Probiotic fermented feed additive for replacing antibiotics to improve milk production rate of dairy cows

Abstract

The invention relates to the field of additives, and discloses a probiotic fermented feed additive for replacing antibiotics to improve the milk yield of dairy cows, and the probiotic fermented feed additive comprises 70-95% of a symbiotic fermentation substrate. 1% to 10% of saccharomyces cerevisiae; 1% to 10% of lactobacillus plantarum; 1% to 5% of bacillus subtilis; 1% to 5% of propionibacterium propionate; the symbiotic fermentation substrate comprises trehalose, glycerol, bacterial exopolysaccharide and organic acid; mycose derived from the symbiotic fermentation substrate is physiologically enriched in cytoplasm of the lactobacillus plantarum, the organic acid in the symbiotic fermentation substrate comprises propionic acid and lactic acid, and the weight ratio of the propionic acid to the lactic acid is between 1.1: 1 and 5: 1. The additive is used for cow breeding instead of antibiotics, the problems of food safety risks and bacterial drug resistance caused by antibiotic residues are radically eradicated, and meanwhile, multiple probiotics such as saccharomyces cerevisiae and lactobacillus plantarum are scientifically proportioned and have a synergistic effect with a symbiotic fermentation substrate.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of additives, in particular to a probiotic fermented feed additive for replacing antibiotics to improve milk yield of dairy cows. BACKGROUND

[0002] In the traditional process of dairy cow breeding, antibiotics are widely used as feed additives to reduce the incidence of diseases and promote the growth of dairy cows. However, long-term abuse of antibiotics can lead to antibiotic residues in milk, which not only affects the quality of milk, but also enters the human body through the food chain, posing a potential threat to human health. At the same time, the continuous use of antibiotics can induce drug resistance in bacteria in the environment, disrupt the microbial ecological balance of the breeding site and the surrounding area, and in addition, antibiotics can interfere with the metabolic activities of normal flora in the rumen of dairy cows, inhibit the proliferation of beneficial microorganisms, and reduce the digestion and absorption efficiency of nutrients in feed, thereby restricting the improvement of milk yield.

[0003] The existing probiotic feed additives on the market mostly use single probiotic strains or simple mixtures of multiple probiotic strains, lack scientific design of symbiotic relationship and functional synergy of flora, resulting in severe loss of activity of probiotics during feed processing, and difficulty in stable colonization and function in the intestinal tract of dairy cows. Most products only focus on regulating the intestinal flora of dairy cows, and do not optimize the ingredients for the core needs of rumen metabolism of dairy cows, so they cannot effectively solve the core problems of improving milk yield and improving milk quality, and the actual application effect cannot meet the needs of large-scale dairy cow breeding. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a probiotic fermented feed additive for replacing antibiotics to improve milk yield of dairy cows, which solves the problem of "the existing probiotic feed additives on the market mostly use single probiotic strains or simple mixtures of multiple probiotic strains, and are difficult to colonize and function stably in the intestinal tract of dairy cows".

[0005] To achieve the above purpose, the present application is implemented by the following technical scheme: a probiotic fermented feed additive for replacing antibiotics to improve milk yield of dairy cows, the final composition comprising 70% to 95% of a symbiotic fermentation substrate; 1% to 10% of Saccharomyces cerevisiae; 1% to 10% of Lactobacillus plantarum; 1% to 5% of Bacillus subtilis; and 1% to 5% of Propionibacterium acidipropionici; the symbiotic fermentation substrate includes trehalose, glycerol, bacterial extracellular polysaccharide and organic acid; the cytoplasm of the Lactobacillus plantarum physiologically enriches trehalose derived from the symbiotic fermentation substrate.

[0006] Preferably, the organic acid in the symbiotic fermentation substrate comprises propionic acid and lactic acid, and the weight ratio of propionic acid to lactic acid is between 1.1:1 and 5:1.

[0007] Preferably, in the symbiotic fermentation substrate, the weight ratio of trehalose to glycerol is between 1:1 and 5:1.

[0008] Preferably, the weight ratio of the biomass of the brewer's yeast to the biomass of the lactobacillus plantarum is between 2:1 and 1:2.

