Wehonor coccus and application thereof in relieving subacute rumen acidosis of ruminants

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a Wehonor coccus strain and application thereof in relieving subacute rumen acidosis of ruminants. The invention relates to a Villonella sp. Strain RGHV1, the Villonella sp. Strain is preserved in China General Microbiological Culture Collection Center on January 21, 2025, and the preservation number of the Villonella sp. Strain is CGMCC NO.33487. The Villonella sp. Strain is a Villonella sp. Strain RGHV1. The Villonella sp. Strain is a Villonella sp. Strain RGHV1. The Villonella sp. Strain RGHV1. The Wehonor coccus RGHV1 is separated from the rumen fluid of Hu sheep, the strain name is undefined and is not fully researched before, and the research on the strain finds that the Wehonor coccus RGHV1 has the characteristics of fast growth and strong lactic acid degradation capacity, can metabolize lactic acid to generate acetic acid and propionic acid, and has the advantages of high yield, high yield and the like. By inoculating the low-concentration strain, the lactic acid level in in-vitro fermentation rumen fluid can be obviously reduced, the generation of VFA (Vitamin Fatty Acid) is promoted, and compared with the peste des petits ruminants subsp.lactis, the peste des petits ruminants subsp.lactis has stronger capability of reducing rumen lactic acid.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Veillonella and application thereof in alleviating subacute rumen acidosis in ruminants. Background Art

[0002] The rumen microbes of ruminants have co-evolved with their hosts, enabling a dynamic balance between acid production and acid utilization during rumen degradation of feed. However, in intensive farming models, the amount of concentrate fed is often increased in pursuit of rapid animal growth or high milk production. High-concentrate feeds ferment rapidly in the rumen, producing large amounts of volatile fatty acids (VFA) and lactic acid that exceed the rumen's absorption and buffering capacity, leading to ruminal acidosis. Among these, subacute ruminal acidosis (SARA) has become a common nutritional metabolic disease that affects animal health and the economic benefits of farming. Developing probiotics for rumen lactic acid-utilizing bacteria may be one of the effective ways to alleviate SARA.

[0003] When animals experience acute acidosis, rumen lactate concentrations can rise to 40-50 mmol / L, or even exceed 120 mmol / L. In animals experiencing SARA, lactate concentrations remain similar to or slightly higher than normal, ranging from 0-5 mmol / L. Although rumen lactate concentrations remain relatively unchanged during SARA, the bacterial composition associated with lactate metabolism has shifted. Restoring rumen microbial homeostasis by inhibiting lactate-producing bacteria and promoting lactate-utilizing bacteria is a key strategy for mitigating SARA. Direct feeding or inoculation of lactate-utilizing bacteria is the most direct way to increase the number of lactate-utilizing bacteria. However, less than 10% of rumen bacteria have been cultured, and the available lactate-utilizing bacteria are limited, primarily Megasphaera elsdenii and Selenomonas ruminantium subsp. lactilytica. Obtaining pure cultures of different lactate-utilizing bacteria through cultivation could provide a more diverse bacterial resource for SARA mitigation. Summary of the Invention

[0004] The present invention provides a Veillonella strain, which has fast growth and strong ability to utilize lactic acid. When inoculated at a low concentration, the strain can promote rumen lactic acid metabolism, regulate rumen fermentation status, and achieve the effect of alleviating SARA.

[0005] The technical solution adopted by the present invention to solve its technical problem is:

[0006] A strain of Veillonella sp., identified as the unidentified species, strain RGHV1, was deposited on January 21, 2025, at the General Microbiology Center of the China Culture Collection Administration (CGMCC, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China) under the accession number CGMCC No. 33487. This strain has a rapid growth rate and a strong ability to break down lactic acid. Even at low concentrations, it can enhance rumen lactic acid metabolism, regulate rumen fermentation, and alleviate SARA.

[0007] A Veillonella gene encoding the Veillonella strain, the complete nucleotide sequence of its 16S rRNA gene is shown in SEQ ID NO.1.

