A novel bacterium for mass production of propionic acid precursor

Abstract

To provide a bacterium that enables enhancement of propionic acid production and reduction of methane production in the rumen of domestic ruminants.SOLUTION: Provided is a bacterium of genus Prevotella having such a property that the proportion of malic acid and lactic acid in organic acids in fermentation products is 20% or higher.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a new bacterium, Prevotella lacticifex, belonging to the genus Prevotella, and a feed additive and feed using the same. [Background technology]

[0002] Feed ingredients such as pasture ingested by ruminant livestock are decomposed and fermented by microorganisms living in the first stomach and converted into organic acids such as acetic acid and propionic acid. Among the organic acids, propionic acid is converted into glucose in the bodies of ruminant livestock and becomes the main energy source for ruminant livestock, so it is a fermentation product that is key to improving livestock feed efficiency. In addition, it is known that propionic acid production in the first stomach competes with methane (a greenhouse gas) production from the perspective of hydrogen utilization, and there are high expectations for enhancing propionic acid production as a technology to reduce methane emissions from ruminant livestock.

[0003] Techniques for increasing propionic acid have been implemented by increasing the amount of high-energy feed such as grain feed and feeding antibacterial substances such as monensin (Non-Patent Document 1). Techniques for reducing methane have also been developed, such as switching to high-energy feed, reducing the number of heads as a result of improving productivity, and using chemical substances such as 3-nitrooxypropanol (3-N0P) (Non-Patent Document 2), but none of these techniques involve live bacterial materials that can change the rumen microflora. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Methanogens: methane producers of the rumen and mitigation strategies (2010) Sarah E Hook, Andre-Denis G Wright, Brian W McBride. Archaea. doi: 10.1155 / 2010 / 945785. [Non-Patent Document 2] Effects of 3-nitrooxypropanol on rumen fermentation, lactational performance, and resumption of ovarian cyclicity in dairy cows (2020) Melgar A., ​​MTHarper, J.Oh, F.Giallongo, MEYoung, TLOtt, S.Duval, ANHristov. Journal of Dairy Science 103(1):410-432. doi: 10.3168 / jds.2019-17085. Summary of the Invention [Problem to be solved by the invention]

[0005] Feeding grain feed has already been established as a common technique, and further increases in feeding can lead to the occurrence of production diseases such as acidosis, so increases in its use are limited. The use of antibacterial substances has been restricted worldwide due to concerns about the emergence of drug-resistant bacteria. There are probiotics that use Bacillus bacteria to improve the rumen fermentation and intestinal microflora of ruminant livestock, such as Calsporin (Asahi Calpis Wellness Co., Ltd.), Bobactin (Miririsan Pharmaceutical Co., Ltd.), and Lumisan (Nissan Synthetic Industries Co., Ltd.). However, all of these are probiotics aimed at improving the balance of the microflora to increase productivity, and are not aimed at modifying specific fermentation such as high propionic acid production or low methane production, so it is not expected to increase propionic acid production or reduce methane production.

[0006] The present invention has been made under such circumstances, and an object of the present invention is to provide a microorganism that enables enhanced propionic acid production and reduced methane production in the rumen of ruminant livestock. [Means for solving the problem]

[0007] As a result of intensive research to solve the above problems, the present inventors have found that bacteria isolated from cows that produce a lot of propionic acid but little methane produce a high proportion of precursors of propionic acid, and among the precursors, produce malic acid and lactic acid in particularly large amounts. Malic acid and lactic acid are metabolized to propionic acid by consuming hydrogen, but methanogens also consume hydrogen to produce methane. Therefore, it is believed that the above bacteria that produce large amounts of malic acid and lactic acid not only increase the amount of propionic acid produced, but also suppress methane production by methanogens by reducing the amount of hydrogen.

