CN114774312A

CN114774312A - Whale bacillus NK01 and application thereof

Info

Publication number: CN114774312A
Application number: CN202210367312.0A
Authority: CN
Inventors: 王淼; 卢迈新; 张紫玥; 范梓健; 衣萌萌; 高风英
Original assignee: Pearl River Fisheries Research Institute CAFS
Current assignee: Pearl River Fisheries Research Institute CAFS
Priority date: 2021-12-01
Filing date: 2022-04-08
Publication date: 2022-07-22
Anticipated expiration: 2042-04-08
Also published as: CN114015613A; CN114774312B

Abstract

The invention discloses a whale bacillus (Cetobacterium) NK01, which is preserved in Guangdong province microorganism strain preservation center at 2 month and 4 days 2021, and the addresses are as follows: the microbial research institute of Guangzhou province, building No. 59, building No. 5, of Michelia furiosa No. 100, Guangzhou City, has a preservation number of GDMCC No. 61502. Cetacea NK01 produces three branched chain amino acids, including valine, leucine, and isoleucine. Glycine, alanine, phenylalanine and proline may also be produced. Therefore, the bacillus whale NK01 has important application value in developing amino acid products.

Description

Whale bacillus NK01 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to whale bacillus NK01 capable of producing branched chain amino acid and application thereof.

Background

Branched-chain amino acids (BCAAs) include valine, leucine, and isoleucine, all of which are essential amino acids, and higher animals cannot synthesize themselves and can only be supplemented with food. The branched chain amino acid accounts for 40% of the essential amino acid, and the sufficient addition amount and the reasonable proportion thereof are important for the normal development of animals. Branched chain amino acid not only can regulate the metabolism of protein and the immunity of organisms, but also can increase the synthesis of protein in the movement of the organisms and reduce the decomposition and damage of muscle protein, is a particularly important nutritional supplement and is widely applied to the industries of medicine, food, feed production and the like. At present, domestic manufacturers are few, the acid production level is low, the market demand can not be met, and hundreds of tons of amino acid transfusion needs to be imported every year only by preparing amino acid transfusion. The branched chain amino acid is formed by fermentation of Corynebacterium glutamicum (Corynebacterium glutamicum) or Escherichia coli (Escherichia coli) and the like which are subjected to mutagenesis or metabolic engineering, and a series of problems of low acid production level, long fermentation period, high by-product and the like exist, so that the breeding of a new strain with good branched chain amino acid production performance and safety performance is very important.

Disclosure of Invention

The invention provides a bacterium, namely cetacea (Cetobacterium) NK01, capable of producing three branched chain amino acids, and capable of producing the three branched chain amino acids simultaneously. The strain is preserved in Guangdong province microorganism strain preservation center at 2021, 2 months and 4 days, addresses: the microbial research institute of Guangdong province, No. 59 building, No. 5 building, of Mieli Zhonglu, Guangzhou, with the collection number GDMCC No. 61502.

A whale bacillus NK01 is obtained by separating from intestinal tracts of healthy tilapia through a fastly-raised anaerobic culture medium, and cultured for 48 hours at 37 ℃, wherein colonies are gray, slightly irregular in round, convex and opaque, and have the diameter of 2.0-3.0 mm. It is intestinal symbiotic bacteria of fish and has high safety.

Cetacea NK01 produces three branched chain amino acids, including valine, leucine, and isoleucine. Glycine, alanine, phenylalanine and proline may also be produced. The whole genome sequencing result shows that 6 genes in the strain participate in anabolic pathways of valine, leucine and isoleucine, and corresponding expression products are valine, leucine and isoleucine respectively. The bacillus whale NK01 has important application value in developing amino acid products.

Drawings

FIG. 1 shows the colony morphology of cetacean NK01 cultured in caustic anaerobic medium;

FIG. 2 is a transmission electron microscope observation image of cetacean bacillus NK 01;

FIG. 3 is a NJ phylogenetic tree of whale bacillus NK01 based on the 16S rRNA gene sequence;

FIG. 4 shows the valine, leucine and isoleucine anabolic pathways of cetacean NK 01.

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 attached drawings.

