CN117070419B - Rumen enterococcus from cow rumen and application of rumen enterococcus as feed additive - Google Patents
Rumen enterococcus from cow rumen and application of rumen enterococcus as feed additive Download PDFInfo
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- CN117070419B CN117070419B CN202311187425.3A CN202311187425A CN117070419B CN 117070419 B CN117070419 B CN 117070419B CN 202311187425 A CN202311187425 A CN 202311187425A CN 117070419 B CN117070419 B CN 117070419B
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- enterococcus
- rumen
- ruminant
- strain
- silage
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Classifications
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Abstract
The invention discloses rumen enterococcus from rumen of dairy cows and application thereof as a feed additive. The invention separates and identifies a new enterococcus strain from cow rumen, named as rumen enterococcus S86.2, and the microorganism preservation number is: GDMCC No. 63263. The separated rumen enterococcus S86.2 can utilize glucose or cellobiose to ferment to produce L-lactic acid and acetic acid, is favorable for inhibiting the growth of harmful microorganisms, maintains the health of the rear intestinal tracts of ruminants, keeps the microecological balance of organisms, can be used as an intestinal probiotics biological resource and can also be used as an inoculant for silage fermentation to improve the silage quality of forage grass, and comprises the following steps: inoculating silage to ferment to improve the nutrition quality and stability of silage, and finally improving the production performance of ruminants; either by adding it directly to the ration of the ruminant to promote ruminant development or to reduce the rate of diarrhea in the ruminant.
Description
Technical Field
The invention relates to a microorganism strain separated from animals, in particular to a rumen enterococcus strain derived from the rumen of cowsEnterococcus ruminicola) And the application of the feed additive in improving the production performance of ruminants or reducing the diarrhea rate, belonging to the field of enterococcus rumbet and the application thereof.
Background
The complex and diverse microbiota inhabiting in the rumen of ruminants is an integral part of the ruminant digestive system. The diversity of the rumen microflora is closely related to the composition of the host ration and dry matter intake. Over the past several decades, many chemical feed additives such as antibiotics, ionophores, methane inhibitors, etc. have been used in ruminant feed to regulate the rumen and intestinal micro-ecosystem to improve host productivity. However, these substances remain in animal products and excessive use of antibiotics can cause bacterial resistance. The chinese agricultural rural part published bulletin 7 in 2019, banning the use of antibiotics in feeds, and finding natural antibiotic additives has become an urgent need for the feed industry. The probiotics can improve the intestinal microecology balance of animal hosts, and has wide application prospect in feed production due to the characteristics of safety, naturalness, greenness and no pollution. Enterococci are one of lactic acid bacteria, a facultative anaerobic gram-positive bacterium present in the intestinal tracts of humans and animals, and the publication 1126 published by the department of agriculture in 2008 shows that china allows the inclusion of enterococci in probiotic species added to feeds. It has been shown that enterococci help to maintain the activity of lactic acid utilizing bacteria, stimulate the growth of ruminal microorganisms, and thereby increase the energy supply of glycogenic propionic acid to ruminant hosts. In addition, enterococcus can also produce antibacterial substances to inhibit the growth of harmful microorganisms and maintain the microecological balance of the organism.
Enterococci are facultative anaerobes, indicating that they not only grow and proliferate in the anaerobic environment of the rumen and intestinal tract, but also survive the fermentation process of silage, through which they are fed to animals, and thus into the gastrointestinal system. High-quality safe silage is an important precondition for guaranteeing the health of ruminants and improving the quality of animal products. Lactic acid bacteria are often used as silage additives for improving silage quality and increasing safety of forage grass. Researches show that the enterococcus inoculation in the silage not only promotes the reproduction and lactic acid content of lactic acid bacteria and obviously improves the nutrition quality and stability, but also improves the production performances of animal feed intake, weight gain, milk production and the like.
Disclosure of Invention
The invention aims at providing a novel enterococcus rumbetEnterococcus ruminicola）；
The second object of the invention is to apply the novel enterococcus ruminalis as a feed additive to improve the quality or stability of silage additives or to improve the productivity of ruminants, reduce the diarrhea rate of ruminants, etc.
In order to achieve the above purpose, the technical scheme adopted by the invention comprises the following steps:
one aspect of the invention provides a new enterococcus strain which is separated and identified from the rumen of dairy cows and named as rumen enterococcusEnterococcus ruminicola) S86.2 strain, whose microorganism deposit number is: GDMCC No. 63263; the classification names are as follows:Enterococcus sp.the method comprises the steps of carrying out a first treatment on the surface of the The preservation time is as follows: 2023, 3 and 12; the preservation units are as follows: the collection of microorganism strains in Guangdong province; the preservation address is: building 5, guangzhou city, first, middle road 100, university, 59, university of Guangdong, academy of sciences of China, and microbiological study.