[0008] The present invention has been completed based on the above findings. That is, the present invention provides the following [1] to

[10] . [1] A bacterium of the genus Prevotella, characterized in having 16S rDNA consisting of a base sequence having 98.7% or more homology to the base sequence shown in SEQ ID NO: 10.

[0009] [2] The Prevotella bacterium according to [1], characterized in that the proportion of malic acid and lactic acid in the organic acids in the fermentation product is 20% or more.

[0010] [3] The Prevotella bacterium according to [1], characterized in that the proportion of succinic acid, malic acid, propionic acid, and lactic acid in the organic acids in the fermentation product is 65% or more.

[0011] [4] The Prevotella bacterium according to [1], characterized in that it has the following properties 1) to 6): 1) It is a Gram-stain-negative rod-shaped bacterium that does not form spores. 2) The catalase test is negative and the organism is obligately anaerobic. 3) When D-glucose, lactose, saccharose, maltose, salicin, L-arabinose, cellobiose, D-mannose, or D-raffinose is added, acid production occurs, but when D-mannitol, glycerol, D-melezitose, D-sorbitol, or D-trehalose is added, acid production does not occur. 4) The major menaquinone is MK11, 5) The most predominant fatty acid in the cells is anteiso-C15:0. 6) The GC content of genomic DNA is 49.4%-49.9%.

[0012] [5] The Prevotella bacterium according to [1], characterized in that the ANI value with the strain identified by accession number NITE BP-03463 is 95% or higher.

[0013] [6] The Prevotella bacterium according to [1], characterized in that the dDDH value with the strain identified by accession number NITE BP-03463 is 70% or more.

[0014] [7] The Prevotella bacterium according to [1], characterized in that it has a 16S rDNA consisting of a base sequence shown in any one of SEQ ID NOs: 1 to 11.

[0015] [8] The Prevotella bacterium according to [1], characterized in that it is a strain identified by accession number NITE BP-03463.

[0016] [9] A feed additive comprising the Prevotella bacterium according to any one of [1] to [8].

[0017]

[10] A feed comprising the Prevotella bacterium according to any one of [1] to [8].

[0018] This specification includes the contents described in the specification and / or drawings of Japanese patent application No. 2021-101574, which is the priority basis of this application. Effect of the Invention

[0019] The present invention provides a novel bacterium, Prevotella lacticifex, belonging to the genus Prevotella. This novel bacterium can be used as a feed additive or in feed to enhance propionic acid production and reduce methane production in the rumen of ruminant livestock. [Brief description of the drawings]

[0020] [Figure 1] Figure showing the proportion of fermentation products of the newly isolated Prevotella bacteria. The left figure shows the proportion of propionic acid and its precursors (fermentation products of the hydrogen consumption pathway), and the right figure shows the proportion of fermentation products of the hydrogen production pathway, such as acetic acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present invention will be described in detail below. The Prevotella bacterium of the present invention is characterized in that it has 16S rDNA consisting of a base sequence that has a homology of 98.7% or more to the base sequence shown in SEQ ID NO:10.

[0022] The base sequence shown in SEQ ID NO: 10 is the base sequence of 16SrDNA of Prevotella strain R6014 described later. R6014 is considered to be a new species of the Prevotella genus, and since a group of strains with 16SrDNA homology of 98.7% or more is generally considered to be the same species, the Prevotella bacteria of the present invention includes a new species of the Prevotella genus. This new species did not have a name at the time of filing of Patent Application No. 2021-101574, which is the basis of the priority of this application (June 18, 2021), but was later named Prevotella lacticifex (Shinkai et al., Int. J. Syst. Evol. Microbiol. 2022;72:005278).

[0023] Whether or not they are of the same species is determined not only by the base sequence of 16SrDNA but also by the homology value of DNA-DNA hybridization, and in general, strains with a homology value of 70% or more in a DNA-DNA hybridization test are determined to be of the same species. Therefore, the Prevotella bacteria of the present invention may be bacteria with a homology value of 70% or more with the Prevotella strain R6014 in a DNA-DNA hybridization test.