Materials and methods

1. Isolation and purification of bacterial species

Placing intestinal contents of Nile tilapia in fastly-cultured anaerobic liquidThe culture medium is subjected to anaerobic culture at 37 ℃ for 48 hours. The obtained culture solution was 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 in a constant-temperature incubator at 37 ℃ for 48 h. And selecting the bacterial colony with single and irregular bacterial colony for repeated purification culture until obtaining the pure strain. And selecting single colonies of the obtained pure strains to perform 16S rRNA gene sequence analysis, morphology and gram staining 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 the screened strains to 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 fastly-cultured anaerobic solid culture medium. 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 staining method under an optical microscope after the single colony is uniformly coated. And observing the shape of the thallus 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 a Merrier anaerobe 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. mu.L of template DNA, 0.5. mu.L of forward and reverse primers, 12.5. mu.L of Mix, 10.5. mu.L of sterilized distilled water, reaction conditions for PCR amplification: 3min at 94 ℃; 30 cycles of 94 ℃ for 30s, 56 ℃ for 30s, and 72 ℃ for 90 s; 10min at 72 ℃. The PCR product was sent to Biotechnology engineering (Shanghai) GmbH 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 in MEGA7.0 software (NJ) and confidence detection is performed by self-construction analysis (bostrap), and self-detection 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 (average weight is 20.5 +/-1.5 g), and 15 tilapia tails are randomly selected from each parallel group. Setting the concentration of the strain to be tested to 1.5 × 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 whale bacillus NK01 to fastidious anaerobic liquid culture medium, anaerobically culturing at 37 deg.C for 24 hr, centrifuging at 4 deg.C and 6000r/min for 5min, and collecting thallus. High quality DNA was extracted with a genome extraction kit and a DNA library was constructed, and single molecule sequencing of DNA was performed using a sequencer PromethION based on the third generation sequencing Technology Oxford Nanopore Technology, while double-end sequencing of the entire genome of the strain was performed using the MGISEQ-2000 system in PE150 read length mode.

2.6 determination of amino acid component produced by whale bacillus NK01

Picking single colony of whale bacillus NK01, inoculating to 200mL fastidious anaerobic liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 48 h. 10mL of the fermentation broth was filtered through a disposable sterilizing filter with a pore size of 0.22 μm, and the filtrate was collected. And (3) determining and analyzing the amino acid components and the content in the filtrate by using a full-automatic amino acid analyzer, and taking the culture medium filtrate without the added strain as a control.

Second, results and analysis

1. Classification and identification of strains

1.1 strains of Bacillus subtilis

The colony morphology of the whale bacillus NK01 on the fastly-cultured anaerobic solid medium (figure 1) is gray, slightly irregular in round shape, convex and opaque, the diameter of the colony is 2.0-3.0mm, and the colony is rod-shaped (figure 2) when observed under a transmission electron microscope. The gram staining results show that the bacteria are gram-negative bacteria.

1.2 physiological and biochemical assays

The results of physiological and biochemical identification of whale bacillus NK01 show that the reactions of gelatinase and glucosidase of the bacillus are positive, the reaction of urease is weak positive, the test of producing indole is positive, the reactions of glucose, sucrose, maltose, salicin, mannitol and trehalose can be utilized, and the reactions of mannitol, lactose, xylose, arabinose, glycerol, cellobiose, melezitose, raffinose, sorbitol, rhamnose and catalase can not be utilized. In contrast to the reported cetacean, cetacean NK01 can utilize gelatin and starch.

TABLE 1 physiological and biochemical characteristics of cetacea NK01

+: positive; -: negative; sex; w: weak positive; blank is no correlation data.

1.316S rRNA Gene sequence analysis

The bacillus NK01 can be basically determined to be bacillus (Cetobacterium) by PCR amplification and 16S rRNA sequencing of the bacillus NK01 to obtain a target sequence, and comparing the target sequence with gene sequences in a database by BLAST (figure 3) and combining the physiological and biochemical characteristics of strains.

2. Safety test

The safety of cetacean NK01 against tilapia was tested by intraperitoneal injection. The injection experiment result shows that the concentration of 100 mu L of tilapia injected by the weight of donor (20.5 +/-1.5) 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 experimental results show that the whale bacillus NK01 is a strain with good safety performance.

