Acetogenic proteinophilic bacteria and application thereof in preparation of feed additive

Abstract

The invention discloses acetogenic proteinophilic bacteria and application thereof in preparation of a feed additive. According to the method, 35 strains with a good fiber degradation function are obtained from Holstein cow rumen fluid through preliminary screening, a bran culture medium is used for screening the 35 strains, it is found that the 25 strains have the capacity of converting a complex carbon source into reducing sugar, the strains of different genus with the strongest function are selected for in-vitro fermentation verification, and the strain with the strongest function is obtained. The utilization efficiency of the strains on the TMR fibers of ruminants is observed, and results show that the acetogenic proteinophilic bacteria (the microbial preservation number is CGMCC No.33516) have the strongest functions on the feed dry matter degradation rate and the fiber degradation rate, the pH value of rumen fermentation liquor can be increased, and the content of acetic acid, isobutyric acid, butyric acid, valeric acid and rumen total volatile fatty acid is remarkably increased. The acetogenic proteinophilic bacterium provided by the invention has an application prospect in the aspects of improving the feed fiber utilization rate or improving the animal digestive ability and the like.

Description

Technical Field

[0001] The present invention relates to proteinophilic bacteria and their applications, in particular to a strain of acetic acid-producing proteinophilic bacteria isolated from the rumen fluid of Holstein cows and its applications in improving animal digestion ability, improving animal gastric environment or promoting the survival and fiber degradation of other fiber-degrading strains, belonging to the field of acetic acid-producing proteinophilic bacteria and their applications. Background Art

[0002] The rumen microbiota of ruminants is rich in composition. Studies have shown that 1 mL of rumen fluid contains 10 9 -10 11 bacteria, 10 3 -10 5 fungi and 10 4 -10 7 protozoa. Among them, bacteria have the largest variety and quantity and are the most metabolically active, with the quantity accounting for about 90% of the total rumen microbiota. Ruminants themselves cannot produce enzymes for digesting and degrading feed fiber substances, and mainly rely on rumen microbiota to produce substances such as volatile fatty acids to provide nutrition for ruminants, while the host provides the environment required for microbial growth. Therefore, it is necessary to systematically study the distribution of rumen microbiota populations and their interactions in fiber degradation from the perspective of rumen microecology to fully exert the potential of rumen microbiota in digesting fiber substances.

[0003] Multiple studies have shown that cellulose-degrading bacteria mainly exist in the rumen of ruminants. The number of cellulose-degrading bacteria in animals that mainly feed on grass and hay is significantly higher than that in animals that mainly feed on concentrated feed. If strains that can efficiently degrade cellulose or improve animal digestion ability can be isolated from the rumen of ruminants, it has important application prospects in improving the feed utilization rate of ruminants or improving animal digestion ability, etc. Summary of the Invention

[0004] One of the objectives of the present invention is to provide a strain of acetic acid-producing proteinophilic bacteria isolated from the rumen of ruminants.

[0005] Another objective of the present invention is to provide a microbial preparation prepared from the above-mentioned acetic acid-producing proteinophilic bacteria.

[0006] The third objective of the present invention is to apply the above-mentioned acetic acid-producing proteinophilic bacteria in aspects such as improving animal digestion ability, improving animal gastric environment or promoting the survival and fiber degradation of other fiber-degrading strains.

[0007] In order to achieve the above objectives, the main technical solutions adopted by the present invention include: On the one hand, the present invention provides a strain of acetic acid-producing proteinophilic bacteria ( Proteiniphilum acetatigenes6076, with the microbial deposit number CGMCC No. 33516; its taxonomic name is: Proteiniphilum acetatigenes ; the deposit date is February 11, 2025; the deposit institution is the General Microbiological Center of the China Committee for Culture Collection of Microorganisms; the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The nucleotide sequence of the 16S rRNA of strain 6076 isolated from the rumen of ruminants in the present invention is shown as SEQ ID No. 1. After alignment on NCBI, the sequence identity is 99.51%. Therefore, this strain 6076 is identified as Proteiniphilum acetatigenes ).

[0009] The Proteiniphilum acetatigenes acetate - loving proteobacterium

[0010] isolated in the present invention, strain 6076, can effectively improve the utilization rate of feed fiber, with the strongest function. It can increase the dry matter degradation rate of feed from 53.45% to 58.49%, an increase of about 9%; increase the fiber degradation rate from 42.15% to 52.24%, an increase of about 24%; at the same time, increase the pH value of the rumen fermentation broth, which is beneficial to the survival and fiber degradation of other fiber - degrading strains in the rumen.