[0024] Although the homology value of DNA-DNA hybridization (DDH) is experimentally determined, the value varies depending on the level of proficiency, and the experimental accuracy is low. Therefore, in recent years, it has become common to obtain a draft genome by DNA sequencing, and use average nucleotide identity (ANI) analysis or digital DNA-DNA hybridization (dDDH) as an alternative method. In general, a group of strains having an ANI value of 95-96% or more is judged to be of the same species, and a group of strains having a dDDH value of 70% or more is judged to be of the same species. For this reason, the Prevotella bacterium of the present invention may be a bacterium having an ANI value of 95% or more or a dDDH value of 70% or more with respect to the Prevotella strain R6014. The present inventors performed ANI analysis according to a known literature (Rodriguez-R LM & Konstantinidis KT (2016). The enveomics collection: a toolbox for specialized analyses of microbial genomes and metagenomes. PeerJ Preprints 4:e1900v1.) and confirmed that the ANI values ​​between the newly isolated strains described below are 98% or higher. In addition, the present inventors performed dDDH analysis according to a known literature (Meier-Kolthoff, JP, Auch, AF, Klenk, H.-P., Goker, M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14:60, 2013.) and confirmed that the dDDH values ​​between the newly isolated strains described below are 89% or higher.

[0025] The Prevotella bacteria of the present invention have the property that the ratio of malic acid and lactic acid in the organic acids (propionic acid, succinic acid, malic acid, lactic acid, acetic acid, formic acid, and butyric acid) in the fermentation product is high. Specifically, the ratio of malic acid and lactic acid in the organic acids is, for example, 20% or more, 25% or more, or 30% or more. A high ratio of malic acid and lactic acid not only supplies a precursor of propionic acid and enhances the production of propionic acid, but also has the favorable effect of suppressing methane production by methanogens by reducing the amount of hydrogen.

[0026] In addition, the Prevotella bacteria of the present invention have the property that the proportion of propionic acid and its precursors (succinic acid, malic acid, and lactic acid) in the organic acids in the fermentation product is high. Specifically, the proportion of propionic acid and its precursors in the organic acids is, for example, 65% or more, 70% or more, or 75% or more. A high proportion of propionic acid and its precursors provides the preferable effect of supplying precursors of propionic acid and enhancing the production of propionic acid.

[0027] Furthermore, the Prevotella bacterium of the present invention has the following properties 1) to 6). 1) It is a Gram-stain-negative rod-shaped bacterium that does not form spores. 2) The catalase test is negative and the organism is obligately anaerobic. 3) When D-glucose, lactose, saccharose, maltose, salicin, L-arabinose, cellobiose, D-mannose, or D-raffinose is added, acid production occurs, but when D-mannitol, glycerol, D-melezitose, D-sorbitol, or D-trehalose is added, acid production does not occur. 4) The major menaquinone is MK11, 5) The most predominant fatty acid in the cells is anteiso-C15:0. 6) The GC content of the genomic DNA is 49.4% to 49.9%. The results of the acid production test in 3) above were obtained using API 20A (bioMérieux), an anaerobic bacteria biochemical identification kit.

[0028] The present inventors have also performed genetic analysis of the newly isolated strains described below and confirmed that these strains have the L-lactate dehydrogenase gene on their genomes. Therefore, the presence of the L-lactate dehydrogenase gene on the genome is also one of the characteristics of the Prevotella bacteria of the present invention.

