CN111961611A - Pediococcus pentosaceus 368 and application thereof - Google Patents
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Abstract
The invention discloses a pediococcus pentosaceus 368 strain and application thereof. The invention relates to pediococcus pentosaceus 368, which is characterized in that: the strain is Pediococcus pentosaceus; the preservation name is Pediococcus pentosaceus LL 2018368; is preserved in China general microbiological culture Collection center, and the preservation address is the institute of microbiology of Zhongkoyao No. 3 Hospital, Xilu No.1 Hospital, North Chen, of Chaozhong, Beijing; the preservation date is as follows: year 2019, month 10, day 21; the preservation number is CGMCC No: 18709. the invention has the advantages that (1) the pediococcus pentosaceus 368 can remarkably inhibit the growth of streptococcus in pig intestines and has disease resistance. (2) Pediococcus pentosaceus 368 can increase average daily gain and average daily feed intake of pigs, and reduce feed-weight ratio, diarrhea rate and constipation rate. (3) The pediococcus pentosaceus 368 can reduce the content of ammonium nitrogen and crude fiber in excrement, reduce ammonium nitrogen pollution and improve the utilization of crude fiber.
Description
Technical Field
The invention relates to the technical field of biology, in particular to pediococcus pentosaceus 368 and application thereof.
Background
The lactobacillus regulates the pH of the intestinal tract through metabolites, thereby promoting the growth of animals, and regulating the structure and relative abundance of intestinal flora, thereby improving the gastrointestinal tract function, improving the growth performance, reducing the morbidity and the like. Pediococcus pentosaceus belongs to the genus Pediococcus of the family Streptococcus, and is capable of fermenting glucose to produce lactic acid and inhibiting pathogenic bacteria such as Aeromonas hydrophila, Listeria monocytogenes, Bacillus cereus, etc. However, no inhibition effect on streptococcus suis has been reported, and at present, many tests for antagonizing pathogenic bacteria are performed in vitro, and whether effective antibacterial effects can be exerted in complex animals has not been reported.
In the livestock and poultry breeding process in China, the abuse of antibiotics easily causes environmental pollution, the residue of antibiotics in biological products, the balance of animal intestinal flora is broken and other problems, and even a series of reactions such as soil environment deterioration and the like; the pediococcus pentosaceus has good acid production and pathogenic bacteria antagonistic properties, can promote nutrient metabolism and absorption, and can be used as a novel feed additive.
At present, pediococcus pentosaceus 368 capable of reducing diarrhea rate and constipation rate of pigs and application thereof are lacked.
Disclosure of Invention
The invention aims to solve the technical problem of providing pediococcus pentosaceus 368 capable of reducing the diarrhea rate and the constipation rate of pigs and application thereof.
In order to achieve the purpose, the invention is realized by the following technical scheme: the invention relates to Pediococcus pentosaceus 368, which is Pediococcus pentosaceus; the preservation name is Pediococcus pentosaceus LL 2018368; is preserved in China general microbiological culture Collection center, and the preservation address is the institute of microbiology of Zhongkoyao No. 3 Hospital, Xilu No.1 Hospital, North Chen, of Chaozhong, Beijing; the preservation date is as follows: year 2019, month 10, day 21; the preservation number is CGMCC No: 18709.
further, the gene sequence of the 16S rDNA of the pediococcus pentosaceus 368 is the nucleotide sequence shown in SEQ ID No. 1.
Further, after aerobic culture is carried out in an MRS culture medium for 72 hours at 35 ℃, bacterial colonies are round and milky, the surface is smooth, the middle is raised, the edges are neat, pigmentation is avoided, the bacterial colonies are easy to pick up, silkiness is avoided, the lactic acid fragrance is provided, gram staining is positive, bacteria are rod-shaped, the bacterial colonies are facultative anaerobic, the bacterial colonies can survive for more than two months when stored in liquid at 4 ℃, and the optimal growth temperature is 30-38 ℃.
The pediococcus pentosaceus 368 microbial inoculum prepared by the pediococcus pentosaceus 368 is provided by the invention.
The preparation method of the pediococcus pentosaceus 368 microbial inoculum comprises the following steps:
(1) preparing strains: inoculating in MRS liquid culture medium, culturing at 32-38 ℃ for 18-20 h, and centrifuging at 8000-10000 rpm to obtain thalli.
(2) The cells were freeze-dried.