The invention identifies a new enterococcus strain separated and identified from cow rumen, and the identification result shows that the separated strain is a new enterococcus strain, the 16S rRNA length is 1633kb, and the nucleotide sequence is shown as SEQ ID No. 3; the sequence is subjected to homology comparison, and the result shows that the strain is a new strain of enterococcus andEnterococcus saigonensisthe closest relationship is, therefore, the strain is named asEnterococcus ruminicola S86.2。
According to the measurement result of acid producing capacity of the strain, the isolated enterococcus rumbet S86.2 can ferment D-mannitol, D-maltose, D-cellobiose, D-glucose, D-lactose, D-trehalose, salicin, gelatin, D-mannose, glycerol and D-sorbitol, and does not ferment D-xylose, D-sucrose, L-arabinose, D-maltose, D-raffinose and L-rhamnose; the enterococcus ruminae S86.2 strain hydrolyzes gelatin and escin to produce indole and catalase.
As a result of measurement of the enzyme activity of the strain, the isolated enterococcus rumbet S86.2 of the present invention has activities of alkaline phosphatase, esterase lipase, leucine aminopeptidase, valine arylamidase, cystine arylamidase, alpha-chymotrypsin, acid phosphatase, naphthol AS-BI phosphate phosphohydrolase, beta-glucosidase and acetylglucosaminidase, and does not have activities of lipase, trypsin, alpha-galactosidase, beta-glucuronidase, alpha-glucosidase, alpha-mannosidase and alpha-fucosidase.
According to the test result of the capability measurement of the strain to produce L-lactic acid and acetic acid, the separated enterococcus rumbet S86.2 can utilize glucose or cellobiose to ferment to produce L-lactic acid and acetic acid, thereby being beneficial to inhibiting the growth of harmful microorganisms, maintaining the intestinal health of ruminants and keeping the microecological balance of organisms. Because the separated rumen enterococcus S86.2 strain is derived from rumen of dairy cows, the strain can utilize extensive polysaccharide substrates to ferment and produce L-lactic acid and acetic acid, thereby improving the utilization rate of ruminant feed and increasing the energy supply of a host.
The separated enterococcus rumbet S86.2 bacterial liquid is added into milk for feeding calves according to a certain proportion, and the result shows that the addition of the enterococcus rumbet S86.2 bacterial liquid increases the weight and daily gain of the calves; the total volatile fatty acid of the rumen is effectively increased, the acetic acid content is increased, and the development of rumen epithelium is further promoted; the length of small intestines such as jejunum, ileum and the like is improved, and the intestinal development of calves is promoted; obviously reduces the calf diarrhea rate and effectively promotes the calf intestinal health.
Thus, another aspect of the invention is the use of the isolated enterococcus ruminalis S86.2 of the invention as silage additive for improving forage silage quality comprising: inoculating the silage with the isolated enterococcus ruminalis S86.2 to improve the nutritional quality and stability of the silage, ultimately increasing or improving the performance of ruminants, comprising: improving feed intake, increasing weight, milk yield, etc.; or directly adding enterococcus ruminalis S86.2 bacteria liquid into the ration of ruminant to improve the production performance of ruminant, including increasing the weight and daily gain of ruminant, and promoting the development of rumen epithelium or intestinal tract of ruminant; or to reduce diarrhea rate in ruminants, etc.
Drawings
FIG. 1 is a phylogenetic tree based on genomic sequences.
FIG. 2 shows the yield of lactic acid produced by fermentation of enterococcus ruminococcus S86.2 using glucose fibrils or cellobiose.
FIG. 3 shows the yield of acetic acid produced by fermentation of enterococcus ruminococcus S86.2 with glucose or cellobiose.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.
EXAMPLE 1 isolation and identification of enterococcus ruminae S86.2
Isolation and purification of enterococcus rumen S86.2: taking rumen fluid of dairy cows, uniformly mixing, filtering by four layers of gauze, subpackaging part of the rumen fluid after filtering into a centrifuge tube for preparing a culture medium, transferring the other part of rumen fluid into an anaerobic serum bottle containing glycerol by using a syringe for separating and culturing microorganisms, and taking all samples back to a laboratory for freezing at-80 ℃.
Anaerobic culture medium was prepared, and specific components are shown in tables 1 to 5. Rumen fluid samples in centrifuge tubes were thawed at room temperature, centrifuged at 13,000Xg at 4℃for 5min and rumen supernatant was collected. Continuous CO feeding of anaerobic culture medium 2 The gas 3 h was deoxygenated, the pH was adjusted to 6.8, and then sterilized at 125℃for 15 minutes. Transferring to an anaerobic incubator to prepare a plate.
