CN114752536A - Romtuz MY01 and application thereof - Google Patents

Romtuz MY01 and application thereof Download PDF

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CN114752536A
CN114752536A CN202210543828.6A CN202210543828A CN114752536A CN 114752536 A CN114752536 A CN 114752536A CN 202210543828 A CN202210543828 A CN 202210543828A CN 114752536 A CN114752536 A CN 114752536A
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tilapia
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roembuz
romboutsia
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王淼
卢迈新
范梓健
张紫玥
衣萌萌
高风英
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Pearl River Fisheries Research Institute CAFS
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Abstract

The invention discloses a Romnez strain MY01 and application thereof. Roembuz strain MY01 was deposited at 13.4.2021 in the Guangdong province Collection of microorganisms with accession number: GDMCC No. 61558. The Romroth bacteria have important application value in developing products for producing amino acids or products for promoting the growth and health of fishes, particularly tilapia.

Description

Romtuz MY01 and application thereof
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a Roembuz strain MY01 for improving growth performance of tilapia and application thereof.
Background
The aquatic probiotics are live bacteria and killed bacteria or certain components of bacterial cells, and are fed or thrown into the aquaculture water body through baits, so that the disease resistance of aquatic animals is improved, the health condition is improved, the growth performance and the feed utilization rate are promoted, and the stress resistance and the overall activity of the animals are improved. The probiotics attract extensive attention in the aquaculture industry due to the advantages of being environment-friendly, safe, effective and the like. Currently, probiotics commonly used in fish farming include bacteria of the genus Bacillus (Bacillus), Lactobacillus (Lactobacillus), Lactococcus (Lactococcus), and Saccharomyces (Saccharomyces). However, the application of the aquatic probiotics has certain blindness. The use of many aquatic probiotic products is not stable, and one of the important reasons for this may be improper selection and use of the probiotic. Many probiotic strains are not isolated from the fish gut, but from the environment or from a warm animal. And the same probiotic may also exhibit different efficacy on different fish species. Because the conditions of probiotic preparations in aquaculture industry for permanent planting of intestinal tracts are different, the probiotic preparations have pertinence in use and are used for separating and screening probiotics with strong pertinence.
A large number of microorganisms are planted in intestinal tracts of fishes, and a nutrition symbiosis relationship exists between the intestinal microorganisms and a host. The intestinal microorganisms can degrade chyme, utilize intestinal nutrients to grow and reproduce, and secrete physiologically active substances such as amino acids, vitamins, fatty acids, digestive enzymes and the like in the metabolic process, so that the growth and health of fishes are facilitated. The research on the screening of the beneficial microorganisms in the intestinal tracts of fishes provides a more efficient probiotic product for the aquatic product industry.
Disclosure of Invention
The invention provides a Romroth bacterium for improving the growth performance of tilapia and application thereof. The strain is preserved in Guangdong province microorganism strain preservation center at 13 months 4 in 2021, named as Romboutsia lituseburensis MY01, with the preservation address as follows: the microbial research institute of Guangzhou province, No. 59 building, No. 5 building, No. 100 institute of Mieli Zhonglu, Guangzhou city, with the preservation number: GDMCC No. 61558.
A Romnez strain MY01 is obtained by separating from intestinal tracts of healthy tilapia by adopting a fastly-cultured anaerobic culture medium, and is cultured for 48 hours at 37 ℃, the colony is white, smooth, semitransparent and round, the diameter is 0.5-1.0mm, and the colony is anaerobic gram-positive bacteria. 16S rRNA gene is PCR amplified by using a universal primer 27F/1492R, and the sequence is sequenced to obtain a target sequence, and NCBI BLAST comparison analysis is carried out, so that the obtained strain has the highest homology with the published Romboutsia litusburgensis. The bacterium has good safety performance.