[0008] A bacterial agent containing the Veillonella of the present invention, comprising the Veillonella strain RGHV1 of the present invention, or a fermentation product thereof. Further, the concentration of Veillonella RGHV1 in the bacterial agent is 10 5 to 10 8 In order to control the cost, the concentration of Veillonella RGHV1 in the bacterial agent was 10 5 Furthermore, the fermentation product of the present invention can be a fermentation product containing Veillonella RGHV1 itself, or a fermentation product not containing Veillonella RGHV1 itself (such as fermentation supernatant).

[0009] A ruminant feed additive comprising the Veillonella or the bacterial agent of the present invention. 5 The added amount of 1000 copies / mL is mixed into conventional animal feed, milk replacer, drinking water, etc. and fed to animals, wherein the animals here are preferably ruminants.

[0010] A use of the Veillonella or the bacterial agent of the present invention in alleviating subacute rumen acidosis in ruminants.

[0011] A use of the Veillonella or the bacterial agent of the present invention in enhancing rumen lactic acid metabolism and regulating rumen fermentation in ruminants.

[0012] Preferably, the ruminants are cattle, sheep and camels.

[0013] An animal medicine prepared from the Veillonella or the bacterial agent of the present invention.

[0014] The beneficial effects of the present invention are:

[0015] The present invention isolated a strain of Veillonella RGHV1 from the rumen fluid of Hu sheep. The strain's species name is undetermined and previously understudied. Studies on this strain have revealed its rapid growth and strong ability to degrade lactic acid, metabolizing lactic acid to produce acetic and propionic acids. Inoculation of low concentrations of this strain significantly reduced lactic acid levels in in vitro fermented rumen fluid and promoted the production of VFAs. This strain has a stronger ability to reduce rumen lactic acid than Lunar Mononas ruminantis subsp. lactolyticus. The Veillonella RGHV1 provided by the present invention can be used as a feed additive for ruminant feeding, effectively regulating rumen fermentation to alleviate SARA. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the growth curve of Veillonella RGHV1;

[0017] Figure 2 This is the lactate metabolism curve of Veillonella RGHV1;

[0018] Figure 3 is a graph of VFA production by Veillonella RGHV1;

[0019] Figure 4 This is a graph showing the effect of Veillonella RGHV1 on lactic acid concentration in an in vitro fermentation system;

[0020] Figure 5 This is a graph showing the effect of Veillonella RGHV1 on the total VFA concentration in the in vitro fermentation system. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described in detail below through specific examples. It should be understood that the implementation of the present invention is not limited to the following examples, and any form of modification and / or change made to the present invention will fall within the scope of protection of the present invention.

[0022] In the present invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used are commercially available or commonly used in the art. The methods in the following examples are all conventional methods in the art unless otherwise specified.

[0023] Unless otherwise specified, the reagents used in the following examples can be purchased from conventional biochemical reagent stores. The formula of GB2M medium is as follows: 1.5 g / L tryptone, 1.47 g / L casein hydrolysate, 0.49 g / L sodium acetate, 9.93 g / L peptone, 6.02 g / L casein peptone, 2.406 g / L soy peptone, 8.127 g / L digested serum powder, 1.1956 g / L bovine liver extract powder, 0.30 g / L polypeptone, 3.7 g / L yeast extract, 2.319 g / L beef extract, 3.306 g / L glucose, 0.15 mL / L Tween 80, 0.15 g / L maltose, 0.15 g / L cellobiose, 0.3306 g / L soluble starch, 0.15 mL / L glycerol, 0.60 g / L K2HPO4, 1.505 g / L KH2PO4, 1.806 g / LNaCl, 0.0903 g / L sodium mercaptoglycolate, 0.2116 g / L cysteine ​​hydrochloride, 1.00 mL / L 0.1% resazurin, 12 mL / L saline solution (saline solution: 0.25 g / L CaCl2·2H2O, 0.50 g / L MgSO4·7H2O, 1.00 g / L K2HPO4, 1.00 g / L KH2PO4, 10.00 g / LNaHCO3, 2.00 g / LNaCl).