Further, in step (1), the MRS medium culture fluid is: peptone 10.0 g; 5.0g of beef extract; 4.0g of yeast powder; 20.0g of glucose; dipotassium phosphate 2.0 g; 2.0g of triammonium citrate; 5.0g of sodium acetate; magnesium sulfate 0.2 g; 0.05g of manganese sulfate; tween 801.0 mL; and 1L of water.
The pediococcus pentosaceus 368 microbial inoculum disclosed by the invention is applied to feed and water.
Further, the effective viable count of the microbial preparation is 1010～1011CFU/g。
Further, the additive amount in the feed is 106～108CFU/g, 10 in drinking water9～1011CFU/L。
Compared with the prior art, the invention has the following advantages:
(1) the pediococcus pentosaceus 368 can remarkably inhibit growth of streptococcus in pig intestines, and has disease resistance.
(2) The pediococcus pentosaceus 368 can improve average daily gain and average daily feed intake of pigs, and reduce feed-weight ratio, diarrhea rate and constipation rate.
(3) The pediococcus pentosaceus 368 can reduce the content of ammonium nitrogen and crude fiber in excrement, reduce ammonium nitrogen pollution and improve the utilization of crude fiber.
(4) The pediococcus pentosaceus 368 can obviously increase important active flavor precursor substances such as N- (1-deoxy-1-fructosyl) proline, N- (1-deoxy-1-fructosyl) glycine and the like related to flavor in products, increase melanin precursor substances such as dopaquinone and the like related to melanin formation, and increase the content of apoptosis factor diphtheria amide negatively related to tumors.
Drawings
FIG. 1 is a 368 colony morphology under a fluorescence microscope according to the present invention.
FIG. 2 is a graph of a species level-based wien diagram analysis of the present invention.
Detailed Description
The invention is further described with reference to the accompanying drawings and specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
The invention relates to pediococcus pentosaceus 368, which is the pediococcus pentosaceus 368; the preservation name is pediococcus pentosaceus 2018LL 368; is preserved in China general microbiological culture Collection center, and the preservation address is the institute of microbiology of Zhongkoyao No. 3 Hospital, Xilu No.1 Hospital, North Chen, of Chaozhong, Beijing; the preservation date is as follows: year 2019, month 10, day 21; the preservation number is CGMCC No: 18709.
the gene sequence of the 16S rDNA of the pediococcus pentosaceus 368 is a nucleotide sequence shown in SEQ ID No. 1.
After aerobic culture is carried out for 72 hours at 35 ℃ in an MRS culture medium, bacterial colonies are round and milky, the surfaces are smooth, the middle part is raised, the edges are neat, pigmentation is avoided, the bacterial colonies are easy to pick up, have no silks, have the fragrance of lactic acid, are positive in gram staining, are rod-shaped, are facultative and anaerobic, can survive for more than two months when stored in liquid at 4 ℃, and have the optimal growth temperature of 30-38 ℃.
The pediococcus pentosaceus 368 microbial inoculum prepared by the pediococcus pentosaceus 368 is provided by the invention.
The preparation method of the pediococcus pentosaceus 368 microbial inoculum comprises the following steps:
(1) preparing strains: inoculating in MRS liquid culture medium, culturing at 32-38 ℃ for 18-20 h, and centrifuging at 8000-10000 rpm to obtain thalli.
(2) The cells were freeze-dried.
In step (1), the MRS culture medium culture solution is: peptone 10.0 g; 5.0g of beef extract; 4.0g of yeast powder; 20.0g of glucose; dipotassium phosphate 2.0 g; 2.0g of triammonium citrate; 5.0g of sodium acetate; magnesium sulfate 0.2 g; 0.05g of manganese sulfate; tween 801.0 mL; and 1L of water.
The pediococcus pentosaceus 368 microbial inoculum disclosed by the invention is applied to feed and water.
The effective viable count of thallus in the microbial preparation is 1010～1011CFU/g。
The additive amount in the feed is 106～108CFU/g, 10 in drinking water9～1011CFU/L。
Example 1
(1) Preparing strains: inoculating 0.1% inoculum size in MRS liquid culture medium, culturing at 38 deg.C for 18h, and centrifuging at 5000rpm to obtain thallus. The MRS culture medium culture solution is as follows: peptone 10.0 g; 5.0g of beef extract; 4.0g of yeast powder; 20.0g of glucose; dipotassium phosphate 2.0 g; 2.0g of triammonium citrate; 5.0g of sodium acetate; magnesium sulfate 0.2 g; 0.05g of manganese sulfate; tween 801.0 mL; and 1L of water.
(2) The cells were freeze-dried.