The Romnez strain MY01 can promote the growth of tilapia, obviously improve the weight of tilapia, and improve the content of tilapia serum lysine, methionine, tryptophan, tyrosine and the like. By adding the Romnez strain MY01 into the feed, the functions of intestinal flora such as anabolism of methionine, tryptophan, tyrosine, phenylalanine, cystine, alanine, valine, leucine and isoleucine can be improved. The whole genome sequencing result shows that 12 genes in the strain participate in the lysine anabolism pathway, 9 genes participate in the phenylalanine, tyrosine and tryptophan anabolism pathway, and corresponding expression products are lysine, phenylalanine and tyrosine respectively. The Romrothz strain has important application value in developing products for producing amino acids or products for promoting the growth and health of fishes, particularly tilapia.
Drawings
FIG. 1 is the colony morphology of Romboutsia MY01 cultured in caustic anaerobic medium;
FIG. 2 is a transmission electron microscopy micrograph of Romboutsia MY 01;
FIG. 3 is a NJ phylogenetic tree of Romboutsia MY01 based on the 16S rRNA gene sequence;
FIG. 4 is the Romboutsia MY01 lysine anabolic pathway;
FIG. 5 is a Romboutsia MY01 phenylalanine, tyrosine, and tryptophan anabolic pathway;
FIG. 6 is a graph of the effect of Romboutsia MY01 on tilapia growth;
FIG. 7 is a graph showing the amino acid content in the serum of tilapia in different groups;
FIG. 8 is a diagram of prediction of the functions of Tax4Fun genes of intestinal flora of tilapia in different groups.
Detailed Description
In order to make the present invention more clear and intuitive for those skilled in the art, the present invention will be further described with reference to the accompanying drawings.
A material and method
1. Isolation and purification of bacterial species
Putting the intestinal contents of the nile tilapia into a fastly-cultured anaerobic liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 48 h. The culture solution obtained is diluted to 10 by gradient dilution-3、10-4、10-5Taking 150 mu l of each diluent, coating the diluent on a fastly-cultured anaerobic solid culture medium, and culturing for 48h in a constant-temperature incubator at 37 ℃. And selecting a colony with a single and irregular colony morphology for repeated purification culture until a pure strain is obtained. Selecting single colony of pure strain for 16S rRNA gene sequence analysis, morphology and gram stain observation.
The formula of the fastly-cultured anaerobic liquid culture medium comprises the following components: 23.0g of mixed peptone, 5.0g of sodium chloride, 1.0g of soluble starch, 0.4g of sodium bicarbonate, 1.0g of glucose, 1.0g of sodium pyruvate, 0.5g of cysteine hydrochloride, 0.01g of hemin, 0.001g of vitamin K, 1.0g of L-arginine, 0.25g of soluble pyrophosphoric acid, 0.5g of sodium succinate, pH7.2 +/-0.2 and 1000mL of double distilled water are uniformly mixed and sterilized for later use.
2. Strain identification
2.1 morphological Observation and staining
And streaking and inoculating the screened strains on a fastly-cultured anaerobic solid culture medium, placing the fastly-cultured anaerobic solid culture medium in a constant-temperature incubator at 37 ℃ for anaerobic culture for 24 hours, and observing the colony morphology of the strains. And selecting a single colony, placing the single colony on a glass slide on which physiological saline is dripped, and observing the thallus morphology by a gram's staining method under an optical microscope after the single colony is uniformly coated. The morphology of the cells was observed by a projection electron microscope.
2.2 physiological and Biochemical assays
The physiological and biochemical indexes of the purified and cultured strains to be detected are detected by using a Merrielia anaerobic bacterium identification kit, and the specific operation is shown in the kit specification.
2.316S rRNA Gene sequence analysis
Extracting bacterial genome DNA of a strain to be detected by using a Beijing tiangen biological bacterial genome DNA extraction kit, and referring to the instruction for specific operation steps. The extracted bacterial genomic DNA was used as a template, and the DNA was purified by primer 27F: 5'-GTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' to amplify the 16S rRNA gene of the bacteria. The reaction system (25. mu.L) was: 1 μ L of template DNA, 0.5 μ L of forward and reverse primers, 12.5 μ L of Mix, 10.5 μ L of sterilized distilled water, and reaction conditions for PCR amplification: 3min at 94 ℃; 30 cycles of 94 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 90 s; 10min at 72 ℃. The PCR product was sent to Biotechnology engineering (Shanghai) Co., Ltd for sequencing. After obtaining the objective series, the NCBI website is logged in, homology analysis is performed by BLAST program in GenBank, 16S rRNA gene sequences of related bacteria are downloaded at the same time, multiple alignment is performed by BioEdit software, molecular phylogenetic trees are constructed by neighbor joining method in MEGA7.0 software (NJ) and confidence detection is performed by self-construction analysis (bostrap), and self-checking data set is 1000 times.
2.4 safety test