TABLE 1 anaerobic Medium composition Table
TABLE 2 anaerobic basal Medium composition Table
TABLE 3 inorganic salt solution 1
TABLE 4 inorganic salt solution 2
TABLE 5 microelements
Rumen fluid samples frozen in anaerobic serum bottles were thawed at room temperature. Sequentially prepared into 10 by using anaerobic PBS solution -3 ，10 -4 ，10 -5 ，10 -6 ，10 -7 0.1ml of the inoculation liquid of the formula (I) is coated on a prepared flat plate for culturing for 48-72 hours at 39 ℃, single bacterial colony is selected, and further streak separation is carried out, so that bacterial purification and expansion culture are realized.
Single colony verification: the picked single colony is placed in a full nutrient medium for culturing at 39 ℃ for 48 hours, DNA is extracted after incubation, then a 27F/1492R primer (27-F5 '-AGA GTT TGA TCC TGG CTC AG-3' (SEQ ID No. 1), 1492-R: 5 '-TAC GGY TAC CTT GTT ACG ACT T-3' (SEQ ID No. 2)) is utilized to amplify the 16SrRNA gene, and the amplified product is subjected to Sanger sequencing, and the colony is verified to be single bacteria according to a sequencing peak diagram without a mixed peak.
And (3) strain identification: the DNA of the single bacteria is purified by a DNA purification kit, and second generation sequencing is performed. The adaptors of the original sequences were removed and quality controlled using trimmonic and fastqc software. The filtered sequences were spliced using MetaHIT software. The quality of the spliced genome was checked by CheckM and the microorganism species identification was performed by GTDB-Tk software.
The results showed that the isolated strain was a novel species of enterococcus, whose 16S rRNA length was 1633kb, and the specific nucleotide sequence was shown in SEQ ID No. 3. The sequence is subjected to homology comparison, and the result shows that the strain is a new strain of enterococcus andEnterococcus saigonensisthe phylogenetic tree is the closest, as shown in FIG. 1, and therefore, this strain is namedEnterococcus ruminicola S86.2。
Test example 1 functional verification test of enterococcus ruminae S86.2
Determination of acid producing ability of bacterial strain
The ruminococcus strain S86.2 was activated on the plate medium described in example 1, and its acid producing ability was determined by using an API 20A test strip, which was operated according to the instructions.
The results of the acid production capacity measurement of enterococcus rumbet S86.2 are shown in Table 6;
table 6 determination of acid generating Capacity of enterococcus ruminococcus strain S86.2
From the test results in Table 6, it can be seen that enterococcus rumbet S86.2 can ferment D-mannitol, D-maltose, D-cellobiose, D-glucose, D-lactose, D-trehalose, salicin, gelatin, D-mannose, glycerol and D-sorbitol, without fermenting D-xylose, D-sucrose, L-arabinose, D-maltose, D-raffinose and L-rhamnose. The enterococcus ruminae S86.2 strain hydrolyzes gelatin and escin to produce indole and catalase.
Strain enzyme Activity assay
Activating the enterococcus ruminae strain S86.2 on a plate culture medium, and determining the enzyme activity by using an API ZAY test strip according to the operation of the specification; the results of the enzyme activity measurement are shown in Table 7:
table 7 determination of the enzyme Activity of the enterococcus ruminococcus strain S86.2
As can be seen from Table 7, enterococcus ruminae S86.2 has alkaline phosphatase, esterase lipase, leucine aminopeptidase, valine arylamidase, cystine arylamidase, alpha-chymotrypsin, acid phosphatase, naphthol AS-BI phosphate phosphohydrolase, beta-glucosidase and acetylglucosaminidase activities, and does not have lipase, trypsin, alpha-galactosidase, beta-glucuronidase, alpha-glucosidase, alpha-mannosidase and alpha-fucosidase activities.
Test example 2 test of L-lactic acid and acetic acid production ability of enterococcus ruminococcus S86.2 Strain
Anaerobic basal medium was prepared, and specific ingredients are shown in table 2. Continuous CO feeding of anaerobic basal culture medium 2 Gas 3 h was deoxygenated, pH adjusted to 6.8, and then transferred to an anaerobic incubator. Glucose medium (0.05 g glucose added to basal medium) and cellobiose medium (0.05 g cellobiose added to basal medium) were prepared. The prepared culture medium was dispensed into a Hungate tube with 5 ml of each tube and sterilized at 125℃for 15 minutes. Inoculating the bacterial liquid according to the bacterial amount of 1%, and incubating at 39 ℃.2 parts of bacterial liquid (1 ml each) were collected during the 0h and exponential growth phases, respectively, 200 μl of 25% metaphosphoric acid was immediately added to 1ml for the measurement of VFA, and all samples were centrifuged at 12000 g at 4℃for 5min, and the supernatant was frozen at-20 ℃.