[0024] Selenomonas ruminantium subspecies lactolyticus (Selenomonas ruminantium) RGHS1, this strain was deposited in the General Microbiology Center of China Culture Collection Administration on January 21, 2025, and its deposit number is: CGMCCNO.33486.

[0025] Example 1 Isolation and Identification of Veillonella RGHV1

[0026] Veillonella RGHV1 was isolated by the following method:

[0027] 1. Rumen fluid was collected from Hu sheep using an oral catheter and stored in anaerobic glycerol vials. After being transported back to the laboratory at 4°C, it was stored at -80°C for long-term storage.

[0028] 2. Thaw the frozen Hu sheep rumen fluid and inoculate it into a Balch tube filled with GB2M medium. Place it horizontally in a shaker at 39°C and 60 rpm and culture for 4 days. After the enrichment is completed, take the bacterial solution and dilute it step by step with fresh GB2M medium. 6 -10 8 The diluted bacterial solution was aliquoted into 96-well cell culture plates, with 160 μL dispensed into each well. The plates were then covered and sealed with parafilm. The 96-well plates were incubated in an anaerobic incubator at 39°C for 4 days. After incubation, 10 μL of the solution from the wells of the 96-well plates with a bacterial count of 30-50% was removed for bacterial identification.

[0029] 3. Take 1 μL of bacterial solution as a template and amplify the 16S rRNA gene using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO. 2) and 1492R (5'-TACGGCTACCTTGTTACGACTT-3', SEQ ID NO. 3). Send the amplified product for sequencing and perform BLAST comparison of the sequencing results against the NCBI database to identify the strain.

[0030] 4. The bacterial liquid identified as Veillonella was streaked and purified three times on GB2M solid medium. The purified single colony was inoculated into GB2M liquid medium for expansion and culture to obtain a pure culture. The pure culture was amplified again to obtain a sequence with a fragment length of 1441 bp, as shown in SEQ ID NO.1. After sequencing and alignment, the unidentified species Veillonella sp. was obtained and named RGHV1. Example 2 Determination of the growth curve, lactate metabolism curve, and VFA metabolism curve of Veillonella RGHV1

[0031] 1. A pure culture of Veillonella RGHV1 was inoculated at a 1% (v / v) ratio into GB2M medium supplemented with 30 mmol / L lactate and cultured horizontally at 39°C, 60 rpm in a shaker. Three replicates were set up for each group. OD600 values ​​were measured at 0, 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, 36, 42, 48, and 60 h, and growth curves were plotted. At 0, 4, 8, 12, 16, 20, 24, 30, 36, 42, 48, and 60 h, 1 mL of the bacterial suspension was aspirated with a syringe. The suspension was centrifuged at 10,000 rpm for 15 min, and the supernatant was collected for determination of lactate and VFA concentrations.

[0032] 2. Growth curve Figure 1 As shown, bacterial growth curves generally can be divided into four phases: hysteresis phase, logarithmic phase, plateau phase, and decay phase. The hysteresis phase of Veillonella RGHV1 is 0-2 hours, and the logarithmic phase is 2-16 hours, when the OD600 reaches above 1.9. After 16 hours, the growth enters the plateau phase and does not enter the decay phase within 60 hours. Veillonella RGHV1 grows rapidly and can achieve a high biomass.

[0033] 3. Lactate metabolism curve Figure 2 As shown in the figure, Veillonella RGHV1 metabolized 28.3mmol / L lactic acid within 16 hours. Veillonella RGHV1 has a strong ability to metabolize lactic acid. The VFAs produced are as follows: Figure 3 As shown, the VFAs produced are mainly acetic acid and propionic acid.