The effective viable count of thallus in the microbial preparation is 1010～1011CFU/g。
Example 2
(1) Preparing strains: inoculating 0.2% inoculum size in MRS liquid culture medium, culturing at 35 deg.C for 20 hr, and centrifuging at 6000rpm to obtain thallus.
(2) The cells were freeze-dried.
The effective viable count of thallus in the microbial preparation is 1010～1011CFU/g。
In step (1), the MRS culture medium culture solution is: peptone 10.0 g; 5.0g of beef extract; 4.0g of yeast powder; 20.0g of glucose; dipotassium phosphate 2.0 g; 2.0g of triammonium citrate; 5.0g of sodium acetate; magnesium sulfate 0.2 g; 0.05g of manganese sulfate; tween 801.0 mL; and 1L of water.
Example 3
(1) Preparing strains: inoculating 0.5% inoculum size in MRS liquid culture medium, culturing at 30 deg.C for 22 hr, and centrifuging at 6000rpm to obtain thallus. (2) The cells were freeze-dried.
The effective viable count of thallus in the microbial preparation is 1010～1011CFU/g。
In the step (1), the MRS agar culture medium culture solution is: peptone 10.0 g; 5.0g of beef extract; 4.0g of yeast powder; 20.0g of glucose; dipotassium phosphate 2.0 g; 2.0g of triammonium citrate; 5.0g of sodium acetate; magnesium sulfate 0.2 g; 0.05g of manganese sulfate; tween 801.0 mL; and 1L of water.
Test example 1
The test was carried out at the scientific academy of livestock and veterinary science of Yunnan province, and 20 healthy fattening pigs with average weight of about 91kg and insignificant weight difference were selected from Diqing Tibetan pigs. Groups were randomized into 2 (1 test group, designated a and 1 control group, designated C) with 2 replicates in each group, each replicate 5. The control group daily ration is the basic daily ration, the test group daily ration is the daily ration, the additive prepared in the embodiment 1 is added into feed, free ingestion and drinking water, conventional immunity and insect expelling are carried out, the disease condition of pigs is observed and recorded every day in the test period, the test period is totally 21 days, and the average daily gain, the daily feed intake, the feed-weight ratio and the survival rate are calculated after the test is finished. The influence of 368 on the growth performance of the fattening pigs in the test results is shown in table 1; the effect of 368 addition on the general composition of feces is shown in table 2:
TABLE 1
TABLE 2
As shown in the data in tables 1-2, after pediococcus pentosaceus 368 is added into the feed, the content of crude fiber and ammonium nitrogen in excrement can be obviously reduced, and the feed has the effects of potentially improving the utilization rate of the crude fiber in the feed and reducing the emission of the ammonium nitrogen. The growth performance data show that the diarrhea rate of the example is only 0.9%, the diarrhea rate is reduced by 50% compared with the control group, the constipation rate is 1.36%, the constipation rate is reduced by 66.8% compared with the control group, and the disease resistance effect is obvious. Meanwhile, the product of the invention also has a certain effect on improving the growth performance of piglets, and compared with a control group, the feed-weight ratio is reduced by 8.64%, the average daily feed intake is increased by 3.38%, and the average daily gain is increased by 13.26%. .
Assay analysis
The otu (operational Taxonomic units) is a unified mark artificially set for a certain classification unit (strain, genus, species, group, etc.) in phylogenetic or population genetics research for the convenience of analysis. The pig manure of the experimental group and the pig manure of the control group are analyzed by adopting a high-throughput sequencing technology, the results are as follows, the analysis result based on the genus level wain diagram is shown in figure 2, the number of bacteria in the intestinal tract of the experimental pig added with the test strain 368 is 188, the number of the bacteria in the intestinal tract of the control group is 177, and the bacteria are 11 fewer than the bacteria in the experimental group, so that the dry bacteria powder additive can improve the types of the bacteria in the intestinal tract of the pig at the genus level.
FIG. 2 is a graph of a Wen chart analysis based on seed level of the present invention, wherein A is a test group; c is a control group. The structural analysis of the intestinal flora at the genus level after 368 addition is shown in table 3:
TABLE 3
The structural analysis of the intestinal flora at the level of the phylum after 368 addition is shown in table 4:
TABLE 4
The data in table 4 show that, after the bacterium 368 of the present invention was added, the relative abundance of firmicutes in the intestinal tract decreased, the relative abundance of bacteroidetes increased, the firmicutes/bacteroidetes ratio decreased, and the relative abundance of spirochetes increased significantly.
