Compound fermentation composition for regulating animal immunity as well as preparation method and application of compound fermentation composition

Abstract

The invention belongs to the technical field of microbial preparations, and particularly relates to a compound fermentation composition for regulating animal immunity and a preparation method and application thereof. The composition is prepared from the following components in parts by weight: 1 to 10 parts of lactobacillus plantarum polysaccharide, 1 to 5 parts of clostridium butyricum, 0.5 to 2 parts of bifidobacterium, 0.5 to 5 parts of beta-glucan, 50 to 70 parts of corn starch and 0.1 to 1 part of silicon dioxide. The lactobacillus plantarum polysaccharide prepared by a specific preparation process, the clostridium butyricum, the bifidobacterium, the beta-glucan, the corn starch and the silicon dioxide are scientifically matched in a specific amount ratio range, so that the synergistic interaction effect of the immunoregulation component, the intestinal micro-ecological regulation component and other functional components is realized; according to the present invention, the IgA content in animal serum can be significantly increased so as to achieve high efficiency and long-term immunoregulation, the maximization of the utilization of each component is achieved, and the safe and effective bioactive substance is finally provided for the improvement of animal immunity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial preparations, and particularly relates to a compound fermentation composition for regulating animal immunity and a preparation method and application thereof. BACKGROUND

[0002] In the field of animal cultivation such as livestock breeding and pet feeding, the immune function of an animal body directly determines the health condition, growth performance and economic benefits of breeding, and is closely related to the safety of animal-derived food. With the deepening of the concept of healthy breeding in the industry, regulating animal immunity by biological regulators has become an important research direction to replace traditional chemical agents. The synergistic application of microbial polysaccharides and probiotics has attracted much attention due to its naturalness and safety.

[0003] Microbial polysaccharides are high-activity macromolecular substances produced by microorganisms during fermentation. Studies have found that they have multiple biological activities such as antioxidant, antiviral, antitumor and immune enhancement, and also have important functions such as anti-aging, blood lipid-lowering, blood sugar-lowering and wound healing promotion, and have become a good biological regulator. In probiotics, polysaccharides, as important secondary metabolites, not only help the colonization and competition of probiotics in the intestinal tract, but also promote the health of the body by regulating the host immune response and improving the balance of intestinal microecology. In addition, microbial polysaccharides have significant advantages in health, safety, environmental protection and cost. Good physicochemical properties and potential functional characteristics make them have broad application prospects in many fields.

[0004] Among many sources of microbial polysaccharides, Lactobacillus plantarum polysaccharide has higher safety than other bacterial-derived exopolysaccharides, and has the advantages of fast fermentation speed, short cultivation period, no restriction by climate and geographical environment, and simple extraction process. At the same time, Lactobacillus plantarum itself has the characteristics of probiotics such as antibacterial, antioxidant, regulation of intestinal homeostasis, improvement of intestinal inflammation and immune regulation, and has a good promoting effect on human health. It has high adaptability to various ecological niches such as the gastrointestinal tract of humans and animals.

[0005] However, the current animal immune regulation field still has defects, which is difficult to meet the efficiency, stability and adaptability requirements in practical application, mainly in the following aspects: first, the existing technology mostly uses single microbial polysaccharide or single probiotics as a regulating factor, the target of single component is limited, which cannot form a multi-pathway and multi-dimensional immune regulation network, resulting in weak immune enhancement effect and short duration; second, although some composite preparations attempt to combine polysaccharide and probiotics, but the synergistic matching design of the action mechanism of the two is not carried out, resulting in simple physical mixing between components, no synergistic effect is formed, and the problems of low probiotic colonization efficiency and insufficient polysaccharide bioavailability are caused; third, the component selection of the existing preparation lacks the adaptability consideration of the physiological characteristics of the animal intestinal tract, for example, the functional carrier capable of promoting the slow release of polysaccharide and probiotics in the intestinal tract, protecting the active ingredients from being destroyed by gastric acid and digestive enzymes is not introduced, resulting in a large amount of degradation of the active ingredients before reaching the target.