[0034] Example 3: Effect of Veillonella RGHV1 on SARA

[0035] 1. Preparation of in vitro gas-producing inoculant. Veillonella RGHV1 and Lunar Mononas ruminantia subsp. lactis RGHS1 (the isolation and purification process for this strain is the same as that for Veillonella RGHV1; this subspecies is an important lactic acid-utilizing bacterium in the rumen and has been shown to alleviate SARA; this strain is used as a positive control in the present invention) frozen at -80°C were inoculated at a 1% (v / v) ratio into GB2M medium and incubated at 39°C at 60 rpm for 15 hours for recovery to obtain a single-bacterial inoculant. Veillonella RGHV1 and Lunar Mononas ruminantia RGHS1 frozen at -80°C were mixed at equal volumes, inoculated at a 1% (v / v) ratio into GB2M medium, and incubated at 39°C at 60 rpm for 15 hours for recovery to obtain a mixed-bacterial inoculant. Real-time fluorescence quantitative PCR was performed using total bacterial quantification primers F (5'-CGGCAACGAGCGCAACCC-3', SEQ ID NO. 4) and R (5'-CCATTGTAGCACGTGTGTAGCC-3', SEQ ID NO. 5), Veillonella RGHV1-specific primers F (5'-TACCGCGTACGATTGAGTTCC-3', SEQ ID NO. 6) and R (5'-TAATCAGACGCAACCCCCTC-3', SEQ ID NO. 7), and Selenomonas ruminantia RGHS1-specific primers F (5'-TGAACGGCCACATTGGAACT-3', SEQ ID NO. 8) and R (5'-TTCGTCCCCGTCAACAGAG-3', SEQ ID NO. 9). The 16S rRNA gene copy number of the strain was calculated based on the quantitative relationship between the total bacterial quantification results and the specific primer quantification results, and normalization was performed to determine the copy number of the strain so that one copy is approximately equal to one colony-forming unit. The number of copies of the strain per ml of Veillonella RGHV1 single-bacterial agent was 2.9×10 8 The number of copies of the strain in each milliliter of Lunamonas ruminantia RGHS1 single bacterial inoculum is 1.1×10 8 The bacterial copy number per milliliter of mixed bacterial inoculum was 5.3×10 8 copies, of which the copy number of Veillonella RGHV1 was 2.3×10 8 copies, and the copy number of Lunamonas ruminantia RGHS1 was 3.0×10 8 copies.

[0036] 2. In vitro gas production evaluation: The in vitro gas production was divided into 6 groups, namely, the blank control group without substrate addition, the CON group with a substrate to crude ratio of 30:70, the SARA group with a substrate to crude ratio of 70:30, and the group with Veillonella RGHV1 added to the SARA group (RGHV1 group, the final concentration of Veillonella RGHV1 single bacterial agent inoculation was 1.0×10 5 copies / mL), the group supplemented with Selenomonas ruminantia RGHS1 based on the SARA group (RGHS1 group, the final inoculation concentration of Selenomonas ruminantia RGHS1 was 1.0×10 5 copies / mL), the co-cultured group of Veillonella RGHV1 and Lunamonas ruminantia RGHS1 based on the SARA group (RGHV1+RGHS1 group, the final concentration of the mixed bacterial inoculum was 1.0×10 5 100 copies / mL), with 4 replicates per group. Before morning feeding, rumen fluid from Hu sheep was collected using an oral catheter and quickly brought back to the laboratory in a preheated thermos cup. The rumen fluid was filtered through four layers of gauze in an anaerobic incubator, and the rumen fluid and artificial saliva were thoroughly stirred and mixed in a ratio of 1:9 to obtain artificial rumen fluid. 50 mL of artificial rumen fluid was dispensed into a gas-producing bottle using a dispenser. Plug in a rubber stopper and cover with an aluminum cap, and gently shake the gas-producing bottle to mix the inoculated bacterial solution and artificial rumen fluid evenly, while trying to avoid the fermentation substrate sticking to the bottle wall. Use a needle to release the gas in the gas-producing bottle, and start fermentation when the air pressure is 0. The gas-producing bottle was placed in a 39°C incubator for fermentation, and the lactic acid concentration and VFA concentration were measured at 2, 4, 6, 8, 12, 24, 36, 48, and 72 hours of fermentation.

