A compound probiotic for preparing and preventing ketosis of dairy cows and application thereof

Abstract

This invention provides a compound probiotic for the preparation and application of treating ketosis in dairy cows, belonging to the field of microbial technology. It is a compound composed of *Westernella* L5, *Pediococcus pentosaceus* G41, and *Lactobacillus paracasei* S1 as active ingredients. Through the synergistic effect of these three probiotics, the energy metabolism of ketotic dairy cows is significantly improved. Specifically, it increases the content of total volatile fatty acids and propionic acid in rumen fluid, reduces the levels of non-esterified fatty acids, β-hydroxybutyrate, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in the blood, and increases blood glucose levels. This invention optimizes the rumen microbiota structure by supplementing the rumen with compound probiotics, increases the production of gluconeogenic precursors such as propionic acid, improves the energy metabolism of dairy cows, alleviates negative energy balance, and ultimately achieves effective prevention and treatment of ketosis in dairy cows.

Description

Technical Field

[0001] This invention discloses a compound probiotic for the preparation of a formula for preventing and treating ketosis in dairy cows and its application. The formula is composed of Weissella L5, Pediococcus pentosaceus G41 and Lactobacillus paracasei S1 and can prevent and treat ketosis in dairy cows. It belongs to the field of microbial technology. Background Technology

[0002] Ketosis is a metabolic disease characterized by high levels of non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHBA), and hypoglycemia. Ketosis poses multiple threats to the dairy industry: reduced production performance and milk quality, a 3-6 fold increase in the risk of infectious and reproductive disorders, and a shortened productive lifespan for dairy cows. Therefore, developing efficient and safe ketosis prevention and control strategies is of great significance for ensuring the health of dairy cows and the sustainable development of the industry.

[0003] Rumen volatile fatty acids (VFAs) are the primary energy source for ruminants, produced by rumen microorganisms fermenting carbohydrates in the diet. They meet approximately 70%-80% of the energy requirements of dairy cows and play a central role in maintaining basal metabolism, lactation, and reproduction. VFAs are the main energy substances produced by rumen microorganisms fermenting feed substrates, and the concentration and ratio of VFAs such as acetic acid, propionic acid, and butyric acid in the rumen directly affect the energy supply and production performance of dairy cows. Increasing VFA levels and reducing the acetic acid / propionic acid ratio through nutritional regulation or microecological intervention can help improve energy metabolism and may become an effective way to prevent and treat ketosis in dairy cows. Summary of the Invention

[0004] This invention provides a compound probiotic for the preparation of a treatment for ketosis in dairy cows and its application. The compound probiotic, composed of Weissella L5, Pediococcus pentosus G41 and Lactobacillus paracasei S1, can effectively prevent and treat ketosis in dairy cows.

[0005] The present invention discloses a compound probiotic for the prevention and treatment of ketosis in dairy cows, wherein the compound probiotic is composed of *Westernella* L5, *Pediococcus pentosaceus* G41, and *Lactobacillus paracasei* S1 in a specific ratio of live bacteria: The viable count ratio of Weissella L5: Pediococcus pentosus G41: Lactobacillus paracasei S1 is 1~3:1~3:1~3; The present invention further optimizes the ratio of viable bacteria content of the three components as follows: The viable cell ratio of Weissella L5: Pediococcus pentosus G41: Lactobacillus paracasei S1 was 3:2:2.

[0006] The effective live bacteria count of the compound probiotics described in this invention is not less than 1×10⁻⁶. 9 CFU / mL.

[0007] The *Weissella* L5 strain described in this invention is deposited at the China General Microbiological Culture Collection Center and classified as *Weissella*. Weissella jogaejeotgali The accession number is CGMCC No.35101, and the accession date is July 4, 2025.

[0008] The aforementioned *Pediococcus pentosaceus* G41 is deposited at the China General Microbiological Culture Collection Center, and is classified and named *Pediococcus pentosaceus*. Pediococcus pentosaceus The accession number is CGMCC No.33270, and the accession date is January 2, 2025.