The experiment sets a strain injection group to be tested and a negative control group, each group is 3 parallels, Nile tilapia is used as experimental fish (the average weight is 22.5 +/-1.3 g), and 15 tilapia tails are randomly selected from each parallel group. Setting three concentrations of the strain to be tested, wherein the three concentrations are respectively 1.5 multiplied by 107CFU/ml、1.5×108CFU/ml and 1.5X 109CFU/ml, injecting each fish in the experimental group and the control group by means of intraperitoneal injection, injecting 100 mu L of each fish in the experimental group, injecting 0.85% normal saline with the same volume in the control group, feeding the fish according to a conventional method during the experimental observation period of 7d, and observing and recording the death number of the fish every day.
2.5 Whole genome sequencing analysis
Inoculating single colony of Romboutsia MY01 into fastidious anaerobic liquid culture medium, anaerobically culturing at 37 deg.C for 24 hr, and centrifuging at 4 deg.C and 6000r/min for 5min to collect thallus. Extracting high-quality DNA by using a genome extraction kit and constructing a DNA library, performing single-molecule sequencing on the DNA by using a sequencer PromethION based on a third-generation sequencing Technology Oxford Nanopore Technology, and simultaneously performing double-end sequencing on the whole genome of the strain by using a MGISEQ-2000 system in a PE150 reading mode.
2.6 influence of Romboutsia MY01 on growth and physiology of tilapia
The experiment is carried out in the pond of the Zhujiang aquatic product research institute of aquatic product science research institute in China. 150 tails of Luofish fries with the mass of about 50g are selected in the experiment and randomly distributed into 6 square net cages, 25 tails per net cage, and the actual water volume of each net cage is 3m3(L × W × H ═ 1m × 2m × 1.5 m). The control group was 1, the MY01 probiotic group (1.0X 10)7cfu/g), 3 replicates per group. Collecting fresh bacterial colonies every day, mixing the bacterial colonies with feed and feeding the bacterial colonies. Bait is thrown twice a day, and the daily bait throwing amount is 4 percent of the body mass. The experimental time was 4 weeks.
TABLE 1 feed formulation
Figure BDA0003650233050000041
After the tilapia is anesthetized in an ice bath, the body surface of the tilapia is washed twice by sterile physiological saline to remove surface dirt. Weighing the dissected tilapia, then drawing blood, collecting serum, collecting intestinal content samples, quickly freezing by liquid nitrogen, and storing in a refrigerator at-80 ℃. And (3) detecting the amino acid content of the serum based on liquid chromatography tandem mass spectrometry (LC-MS/MS), and performing 16S rRNA sequencing analysis on the flora of the intestinal contents.
Two results and analysis
1. Classification and identification of strains
1.1 strains
The colony morphology of the Romboutsia MY01 on the fastidious anaerobic solid culture medium is white, smooth, semitransparent and circular, the diameter of the colony is 0.5-1.0mm, and the colony is anaerobic gram-positive bacteria. The cells were rod-shaped when observed under a transmission electron microscope (FIG. 1; FIG. 2). The gram staining result shows that the bacterium is a gram positive bacterium.
1.2 physiological and biochemical assays
The physiological and biochemical identification results of Romboutsia MY01 (Table 2) show that the bacterium is positive in gelatinase and glucosidase reaction, positive in urease reaction and negative in indole production test, can utilize glucose, sucrose, maltose and trehalose, and cannot utilize mannitol, lactose, xylose, arabinose, glycerol, cellobiose, melezitose, raffinose, sorbitol, rhamnose, salicin, mannitol and catalase reaction.
TABLE 2 physiological and biochemical characteristics of Romboutsia MY01
Figure BDA0003650233050000051
Figure BDA0003650233050000061
+: positive; -: and (4) negativity.
1.316S rRNA Gene sequence analysis
The Romboutsia MY01 is amplified by PCR, 16S rRNA is sequenced to obtain a target sequence, and the target sequence is compared with gene sequences in a database by BLAST (figure 3), so that MY01 can be basically determined to be a bacterium in the Romboutsia genus by combining the physiological and biochemical characteristics of the strain.
2. Safety test
The safety of Romboutsia MY01 against tilapia was tested by intraperitoneal injection. The injection experiment result shows that the concentration of 100 mu L of tilapia injected into the tilapia with the weight of 22.5 +/-1.3 g is 1.5 multiplied by 107CFU/ml、1.5×108CFU/ml and 1.5X 109No death or other abnormal conditions appeared under the condition of CFU/mL bacterial liquid. The results of the experiments show that the Romboutsia MY01 is a strain with good safety performance.
3. Whole genome sequencing