[0037] 3. Changes in lactic acid concentration in the in vitro gas production system Figure 4The lactate concentrations in each group varied between 0 and 5 mmol / L. Except for the CON group, which showed a gradual decrease in lactate concentration, the lactate concentrations in the other groups showed an increase first and then a decrease. The lactate concentrations in the RGHV1 group and the RGHV1+RGHS1 group reached their highest concentrations of 1.6 mmol / L and 2.1 mmol / L, respectively, at the 4th hour of fermentation and then began to decrease. The lactate concentrations in the SARA group and the RGHS1 group both reached their highest concentrations of 4.2 mmol / L at the 8th hour of fermentation. During the 4th to 12th hour of fermentation, lactate concentrations were significantly lower than those in the SARA and RGHS1 groups (P < 0.05). After 8 hours of fermentation, lactate concentrations in the RGHV1 group decreased to 0.4 mmol / L, which was not significantly different from the CON group (P > 0.05). After 12 hours of fermentation, lactate concentrations in the RGHV1+RGHS1 group decreased to 0.1 mmol / L, which was not significantly different from either the CON or RGHV1 groups (P > 0.05). After 24 hours of fermentation, lactate concentrations in all groups were below 0.2 mmol / L. Veillonella RGHV1 has a strong ability to enhance ruminal lactate metabolism and regulates ruminal lactate metabolism more effectively than a co-culture of Veillonella RGHV1 and Lunar Newcomenas ruminantia RGHS1, and more effectively than the positive control Lunar Newcomenas ruminantia RGHS1.

[0038] 4. At 72h of fermentation, the total VFA concentration in the in vitro gas production system is as follows: Figure 5 After 72 hours of fermentation, the total VFA concentration in the RGHV1 group was significantly higher than that in the RGHS1 group, SARA group, RGHV1+RGHS1 group, and CON group (P < 0.05). The total VFA concentration in the RGHS1 group was significantly higher than that in the SARA group, RGHV1+RGHS1 group, and CON group (P < 0.05). The total VFA concentration in the RGHV1+RGHS1 group did not differ significantly from that in the SARA group or CON group (P > 0.05). The concentrations of various VFAs in the in vitro gas production system at 72 hours of fermentation are shown in Table 1. After 72 hours of fermentation, the concentrations of all VFAs in the RGHV1 group were significantly higher than those in the SARA group and the RGHV1+RGHS1 group (P < 0.05). Except for propionic acid, which was numerically higher in the RGHS1 group (P > 0.05), all other VFA concentrations were significantly higher in the RGHS1 group (P < 0.05). The concentrations of all VFAs in the RGHS1 group were numerically higher than those in the SARA group (P > 0.05). The concentrations of isobutyric, valeric, and isovaleric acids in the RGHV1+RGHS1 group were significantly higher than those in the SARA group (P < 0.05), while the concentrations of acetic, propionic, and butyric acids were not significantly different from those in the SARA group (P > 0.05). Veillonella RGHV1 promoted VFA production and had a strong ability to regulate rumen fermentation, a capability that was stronger than that of a co-culture of Veillonella RGHV1 and Lunaromonas ruminantia RGHS1, and stronger than that of Lunaromonas ruminantia RGHS1.

[0039] Table 1

[0040]

[0041] abcd Indicates significant differences among groups in the same row (P<0.05); SEM is standard error.

[0042] Example 4 Comparison of the SARA-mitigating effects of Veillonella RGHV1 and other lactic acid decomposing bacteria

[0043] 1. Studies by Wiryawan et al. [1], Long Liming et al. [2], and Mao Shengyong et al. [3] have shown that Lunamonas ruminans, especially Lunamonas ruminans lactolyticus, can promote the degradation of rumen lactic acid and alleviate rumen acidosis. In the present invention, Lunamonas ruminans RGHS1 was used as a positive control and a low concentration of 1.0×10 5 Copies / mL of Selenomonas ruminantia RGHS1 did not significantly regulate rumen lactate, while the same concentration of Veillonella RGHV1 significantly reduced rumen lactate and promoted the production of various VFAs. This suggests that low concentrations of Veillonella RGHV1 can effectively regulate rumen fermentation and alleviate SARA.