[0009] Preferably, the bacterial extracellular polysaccharide comprises at least one selected from polyγ-glutamic acid, glucan, fructan, and any combination thereof.

[0010] A method for preparing a probiotic fermented feed additive to replace antibiotics for improving milk production in dairy cows includes the following steps: S1. Protective agent-induced fermentation step: First, prepare the raw materials according to the following proportions: 25.0%~40.0% Saccharomyces cerevisiae, 25.0%~40.0% Lactobacillus plantarum, 15.0%~20.0% Bacillus subtilis, and 15.0%~20.0% Propionibacterium propionate. Inoculate Saccharomyces cerevisiae into a high osmotic pressure liquid culture medium for cultivation to obtain the first-stage fermentation broth rich in endogenous trehalose and glycerol. S2, Core microbial stress acclimatization steps: Inoculate Lactobacillus plantarum and Bacillus subtilis into the first stage fermentation broth for culture, so that Lactobacillus plantarum actively absorbs and enriches trehalose in its cytoplasm to obtain the second stage fermentation broth; S3, Functional microbial community symbiotic fermentation step: Propionibacterium propionate is inoculated into the second stage fermentation broth for symbiotic culture to obtain the third stage fermentation broth containing all target microbial communities and their metabolites; S4. Overall drying step: The third-stage fermentation broth is dried as a whole to obtain the composition.

[0011] Preferably, the high osmotic pressure liquid culture medium described in S1 is a culture medium with whey powder or molasses as the main carbon source.

[0012] Preferably, the stress acclimatization described in S2 refers to the process of inducing Lactobacillus plantarum to develop intrinsic stress tolerance by utilizing the high concentration of trehalose and glycerol produced by yeast in the fermentation broth of the first stage.

[0013] Preferably, the symbiotic culture described in S3 includes the metabolic process by which Propionibacterium propionate utilizes lactic acid produced by Lactobacillus plantarum and converts it into propionic acid.

[0014] Preferably, the overall drying step described in S4 employs spray drying or fluidized bed drying, and the entire process does not involve any microencapsulation treatment that physically encapsulates the bacterial cells with the exogenous wall material.

[0015] This invention provides a probiotic fermented feed additive that can replace antibiotics to improve milk production in dairy cows. It has the following beneficial effects: 1. This invention uses additives to replace antibiotics in dairy farming, eliminating the food safety risks and bacterial resistance problems caused by antibiotic residues at the source. At the same time, through the scientific formulation of multiple probiotic strains such as brewer's yeast and Lactobacillus plantarum, which work synergistically with the symbiotic fermentation substrate, it can maintain the balance of the rumen and intestinal microecology of dairy cows, reduce the incidence of intestinal diseases, and ensure the health of dairy cows, which is in line with the development requirements of modern green and pollution-free animal husbandry.

[0016] 2. This invention provides dairy cows with key precursors for the synthesis of milk fat and lactose through the symbiotic metabolism of functional microbial communities. At the same time, the trehalose and bacterial extracellular polysaccharides in the symbiotic fermentation substrate can enhance the dairy cows' ability to digest and absorb feed. This not only significantly increases the milk yield of dairy cows, but also effectively increases the content of milk fat and lactose in milk, thereby improving milk quality and creating higher economic benefits for dairy farms.

[0017] 3. This invention enables Lactobacillus plantarum to actively accumulate trehalose through stress domestication, and combined with spray drying or fluidized bed drying processes, it forms a dual protection mechanism for probiotics, which greatly improves the activity and stability of probiotics during processing, transportation and storage; and the symbiotic system design among the bacterial communities avoids the problem of functional limitations of a single strain, ensuring that the product can continue to play a role in dairy cows, and improving the reliability and durability of the application effect. Attached Figure Description

[0018] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1: Please see the appendix Figure 1 This invention provides a probiotic fermented feed additive that can replace antibiotics to improve milk production in dairy cows. The final components, by dry matter weight, include: 85% symbiotic fermentation substrate, 5% Saccharomyces cerevisiae, 5% Lactobacillus plantarum, 2.5% Bacillus subtilis and 2.5% Propionibacterium propionate.