By aligning the KEGG library, 12 genes (indicated by bold line boxes) of this strain were involved in the lysine anabolism pathway (fig. 4) and 9 genes (indicated by bold line boxes) were involved in the phenylalanine, tyrosine and tryptophan anabolism pathway (fig. 5) in the romboudia MY01, and the corresponding expression products were lysine, phenylalanine and tyrosine, respectively.
Comparing MY01 with the reference genome of Romboutsia lituseberensis with the greatest similarity at NCBI, the Average Nucleotide similarity (ANI) was below 95%, since ANI values between species are generally above 95%, and thus MY01 may be a new species of the Romboutsia genus.
TABLE 2 comparison of the mean nucleotide similarity of Romboutsia MY01 to Romboutsia lituseburensis genome at NCBI
Figure BDA0003650233050000071
4. Influence of feeding Romboutsia MY01 on growth and physiology of tilapia
Romboutsia MY01 can promote the growth of tilapia (figure 6), and significantly improve the weight of tilapia and the weight of tilapia after evisceration and significantly improve the content of lysine, methionine, tryptophan and tyrosine in tilapia serum after 4 weeks of probiotic feeding (figure 7; Table 3).
TABLE 3 Effect of MY01 on serum amino acid composition of Tilapia mossambica
Figure BDA0003650233050000072
By adding Romboutsia MY01 to the feed, the functions of intestinal flora such as anabolism of methionine, tryptophan, tyrosine, phenylalanine, cystine, alanine, valine, leucine, isoleucine and the like can be improved (FIG. 8).
In the present invention, the sequence of Romboutsia MY 01:
CTTCTTCGGAAGAGAGCGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCCT GTACACACGGATAACATACCGAAAGGTATGCTAATACGGGATAACATACTTTTATCGC ATGGTAGAAGTATCAAAGCTCCGGCGGTACAGGATGGACCCGCGTCTGATTAGCTAG TTGGTAAGGTAACGGCTTACCAAGGCGACGATCAGTAGCCGACCTGAGAGGGTGAT CGGCCACATTGGAACTGAGACACGGTCCAAACTCCTACGGGAGGCAGCAGTGGGG AATATTGCACAATGGGCGAAAGCCTGATGCAGCAACGCCGCGTGAGCGATGAAGGC CTTCGGGTCGTAAAGCTCTGTCCTCAAGGAAGATAATGACGGTACTTGAGGAGGAA GCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCTAGCGTTATCC GGAATTACTGGGCGTAAAGGGTGCGTAGGTGGTTTCTTAAGTCAGAAGTGAAAGGC TACGGCTCAACCGTAGTAAGCTTTTGAAACTAAGAGACTTGAGTGCAGGAGAGGAG AGTAGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAATACCAGTTGC GAAGGCGGCTCTCTGGACTGTAACTGACACTGAGGCACGAAAGCGTGGGGAGCAA ACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTACTAGCTGTCGGGG GTTACCCCCCTCGGTGGCGCAGCTAACGCATTAAGTACTCCGCCTGGGAAGTACGCT CGCAAGAGTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGTAGCGGAGCATG TGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTAAGCTTGACATCCTTTTGACCT CTCCCTAATCGGAGATTTCCCTTCGGGGACAGAAGTGACAGGTGGTGCATGGTTGTC GTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGCC TTTAGTTGCCAGCATTAAGTTGGGCACTCTAGAGGGACTGCCAGGGATAACCTGGAG GAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGCTTAGGGCTACACACGTGC TACAATGGGTGGTACAGAGGGCGGCCAAGTCGTGAGGCGGAGCTAATCCCTTAAAG CCATTCTCAGTTCGGATTGTAGGCTGAAACTCGCCTACATGAAGCTGGAGTTACTAG TAATCGCAGATCAGAATGCTGCGGTGAATGCGTTCCCGGGTCTTGTACACACCGCCC GTCACACCACGGGAGTTGGAGGCGCCCGAAGCCGGATAGCTAACCTTTGGAAGC
sequence listing
<110> Zhujiang aquatic research institute of Chinese aquatic science research institute
<120> Romtuzzium MY01 and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1349
<212> DNA
<213> Rombutz bacteria (Romboutsia lituseberensis)
<400> 1
cttcttcgga agagagcggc ggacgggtga gtaacgcgtg ggtaacctgc cctgtacaca 60
cggataacat accgaaaggt atgctaatac gggataacat acttttatcg catggtagaa 120
gtatcaaagc tccggcggta caggatggac ccgcgtctga ttagctagtt ggtaaggtaa 180
cggcttacca aggcgacgat cagtagccga cctgagaggg tgatcggcca cattggaact 240
gagacacggt ccaaactcct acgggaggca gcagtgggga atattgcaca atgggcgaaa 300
gcctgatgca gcaacgccgc gtgagcgatg aaggccttcg ggtcgtaaag ctctgtcctc 360
aaggaagata atgacggtac ttgaggagga agccccggct aactacgtgc cagcagccgc 420
ggtaatacgt agggggctag cgttatccgg aattactggg cgtaaagggt gcgtaggtgg 480
tttcttaagt cagaagtgaa aggctacggc tcaaccgtag taagcttttg aaactaagag 540
acttgagtgc aggagaggag agtagaattc ctagtgtagc ggtgaaatgc gtagatatta 600
ggaggaatac cagttgcgaa ggcggctctc tggactgtaa ctgacactga ggcacgaaag 660
cgtggggagc aaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtactag 720
ctgtcggggg ttacccccct cggtggcgca gctaacgcat taagtactcc gcctgggaag 780
tacgctcgca agagtgaaac tcaaaggaat tgacggggac ccgcacaagt agcggagcat 840
gtggtttaat tcgaagcaac gcgaagaacc ttacctaagc ttgacatcct tttgacctct 900
ccctaatcgg agatttccct tcggggacag aagtgacagg tggtgcatgg ttgtcgtcag 960
ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttgc ctttagttgc 1020
cagcattaag ttgggcactc tagagggact gccagggata acctggagga aggtggggat 1080
gacgtcaaat catcatgccc cttatgctta gggctacaca cgtgctacaa tgggtggtac 1140
agagggcggc caagtcgtga ggcggagcta atcccttaaa gccattctca gttcggattg 1200
taggctgaaa ctcgcctaca tgaagctgga gttactagta atcgcagatc agaatgctgc 1260
ggtgaatgcg ttcccgggtc ttgtacacac cgcccgtcac accacgggag ttggaggcgc 1320
ccgaagccgg atagctaacc tttggaagc 1349
<210> 2
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gtttgatcct ggctcag 17
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tacggctacc ttgttacgac tt 22