[0009] The aforementioned *Lactobacillus paracasei* S1 is deposited at the China General Microbiological Culture Collection Center, and is classified and named *Lactobacillus paracasei*. Lactobacillus paracasei The accession number is CGMCC No.33269, and the accession date is January 2, 2025.

[0010] The product is an oral preparation, including tablets, capsules, granules, or suspensions.

[0011] The product can increase the content of VFAs and propionic acid in rumen fluid, reduce the levels of NEFA, BHBA, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood, and increase GLU levels.

[0012] Compared to the strains used in existing animal probiotic preparations, *Westernella* L5, *Pediococcus pentosaceus* G41, and *Lactobacillus paracasei* S1, isolated from the normal flora of the rumen of dairy cows, can ensure rapid growth and colonization after entering the rumen when used as probiotics in production, thus facilitating their probiotic functions. The combined use of *Westernella* L5, *Pediococcus pentosaceus* G41, and *Lactobacillus paracasei* S1 can, on the one hand, increase VFA (vitamin-free fatty acids) production, and on the other hand, reduce the acetic acid / propionic acid ratio in the rumen, thereby improving the negative energy balance in dairy cows and playing a role in preventing ketosis.

[0013] This invention increases the content of total volatile fatty acids and propionic acid in rumen fluid, reduces the levels of non-esterified fatty acids, β-hydroxybutyrate, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in the blood, and increases blood glucose levels. By supplementing the rumen of dairy cows with compound probiotics, this invention optimizes the rumen microbiota structure, increases the production of gluconeogenic precursors such as propionic acid, improves the energy metabolism of dairy cows, alleviates negative energy balance, and ultimately achieves effective prevention and treatment of ketosis in dairy cows.

[0014] The positive effects of this invention are as follows: The compound probiotics composed of *Westernella* L5, *Pediococcus pentosaceus* G41, and *Lactobacillus paracasei* S1, through their synergistic effect, can effectively increase the content of VFAs and propionic acid in the rumen fluid of dairy cows, reduce the acetic acid / propionic acid ratio, and significantly reduce the levels of NEFA, BHBA, ALT, and AST in the blood, while increasing GLU levels. This effectively improves the energy metabolism of dairy cows, reduces the burden on the liver, and thus achieves the prevention and treatment of ketosis in dairy cows. The three strains involved in this invention are all isolated from the rumen fluid of healthy dairy cows, are endogenous lactic acid bacteria in dairy cows, have high safety, and no side effects. They can exert their effects through oral administration, avoiding the inconvenience of traditional intravenous glucose supplementation therapy and the metabolic interference risks of hormonal drugs. This makes them suitable for long-term or early intervention during the peripartum period, and the resulting drug can effectively prevent and treat ketosis in dairy cows. Attached Figure Description

[0015] Figure 1 The results of the determination of VFAs and propionic acid content in bovine rumen fluid provided in Example 2 of the present invention are shown in Figure 2. a represents the VFAs content in bovine rumen fluid; b represents the propionic acid content in bovine rumen fluid. Figure 2 The results of measuring the levels of NEFA, BHBA, and GLU in the blood of dairy cows provided in Example 2 of the present invention are shown below; a) represents the NEFA level in the blood of dairy cows; b) represents the BHBA level in the blood of dairy cows; and c) represents the GLU level in the blood of dairy cows. Figure 3 The results of ALT and AST activities in bovine serum provided in Example 2 of this invention are shown below; a represents bovine serum ALT activity; b represents bovine serum AST activity. Figure 4 The results of the determination of VFAs and propionic acid content in bovine rumen fluid provided in Example 3 of the present invention are shown in Figure 3; a represents the VFAs content in bovine rumen fluid; b represents the propionic acid content in bovine rumen fluid. Figure 5 The results of measuring the levels of NEFA, BHBA, and GLU in the blood of dairy cows provided in Example 3 of the present invention are shown below; a) represents the NEFA level in the blood of dairy cows; b) represents the BHBA level in the blood of dairy cows; c) represents the GLU level in the blood of dairy cows. Figure 6 The results of ALT and AST activities in bovine serum provided in Example 3 of the present invention are shown below; a represents bovine serum ALT activity; b represents bovine serum AST activity. Figure 7 The incidence of ketosis one week postpartum was compared between the control group and the compound probiotic group provided in Example 3 of this invention. Figure 1 , 2 Different letters (a, b, c) at the top of columns 1, 2, 3, 4, 5, and 6 indicate significant differences between groups (P < 0.05), while the same letter indicates no significant differences (P > 0.05). Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0017] The specific implementation of the present invention will be described in detail below with reference to specific embodiments. Example 1