[0006] In view of the above deficiencies of the prior art, it is urgent to develop a composite fermentation composition with scientific component ratio, significant synergistic effect, adaptation to the physiological characteristics of the animal intestinal tract and controllable safety, so as to realize the efficient and stable regulation of animal immunity. SUMMARY

[0007] The purpose of the present application is to overcome the deficiencies of the prior art and provide a composite fermentation composition for regulating animal immunity and a preparation method thereof, so as to effectively improve the animal immunoglobulin IgA level, and further provide a green and healthy composite biological active substance solution for animal immunity improvement.

[0008] To achieve the above purpose, the technical solution adopted by the present application includes: In a first aspect, the present application provides a composite fermentation composition for regulating animal immunity, which comprises the following components by weight: Lactobacillus plantarum polysaccharide 1-10 parts, Clostridium butyricum 1-5 parts, Bifidobacterium 0.5-2 parts, beta-glucan 0.5-5 parts, corn starch 50-70 parts and silicon dioxide 0.1-1 part.

[0009] The present application scientifically matches Lactobacillus plantarum polysaccharide, Clostridium butyricum, Bifidobacterium, beta-glucan, corn starch and silicon dioxide, and realizes the synergistic effect of immune regulation components, intestinal microecological regulation components and other functional components. Among them, the core functional components Lactobacillus plantarum polysaccharide, Clostridium butyricum, Bifidobacterium and beta-glucan cooperate with each other to form a multi-level synergistic mechanism - Lactobacillus plantarum polysaccharide and beta-glucan not only synergistically activate macrophages to secrete ROS, NO and IL-1β to enhance immune response, but also induce IL-10 secretion to avoid excessive inflammation, and both of them as high-hydrophilic polysaccharides can form a "polysaccharide microcapsule" on the surface of live bacteria, helping Clostridium butyricum and Bifidobacterium resist gastric acid and high temperature stress and protect the activity of live bacteria; Clostridium butyricum relies on corn starch to slowly supply sugar to realize high-yield butyric acid, butyric acid not only promotes Treg cell proliferation by activating GPR109A receptor to further improve IL-10 level, forming a positive feedback of "immune tolerance-anti-inflammatory", but also provides energy for intestinal epithelial cells to strengthen tight junction protein expression, while Bifidobacterium can improve intestinal peristalsis and component absorption rate, and at the same time, it promotes the proliferation of goblet cells together with corn starch, cooperating with the secretion of mucin Muc2 induced by Lactobacillus plantarum polysaccharide, to build a solid physical barrier of the intestinal tract, reduce endotoxin into blood and systemic inflammation; The auxiliary component corn starch not only provides energy for Clostridium butyricum, but also serves as an anti-caking agent together with silicon dioxide to ensure that the composition remains loose during filling, transportation and shelf life, and to ensure product stability and effectiveness.

[0010] In summary, the present application can realize efficient and long-acting immune regulation and maximize the use of components, and provides a safe, healthy and effective biological active substance for improving animal immunity.

[0011] Preferably, the compound fermentation composition comprises the following components in parts by weight: Lactobacillus plantarum polysaccharide 4-6 parts, Clostridium butyricum 1.5-2.5 parts, Bifidobacterium 0.8-1.2 parts, beta-glucan 1-3 parts, corn starch 55-65 parts and silicon dioxide 0.4-0.8 parts.

[0012] Preferably, the compound fermentation composition comprises the following components in parts by weight: Lactobacillus plantarum polysaccharide 5 parts, Clostridium butyricum 2 parts, Bifidobacterium 1 part, beta-glucan 2 parts, corn starch 60 parts and silicon dioxide 0.6 parts.

[0013] It is found through experiments that when the components are compounded in the above optimal ratio, the content of animal immunoglobulin IgA can be maximally improved, so as to fully exert the optimal immune enhancing effect of the compound fermentation composition.