According to the analysis results on the genus level, as shown in table 3, after pediococcus pentosaceus 368 is added to the feed of fattening pigs, the relative abundance of streptococcus in the intestinal tract is greatly reduced, the streptococcus is common bacteria in the intestinal tract of pigs, the streptococcus is low in relative abundance and is not dominant bacteria in the intestinal tract, and if the streptococcus is dominant bacteria, the streptococcus is infected in the pig farm but does not attack the pig farm. According to literature, streptococcus in pig intestines is partially pathogenic bacteria, is common pathogenic bacteria in a breeding process, can reduce growth performance and improve morbidity, is not found to have pathogenic symptoms by observing feeding and mental states of pigs at the beginning of an experiment, but is found to have abnormal relative abundance of streptococcus by flora diversity analysis, is frequently found in the breeding process, and is found to be capable of greatly reducing the abundance of streptococcus in intestines and improving the relative abundance of lactobacillus after being fed to pediococcus pentosaceus 368 in the example, and simultaneously can be capable of remarkably improving the relative abundance of clostridium, Lachnospiraceae _ 1014 b _ group and terrestris which are negatively related to intestinal inflammation. Wherein Lachnospiraceae is associated with host mucosal integrity, bile acid metabolism and polysaccharide breakdown, and is negatively associated with intestinal diseases. The data in table 5 show that the relative abundance of firmicutes in the intestinal tract decreases, the relative abundance of bacteroidetes increases, the firmicutes/bacteroidetes ratio decreases, and the relative abundance of spirochetes increases significantly after the addition of the bacterium 368 of the present invention.
The differential metabolite data in table 5 show that diphtheria amide, 2 hydroxytetradecanoic acid, Furanofukinin, dopaquinone, gestriene, ptatomide B, ketone, N- (1-deoxy-1-fructosyl) proline and N- (1-deoxy-1-fructosyl) glycine are all significantly or very significantly increased in the intestine of fattening pigs after addition of the product of the invention. N- (1-deoxy-y-1-fructosyl) proline and N- (1-deoxy-y-1-fructosyl) glycine are important active flavor precursors and are related to the flavor in the product; diphtheria amide is inversely related to tumorigenesis, 2 hydroxytetradecanoic acid is related to fat metabolism, and Furanofukinin is vitamin B2A precursor substance; dopaquinone is a melanin precursor substance and is involved in melanin formation, and therefore it is understood that 368 is added to significantly increase biomarkers related to flavor formation, VB2, and melanin-related substance synthesis.
Differential metabolite analysis is shown in table 5:
TABLE 5
Note: FC (D/C) is the fold differential expression (fold change) of this metabolite between the two groups. C: expression level of the metabolite in control group X, D: the expression level of the metabolite in the experimental group Y was controlled by C.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification, and equivalents thereof.
Sequence listing
<110> department of sciences of animal husbandry and veterinary science of Yunnan province
<120> Pediococcus pentosaceus 368 and application thereof
<130> 2020
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1456
<212> DNA
<213> Artificial sequence (16S rDNA Gene sequence)
<400> 1
ttagacggct agctcctaaa aggttacccc accggctttg ggtgttacaa actctcatgg 60
tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga 120
ttactagcga ttccgacttc gtgtaggcga gttgcagcct acagtccgaa ctgagaatgg 180
ttttaagaga ttagcttaac ctcgcggtct cgcgactcgt tgtaccatcc attgtagcac 240
gtgtgtagcc caggtcataa ggggcatgat gatttgacgt cgtccccacc ttcctccggt 300
ttgtcaccgg cagtctcact agagtgccca acttaatgct ggcaactart aataagggtt 360
gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc 420
acctgtcatt ctgtccccga agggaacctc taatctctta gactgtcaga agatgtcaag 480
acctggtaag gttcttcgcg tagcttcgaa ttaaaccaca tgctccaccg cttgtgcggg 540
cccccgtcaa ttcttttgag tttcaacctt gcggycgtac tccccaggcg gattacttaa 600
tgcgttagct gcagcactga agggcggaaa ccctcyaaca cttagtaatc atcgtttacg 660