[0011] In a preferred specific embodiment of the present invention, the

[0012] acetate - loving proteobacterium 6076 is applied to improve the digestive ability of animals.

[0013] In a preferred specific embodiment of the present invention, the

[0014] acetate - loving proteobacterium 6076 is applied to improve the gastric environment of animals.

[0015] In a preferred specific embodiment of the present invention, the

[0016] acetate - loving proteobacterium 6076 is applied to promote the survival and fiber degradation of other fiber - degrading strains.Another aspect of the present invention is the use of the acetogenic proteinophilic bacterium 6076 and the microbial preparation containing the acetogenic proteinophilic bacterium 6076 in the preparation of animal feed additives.

[0017] In a preferred specific embodiment of the present invention, the animal is a ruminant; among them, the ruminant is a Holstein cow.

[0018] Detailed description of the overall technical solution of the present invention The present invention uses the culturomics method. For the rumen fluid of Holstein cows, 8 solid media are used to directly isolate and culture the freshly collected rumen fluid. At the same time, M10 liquid medium is used to enrich and culture the rumen fluid for up to 28 days. The obtained strains are screened for fiber degradation function to screen strict anaerobic strains from the rumen environment and verify their fiber degradation function.

[0019] From the results of screening on the sodium carboxymethyl cellulose plate, it can be seen that about 50% of the strains have preliminary fiber degradation function, but only 35 strains have a transparent degradation circle diameter greater than 9 mm, indicating that they have good cellulose utilization function.

[0020] The present invention further uses a bran medium to screen 35 strains with stronger functions and finds that 25 strains can effectively utilize bran and degrade it into reducing sugars, indicating that these 25 strains have the ability to convert complex carbon sources into reducing sugars.

[0021] Further, the strains with the strongest functions from different genera are selected for in vitro fermentation verification to observe their fiber utilization efficiency for the TMR of ruminants. The results show that Proteiniphilum acetatigenes 6076, Actinomyces glycerinitolerans 6345 and Caldibacillus pasinlerensis 5847 can all effectively improve the feed fiber utilization rate, and Proteiniphilum acetatigenes the function of strain 6076 is the strongest, which can increase the dry matter degradation rate of the feed from 53.45% to 58.49%, an increase of about 9%, and the fiber degradation rate from 42.15% to 52.24%, an increase of about 24%. At the same time, it increases the pH value of the fermentation broth, which is more conducive to the survival and fiber degradation of other fiber-degrading strains in the rumen.

[0022] The present invention initially screens 35 strains with good fiber degradation function from the rumen fluid of Holstein cows, uses a bran medium to screen these 35 strains, and finds that 25 strains have the ability to convert complex carbon sources into reducing sugars. The present invention further selects the strains with the strongest functions from different genera for in vitro fermentation verification to observe the fiber utilization efficiency of these strains for the TMR of ruminants. The results show that the acetogenic proteinophilic bacterium ( Proteiniphilum acetatigenes)6076 (with a microbial deposit number of CGMCC No. 33516) has the strongest function on the dry matter degradation rate and fiber degradation rate of feed, and can increase the pH value of rumen fermentation broth, significantly increase the contents of acetic acid, isobutyric acid, butyric acid, valeric acid, and the total content of volatile fatty acids in the rumen. The acetic acid-producing protein bacterium provided by the present invention ( Proteiniphilum acetatigenes )6076 has an application prospect in improving the utilization rate of feed fiber or improving the digestive ability of animals, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the number of strains used for screening on the sodium carboxymethylcellulose plate; among them, there are 101 strains from the Streptococcus genus, 98 strains from the Enterococcus genus, and 51 strains from the Proteiniphilum genus.

[0024] Figure 2 is the screening result of the number of strains with the ability to degrade sodium carboxymethylcellulose; among them, 163 strains can produce a clear degradation zone on the sodium carboxymethylcellulose plate and have a preliminary fiber degradation function, and 197 strains do not produce a clear degradation zone on the sodium carboxymethylcellulose plate and do not have a preliminary fiber degradation function.