The results of the experiment are shown in FIGS. 2 and 3, and it can be seen from the results of the experiment that enterococcus ruminarum S86.2 can produce L-lactic acid and acetic acid by fermentation using glucose and cellobiose.
Test example 3 test of enterococcus ruminae S86.2 for promoting calf growth and development
Test method
Experimental animal management: selecting about 24 Chinese Holstein cow male calves with the age of 10 days, dividing the Chinese Holstein cow male calves into 3 treatment groups according to a random complete group design, wherein the weight difference of the primary calves among groups is not obvious. The calf is raised in a single circle. The calves are fed with fresh milk before weaning, and the specific feeding amount is as follows: before 30 days of age, the milk feeding amount is 10% of the weight of the milk; 31-50 days old, and the milk feeding amount is 4 kg/d; calves older than 50 days are 2 kg/d. The test starts by adding the feed, and the feed can be eaten and drunk freely.
And (3) test design: calves are divided into three treatment groups, which are respectively: test group 1, test group 2, control group. Test group 1 and 2 calves fed with ruminococcus S86.2 bacteria solution (bacterial load 1×10) 10 cell/mL), the control group was not fed with bacterial liquid. The bacterial liquid feeding bacterial liquid amount of the test group 1 is added by 0.5 percent of the weight of the calf feeding dry matter, the bacterial liquid feeding bacterial liquid amount of the test group 2 is added by 1% of the weight of the calf fed dry matter, and is directly added into milk.
Detecting the index: 1) The calf body weight was measured at trial start 1, d and at 7-d intervals after trial start. 2) Every 2 d calves feed intake was recorded. 3) Slaughtering 8 weeks after weaning.
Statistics and analysis: the basic data were consolidated with Excel and the data analysis was performed using SAS, with the results expressed as least squares mean and the significance level p <0.05.
Test results
The test results are shown in tables 8-11, and according to the test results, the addition of the enterococcus rumbet S86.2 bacterial liquid increases the weight and daily gain of calves; the total volatile fatty acid of the rumen is increased, the acetic acid content is increased, and the development of rumen epithelium can be promoted; the length of small intestines such as jejunum, ileum and the like is improved, which shows that the bacterial liquid promotes the development of intestinal tracts; the diarrhea rate of calves is reduced, which shows that the bacterial liquid promotes intestinal health.
TABLE 8 influence of addition of enterococcus rumbet S86.2 bacteria solution on daily weight gain of calves
Note that: the same row shoulder notes with different lower case letters indicate significant differences (P < 0.05)
TABLE 9 changes in calf rumen fermentation after addition of enterococcus rumbet S86.2 bacteria solution
Note that: the same row shoulder-notes with different lower case letters indicate significant differences (P < 0.05).
TABLE 10 Effect of adding enterococcus rumbet S86.2 bacterial liquid on calf intestinal length
Note that: different letters from shoulder-injection of the same row indicate significant differences (P < 0.05).
TABLE 11 Effect of adding enterococcus rumbet S86.2 bacteria solution on calf diarrhea
Note that: different letters from shoulder-injection of the same row indicate significant differences (P < 0.05).
Claims (9)
1. Enterococcus genus isolated from cow rumenEnterococcus sp.) S86.2 strain, characterized in that the microorganism deposit number is: GDMCC No. 63263.
2. Use of the enterococcus S86.2 strain according to claim 1 as a feed additive for ruminants.
3. The use according to claim 2, characterized in that it comprises: the enterococcus S86.2 strain is inoculated with silage and then fermented to improve the yield of L-lactic acid or acetic acid of the silage.
4. The use according to claim 2, characterized in that it comprises: inoculating the enterococcus S86.2 strain into silage for fermentation, and feeding the silage to ruminants to improve the production performance of the ruminants; wherein said increasing the productivity of the ruminant comprises increasing the weight of the ruminant; the ruminant is a dairy cow.
5. The use according to claim 2, characterized in that it comprises: the enterococcus S86.2 strain is directly added into the ration of ruminant as a feed additive to promote the development of the ruminant or reduce the diarrhea rate of the ruminant; the ruminant is a dairy cow.
6. The use according to claim 5, wherein said promoting ruminant development comprises increasing ruminant weight and daily gain, and/or promoting ruminant rumen epithelial development or intestinal development; the ruminant is a dairy cow.
7. The use according to claim 2, wherein the ruminant is a calf.
8. Use of the enterococcus S86.2 strain according to claim 1 for the fermentative production of L-lactic acid and acetic acid, comprising: glucose or cellobiose is used as a fermentation substrate, and the enterococcus S86.2 strain of claim 1 is inoculated for fermentation.
9. A ruminant feed comprising the enterococcus S86.2 strain of claim 1.
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