[0029] Examples of strains included in the Prevotella bacteria of the present invention include R5003, R5019, R5025, R5027, R5052, R5064, R5067, R5076, R5107, R6014, and R6025 isolated by the present inventors. Among these, R6014 has been deposited internationally as follows:

[0030] 1) Name and address of the international depositary institution Name: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center Address: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan Postal code: 292-0818 2) Domestic deposit date: April 20, 2021 3) Domestic accession number: NITE P-03463 4) Transfer date to international deposit: May 6, 2022 5) International accession number: NITE BP-03463

[0031] Since it is also possible to identify the strains by the base sequence of the 16SrDNA, the following strains are available: a strain having a 16SrDNA consisting of the base sequence shown in SEQ ID NO: 1 (the base sequence of the 16SrDNA of R5003), a strain having a 16SrDNA consisting of the base sequence shown in SEQ ID NO: 2 (the base sequence of the 16SrDNA of R5019), a strain having a 16SrDNA consisting of the base sequence shown in SEQ ID NO: 3 (the base sequence of the 16SrDNA of R5025), a strain having a 16SrDNA consisting of the base sequence shown in SEQ ID NO: 4 (the base sequence of the 16SrDNA of R5027), a strain having a 16SrDNA consisting of the base sequence shown in SEQ ID NO: 5 (the base sequence of the 16SrDNA of R5052), a strain having a 16SrDNA consisting of the base sequence shown in SEQ ID NO: 6 (the base sequence of the 16SrDNA of R5064), The strains included in the Prevotella bacterium of the present invention can also be strains having 16SrDNA consisting of the base sequence shown in SEQ ID NO: 7 (the base sequence of the 16SrDNA of R5067), strains having 16SrDNA consisting of the base sequence shown in SEQ ID NO: 8 (the base sequence of the 16SrDNA of R5076), strains having 16SrDNA consisting of the base sequence shown in SEQ ID NO: 9 (the base sequence of the 16SrDNA of R5107), strains having 16SrDNA consisting of the base sequence shown in SEQ ID NO: 10 (the base sequence of the 16SrDNA of R6014), and strains having 16SrDNA consisting of the base sequence shown in SEQ ID NO: 11 (the base sequence of the 16SrDNA of R6025).

[0032] The Prevotella bacterium of the present invention has the excellent effects of enhancing propionic acid production and suppressing methane production, and therefore can be used as a feed additive or feed by allowing the bacterium to settle in the digestive tract of animals.

[0033] The feed additive of the present invention can be prepared by freeze-drying the Prevotella bacteria of the present invention and adding other ingredients as necessary, in the same manner as known live bacteria preparations. Alternatively, the culture solution of the Prevotella bacteria of the present invention may be used as a feed additive as it is without freeze-drying.

[0034] The feed of the present invention can be prepared by adding the Prevotella bacterium of the present invention (freeze-dried bacteria, bacterial culture solution, etc.) to general feed.

[0035] The feed additive and feed of the present invention may be fed to any animal, but is preferably fed to ruminant livestock such as dairy cows, beef cattle, sheep, goats and the like. EXAMPLES

[0036] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0037] Example 1: Isolation and identification of strains The novel Prevotella bacteria characteristic of low methane producing cows were isolated from two Holstein dairy cows managed by the Livestock Research Department of the National Agriculture and Food Research Organization. Ruminal fluid was collected orally using a ruminal fluid collection probe (Luminar, Sanshin Kogyo), filtered through triple-layered sterile gauze, and then diluted with anaerobic dilution solution (Bryant & Burkey, 1953). The diluted bacterial suspension was immediately inoculated onto improved YTR agar medium to prepare roll tubes.

[0038] The preparation of YTR agar medium was performed according to the method of Bryant & Burkey (1953). The composition of the modified YTR agar medium is as follows: per liter: yeast extract, 1.2 g (Oxoid); Bacto peptone, 2 g (Difco); mineral solution I, 75 mL; mineral solution II, 75 mL; bovine serum albumin solution prepared for medium, 300 mL; glucose, 1.5 g; cellobiose, 1.5 g; L-cysteine ​​hydrochloride, 3 g; 0.1% resazurin solution, 1 mL; 0.5 g / L hemin solution, 10 mL; 8% NaHCO 3solution, 50 mL; bacto agar, 1.2 g (Difco); distilled water, 500 mL. Hemin solution was prepared as follows: 50 mg of hemin was dissolved in 1 ml of 1N NaOH and diluted with 100 ml of distilled water. Mineral solutions I and II are described by Bryant and Burkey (1953).