[0021] In this embodiment, the weight ratio of Saccharomyces cerevisiae to Lactobacillus plantarum is 1:1; the weight ratio of trehalose to glycerol in the symbiotic fermentation substrate is 3:1; the weight ratio of propionic acid to lactic acid in the symbiotic fermentation substrate is 2.5:1; and in this embodiment, poly-γ-glutamic acid produced by Bacillus subtilis biomass is used as a structural component of the symbiotic fermentation substrate.

[0022] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that the proportion of symbiotic fermentation substrate in this comparative example is adjusted from 85% in Example 1 to 90%, and the total proportion of the four probiotics is reduced proportionally from 15% to 10% (4% Saccharomyces cerevisiae, 4% Lactobacillus plantarum, 1% Bacillus subtilis, and 1% Propionibacterium propionitum). The remaining parameters are the same as in Example 1.

[0023] Example 2: Please see the appendix Figure 1 This invention provides a probiotic fermented feed additive that can replace antibiotics to improve milk production in dairy cows. By dry weight, it comprises: The symbiotic fermentation substrate consists of 93% *Saccharomyces cerevisiae*, 2% *Lactobacillus plantarum*, 1.5% *Bacillus subtilis*, and 1.5% *Propionibacterium propionatum*. The weight ratio of propionic acid to lactic acid in the symbiotic fermentation substrate is 4.5:1. The weight ratio of trehalose to glycerol in the symbiotic fermentation substrate is 4:1. In this embodiment, glucan produced by the *Bacillus subtilis* flora is used as a substrate structural component.

[0024] Comparative Example 2: The difference between Comparative Example 2 and Example 2 is that the weight ratio of propionic acid to lactic acid in the symbiotic fermentation substrate in this comparative example is adjusted from 4.5:1 in Example 2 to 1.5:1 (still at the lower limit of the general range of 1.1:1-5:1). The other parameters (93% symbiotic fermentation substrate, 2% Saccharomyces cerevisiae, 2% Lactobacillus plantarum, 1.5% Bacillus subtilis, 1.5% Propionibacterium propionitum, trehalose:glycerol 4:1, and bacterial extracellular polysaccharide is glucan) are consistent with Example 2.

[0025] Example 3: Please see the appendix Figure 1 This invention provides a probiotic fermented feed additive that can replace antibiotics to improve milk production in dairy cows. By dry matter weight, it contains… The symbiotic fermentation substrate consisted of 77% *Saccharomyces cerevisiae* biomass, 6% *Saccharomyces cerevisiae* biomass, 9% *Lactobacillus plantarum* biomass, 4% *Bacillus subtilis* biomass, and 4% *Propionibacterium propionitum* biomass. The weight ratio of trehalose to glycerol in the symbiotic fermentation substrate was 1.5:1. In this example, the composite EPS of polyγ-glutamic acid and fructan produced by the *Bacillus subtilis* flora was used as the substrate structural component. The weight ratio of propionic acid to lactic acid in the symbiotic fermentation substrate was 1.2:1. Comparative Example 3: The difference between Comparative Example 3 and Example 3 is that the type of bacterial extracellular polysaccharide in this comparative example is changed from polyγ-glutamic acid + fructan composite EPS in Example 3 to single glucan. The remaining parameters (77% symbiotic fermentation substrate, 6% Saccharomyces cerevisiae, 9% Lactobacillus plantarum, 4% Bacillus subtilis, 4% Propionibacterium propionitum, trehalose:glycerol 1.5:1, propionic acid:lactic acid 1.2:1) are the same as in Example 3.

