Proteus aceticus and its application in preparing feed additive

Abstract

The application discloses acetic acid-producing proteobacteria and application thereof in preparation of feed additives. 35 strains with good fiber degradation function are obtained from Holstein cow rumen fluid through preliminary screening, and bran medium is used for screening the 35 strains, and it is found that 25 strains have the ability of converting complex carbon source into reducing sugar, and the most powerful strains of different genera are selected for in-vitro fermentation verification, and the fiber utilization efficiency of the strains on ruminant TMR is observed, and it is found that the acetic acid-producing proteobacteria (microbial preservation number is CGMCC No. 33516) has the most powerful function on feed dry matter degradation rate and fiber degradation rate, and can improve rumen fermentation liquid pH value, and significantly improve the content of acetic acid, isobutyric acid, butyric acid, valeric acid and total rumen volatile fatty acid. The acetic acid-producing proteobacteria provided by the application has application prospect in improving feed fiber utilization rate or improving animal digestion capacity.

Description

TECHNICAL FIELD

[0001] The present application relates to proteinophilic bacteria and its application, especially to a strain of acetic acid-producing proteinophilic bacteria isolated from the rumen fluid of Holstein cow and its application in improving the digestive capacity of animals, improving the stomach environment of animals or promoting the survival and fiber degradation of other fiber-degrading strains, belonging to the field of acetic acid-producing proteinophilic bacteria and its application. BACKGROUND

[0002] The rumen microflora of ruminants is rich in composition, and 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 which the species and quantity of bacteria are the most, and the metabolism is the most active, and the quantity accounts for about 90% of the total amount of rumen microorganisms. Ruminants cannot produce enzymes for digesting and degrading feed fiber substances, and mainly rely on rumen microorganisms to produce volatile fatty acids and other substances to provide nutrition for ruminants, and the host provides the required environment for the growth of microorganisms. Therefore, it is necessary to systematically study the distribution of rumen microbial population and its interaction in the degradation of fiber substances from the perspective of rumen microecology, so as to fully exert the potential of rumen microorganisms in digesting fiber substances.

[0003] Many studies have shown that cellulose-degrading bacteria mainly exist in the rumen of ruminants, and the number of cellulose-degrading bacteria in animals mainly feeding on grass and hay is significantly higher than that in animals mainly feeding on concentrated feed. If a strain of high-efficiency cellulose-degrading bacteria or improving the digestive capacity of animals can be isolated from the rumen of ruminants, it has important application prospects in improving the feed utilization rate of ruminants or improving the digestive capacity of animals. SUMMARY

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

[0005] The second purpose of the present application is to provide a microbial preparation prepared from the acetic acid-producing proteinophilic bacteria.

[0006] The third purpose of the present application is to apply the acetic acid-producing proteinophilic bacteria to improve the digestive capacity of animals, improve the stomach environment of animals or promote the survival and fiber degradation of other fiber-degrading strains.

[0007] In order to achieve the above purposes, the main technical solutions adopted by the present application include:

[0008] The present application provides a strain of acetic acid-producing proteinophilic bacteria (Proteobacterium) in one aspect. Proteiniphilum acetatigenesMicrobial accession number 6076 is CGMCC No. 33516; its classification and nomenclature are as follows: Proteiniphilum acetatigenes The deposit date is February 11, 2025; the depositary institution is the China General Microbiological Culture Collection Center; the depositary address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The nucleotide sequence of the 16S rRNA of strain 6076, isolated from the rumen of ruminants in this invention, is shown in SEQ ID No. 1. Sequence alignment on NCBI showed 99.51% identity. Therefore, strain 6076 was identified as an acetic acid-producing proteinophilic bacterium. Proteiniphilum acetatigenes ).

[0010] The acetic acid-producing proteinophilic bacteria isolated in this invention ( Proteiniphilum acetatigenes 6076 can effectively improve feed fiber utilization, with the strongest function. It can increase the feed dry matter degradation rate 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 rumen fermentation broth, which is beneficial to the survival of other fiber-degrading strains in the rumen and fiber degradation.

[0011] Another aspect of the present invention is to apply the acetic acid-producing proteophile 6076 to improving animal digestive capacity, improving the gastric environment of animals, or promoting the survival of other fiber-degrading strains and fiber degradation.

[0012] In a preferred embodiment of the present invention, the acetic acid-producing proteophile 6076 is used to improve the digestive capacity of animals.

[0013] In a preferred embodiment of the present invention, improving animal digestibility means improving feed fiber utilization; wherein, improving feed fiber utilization means improving feed dry matter degradation rate or neutral detergent fiber degradation rate.

[0014] In a preferred embodiment of the present invention, the acetic acid-producing proteophile 6076 is applied to improve the gastric environment of animals.