[0014] Preferably, the preparation method of the Lactobacillus plantarum polysaccharide comprises the following steps: S1. Inoculate Lactobacillus plantarum into a seed culture medium to obtain a seed liquid, and then inoculate the seed liquid into a fermentation culture medium to obtain a fermentation broth. S2. Mix the fermentation broth with the buffer solution, and then perform homogenization and cell wall disruption treatment to obtain the cell wall disruption solution; S3. Spray-dry the cell wall-breaking solution to obtain spray-dried powder, which is the Lactobacillus plantarum polysaccharide.

[0015] Preferably, in step S2, the specific conditions for homogenization and cell wall disruption are: homogenization for 10-20 minutes at a pressure of 800-1000 MPa and a temperature of 20-30°C, repeated 1-3 times; the mass concentration of the fermentation broth in the buffer solution is 5-10%.

[0016] Experimental studies have shown that the optimized parameters and process described above can effectively break down the cell walls of *Lactobacillus plantarum* to release intracellular polysaccharides, thereby increasing the polysaccharide extraction rate. Simultaneously, the mild and controllable homogenized environment and buffer system effectively reduce damage to live bacteria, preserving sufficient viable cells within the system. This results in *Lactobacillus plantarum* polysaccharides possessing both high content and synergistic advantages with live bacteria. When this *Lactobacillus plantarum* polysaccharide is combined with components such as *Clostridium butyricum*, *Bifidobacterium*, and β-glucan, the high polysaccharide content can directly exert immunomodulatory effects, while the remaining live bacteria can form a probiotic synergistic community with *Clostridium butyricum* and *Bifidobacterium*, further enhancing the intestinal microecological regulatory capacity. Combined with the immune-activating effect of β-glucan, the complementary and synergistic effects of each component are achieved, significantly improving the probiotic efficacy of the compound fermentation composition and ensuring optimal results in regulating animal immunity.

[0017] Preferably, in step S2, the buffer solution is a PBS buffer with a pH of 7.0-7.2 and a concentration of 0.05-0.15M.

[0018] Preferably, the *Lactobacillus plantarum* polysaccharide is loaded with live *Lactobacillus plantarum* bacteria; based on the mass of the *Lactobacillus plantarum* polysaccharide component, the concentration of live *Lactobacillus plantarum* bacteria in the *Lactobacillus plantarum* polysaccharide component is not less than 4 billion CFU / g.

[0019] Maintaining a sufficient and effective concentration of viable *Lactobacillus plantarum* bacteria in the polysaccharide component is a prerequisite for ensuring its stable colonization in the animal intestine and its probiotic function. Sufficient viable bacteria can regulate the balance of the intestinal flora structure, providing a favorable microecological environment for the construction of the intestinal mucosal immune barrier. Based on this, the probiotic effect of *Lactobacillus plantarum* can be fully released, thereby forming a complementary and synergistic immune regulatory network with components such as *Clostridium butyricum* and *Bifidobacterium*, ensuring the overall efficacy of the composition of this invention is highly efficient.

[0020] Preferably, in step S2, the inlet air temperature of the spray drying process is 130-140°C, and the outlet air temperature is 70-80°C.

[0021] In the method for preparing Lactobacillus plantarum polysaccharide, step S1 can be implemented using conventional techniques in the art. The specific culture medium components and suitable growth conditions selected in this invention are as follows: The seed culture medium comprises the following components by weight percentage: glucose 1-3%, peptone 0.5-2%, beef extract 0.5-3%, yeast extract 0.4-0.8%, dipotassium hydrogen phosphate 0.1%-0.4%, magnesium sulfate 0.03-0.06%, manganese sulfate 0.2-0.5%, calcium carbonate 1-3%, Tween 80 0.5-2%, diammonium hydrogen citrate 1-3%, sodium acetate 4-6%, and the balance being water; culture conditions: culture temperature 35-38℃, culture time 20-28h.