[0026] Experimental ingredient table

[0027] Experimental ratio table

[0028] Based on the above, the present invention also provides a method for preparing a probiotic fermented feed additive to replace antibiotics for improving milk production in dairy cows, comprising the following steps: S1. Protective agent-induced fermentation steps: First, prepare the raw materials according to the following proportions: 25.0%~40.0% Saccharomyces cerevisiae, 25.0%~40.0% Lactobacillus plantarum, 15.0%~20.0% Bacillus subtilis, and 15.0%~20.0% Propionibacterium propionate. Inoculate the Saccharomyces cerevisiae in a high-osmotic pressure liquid culture medium to obtain a first-stage fermentation broth rich in endogenous trehalose and glycerol. The high-osmotic pressure environment can precisely trigger the stress protection mechanism of Saccharomyces cerevisiae, enabling it to preferentially synthesize and accumulate endogenous trehalose and glycerol. At the same time, the growth and reproduction process of Saccharomyces cerevisiae in this culture medium will also synchronously produce a small amount of auxiliary nutrients, providing a basic nutritional environment for subsequent inoculation of the microbial community.

[0029] S2. Core microbial stress acclimatization steps: Lactobacillus plantarum and Bacillus subtilis are inoculated into the first-stage fermentation broth for cultivation, allowing Lactobacillus plantarum to actively absorb and enrich trehalose in its cytoplasm, thus obtaining the second-stage fermentation broth. Lactobacillus plantarum exhibits specificity in its active absorption of trehalose, and its cytoplasmic transport proteins are activated in a high-concentration trehalose environment, ensuring efficient enrichment of trehalose. Meanwhile, Bacillus subtilis secretes a small amount of extracellular enzymes during this process, which can slightly degrade some macromolecules in the fermentation broth, providing more easily absorbed small-molecule nutrients for the growth of Lactobacillus plantarum.

[0030] S3. Functional microbial symbiotic fermentation steps: Propionibacterium propionate is inoculated into the second-stage fermentation broth for symbiotic culture to obtain the third-stage fermentation broth containing all target microbial communities and their metabolites. Propionibacterium propionate cannot directly utilize some of the carbon sources in the fermentation broth and needs to rely on lactic acid produced by Lactobacillus plantarum as the main carbon source for metabolism, forming a synergistic metabolic chain from Lactobacillus plantarum producing lactic acid to Propionibacterium propionate converting lactic acid into propionic acid. This symbiotic relationship can also promote signal exchange between microbial communities and maintain the dynamic balance of the number of microbial communities in the fermentation system.

[0031] S4. Overall drying step: The third-stage fermentation broth is dried as a whole to obtain the composition. The overall drying method can avoid the separation of microbial community and metabolites in the fermentation broth, and ensure that the proportion of each component in the final product is consistent with that of the fermentation broth. During the drying process, temperature and humidity must be strictly controlled to prevent high temperature from damaging the activity of microbial community and the structure of heat-sensitive metabolites.

[0032] The high osmotic pressure liquid culture medium described in S1 is a culture medium with whey powder or molasses as the main carbon source; whey powder is rich in nutrients such as lactose and whey protein, which can provide sufficient carbon source for brewer's yeast and naturally create a high osmotic pressure environment; molasses contains rich sucrose, minerals and vitamins, which can enhance the metabolic activity of brewer's yeast and increase the production of endogenous trehalose and glycerol.

[0033] The stress acclimatization described in S2 refers to the process of inducing Lactobacillus plantarum to develop intrinsic stress tolerance by utilizing the high concentration of trehalose and glycerol produced by yeast in the fermentation broth of the first stage. The high concentration of trehalose and glycerol acts as a stress signal, prompting Lactobacillus plantarum to synthesize stress-related proteins, which can protect the stability of the cell membrane and enzyme system. The formation of this intrinsic tolerance enables Lactobacillus plantarum to better tolerate harsh conditions such as drying and storage.

[0034] The symbiotic culture described in S3 includes the metabolic process by which Propionibacterium propionate utilizes lactic acid produced by Lactobacillus plantarum and converts it into propionic acid. Propionibacterium propionate uses enzymes such as lactate dehydrogenase in its body to gradually convert lactic acid into propionic acid. This process is also accompanied by the generation of a small amount of acetic acid. The two organic acids work together to adjust the pH of the fermentation broth to a suitable range. Propionic acid, as a key functional substance in the rumen of dairy cows, is an important precursor for the subsequent synthesis of milk fat and lactose by dairy cows.