[0015] In a preferred embodiment of the present invention, the improvement of the animal's gastric environment is achieved by increasing the pH value, or increasing the content of acetic acid, isobutyric acid, butyric acid, or valeric acid, or increasing the total volatile fatty acid content.

[0016] In a preferred embodiment of the present invention, the acetic acid-producing proteinophilic bacterium 6076 is used to promote the survival of other fiber-degrading strains and fiber degradation.

[0017] Another aspect of the present application is the use of the acetogenic proteobacterium 6076 and the microbial preparation containing the acetogenic proteobacterium 6076 in the preparation of an animal feed additive.

[0018] In a preferred embodiment of the present application, the animal is a ruminant; wherein the ruminant is a Holstein cow.

[0019] Detailed description of the overall technical solution of the present invention

[0020] The present application uses a culture-omics method, and uses 8 solid culture media to directly separate and culture the freshly collected rumen fluid, and uses M10 liquid culture medium to enrich and culture the rumen fluid for 28 days, and the obtained strains are subjected to fiber degradation function screening, so as to screen strict anaerobic strains from the rumen environment and verify the fiber degradation function thereof.

[0021] It can be known from the screening result on the sodium carboxymethyl cellulose flat plate that about 50% of the strains have a preliminary fiber degradation function, but only 35 strains have a transparent degradation circle diameter greater than 9 mm, indicating that the strains have a good cellulose utilization function.

[0022] The present application further uses bran medium to screen the 35 strains with strong function, and finds that 25 strains can effectively utilize bran and degrade the bran into reducing sugar, indicating that the 25 strains have the ability to convert complex carbon sources into reducing sugar.

[0023] The strongest functional strains of different genera are further selected for in-vitro fermentation verification, and the fiber utilization efficiency of the strains on the TMR of ruminants is observed. Proteiniphilum acetatigenes 6076, Actinomyces glycerinitolerans 6345 and Caldibacillus pasinlerensis 5847 can effectively improve the fiber utilization rate of feed, and Proteiniphilum acetatigenes The 6076 strain has the strongest function, can increase the dry matter degradation rate of feed from 53.45% to 58.49%, about 9%, and can increase the fiber degradation rate from 42.15% to 52.24%, about 24%, and at the same time, can increase the pH value of the fermentation liquor, which is more conducive to the survival and fiber degradation of other fiber-degrading strains in the rumen.

[0024] The present application obtains 35 strains with good fiber degradation function from the rumen fluid of Holstein cows through preliminary screening, uses bran medium to screen the 35 strains, finds that 25 strains have the ability to convert complex carbon sources into reducing sugar, and further selects the strongest functional strains of different genera for in-vitro fermentation verification, and observes the fiber utilization efficiency of the strains on the TMR of ruminants, and finds that the acetogenic proteobacterium (Acetogenium Proteiniphilum acetatigenesMicrobial culture strain 6076 (its microbial preservation number is CGMCC No. 33516) exhibits the strongest function in reducing feed dry matter and fiber degradation rates, and can also increase the pH value of rumen fermentation broth, significantly increasing the content of acetic acid, isobutyric acid, butyric acid, valeric acid, and total volatile fatty acids in the rumen. The acetic acid-producing proteinophilic bacteria (Acetophilus) provided by this invention... Proteiniphilum acetatigenes 6076 has promising applications in improving feed fiber utilization or enhancing animal digestive capacity. Attached Figure Description

[0025] Figure 1 The number of strains used for screening on sodium carboxymethyl cellulose plates; of which, from Streptococcus There are 101 strains of bacteria, from Enterococcus There are 98 strains of bacteria, from Proteiniphilum There are 51 strains of bacteria in the genus.

[0026] Figure 2 The results show the number of strains capable of degrading sodium carboxymethyl cellulose. Among them, 163 strains produced transparent degradation zones on sodium carboxymethyl cellulose plates, indicating preliminary fiber degradation function, while 197 strains did not produce transparent degradation zones on sodium carboxymethyl cellulose plates, indicating no preliminary fiber degradation function.

[0027] Figure 3 The diameter of the transparent degradation zone produced on sodium carboxymethyl cellulose plates was determined for 163 bacterial strains isolated from bovine rumen fluid. Among them, 79 strains had transparent degradation zones with a diameter less than 4 mm, 49 strains had transparent degradation zones with a diameter between 4 mm and 9 mm, and 35 strains had transparent degradation zones with a diameter greater than 9 mm.

[0028] Figure 4 OD strains for bran degradation 600 nm value; among them, 25 strains of bacteria can degrade wheat bran into reducing sugars and have the ability to degrade complex carbon sources; 10 strains of bacteria cannot degrade wheat bran into reducing sugars and do not have the ability to degrade complex carbon sources.