[0018] Isolation and Culture: Fresh rumen fluid was collected from healthy adult dairy cows at a dairy farm in Changchun City, Jilin Province, using rumen tubes. After filtration through sterile gauze, 1 mL of the supernatant was serially diluted 10-fold, and 10 samples were selected... -4 100 μL of the diluted solution was evenly spread onto MRS-CaCO3 solid medium. After incubation at 37℃ for 24 h, single colonies with typical calcium dissolution zones were selected and purified by streak plating. This operation was repeated 3-4 times until the colony morphology and microscopic observation were completely consistent, thus obtaining a pure culture.

[0019] Screening of high-VFAs-producing lactic acid bacteria: This invention evaluated the in vitro VFAs-producing performance of several isolated rumen-derived lactic acid bacteria strains. By comparing the total VFAs yield and composition in the fermentation broth of each strain, three lactic acid bacteria strains with significant acid-producing advantages were successfully screened and named L5, G41, and S1, respectively, for subsequent research. Preservation: Select purified L5, G41 and S1 single colonies and inoculate them into MRS liquid medium. After culturing at 37℃ for 24 h, obtain bacterial suspension. Take 1 mL of bacterial suspension and centrifuge in a 1.5 mL centrifuge tube (3000 rpm, 5 min). Discard the supernatant. Take the bacterial sludge, add sterile physiological saline, centrifuge (3000 rpm, 5 min) and wash to obtain washed bacterial cells. Repeat the above operation 3 times. Add sterile 50% glycerol to the obtained bacterial cells and store at -80℃. 16S rRNA gene sequence homology analysis: Purified L5, G41 and S1 single colonies were picked and inoculated into MRS liquid medium. After incubation at 37℃ for 24 h, 1 mL of bacterial solution was sent to Jilin Kumei Biotechnology Co., Ltd. for 16S rRNA sequencing. The obtained gene sequences were compared with BLAST homology in the NCBI database. L5 was identified as Weisslerella. Weissella jogaejeotgaliIt was named Weissella L5. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35101 and deposit date of July 4, 2025. The nucleotide sequence of the 16S rRNA of this strain is shown in SEQ ID NO. 1. G41 was identified as Pediococcus pentosaceus. Pediococcus pentosaceus It was named Pediococcus pentosaceus G41. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33270 and deposit date of January 2, 2025. The nucleotide sequence of the 16S rRNA of this strain is shown in SEQ ID NO. 2. S1 was identified as Lactobacillus paracasei. Lactobacillus paracasei The strain was named *Lactobacillus paracasei* S1. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33269 and deposit date of January 2, 2025. The nucleotide sequence of the 16S rRNA of this strain is shown in SEQ ID NO. 3.