[0025] Figure 3 is the diameter size of the clear degradation zone produced by 163 strains isolated from cow rumen fluid on the sodium carboxymethylcellulose plate; among them, the clear degradation zone diameter of 79 strains is less than 4 mm, the clear degradation zone diameter of 49 strains is between 4 mm and 9 mm, and the clear degradation zone diameter of 35 strains is greater than 9 mm.

[0026] Figure 4 is the OD 600 nm value of the bran-degrading strain; among them, 25 strains can degrade bran into reducing sugars and have the ability to degrade complex carbon sources; 10 strains cannot degrade bran into reducing sugars and do not have the ability to degrade complex carbon sources.

[0027] Figure 5 is the dry matter degradation rate and fiber degradation rate of the strain after in vitro fermentation for 48 h. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, it should be understood that the described embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements all fall within the protection scope of the present invention.

[0029] Example 1 Isolation, Identification and Screening of Fiber-Degrading Strains 1 Test Method 1.1 Strain Isolation Collect rumen fluid from 4 healthy late-lactation Holstein cows with similar lactation days (242 ± 34 days) and first calving from Youyuan Runze Ranch in Shunyi District, Beijing. Transport the rumen fluid back to the laboratory on the same day and separate it. Use 8 kinds of culture media: Columbia blood agar medium (CA medium, Sigma-Aldrich, catalog number is BCCG9917), brain heart infusion medium (BHI medium, Solarbio, catalog number is LA0360), PA medium (prepared by oneself, reference is Dai X, Hackmann TJ, Lobo RR, Faciola AP. Lipopolysaccharide stimulates the growth of bacteria that contribute to ruminal acidosis. Ercolini D, editor. Appl Environ Microbiol. 2020;86:e02193-19.), PB medium (prepared by oneself, reference is: Dai X, Hackmann TJ, Lobo RR, Faciola AP. Lipopolysaccharide stimulates the growth of bacteria that contribute to ruminal acidosis. Ercolini D, editor. Appl Environ Microbiol. 2020;86:e02193-19.), MBA medium (prepared by oneself, reference is Dewanckele L, Jeyanathan J, Vlaeminck B, Fievez V. Identifying and exploring biohydrogenating rumen bacteria with emphasis on pathways including trans-10 intermediates. BMC Microbiol. 2020;20:198.), MBB medium (prepared by oneself, reference is: Dewanckele L, Jeyanathan J, Vlaeminck B, Fievez V. Identifying and exploring biohydrogenating rumen bacteria with emphasis on pathways including trans-10 intermediates. BMC Microbiol. 2020;20:198.), directly isolate the rumen fluid with YCFA medium (prepared by oneself, reference: Chang Y, Hou F, Pan Z, Huang Z, Han N, Bin L, et al. Optimization of culturomics strategy in human fecal samples. Front Microbiol. 2019;10:2891.) and Wilkins-Chalgren anaerobic medium (WIL medium, Solarbio, product number is LA4420). Enrich the rumen fluid with M10 liquid medium (prepared by oneself, reference: Hailemariam S, Zhao S, Wang J. Complete genome sequencing and transcriptome analysis of nitrogen metabolism of succinivibrio dextrinosolvens strain Z6 isolated from dairy cow rumen. Front Microbiol. 2020;11:1826.) for 28 days. During this period, collect the enriched rumen fluid on the 7th day, 14th day, and 28th day, and use M10 solid medium (prepared by oneself, reference: Hailemariam S, Zhao S, Wang J. Complete genome sequencing and transcriptome analysis of nitrogen metabolism of succinivibrio dextrinosolvens strain Z6 isolated from dairy cow rumen. Front Microbiol. 2020;11:1826.) to isolate the enriched rumen fluid. After evenly coating, place the plate in an anaerobic culture bag (equipped with an anaerobic gas generation bag) and culture it in an incubator at 39°C for 3 - 5 days. After the strain grows, use the quadrant streak method to purify the strain on BHI and M10 solid media and then perform 16S rRNA identification.