[0035] The overall drying step described in S4 employs either spray drying or fluidized bed drying, and the entire process does not involve any microencapsulation treatment that physically encapsulates the bacteria with exogenous wall materials. Spray drying atomizes the fermentation broth into tiny droplets, rapidly dehydrating them in a high-temperature airflow, thus minimizing the time the bacteria are exposed to high temperatures. Fluidized bed drying uses hot air to keep the fermentation broth particles in a fluidized state, resulting in uniform drying and preventing clumping. By not using exogenous wall materials for encapsulation, the problem of wall materials being difficult to degrade in the dairy cow's intestines is avoided, ensuring that the product's effective components are directly absorbed and utilized.

[0036] To facilitate understanding of the present invention, a ratio table is also provided for comparison: Experimental Data Table

[0037] Experimental conclusion: Examples 1-3 are optimized formulations designed for different farming needs, exhibiting excellent overall performance with distinct focuses: Example 1, a balanced formulation, comprises 85% symbiotic fermentation substrate and 15% total probiotics, including 5% Saccharomyces cerevisiae, 5% Lactobacillus plantarum, 2.5% Bacillus subtilis, and 2.5% Propionibacterium propionate. The weight ratio of trehalose to glycerol is 3:1, and the weight ratio of propionic acid to lactic acid is 2.5:1. Polygamma-glutamic acid is used as the bacterial extracellular polysaccharide. This design ensures no significant weaknesses in any aspect of performance, achieving an 80% survival rate of the probiotics after drying, and promoting intestinal colonization in dairy cows. With a survival rate of 55%, this product ensures stable colonization of live bacteria to regulate the intestinal microecology and achieves an average daily milk yield of 27.25 kg / head through synergistic microbial metabolism, a 9% increase compared to the conventional benchmark of 25 kg / head. The milk fat percentage is 4.1%, a 0.6 percentage point increase compared to the benchmark of 3.5%. At the same time, the product forms homogeneous particles due to the binding effect of polyγ-glutamic acid. Under 30-day storage conditions at 25℃, the live bacteria retention rate is 72%, and the incidence of intestinal diseases in dairy cows is only 2.5% after 30 days, a 68.75% reduction compared to the conventional 8%. This meets the basic needs of most large-scale farms for "stable production and improved quality". Example 2 is a high-metabolism formula focusing on milk fat enhancement. The proportion of symbiotic fermentation substrate is increased to 93%, and the total proportion of probiotics is reduced to 7%. The weight ratio of trehalose to glycerol is 4:1 to enhance the protective effect through high trehalose content. The weight ratio of propionic acid to lactic acid is 4.5:1 to provide sufficient precursors for milk fat synthesis through high propionic acid content. Glucan is used as the bacterial extracellular polysaccharide. This design makes it outstanding in milk fat enhancement and stability. The survival rate of probiotics after drying is 83%, and the viable bacteria retention rate is 78% under 30-day storage at 25°C, which is the highest among all groups. Glucan gives the product high hygroscopic resistance, ultimately achieving an average daily milk yield of 27.6 kg / head, an increase of 10.4% compared to the baseline, and a milk fat percentage of 4.5%, an increase of 1 percentage point compared to the baseline, which is the highest among all groups. Even with a low proportion of probiotics, the rumen needs of dairy cows can still be met through precise supply of metabolites. Example 3 focuses on a high-live-bacteria formula that regulates the gut microbiota. The proportion of symbiotic fermentation substrate is reduced to 77%, while the total proportion of probiotics is increased to 23%, with Lactobacillus plantarum accounting for 9%, the highest among all groups. The weight ratio of trehalose to glycerol is 1.5:1 to promote the absorption of live bacteria with the help of high glycerol. A complex bacterial extracellular polysaccharide of polygamma-glutamic acid and fructan is used, which optimizes the colonization ability of live bacteria. The survival rate of probiotics after drying is 85%, the highest among all groups. The three-dimensional network water-holding structure formed by the composite EPS can protect the live bacteria against gastric acid. The intestinal colonization rate of dairy cows reaches 62%, the highest among all groups. The final average daily milk yield is 27.5 kg / head, an increase of 10% compared to the baseline. The milk fat percentage is 4.2%, an increase of 0.7 percentage points compared to the baseline. The incidence of intestinal diseases in dairy cows after 30 days is 2.3%, the lowest among all groups, which can effectively improve the intestinal health of dairy cows.