[0039] After incubation of the roll tube at 37°C for 48-72 hours, the resulting colonies were picked and inoculated onto a new improved YTR agar medium. After bacterial growth, DNA was extracted and the 16S rRNA gene was amplified by PCR using Takara Ex Taq HS (Takara) and the primer set 27f (5'-AGAGTTTGATCMTGGCTCAG-3') (SEQ ID NO: 12) and 1492r (5'-GGYTACCTTGTTACGACTT-3') (SEQ ID NO: 13). The PCR product was confirmed to be specifically amplified by electrophoresis, purified (ExoSAP-IT, Applied Biosystems), and sequenced (Eurofins Genomics Co.ltd.). The obtained sequence was compared with the sequence of pyrosequence detected as a low methane producing cow-specific Prevotella genus and the sequence of the clone detected by the clone library analysis of the full-length 16S rRNA, and it was confirmed to be the target bacterial strain.

[0040] The isolated novel Prevotella strains were named R5003, R5019, R5025, R5027, R5052, R5064, R5067, R5076, R5107, R6014, and R6025, and the 16S rDNA sequences of these strains are shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11, respectively.

[0041] Example 2: Cultivation of strains and fermentation product analysis The type strains (Prevotella ruminicola JCM8958, P. bryantii DSM11371, and Butyrivibrio fibrisolvens JCM6563) were purchased from a bacterial strain depository. The type strains and the isolated novel Prevotella strains were inoculated onto modified YTR slant agar medium and serially cultured at 37°C under anaerobic conditions.

[0042] Glucose medium (a modified PYG medium as described in DSMZ medium 104, omitting beef extract and Tween 80) was used to monitor growth and measure fermentation products. Strains were grown in early-mid logarithmic phase (OD 660 0.2 ml of bacterial growth medium with a β-glucose concentration of 0.01% (β = 0.3-0.5) was inoculated into 5 ml of fresh glucose medium and subcultured. The pre-preculture, preculture, and main culture were performed while maintaining high growth activity. Bacterial growth was measured every 10 minutes using a turbidity meter (OD-Monitor C&T, TAITEC Co. Ltd.) installed in a shaking incubator (120 rpm, Bio-Shaker BR-40LF, TAITEC Co. Ltd.), and the fermentation products were collected from the stationary phase medium 44-48 hours after the start of culture and analyzed. The culture was repeated three times. The culture solution was purified, and the organic acids in the culture solution were comprehensively quantified using an HPLC organic acid analysis system (SHIMADZU Co. Ltd.).

[0043] The proportion of organic acids in the culture medium of each strain is shown in Figure 1. In the culture medium of the new Prevotella genus isolate, the proportion of propionic acid and its precursors (succinic acid, malic acid, and lactic acid) was high, and the proportion of fermentation products of the hydrogen production pathway (acetic acid, formic acid, and butyric acid) was low. In addition, the new Prevotella genus isolate produced more malic acid and lactic acid than the type strain.

[0044] [References] Bryant MP & Burkey LA (1953) Cultural methods and some characteristics of some of the more numerous groups of bacteria in the bovine rumen. J Dairy Sci 36: 205-217.

[0045] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. [Industrial Applicability]

[0046] The present invention may be industrially used as a probiotic agent that changes the ruminal fermentation of ruminant livestock such as dairy cows, beef cattle, sheep, and goats to propionic acid fermentation. In the future, it may be used as an agent for reducing methane generated from fermentation in the stomach of cattle.

Claims

1. A method for using a bacterium of the genus Prevotella derived from the digestive tract of ruminants, characterized in that it has 16S rDNA consisting of a base sequence that has 98.7% or more homology to the base sequence shown in SEQ ID NO: 10, said method being used as feed or a feed additive for cattle.

