacteria feed according to claim 1, characterized in that, The composite trace elements include one or more of magnesium, manganese, iron, and zinc.

7. The processing method for an antibody-enhancing beneficial bacteria feed according to claim 1, characterized in that, The buffer salt is selected from one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and calcium carbonate.

8. The processing method for an antibody-enhancing beneficial bacteria feed according to claim 1, characterized in that, The filtration process uses a 100-300 mesh filter medium.

9. The processing method for an antibody-enhancing beneficial bacteria feed according to claim 1, characterized in that, The homogenization process pressure is 5–10 MPa.