[0022] The fermentation medium comprises the following components by weight percentage: glucose 6-10%, yeast extract 0.6-0.9%, dipotassium hydrogen phosphate 0.05-0.2%, magnesium sulfate 0.04-0.08%, manganese sulfate 0.02-0.05%, calcium carbonate 1-5%, ammonium chloride 0.5-2%, diammonium hydrogen citrate 0.1-0.3%, sodium acetate 0.4-0.6%, and the balance being water; culture conditions: culture temperature 35-38℃, culture time 20-28h.

[0023] Secondly, the present invention provides the application of the aforementioned compound fermentation composition in the preparation of products that regulate animal immunity.

[0024] Preferably, the regulation of animal immunity specifically manifests as increasing the content of immunoglobulin IgA in the serum or mucosal tissue of the animal.

[0025] Immunoglobulin A (IgA) is a core effector molecule of mucosal immunity in animals, mainly distributed on the mucosal surfaces of the respiratory, digestive, and reproductive tracts, forming the first line of defense against pathogen invasion. Its concentration directly determines the strength of the animal's mucosal immune function. The composite fermentation composition described in this invention, when applied to animal immune regulation, can precisely increase serum IgA levels, thereby strengthening the animal's defense capabilities from the source of pathogen invasion. Compared to traditional methods, it offers a more direct and efficient immune protection effect.

[0026] Thirdly, the present invention provides a method for preparing the aforementioned composite fermentation composition, comprising the following steps: All components are mixed to obtain a mixture, which is then dried at 50-60°C until the moisture content is 5-10%, thus obtaining the composite fermentation composition.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: The compound fermentation composition for regulating animal immunity provided by this invention scientifically combines *Lactobacillus plantarum* polysaccharide obtained through a specific preparation process with *Clostridium butyricum*, *Bifidobacterium*, β-glucan, corn starch, and silicon dioxide within a specific ratio range. This achieves a synergistic effect between immunomodulatory components, intestinal microecological regulatory components, and other functional components. This synergistic effect not only significantly increases the IgA content in animal serum, achieving efficient and long-lasting immunomodulation, but also maximizes the utilization of each component. Ultimately, it provides safe, healthy, and effective bioactive substances for enhancing animal immunity. Compared with single-component or non-scientifically formulated compositions, it has superior immunomodulatory effects and application value, and can be widely applied to the immune enhancement needs in the animal husbandry field. Attached Figure Description

[0028] Figure 1 This is an image of the finished product of the compound fermentation composition described in Example 1. Detailed Implementation

[0029] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0030] Unless otherwise specified, all raw materials used in the following examples and comparative examples are commercially available and commonly used raw materials.

[0031] The Clostridium butyricum was purchased from Jinan Jinyuyuan Biotechnology Co., Ltd.; the Bifidobacterium was purchased from Shandong Pingao Pharmaceutical Co., Ltd.; the β-glucan was purchased from Wuhan Baixing Biotechnology Co., Ltd.; and the silica was purchased from Anhui Weimao Biotechnology Co., Ltd.

[0032] Examples 1-5 Examples 1-5 provide a compound fermentation composition for regulating animal immunity, the formulation of which is shown in Table 1. The preparation method of the compound fermentation composition includes the following steps: All components are mixed to obtain a mixture, which is then dried at 55°C until the moisture content is 8%, thus obtaining the composite fermentation composition.