[0030] 1.2 Primary screening of fiber-degrading strains 360 strains of bacteria that had been isolated and preserved were cultured in BHI liquid medium, and purified using BHI solid medium. The purified strains were enriched again using BHI liquid medium to obtain a bacterial solution. 1 mL of the bacterial solution (OD value above 0.9) was centrifuged, and the centrifuged bacterial cells were picked up and spot-inoculated onto a sodium carboxymethylcellulose plate. After anaerobic incubation at 39 °C for 72 h, 1 mg / mL congo red solution was poured in for staining for 10 min. The congo red solution was then poured off, and 1 mol / L NaCl solution was added to the plate and soaked for 15 min to wash away the stain. The presence or absence of a hydrolysis zone around the colonies was observed to preliminarily judge whether the strain had the ability to degrade fiber. The hydrolysis zone diameter of the strains that could produce a clear hydrolysis zone was accurately measured using a vernier caliper, and bacteria with a hydrolysis zone diameter d > 9.0 mm were selected for further screening according to the size of the clear zone.

[0031] 1.3 Rescreening of fiber-degrading strains 35 strains of bacteria with a transparent degradation zone diameter greater than 9 mm were cultured in BHI medium. 1 mL of the bacterial solution (OD 540 nm value above 0.9) was centrifuged to discard the supernatant to obtain the bacterial cells, which were resuspended with sterile water and the OD value was adjusted to 0.15. The glucose in the BHI medium was replaced with an equal amount of wheat bran to prepare a wheat bran liquid medium. 1% of the bacterial suspension was inoculated into the wheat bran liquid medium and anaerobically shaken and cultured at 39 °C for 5 days. The amount of reducing sugar produced in the medium was measured using the DNS method to judge the degradation ability of the strain to complex carbon sources.

[0032] 2 Test results 2.1 Results of identifying strains by 16S rRNA The 16S rRNA of strain 6076 was sequenced, and its nucleotide sequence is shown as SEQ ID No.1. It was compared on NCBI, and the identity of the sequence with Proteiniphilum acetatigenes was 99.51%. Strain 6076 was identified as Proteiniphilum acetatigenes ( Proteiniphilum acetatigenes ), hereinafter referred to as P. acetatigenes 6076).

[0033] Strain P. acetatigenes

[0034] 2.2 Preliminary screening results of fiber-degrading strains A total of 360 strains of bacteria from 27 genera were selected for preliminary screening. Among them, 101 strains were from Streptococcus genus, 98 strains were from Enterococcus genus, and 51 strains were from Proteiniphilum genus ( Figure 1 ). The preliminary screening results showed that 163 strains (including strain 6076) could produce clear degradation zones on carboxymethyl cellulose sodium plates (CMC-Na plates), indicating preliminary fiber-degrading ability ( Figure 2 ). Among them, the diameter of the clear degradation zone of 79 strains was less than 4 mm, the diameter of the clear degradation zone of 49 strains was between 4 mm and 9 mm, and the diameter of the clear degradation zone of 35 strains (including strain 6076) was greater than 9 mm ( Figure 3 ).

[0035] 2.3 Rescreening results of fiber-degrading strains The complex carbon source degradation ability of 35 strains with a clear degradation zone diameter of more than 9 mm for cellulose was further detected using bran liquid medium. Among them, 25 strains, including strain P. acetatigenes 6076, could significantly degrade bran into reducing sugars, indicating the ability to degrade complex carbon sources ( Figure 4 ).

[0036] Experimental example 1 Degradation ability test of strains isolated from dairy cow rumen fluid on feed fiber 1 Test method 1.1 Fermentation substrates and additives Fermentation substrate: TMR diet (collected from Modern Animal Husbandry (Xinle) Co., Ltd., Jimu Village, Xinle City, Shijiazhuang City, Hebei Province).

[0037] Additives: B. subtilis 5632, S. equinus 5640, S. pluranimalium 5823, S. infantarius 6074, L. delbrueckii 5812, P. mucosa 6360, S. condimenti 5845, E. gallinarum 6392, A. glycerinitolerans 6345, E. lactis 6088, C. pasinlerensis 5847, L. pontis 5897, P. acetatigenes 6076.

[0038] Concentration of additives: 1×107 CFU / mL.

[0039] 1.2 Collection of rumen fluid and preparation of buffer solution Three healthy Holstein dairy cows with similar body weights (550 ± 50 kg) and lactation days (136 ± 37 d) and equipped with permanent rumen fistulas were selected as rumen fluid donor cows at the Changping Animal Experiment Base of the Institute of Animal Science, Chinese Academy of Agricultural Sciences. The cows were fed twice a day (at 7:00 and 19:00) and had free access to water. Rumen contents were collected through the rumen fistula 1 h before morning feeding, mixed, placed in a thermos flask, and quickly taken back to the laboratory. The mixture was filtered through four layers of gauze (while passing CO2), and the whole operation was carried out in a 39 °C water bath and should be completed in the shortest possible time. At the same time, buffer solution was prepared. After preparation, CO2 was continuously passed until the pH value reached 6.8 - 7.0, and then it was placed in a 39 °C water bath for constant temperature standing and standby.