[0029] Figure 5 The dry matter degradation rate and fiber degradation rate of the strain after 48 h of in vitro fermentation. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0031] Example 1 Isolation, identification and screening of fiber-degrading bacterial strains

[0032] 1 Test method

[0033] 1.1 Isolation of strains

[0034] Rumen fluid was collected from 4 healthy and late-lactation Holstein cows (242 ± 34 days in milk) at the same day from Youyuanrunze Ranch in Shunyi District, Beijing. The rumen fluid was transported back to the laboratory and separated on the same day. Eight media were used: Columbia blood agar medium (CA medium, Sigma-Aldrich, Cat# BCCG9917), brain heart infusion medium (BHI medium, Solarbio, Cat# LA0360), PA medium (self-prepared, 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 (self-prepared, 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 (self-prepared, 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 (self-prepared, 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.).YCFA medium (self-prepared, 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 bacteria medium (WIL medium, Solarbio, Cat# LA4420) were used to directly isolate the rumen fluid. M10 liquid medium (self-prepared, 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.) was used to enrich the rumen fluid for 28 days, and the enriched rumen fluid was collected at the 7th day, 14th day and 28th day. M10 solid medium (self-prepared, 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.) was used to isolate the enriched rumen fluid. After uniform coating, the plates were placed in an anaerobic culture bag (with an anaerobic gas production bag) and incubated in a 39°C incubator for 3-5 days. After the strains grew, the strains were purified on BHI and M10 solid medium using four-zone streaking method, and then 16S rRNA identification was performed.

[0035] 1.2 Initial screening of fiber-degrading strains

[0036] The 360 strains of bacteria which have been isolated and preserved were liquid cultured using BHI liquid medium, purified using BHI solid medium, and then enriched to obtain a bacterial solution using BHI liquid medium again. 1 mL of the bacterial solution (OD value reached 0.9 or above) was centrifuged, and the centrifuged bacterial body was dipped and inoculated on a sodium carboxymethyl cellulose plate, which was anaerobically cultured at 39°C for 72 h. Then, 1 mg / mL Congo red solution was used for dyeing for 10 min, the Congo red solution was poured out, 1 mol / L NaCl solution was added to the plate for soaking for 15 min to wash off the dye, and whether there was a hydrolysis ring around the colony was observed to preliminarily determine whether the strain had the ability of fiber degradation. The strains which could produce transparent hydrolysis rings were accurately measured for the diameter of the hydrolysis ring using a vernier caliper, and the strains with a transparent ring diameter d>9.0 mm were further screened.

[0037] 1.3 Fiber-degrading strain rescreening

[0038] The 35 strains of bacteria with a transparent degradation ring diameter greater than 9 mm were cultured in BHI medium, 1 mL of the bacterial solution (OD value reached 0.9 or above) was centrifuged to obtain the bacterial body, and the bacterial body was resuspended using sterile water and adjusted to an OD value of 0.15. The glucose in the BHI medium was replaced with an equal amount of bran to configure a bran liquid medium. 1% bacterial suspension was inoculated in the bran liquid medium and anaerobically shaken and cultured at 39°C for 5 days, and the amount of reducing sugar produced in the medium was determined using the DNS method to determine the degradation ability of the strain to complex carbon sources. 540 nm value reached 0.9 or above) was centrifuged to obtain the bacterial body, and the bacterial body was resuspended using sterile water and adjusted to an OD value of 0.15. The glucose in the BHI medium was replaced with an equal amount of bran to configure a bran liquid medium. 1% bacterial suspension was inoculated in the bran liquid medium and anaerobically shaken and cultured at 39°C for 5 days, and the amount of reducing sugar produced in the medium was determined using the DNS method to determine the degradation ability of the strain to complex carbon sources.

[0039] 2 Test results

[0040] 2.1 16S rRNA strain identification results

[0041] The 16S rRNA of the strain 6076 was sequenced, and the nucleotide sequence is shown in SEQ ID No. 1. The sequence was compared on NCBI, and the sequence identity with the Acetogenium proteophilum was 99.51%. The strain 6076 was identified as Acetogenium proteophilum (hereinafter referred to as 6076). Proteiniphilum acetatigenes P. acetatigenes

[0042] Strain P. acetatigenes ​​

[0043] 2.2 Primary screening results of the fiber-degrading strains

[0044] 360 strains from 27 genera were selected for the primary screening, among which 101 strains were from Streptococcus genus, 98 strains were from Enterococcus genus, and 51 strains were from Proteiniphilum genus. Figure 1 The primary screening results showed that 163 strains (including strain 6076) could produce transparent degradation circles on the sodium carboxymethyl cellulose plate (CMC-Na plate), and had the preliminary fiber-degrading function. Figure 2 Among them, the transparent degradation circle diameters of 79 strains were less than 4 mm, the transparent degradation circle diameters of 49 strains were between 4 mm and 9 mm, and the transparent degradation circle diameters of 35 strains (including strain 6076) were greater than 9 mm. Figure 3