gcatggacta ccagggtatc taatcctgtt cgctacccat gctttcgagc ctcagcgtca 720
gttgcagacc agacagycgc cttcgccact ggtgttcttc catatatcta cgcatttcac 780
cgctacacat ggagttccac tgtcctcttc tgcactcaag tctcccagtt tccaatgmac 840
ttcttcggtt gagccgaagg ctttcacatt agacttaaaa gaccgcctgc gctcgcttta 900
cgcccaataa atccggataa cgcttgccac ctacgtatta ccgcggctgc tggcacgtag 960
ttagccgtgg ctttctggtt aaataccgtc actgggtaaa cagttactct tacccacgtt 1020
cttctttaac aacagagctt tacgagccga aacccttctt cactcacgcg gcgttgctcc 1080
atcagacttg cgtccattgt ggaagattcc ctactgctgc ctcccgtagg agtctgggcc 1140
gtgtctcagt cccaatgtgg ccgattaccc tctcaggtcg gctacgtatc actgccttgg 1200
tgagccttta cctcaccaac tagctaatac gccgcgggtc catccagaag tgatagcaga 1260
gccatctttt aaaagaaaac catgcggttt tctctattat acggtattag catctgtttc 1320
caggtgttat cccctacttc tgggcaggtt acccacgtgt tactcacccg ttcgccactc 1380
acttcgtgtt aaaatctcaa tcagtacaag tacgtcataa tcaattaacg gaagttcgtt 1440
cgacttgcat gtatag 1456
Claims (9)
1. Pediococcus pentosaceus 368, characterized by: the strain is pediococcus pentosaceus 368; the preservation name is Pediococcus pentosaceus LL 2018368; is preserved in China general microbiological culture Collection center, and the preservation address is the institute of microbiology of Zhongkoyao No. 3 Hospital, Xilu No.1 Hospital, North Chen, of Chaozhong, Beijing; the preservation date is as follows: year 2019, month 10, day 21; the preservation number is CGMCC No: 18709.
2. pediococcus pentosaceus 368 according to claim 1, wherein: the gene sequence of the 16S rDNA of the pediococcus pentosaceus 368 is a nucleotide sequence shown in SEQ ID No. 1.
3. Pediococcus pentosaceus 368 according to claim 1, wherein: after aerobic culture is carried out for 72 hours at 35 ℃ in an MRS culture medium, bacterial colonies are round and milky, the surfaces are smooth, the middle part is raised, the edges are neat, pigmentation is avoided, the bacterial colonies are easy to pick up, have no silks, have the fragrance of lactic acid, are positive in gram staining, are rod-shaped, are facultative and anaerobic, can survive for more than two months when stored in liquid at 4 ℃, and have the optimal growth temperature of 30-38 ℃.
4. A pediococcus pentosaceus 368 bacterial agent produced by the pediococcus pentosaceus 368 according to claim 1.
5. A method for preparing a Pediococcus pentosaceus 368 bacterial agent as claimed in any one of claims 1 to 4, comprising the steps of:
(1) preparing strains: inoculating in MRS liquid culture medium, culturing at 32-38 ℃ for 18-20 h, and centrifuging at 8000-10000 rpm to obtain thalli.
(2) The cells were freeze-dried.
6. A method for producing a bacterial agent of Pediococcus pentosaceus 368 according to claim 5, comprising: in step (1), the MRS culture medium culture solution is: peptone 10.0 g; 5.0g of beef extract; 4.0g of yeast powder; 20.0g of glucose; dipotassium phosphate 2.0 g; 2.0g of triammonium citrate; 5.0g of sodium acetate; magnesium sulfate 0.2 g; 0.05g of manganese sulfate; tween 801.0 mL; and 1L of water.
7. Use of the pediococcus pentosaceus 368 bacterial agent of claim 2 in feed, water, and health care and therapeutic drugs.
8. The use according to claim 7, wherein the microbial preparation has an effective viable count of 1010～1011CFU/g。
9. The use of claim 7, wherein the feed additive is present in an amount of 106～108CFU/g, 10 in drinking water9～1011CFU/L。
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|---|---|---|---|---|
| CN113980848A (en) * | 2021-11-01 | 2022-01-28 | 河北科技大学 | Pediococcus pentosaceus SBC5 and application thereof |
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| CN116445367A (en) * | 2023-06-05 | 2023-07-18 | 中科嘉亿营养医学(山东)微生态研究院有限公司 | Pediococcus pentosaceus JYPR 9330 for improving oral health as well as microbial inoculum and application thereof |
| CN116445367B (en) * | 2023-06-05 | 2023-08-22 | 中科嘉亿营养医学(山东)微生态研究院有限公司 | Pediococcus pentosaceus JYPR 9330 for improving oral health as well as microbial inoculum and application thereof |
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| Publication number | Publication date |
|---|---|
| CN113462605A (en) | 2021-10-01 |
| CN113462605B (en) | 2023-03-21 |
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