[0020] Preparation of *Weissella vesicanthizobium* L5, *Pediococcus pentosaceus* G41, and *Lactobacillus paracasei* S1 cells The isolated *Westernella* L5, *Pediococcus pentosaceus* G41, and *Lactobacillus paracasei* S1 were inoculated at a 10% inoculum into fresh MRS liquid medium and cultured at 37°C under anaerobic conditions for 24 h. After centrifugation, the bacterial cells were collected and the supernatant was discarded. The bacterial cells were washed twice with PBS buffer and resuspended in 0.9% physiological saline before gavage. The bacterial concentration was adjusted to no less than 1 × 10⁻⁶. 9 CFU / mL.

[0021] Preparation of compound probiotics Take the *Westernella* L5, *Pediococcus pentosaceus* G41, and *Lactobacillus paracasei* S1 suspensions prepared in the above operations, and mix them in aseptic containers at different ratios (see Table 1 for specific ratios) to achieve a final bacterial concentration of 1×10⁻⁶. 9 CFU / mL, and incubated at 37℃ for 24 h. After incubation, the fermentation broth of each group was collected by centrifugation, and the supernatant was collected. The content of VFAs in the fermentation broth was determined by gas chromatography, and the relative level of VFAs in different groups was calculated (based on the compound probiotic group).

[0022] result The acid production effects of each group are shown in Table 1. When Weisslerella L5, Pediococcus pentosaceus G41, and Lactobacillus paracasei S1 were combined in pairs to prepare compound probiotics, the content of VFAs in the fermentation broth was not significantly different from that of the single strains. When the viable cell ratio of Weisslerella L5, Pediococcus pentosaceus G41, and Lactobacillus paracasei S1 was 3:2:2, the content of VFAs in the supernatant of the culture medium was significantly higher than that of the single strains and other compound groups, indicating that the synergistic effect between strains was optimal under this ratio, which is the optimal ratio of the present invention.

[0023] Table 1. Relative levels of VFAs in the supernatant of different culture media groups

[0024] Note: In the table, the ratio of Weissella L5 + Pediococcus pentosaceus G41 + Lactobacillus paracasei S1 in the compound probiotics is 3:2:2, and the ratio of Weissella L5 + Pediococcus pentosaceus G41, Weissella L5 + Lactobacillus paracasei S1, and Pediococcus pentosaceus G41 + Lactobacillus paracasei S1 is 1:1. Different letters (a, b) in the table indicate significant differences between groups (P < 0.05), and the same letter indicates no significant differences (P > 0.05). Example 2 Evaluation of the therapeutic effect of compound probiotics on ketosis in dairy cows:

[0025] Experimental animals: Blood samples were collected from the tail vein of dairy cows 3-9 days postpartum to detect BHBA levels in the blood. Fifty dairy cows with blood BHBA concentrations greater than 1.2 mmol / L and without diseases such as abomasal displacement, mastitis, retained placenta, or postpartum paralysis were selected and randomly divided into four groups: ketosis group (ketosis cows + saline), Weissella L5 group (ketosis cows + Weissella L5), Pediococcus pentosaceus G41 group (ketosis cows + Pediococcus pentosaceus G41), Lactobacillus paracasei S1 group (ketosis cows + Lactobacillus paracasei S1), and compound probiotic group (ketosis cows + compound probiotics), with 10 cows in each group. Dosage regimen: Cows in the *Vibrio vulnificus* L5 group, *Pediococcus pentosaceus* G41 group, *Lactobacillus paracasei* S1 group, and the compound probiotic group were administered the bacterial suspension prepared in Example 1 via gavage before morning feeding. Cows in the ketosis group were administered an equal volume of physiological saline via gavage. The bacterial suspension was prepared using 0.9% physiological saline as a solvent, and the total viable bacteria concentration was adjusted to not less than 1 × 10⁻⁶. 9 The dosage was 500 mL per head per oral gavage. After grouping, the cows were administered the medication continuously for two weeks, once every other day (i.e., once every 48 hours), for a total of 7 doses. During the administration period, the cows had free access to feed and water, and the feeding and management conditions were kept consistent across all groups. Rumen fluid collection and testing: Rumen fluid was collected from dairy cows the morning before feeding, the day after the last administration of medication. The fluid was filtered through four layers of gauze and stored at -20°C for later use. The content of VFAs and propionic acid in the rumen fluid was determined. Blood collection and testing: Blood samples were collected from dairy cows the morning before feeding, the day after the last administration of medication. After standing for 4 hours, the samples were centrifuged and serum was separated. The levels of BHBA, NEFA, GLU, ALT, and AST in the blood were measured.