[0033] Table 1. Formulation table (parts by weight) of the compound fermentation compositions described in Examples 1-5 The preparation method of Lactobacillus plantarum polysaccharide described in Examples 1-3 includes the following steps: (1) Inoculate Lactobacillus plantarum into a seed culture medium containing 2% glucose, 1% peptone, 1% beef extract, 0.5% yeast extract, 0.2% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.3% manganese sulfate, 2% calcium carbonate, 1% Tween 80, 2% diammonium hydrogen citrate, and 5% sodium acetate, and ferment at 35°C for 24 hours to obtain seed liquid. (2) The seed culture from step (1) was transferred to a fermentation medium containing 8% glucose, 0.8% yeast extract, 0.1% dipotassium hydrogen phosphate, 0.06% magnesium sulfate, 0.03% manganese sulfate, 3% calcium carbonate, 1% ammonium chloride, 0.2% diammonium hydrogen citrate, and 0.5% sodium acetate. Fermentation was carried out at 35°C for 24 hours to obtain the fermentation broth. (3) Place the fermentation broth from step (2) into a high-pressure homogenizer for cell disruption. The cell disruption process is as follows: 900 MPa, homogenization time 15 min, homogenization times 2, bacterial concentration 7% (7 g wet cells / 100 mL buffer, the buffer is 0.1 M PBS buffer with pH 7.0), and temperature 20-30℃. (4) The cell wall breaking solution obtained in step (3) is sprayed dry by a spray dryer (spray drying process: air inlet temperature 135℃, air outlet temperature 75℃) to obtain spray-dried powder, which is plant lactobacillus polysaccharide (of which the effective live bacteria count is not less than 4 billion CFU / g).

[0034] The difference between the preparation method of Lactobacillus plantarum polysaccharide in Example 4 and Example 1 lies only in the cell wall breaking process. The cell wall breaking process in this example is as follows: 800 MPa, homogenization time 10 min, homogenization times 1, bacterial concentration 10%, and temperature 30℃.

[0035] The difference between the preparation method of Lactobacillus plantarum polysaccharide in Example 5 and Example 1 lies only in the cell wall breaking process. The cell wall breaking process in this example is as follows: 1000 MPa, homogenization time 20 min, homogenization times 3, bacterial concentration 5%, and temperature 20℃.

[0036] Comparative Examples 1-8 Comparative Examples 1-8 provide a composite fermentation composition, the formulation of which is shown in Table 2. The preparation method of the composite fermentation composition and the related *Lactobacillus plantarum* polysaccharide is the same as in Example 1.

[0037] Table 2 shows the formulations (parts by weight) of the compound fermentation compositions described in Comparative Examples 1-8. Comparative Example 9 This comparative example provides a compound fermentation composition, which differs from Example 1 only in that an equal amount of *Lactobacillus plantarum* is used to replace *Lactobacillus plantarum* polysaccharide, while the remaining components and amounts are the same as in Example 1.

[0038] Comparative Example 10 This comparative example provides a compound fermentation composition, which differs from Example 1 only in that an equal amount of Lactobacillus is used to replace Bifidobacterium plantarum, while the remaining components and amounts are the same as in Example 1.

[0039] Example 1 This efficacy example uses the composite fermentation compositions prepared in Examples 1-5 and Comparative Examples 1-10 as test samples. The efficacy of these compositions in regulating animal immunity was tested by measuring the serum IgA content in mice. The specific method is as follows: Eighty-five SPF-grade female mice were randomly divided into 17 groups of five each. After 7 days of acclimatization, one group served as control group 1, receiving no compound fermentation composition; another group served as control group 2, receiving only the *Lactobacillus plantarum* polysaccharide prepared in Example 1; and the other 15 groups received the compound fermentation compositions prepared in Examples 1-5 and Comparative Examples 1-10, dissolved in water (10% concentration), via gavage. After 30 days of feeding, serum IgA levels were measured in the mice, and the results are shown in Table 3.

[0040] Table 3 Immunoglobulin A (IgA) is a core indicator of mucosal immunity in animals, and its content directly reflects the strength of the body's local immune defense capabilities. The experimental data show that the IgA content in Examples 1-5 was significantly higher than that in the control group and all comparative examples, confirming that the composition of this invention has a significant regulatory effect on animal immunity.