[0038] Comparative Examples 1-3 were control groups where a single parameter deviated from the optimized formula. All three examples showed significant performance decline due to core design flaws. Comparative Example 1 corresponds to Example 1, where only the proportion of the symbiotic fermentation substrate was increased from 85% to 90%, and the total proportion of probiotics was proportionally reduced from 15% to 10% (specifically, 4% *Saccharomyces cerevisiae*, 4% *Lactobacillus plantarum*, 1% *Bacillus subtilis*, and 1% *Propionibacterium propionate*). All other parameters remained unchanged. However, due to insufficient live probiotic counts, a chain reaction of problems occurred, resulting in a 75% survival rate of the dried probiotics and a mere 35% colonization rate in the dairy cow's intestines, a significant decrease compared to Example 1. The milk yield decreased by 36.4%. Insufficient lactic acid secreted by *Lactobacillus plantarum* led to a shortage of metabolic substrates for *Propionibacterium propionate*, resulting in an average daily milk yield of only 20.44 kg / head, a 25% decrease compared to Example 1. The milk fat percentage was 3.7%, a 0.4 percentage point decrease compared to Example 1. At the same time, the reduction in *Bacillus subtilis* led to insufficient polyγ-glutamic acid, resulting in a loose and easily clumped product. Under 30-day storage conditions at 25°C, the viable bacteria retention rate was 65%, a 7 percentage point decrease compared to Example 1. The incidence of intestinal diseases in dairy cows increased to 5.5%, a 120% increase compared to Example 1. Comparative Example 2 corresponds to Example 2, except that the weight ratio of propionic acid to lactic acid was reduced from 4.5:1 to 1.5:1, while the other parameters remained unchanged. Due to the insufficient amount of the core metabolite propionic acid, the milk fat percentage dropped directly back to the baseline of 3.5%, a decrease of 1 percentage point compared to Example 2. The insufficient propionic acid also led to a tendency for the rumen pH to become acidic and the activity of fiber-decomposing bacteria to be inhibited, resulting in a decrease in the average daily milk production to 25.85 kg / head, a decrease of 6.3% compared to Example 2. Although the survival rate of probiotics and storage stability were similar to those of Example 2, the core value of the high-metabolic formula was lost. Comparative Example 3 corresponds to Example 3, except that only a single glucan was used to replace the complex bacterial extracellular polysaccharide, while the other parameters remained unchanged. Because the single glucan could not form a three-dimensional protective structure, the survival rate of the probiotics after drying plummeted to 49%, a decrease of 42.4% compared to Example 3. The intestinal colonization rate of live bacteria after being eroded by gastric acid was only 6.2%, a decrease of 90% compared to Example 3. The final average daily milk yield was 26 kg / head, a decrease of 5.5% compared to Example 3. The milk fat percentage was 3.7%, a decrease of 0.5 percentage points compared to Example 3. The incidence of intestinal diseases in dairy cows increased to 6.2%, an increase of 170% compared to Example 3, completely losing the intestinal regulatory advantages of the high live bacteria formula.

[0039] In summary, we can conclude that the formulation design of this probiotic fermented feed additive is highly correlated. The proportion of symbiotic fermentation substrate, the proportion of probiotics, the ratio of trehalose to glycerol, the ratio of propionic acid to lactic acid, and the type of bacterial extracellular polysaccharides are all key parameters. Any deviation of any parameter from the optimization range will lead to a significant decrease in performance. Among them, the balanced design of Example 1 is suitable for general aquaculture needs, the high-metabolism design of Example 2 is suitable for scenarios that pursue increased milk fat, and the high-live bacteria design of Example 3 is suitable for farms with prominent intestinal health problems. All three can effectively replace antibiotics, achieving a dual improvement in milk yield and quality while ensuring milk safety. The deficiencies in the proportions further confirm the necessity of the synergistic effect of various parameters in the optimized formulation, providing a scientific basis for large-scale application.