[0045] 2.3 Secondary screening results of the fiber-degrading strains

[0046] Further, the 35 strains with the transparent degradation circle diameters greater than 9 mm were detected for the complex carbon source degradation ability using the bran liquid medium. Among them, 25 strains (including strain 6076) could significantly degrade the bran into reducing sugar, and had the ability to degrade the complex carbon source. P. acetatigenes Figure 4

[0047] Test Example 1 Test on the feed fiber degradation ability of the strains isolated from the rumen fluid of dairy cows

[0048] 1 Test method

[0049] 1.1 Fermentation substrate and additives

[0050] Fermentation substrate: TMR daily ration (collected from Shijiazhuang Xingle Modern Animal Husbandry (Xingle) Co., Ltd. in Hebei Province).

[0051] 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.

[0052] Concentration of additives: 1 x 10 7 CFU / mL.

[0053] 1.2 Rumen fluid collection and buffer preparation

[0054] Three healthy Holstein cows with similar body weight (550 ± 50 kg) and lactation age (136 ± 37 d) and fitted with permanent rumen fistula were selected as the rumen fluid donors at the Changping Animal Experiment Base of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences. The cows were fed twice a day (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, and placed in a thermos bottle for rapid transport to the laboratory. The contents were filtered through 4 layers of gauze while CO2 was being introduced. The entire operation was carried out in a 39°C water bath and should be completed in the shortest possible time. At the same time, the buffer was prepared. After preparation, CO2 was continuously introduced until the pH value reached 6.8-7.0. The buffer was placed in a 39°C water bath for constant temperature and was ready for use.

[0055] 1.3 Fermentation method and group setting

[0056] About 0.75 g of fermentation substrate was accurately weighed into a 100 mL anaerobic fermentation bottle. When inoculating, 45 mL of preheated liquid medium and 15 mL of fresh rumen fluid filtered through 4 layers of gauze were quickly added to each bottle. CO2 was continuously introduced into the bottle for 5 s, then the bottle stopper was immediately added, and each fermentation bottle was placed in a 39°C constant temperature incubator for continuous culture for 24 h. The experiment was set up in 11 groups, including the CON group (blank control) and each strain test group, with 4 replicates for each group.

[0057] 1.4 Sample collection and preparation

[0058] The fermentation program was terminated after 48 h of fermentation, and the pH value of the fermentation broth was immediately determined. The fermentation broth was filtered through a nylon bag to separate the solid phase, and the remaining feed was dried at 65°C for determination of dry matter, neutral detergent fiber, and acid detergent fiber. Part of the fermentation broth was collected and stored at -20°C for determination of volatile fatty acids (VFA) and NH3-N.

[0059] 2 Test results

[0060] The 25 strains of bran-degrading bacteria were from 10 genera. The most functional strains from different genera were selected for in vitro fermentation to determine their effects on feed fiber degradation efficiency in a simulated rumen environment.

[0061] The test results are as follows Figure 5The results showed that the in vitro fermentation of different strains with TMR as substrate P. acetatigenes 6076 and Actinomyces glycerinitolerans 6345 could significantly improve the dry matter degradability and neutral detergent fiber degradability of feed (P<0.05), P <0.05), Caldibacillus pasinlerensis 5847 could significantly improve the neutral detergent fiber degradability of feed (P<0.05). P <0.05). Among them, P. acetatigenes 6076 could increase the dry matter degradability of feed from 53.45% to 58.49%, about 9%; the fiber degradability from 42.15% to 52.24%, about 24%.

[0062] Table 1 in vitro fermentation determination P. acetatigenes Effect of 6076 on rumen fermentation parameters

[0063]

[0064] P. acetatigenes Effect of 6076 on rumen fermentation parameters as shown in Table 1, compared with the control group, the addition of P. acetatigenes 6076 could significantly improve the pH value of fermentation liquor and significantly improve the content of acetic acid, isobutyric acid, butyric acid and valeric acid, and significantly improve the total volatile fatty acid content of rumen, but had no significant effect on the ratio of propionic acid to acetic acid.

Claims

1. A strain of the acetic acid producing bacterium (Alistipes sp. Proteiniphilum acetatigenes ) 6076, characterized in that, The microbial preservation number thereof is CGMCC No. 33516.

2. A microbial preparation containing the Proteiniphilum acetatigenes 6076 according to claim 1.

3. Use of the Proteiniphilum acetatigenes 6076 according to claim 1 or the microbial preparation according to claim 2 in the preparation of an animal feed additive.

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

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

6. Use according to claim 5, characterized in that, The improvement of the digestive capacity of animals includes improvement of the utilization rate of feed fiber.

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

8. Use according to claim 3, characterized in that, The feed additive is a feed additive having the function of improving the stomach environment of animals.

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

Images