[0040] 1.3 Fermentation method and group setting Approximately 0.75 g of fermentation substrate was accurately weighed into a 100 mL anaerobic fermentation bottle. During inoculation, 45 mL of preheated liquid medium and 15 mL of fresh rumen fluid filtered through four layers of gauze were quickly added to each bottle. After continuously passing CO2 into the bottle for 5 s, the bottle stopper was immediately added, and each fermentation bottle was placed in a 39 °C incubator for continuous culture for 24 h. A total of 11 groups were set up in the experiment, including the CON group (blank control) and experimental groups of each strain, with 4 replicates in each group.

[0041] 1.4 Sample collection and preparation After 48 h of fermentation culture, the fermentation procedure was terminated. The pH value of the fermentation broth was immediately measured. After the fermentation broth was filtered through a nylon bag to obtain the solid phase of the fermentation broth, the remaining feed was dried at 65 °C for the determination of dry matter, neutral detergent fiber, and acid detergent fiber. Part of the fermentation broth was collected and stored frozen at -20 °C for the determination of volatile fatty acids (VFA) and NH3-N.

[0042] 2 Experimental results Twenty-five strains of bacteria that could degrade wheat bran belonged to 10 genera. The strains with the strongest functions from different genera were selected for in vitro fermentation to determine their effects on the degradation efficiency of feed fiber in a simulated rumen environment.

[0043] The experimental results were as Figure 5 shown. Using TMR as the substrate, in vitro fermentation of different strains was carried out, and it was found that P. acetatigenes 6076 and Actinomyces glycerinitolerans 6345 could significantly increase the degradation rates of feed dry matter and neutral detergent fiber compared with the control group ( P <0.05), Caldibacillus pasinlerensis5847 can significantly increase the degradation rate of neutral detergent fiber in feed ( P <0.05). Among them, P. acetatigenes 6076 can increase the degradation rate of feed dry matter from 53.45% to 58.49%, an increase of about 9%; the fiber degradation rate increases from 42.15% to 52.24%, an increase of about 24%.

[0044] Table 1 In vitro fermentation assay P. acetatigenes Effect of 6076 on rumen fermentation parameters

[0045] P. acetatigenes The effect of 6076 on rumen fermentation parameters is shown in Table 1. Compared with the control group, after adding P. acetatigenes 6076, it can significantly increase the pH value of the fermentation broth and significantly increase the contents of acetic acid, isobutyric acid, butyric acid and valeric acid. At the same time, it significantly increases the total volatile fatty acid content in the rumen and has no significant effect on the acetic acid to propionic acid ratio.

Claims

1. A proteinophilic acetic acid-producing bacterium ( Proteiniphilum acetatigenes ), characterized in that Its microbial deposit number is CGMCC No. 33516.

2. A microbial preparation containing the proteiniphilic acetogenic bacterium 6076 described in claim 1.

3. Use of the proteiniphilic acetogenic bacterium 6076 described in claim 1 and the microbial preparation described in claim 2 in the preparation of an animal feed additive.

4. The application according to claim 3, characterized in that, The animal is a ruminant.

5. The application according to claim 3, characterized in that, The animal feed additive is a feed additive having the function of improving the digestion ability of animals or degrading cellulose.

6. The application according to claim 5, wherein The improvement of the digestion ability of animals includes improving the utilization rate of feed fiber.

7. The application according to claim 6, characterized in that, The improvement of the utilization rate of feed fiber is to increase the dry matter degradation rate or neutral detergent fiber degradation rate of the feed.

8. The application according to claim 3, wherein The feed additive is a feed additive having the function of improving the gastric environment of animals.

9. The application according to claim 8, characterized in that, The improvement of the gastric environment of animals includes increasing the rumen pH value; or increasing the acetic acid content, isobutyric acid content, butyric acid content, valeric acid content or total volatile fatty acid content.

10. The application according to claim 3, characterized in that, The feed additive is a feed additive having the function of promoting the survival and fiber degradation of fiber-degrading strains.

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