[0040] Although embodiments of the invention have been shown and described, 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 invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A probiotic fermented feed additive for improving milk production in dairy cows as an alternative to antibiotics, characterized in that, The final composition, based on dry matter weight, includes: 70% to 95% symbiotic fermentation substrate; 1% to 10% Saccharomyces cerevisiae; 1% to 10% Lactobacillus plantarum; 1% to 5% Bacillus subtilis; 1% to 5% Propionibacterium propionitum; The symbiotic fermentation substrate includes trehalose, glycerol, bacterial extracellular polysaccharides, and organic acids; The cytoplasm of the plant lactobacillus contains physiologically enriched trehalose derived from the symbiotic fermentation substrate.

2. The probiotic fermented feed additive according to claim 1, used to replace antibiotics and improve milk production in dairy cows, is characterized in that, The organic acids in the symbiotic fermentation substrate include propionic acid and lactic acid, wherein the weight ratio of propionic acid to lactic acid is between 1.1:1 and 5:

1.

3. The probiotic fermented feed additive according to claim 1, used to replace antibiotics and improve milk production in dairy cows, is characterized in that... In the symbiotic fermentation substrate, the weight ratio of trehalose to glycerol is between 1:1 and 5:

1.

4. The probiotic fermented feed additive according to claim 1, used to replace antibiotics and improve milk production in dairy cows, is characterized in that... The weight ratio of the biomass of the brewer's yeast to the biomass of the lactobacillus plantarum is between 2:1 and 1:

2.

5. A probiotic fermented feed additive according to claim 1 for improving milk production in dairy cows, characterized in that, The bacterial extracellular polysaccharide contains at least one selected from polyγ-glutamic acid, glucan, fructan, and any combination thereof.

6. A method for preparing a probiotic fermented feed additive to replace antibiotics for improving milk production in dairy cows, comprising the probiotic fermented feed additive as described in claims 1-5, characterized in that, Includes the following steps: S1. Protective agent-induced fermentation step: First, prepare the raw materials according to the following proportions: 25.0%~40.0% Saccharomyces cerevisiae, 25.0%~40.0% Lactobacillus plantarum, 15.0%~20.0% Bacillus subtilis, and 15.0%~20.0% Propionibacterium propionate. Inoculate Saccharomyces cerevisiae into a high osmotic pressure liquid culture medium for cultivation to obtain the first-stage fermentation broth rich in endogenous trehalose and glycerol. S2, Core microbial stress acclimatization steps: Inoculate Lactobacillus plantarum and Bacillus subtilis into the first stage fermentation broth for culture, so that Lactobacillus plantarum actively absorbs and enriches trehalose in its cytoplasm to obtain the second stage fermentation broth; S3, Functional microbial community symbiotic fermentation step: Propionibacterium propionate is inoculated into the second stage fermentation broth for symbiotic culture to obtain the third stage fermentation broth containing all target microbial communities and their metabolites; S4. Overall drying step: The third-stage fermentation broth is dried as a whole to obtain the composition.

7. A probiotic fermented feed additive according to claim 6, used to replace antibiotics and improve milk production in dairy cows, characterized in that, The high osmotic pressure liquid culture medium described in S1 is a culture medium with whey powder or molasses as the main carbon source.

8. A probiotic fermented feed additive according to claim 6, used to replace antibiotics and improve milk production in dairy cows, characterized in that, The stress acclimatization described in S2 refers to the process of inducing Lactobacillus plantarum to develop intrinsic stress tolerance by utilizing the high concentration of trehalose and glycerol produced by yeast in the fermentation broth of the first stage.

9. A probiotic fermented feed additive according to claim 6, used to replace antibiotics and improve milk production in dairy cows, characterized in that, The symbiotic culture described in S3 includes the metabolic process by which Propionibacterium propionate utilizes lactic acid produced by Lactobacillus plantarum and converts it into propionic acid.

10. A probiotic fermented feed additive according to claim 6, used to replace antibiotics and improve milk production in dairy cows, characterized in that, The overall drying step described in S4 employs spray drying or fluidized bed drying, and the entire process does not involve any microencapsulation treatment that physically encapsulates the bacteria with the exogenous wall material.

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