[0044] 2. Kung et al. [4] showed that the final inoculation concentration in the in vitro rumen fermentation system was 8.7×10 6 CFU / mL of Megasphaera elsdenii B159 can effectively reduce the concentration of lactic acid in the acute acidosis model and maintain the lactic acid concentration below 2 mmol / L; while reducing the inoculum size of Megasphaera elsdenii B159 to 8.7×10 5 CFU / mL, the lactic acid concentration will rise to 25mmol / L, and the effect of relieving acidosis will be weakened. Xia Guangliang [5] showed that in the in vitro rumen acidosis model, the final concentration of about 3.3×10 6 CFU / mL of Megasphaera elsdenii ATCC25940 can reduce lactic acid concentration and alleviate rumen acidosis. In the present invention, the final inoculation concentration of Veillonella RGHV1 is 1.0×10 5 copies / mL, which is at a low level compared with the literature, can show a good effect of reducing rumen lactic acid concentration and alleviating acidosis.

[0045] In summary, Veillonella RGHV1 has the ability to grow rapidly and metabolize lactic acid. Inoculating a low concentration of this bacterium can enhance the lactic acid decomposition ability of the rumen, promote the production of various VFAs, and regulate rumen fermentation. The effect is better than the co-culture of Veillonella RGHV1 and Lunamonas ruminans RGHS1, and better than the commonly used Lunamonas ruminans lactolyticus subspecies. Therefore, it can be used to alleviate SARA in ruminants. The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0046] The above is a detailed introduction to a strain of Veillonella provided by the present invention and its application in alleviating SARA. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only intended to help understand the method of the present invention and its core concept. It should be noted that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0047] References

[0048] [1] Wiryawan KG, and Brooker JD (1995). Probiotic control oflactateaccumulation in acutely grain-fed sheep[J]. Australian Journal of AgriculturalResearch, 46(8):1555-1568.

[0049] [2] Long Liming, Mao Shengyong, Su Yong, Zhu Weiyun (2008). Isolation and identification of a rumen-derived lactic acid-utilizing bacterium and its in vitro metabolic characteristics [J]. Acta Microbiologica Sinica, 12: 1571-1577.

[0050] [3] Mao Shengyong, Long Liming, Zhu Weiyun (2010). In vitro study on the effects of Lucanthias ruminants and its combination with yeast on rumen microbial fermentation [J]. Acta Prataculturae Sinica, 19(4): 176-186.

[0051] [4]Kung L.Jr.,and Hession AO(1995)Preventing in vitro lactateaccumulation in ruminal fermentations by inoculation with Megasphaeraelsdenii[J].Journal ofAnimal Science,73(1),250-256.

[0052] [5] Xia Guangliang (2020). Lactic acid utilization mechanism of lactic acid metabolizing bacteria in the rumen and its regulatory effect on ruminal acidosis [M]. Yangzhou University.

Claims

1. A strain of Veillonella sp., which is Veillonella sp. strain RGHV1. The strain was deposited at the General Microbiology Center of the China Culture Collection Administration on January 21, 2025, and its deposit number is: CGMCCNO.33487.

2. A bacterial agent, characterized in that The bacterial agent comprises the Veillonella according to claim 1, or a fermentation product thereof.

3. The microbial agent according to claim 2, characterized in that The concentration of Veillonella strain RGHV1 in the bacterial agent is 10 5 to 10 8 copies / mL.

4. A ruminant feed additive comprising the Veillonella according to claim 1 or the bacterial agent according to claim 2.

5. Use of the Veillonella according to claim 1 or the bacterial agent according to claim 2 in alleviating subacute ruminal acidosis in ruminants.

6. Use of the Veillonella according to claim 1 or the bacterial agent according to claim 2 for enhancing rumen lactic acid metabolism and regulating rumen fermentation in ruminants.

7. The use according to claim 5 or 6, characterized in that The ruminants are cattle, sheep and camels.

8. An animal medicine prepared from the Veillonella according to claim 1 or the bacterial agent according to claim 2.

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