[0026] Rumen fermentation index results as follows Figure 1 As shown, compared with ketotic cows administered physiological saline, the rumen contents of VFAs and propionic acid were significantly increased in ketotic cows administered Weissella L5, Pediococcus pentosaceus G41, Lactobacillus paracasei S1, and a compound probiotic.

[0027] Results of dairy cow energy metabolism indicators as follows Figure 2 As shown, compared with ketotic cows administered saline, ketotic cows administered Weissella L5, Pediococcus pentosaceus G41, Lactobacillus paracasei S1, and a compound probiotic showed significantly lower levels of NEFA and BHBA in their blood, while GLU levels were significantly higher.

[0028] Results of liver function indicators in dairy cows as follows Figure 3 As shown, compared with ketotic dairy cows administered physiological saline, the ALT and AST activities in the blood of ketotic dairy cows administered Weissella L5, Pediococcus pentosaceus G41, Lactobacillus paracasei S1, and compound probiotics were significantly reduced. Compared with ketotic dairy cows administered Weissella L5, Pediococcus pentosaceus G41, and Lactobacillus paracasei S1, the serum ALT and AST activities of ketotic dairy cows administered compound probiotics were significantly reduced.

[0029] In summary, the compound probiotics described in this invention can optimize rumen fermentation patterns, increase VFA production, provide dairy cows with more energy metabolism substrates, thereby reducing blood NEFA and BHBA levels and effectively alleviating the negative energy balance in ketotic dairy cows. At the same time, the reduction in ALT and AST levels suggests that the compound probiotics have a protective effect on liver function and can reduce liver damage in ketotic dairy cows.

[0030] Compared with single strains, the compound probiotics described in this invention have significant advantages in increasing VFA production, improving energy metabolism indicators, and protecting liver function, exhibiting a clear synergistic effect. This indicates that the compound probiotics can effectively alleviate negative energy balance in dairy cows and have broad application prospects in the treatment of ketosis in dairy cows. Example 3 Evaluation of the preventive effect of compound probiotics on ketosis in dairy cows:

[0031] Experimental animals: Sixty healthy dairy cows two weeks before calving were randomly selected from the pasture and randomly divided into two groups: a control group (healthy dairy cows + saline) and a compound probiotic group (healthy dairy cows + compound probiotics), with 30 cows in each group.

[0032] Dosage regimen: Cows in the compound probiotic group were administered the compound probiotic suspension prepared in Example 1 via gavage before morning feeding, while cows in the control group were administered an equal volume of physiological saline via gavage. The bacterial suspension was prepared using 0.9% physiological saline as a solvent, and the total viable bacteria concentration was adjusted to 1×10⁻⁶. 9 The dosage was 500 mL per head per oral gavage. After grouping, the cows were administered the medication continuously for two weeks, once every other day (i.e., once every 48 hours), for a total of 7 doses. During the administration period, the cows had free access to feed and water, and all groups maintained the same feeding and management conditions.

[0033] Statistical analysis of ketosis incidence: Blood samples were collected from the tail vein of dairy cows on the morning of the 7th day postpartum before feeding. After standing for 4 hours, the samples were centrifuged, and the serum was separated to determine the BHBA concentration. The incidence of ketosis in each group of dairy cows was calculated using BHBA > 1.2 mmol / L as the diagnostic criterion.