[0041] In control group 2, mice were fed only with a single type of *Lactobacillus plantarum* polysaccharide. The IgA content was slightly higher than in control group 1, but significantly lower than in the example. In comparative examples 1-6, when any one or two of *Lactobacillus plantarum* polysaccharide, *Clostridium butyricum*, *Bifidobacterium*, and β-glucan were missing, and the total amount of the four key components remained consistent with example 1, the IgA content in mouse serum was significantly lower than in example 1. The effect was worst in comparative example 1 when *Lactobacillus plantarum* polysaccharide was missing, confirming that *Lactobacillus plantarum* polysaccharide is the core active ingredient of this invention and the basis for immune regulation. The other three components are necessary for achieving optimal immune effects; the absence of any one component will lead to a break in the synergistic effect and a significant decrease in immune regulation. In comparative examples 7-8, when the ratio of the four core components exceeded the limits of this invention and the total amount was higher than in example 1, the IgA content was also lower than in the example. This indicates that the component ratios defined in this invention are key to achieving optimal synergistic effects. Blindly increasing the total amount or deviating from the reasonable ratio will not only fail to improve the effect but may also weaken the synergistic effect due to an imbalance in the proportions between components. In Comparative Example 9, when Lactobacillus plantarum was used to replace the polysaccharide of Lactobacillus plantarum, the IgA content decreased to a certain extent compared with the example, indicating that directly using the strain could not achieve the targeted immunomodulatory effect of the polysaccharide prepared by the specific process of the present invention, thus leading to a decrease in the overall efficacy of the composition. In Comparative Example 10, when Lactobacillus, which is also an intestinal probiotic, was used to replace Bifidobacterium, its IgA content also decreased, confirming that the key components of the present invention cannot be simply replaced, otherwise the synergistic effect between components will be affected, thus leading to a decrease in the efficacy of regulating animal immunity.

[0042] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A compound fermentation composition for regulating animal immunity, characterized in that, The compound fermentation composition comprises the following components in parts by weight: 1-10 parts of Lactobacillus plantarum polysaccharide, 1-5 parts of Clostridium butyricum, 0.5-2 parts of Bifidobacterium, 0.5-5 parts of β-glucan, 50-70 parts of corn starch, and 0.1-1 parts of silicon dioxide.

2. The compound fermentation composition according to claim 1, characterized in that, The compound fermentation composition comprises the following components in parts by weight: 4-6 parts of Lactobacillus plantarum polysaccharide, 1.5-2.5 parts of Clostridium butyricum, 0.8-1.2 parts of Bifidobacterium, 1-3 parts of β-glucan, 55-65 parts of corn starch, and 0.4-0.8 parts of silicon dioxide.

3. The compound fermentation composition as described in claim 2, characterized in that, The compound fermentation composition comprises the following components in parts by weight: 5 parts of Lactobacillus plantarum polysaccharide, 2 parts of Clostridium butyricum, 1 part of Bifidobacterium, 2 parts of β-glucan, 60 parts of corn starch, and 0.6 parts of silicon dioxide.

4. The compound fermentation composition according to any one of claims 1-3, characterized in that, The preparation method of the Lactobacillus plantarum polysaccharide includes the following steps: S1. Inoculate Lactobacillus plantarum into a seed culture medium to obtain a seed liquid, and then inoculate the seed liquid into a fermentation culture medium to obtain a fermentation broth. S2. Mix the fermentation broth with the buffer solution, and then perform homogenization and cell wall disruption treatment to obtain the cell wall disruption solution; S3. Spray-dry the cell wall-breaking solution to obtain spray-dried powder, which is the Lactobacillus plantarum polysaccharide.

5. The compound fermentation composition according to claim 4, characterized in that, In step S2, the specific conditions for the homogenization and cell wall disruption treatment are as follows: homogenization for 10-20 minutes at a pressure of 800-1000 MPa and a temperature of 20-30°C, repeated 1-3 times; the mass concentration of the fermentation broth in the buffer solution is 5-10%.

6. The use of the compound fermentation composition according to any one of claims 1-5 in the preparation of products that regulate animal immunity.

7. The application as described in claim 6, characterized in that, The regulation of animal immunity specifically manifests as increasing the content of immunoglobulin IgA in the serum or mucosal tissue of the animal.

8. The method for preparing the compound fermentation composition according to any one of claims 1-5, characterized in that, Includes the following steps: All components are mixed to obtain a mixture, which is then dried at 50-60°C until the moisture content is 5-10%, thus obtaining the composite fermentation composition.

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