2. A method for using the Prevotella bacterium derived from the digestive tract of a ruminant as described in claim 1, characterized in that when cultured in a medium containing glucose, the ratio of malic acid and lactic acid in the organic acids in the fermentation product is 20% (mol / mol) or more, said method being used as feed or a feed additive for cattle.

3. A method for using the Prevotella genus bacterium derived from the digestive tract of a ruminant as described in claim 1, characterized in that when cultured in a medium containing glucose, the proportion of succinic acid, malic acid, propionic acid, and lactic acid in the organic acids in the fermentation product is 65% (mol / mol) or more, said method being used as feed or a feed additive for cattle.

4. A method for using the Prevotella bacterium according to claim 1, characterized in that it has all of the following properties 1) to 4): 1) It is a Gram-stain-negative rod-shaped bacterium that does not form spores. 2) The catalase test is negative and the organism is obligately anaerobic. 3) It produces acid when D-glucose, lactose, saccharose, maltose, salicin, L-arabinose, cellobiose, D-mannose, or D-raffinose is added, but it does not produce acid when D-mannitol, glycerol, D-melezitose, D-sorbitol, or D-trehalose is added. 4) The GC content of the genomic DNA is 49.4% to 49.9%.

5. A method for using the Prevotella bacterium derived from the digestive tract of a ruminant as described in claim 1, characterized in that the ANI value with the strain identified by accession number NITE BP-03463 is 95% or more, said method being used as feed or a feed additive for cattle.

6. A method for using the Prevotella bacterium derived from the digestive tract of a ruminant as described in claim 1, characterized in that the dDDH value with the strain identified by accession number NITE BP-03463 is 70% or more, said method being used as feed or a feed additive for cattle.

7. A method for using the Prevotella bacterium derived from the digestive tract of a ruminant as described in claim 1, characterized in that it has a 16S rDNA consisting of a base sequence shown in any of SEQ ID NOs: 1 to 11, said method being used as feed or a feed additive for cattle.

8. A method for using the Prevotella bacterium derived from the digestive tract of ruminants according to claim 1, characterized in that the bacterium is a strain identified by accession number NITE BP-03463, as feed or a feed additive for cattle.

9. A feed additive for cattle, comprising the bacterium of the genus Prevotella derived from the digestive tract of a ruminant according to any one of claims 1 to 8.

10. A feed for cattle, comprising the Prevotella bacterium derived from the digestive tract of a ruminant according to any one of claims 1 to 8.

11. A bacterium of the genus Prevotella derived from the digestive tract of ruminants, characterized in that it has a 16S rDNA consisting of a base sequence that has 98.7% or more homology to the base sequence shown in SEQ ID NO:10, and is for use as feed or a feed additive for cattle.

12. The bacterium of the genus Prevotella according to claim 11, characterized in that it has all of the following properties 1) to 4): 1) It is a Gram-stain-negative rod-shaped bacterium that does not form spores. 2) The catalase test is negative and the organism is obligately anaerobic. 3) It produces acid when D-glucose, lactose, saccharose, maltose, salicin, L-arabinose, cellobiose, D-mannose, or D-raffinose is added, but it does not produce acid when D-mannitol, glycerol, D-melezitose, D-sorbitol, or D-trehalose is added. 4) The GC content of the genomic DNA is 49.4% to 49.9%.

13. A Prevotella bacterium derived from the digestive tract of a ruminant as described in claim 11, characterized in that the ANI value with the strain identified by accession number NITE BP-03463 is 95% or more, and the bacterium is for use as feed or a feed additive for cattle.

14. A Prevotella bacterium derived from the digestive tract of a ruminant as described in claim 11, characterized in that the dDDH value with the strain identified by accession number NITE BP-03463 is 70% or more, and the bacterium is for use as feed or a feed additive for cattle.

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