[0034] Rumen fluid collection and testing: Rumen fluid was collected from dairy cows on the morning of the 7th day postpartum before feeding. After filtration through four layers of gauze, the filtrate was stored at -20℃ for later use. The content of VFAs and propionic acid in the rumen fluid was determined.

[0035] Blood collection and testing: Blood samples were collected from the tail vein of dairy cows on the morning of the 7th day after calving, before feeding. After standing for 4 hours, serum was separated and the levels of BHBA, NEFA, GLU, ALT and AST in the blood were tested.

[0036] Results of rumen fermentation indicators in dairy cows as follows Figure 4 As shown, compared with the control group, the contents of VFAs and propionic acid in the rumen of dairy cows that were given compound probiotics by gavage were significantly increased; Results of dairy cow energy metabolism indicators as follows Figure 5 As shown, compared with the control group, the levels of NEFA and BHBA in the blood of dairy cows that were administered compound probiotics were significantly reduced, while the level of GLU was significantly increased. Results of liver function indicators in dairy cows as follows Figure 6As shown, compared with the control group, the blood ALT and AST activities of dairy cows that were administered compound probiotics were significantly reduced. The incidence rate of ketosis in dairy cows is as follows: Figure 7 As shown, the incidence of ketosis in the control group was 16.67%, while the incidence of ketosis in dairy cows that were given compound probiotics was only 6.67%. The above results indicate that the compound probiotic can optimize rumen fermentation patterns, increase VFA production, reduce NEFA and BHBA levels in dairy cows, increase GLU levels, and effectively alleviate the negative energy balance in periparturient dairy cows.

[0037] In summary, the results of in vivo experiments on dairy cows show that oral administration of the compound probiotics described in this invention can effectively increase rumen VFA production, improve the energy metabolism status of dairy cows, and reduce liver damage, effectively preventing and treating ketosis in dairy cows.

[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound probiotic, characterized in that: Composed of a mixture of Weissella L5, Pediococcus pentosaceus G41 and Lactobacillus paracasei S1 in a specific ratio: The viable count ratio of Weissella L5: Pediococcus pentosus G41: Lactobacillus paracasei S1 is 1~3:1~3:1~3; The aforementioned Weissella L5 is deposited at the China General Microbiological Culture Collection Center of the China Microbiological Culture Collection Committee and is classified and named Weissella. Weissella jogaejeotgali The accession number is CGMCC No. 35101, and the accession date is July 4, 2025; The aforementioned *Pediococcus pentosaceus* G41 is deposited at the China General Microbiological Culture Collection Center, and is classified and named *Pediococcus pentosaceus*. Pediococcus pentosaceus The accession number is CGMCC No. 33270, and the accession date is January 2, 2025; The aforementioned *Lactobacillus paracasei* S1 is deposited at the China General Microbiological Culture Collection Center, and is classified and named *Lactobacillus paracasei*. Lactobacillus paracasei The accession number is CGMCC No.33269, and the accession date is January 2, 2025.

2. The compound probiotic as described in claim 1, characterized in that: The viable cell ratio of Weissella L5: Pediococcus pentosus G41: Lactobacillus paracasei S1 was 3:2:

2.

3. A compound probiotic as described in claim 1 or 2, characterized in that: The effective live bacteria count of the compound probiotics is not less than 1×10⁻⁶. 9 CFU / mL.

4. A method for preparing a compound probiotic as described in claim 1 or 2, characterized in that... Includes the following steps: Suspensions of *Westernella* L5, *Pediococcus pentosaceus* G41, and *Lactobacillus paracasei* S1 were taken separately and mixed according to the live bacteria content ratio to achieve a final bacterial concentration of 1×10⁻⁶. 9 The concentration of CFU / mL was increased and the mixture was incubated at 37℃ for 24 h. After the incubation period, the fermentation broth was collected.

5. The application of the compound probiotic as described in claim 1 or 2 in the preparation of a formulation for preventing and treating ketosis in dairy cows.

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