Claims (10)

1. Roembuz strain MY01, which was deposited at 13.4.2021 in the Guangdong province Collection of microorganisms, accession number: GDMCC No. 61558.
2. Use of the bacterium rembuzox MY01 as an amino acid producing bacterium in the production of lysine and/or phenylalanine and/or tyrosine.
3. Application of Roembuz strain MY01 in preparation of products for promoting fish growth.
4. The use according to claim 3, wherein the fish is tilapia.
5. Use according to claim 3, wherein the promotion of fish growth is manifested by an increase in fish weight.
6. Use according to claim 3, wherein the product is a microbial preparation or a feed additive.
7. Use according to claim 3, characterized in thatCharacterized in that the content of the Romnez fungus MY01 in the product is 1.0 multiplied by 107cfu/g。
8. A product contains Rombuz bacteria MY 01.
9. The product of claim 8, wherein the product is a microbial preparation or a feed additive or a feed.
10. The method for culturing the Roembuz strain MY01 is characterized in that the Roembuz strain MY01 is inoculated on a fastly-cultured anaerobic solid culture medium and then is cultured in a constant-temperature incubator at 37 ℃.
CN202210543828.6A 2022-05-18 2022-05-18 Romtuz MY01 and application thereof Pending CN114752536A (en)

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WO2021119729A1 (en) * 2019-12-19 2021-06-24 University Of The Sunshine Coast Method of increasing the productivity of a non-ruminant animal

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WO2021119729A1 (en) * 2019-12-19 2021-06-24 University Of The Sunshine Coast Method of increasing the productivity of a non-ruminant animal
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