3. Whole genome sequencing

By aligning the KEGG library, 6 genes in cetacean NK01 were involved in valine, leucine and isoleucine anabolic pathways, and the corresponding expression products were valine, leucine and isoleucine, respectively (fig. 4).

Comparing NK01 with the largest similarity in NCBI of the reference genome of whale's funeralis, the Average Nucleotide similarity (ANI) was below 95%, because the ANI value between species is generally above 95%, therefore NKO1 may be a new species of whale.

TABLE 2 comparison of the average nucleotide similarity of the whale NK01 and whale Souleiella genome on NCBI

NCBI Strain (reference genome)	ANI estimation
		Cetobacterium_somerae/GCF_902375135.fna	88.9613
Cetobacterium_somerae/GCF_000479045.fna	88.9613
		Cetobacterium_somerae/GCA_905206145.fna	90.1069

4. Determination of amino acid components produced by whale bacillus NK01

The detection result shows that the whale bacillus NK01 produces three branched-chain amino acids including valine, leucine and isoleucine. Glycine, alanine, phenylalanine and proline may also be produced.

TABLE 3 amino acid components and contents produced by cetacean NK01

Amino acid component	Content (mg/L)
		Leucine (Leu)	140
Isoleucine (Ile)	130
		Valine (Val)	120
Alanine (Ala)	220
		Glycine (Gly)	100
Proline (Pro)	40
		Phenylalanine (Phe)	30

The sequence of the bacillus cetamicus NK01 is as follows:

ATTCGATTTACCTTCGGGTAATGAGGATGGCGGACGGGTGAGTAACGCGTAAGGAACTTGCCTCTTGGTCTGGGACAACTGTTGGAAACGACAGCTAATACCGGATATTATGAGATTCTCGCATGGGAAACTTATGAAAGCTATATGCGCCAAGAGAGAGCCTTGCGTTCCATTAGCTAGTTGGTGGGGTAACGGCCCACCAAGGCGACGATGGATAGCCGGCCTGAGAGGGTGAACGGCCACAAGGGGACTGAGACACGGCCCTTACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCCACAAGCCTGATCCAGCAATTCTGTGTGCACGATGAAGGTCTTCGGATTGTAAAGTGCTTTCAGTTGGGAAGAAGAAAGTGACGGTACCAACAGAAGAAGCGACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTCGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGCGTCTAGGCGGAAAAATAAGTCTGATGTTAAAATGCGGGGCTCAACTCCGTATTGCGTTGGAAACTGTTTTTCTAGAGTACTGGAGAGGTGGGCGGAACTACAAGTGTAGAGGTGAAATTCGTAGATATTTGTAGGAATGCCGATGGAGAAGTCAGCTCACTGGACAGATACTGACGCTGAAGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATCACTAGGTGTTGGGGGGTCGAACCTCAGCGCCCAAGCTAACGCGATAAGTGATCCGCCTGGGGAGTACGCACGCAAGTGTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGACGCAACGCGAGAAACCTTACCAGCGTTTGACATCCTAAGAAGTTTCCAGAGATAGATTCGTGCCGGCTTGCCGGAACTTAGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTATGTTGCTACCATTAAGTTGAGCACTCATGCGATACTGCCTGCGATGAGCAGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATACGCTGGGCTACACACGTGCTACAATGGGCAGTACAGAGAGTTGCCAACCCGCGAGGGTGAGCTAATCTCTTAAAGCTGTTCTTAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGCAAATCAGCATGTTGCGGTGAATACGTTCTCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTGGTTGCACCTGAAGTAGCAGGCCTAACCGTAAGG。

the embodiments described above are presented to facilitate one of ordinary skill in the art to understand and practice the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should make improvements and modifications to the present invention based on the disclosure of the present invention within the protection scope of the present invention.

Sequence listing

<110> Zhujiang aquatic product institute of China aquatic product science institute

<120> whale bacillus NK01 and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1363

<212> DNA

<213> whale bacillus (Cetobacterium)

<400> 1

attcgattta ccttcgggta atgaggatgg cggacgggtg agtaacgcgt aaggaacttg 60

cctcttggtc tgggacaact gttggaaacg acagctaata ccggatatta tgagattctc 120

gcatgggaaa cttatgaaag ctatatgcgc caagagagag ccttgcgttc cattagctag 180

ttggtggggt aacggcccac caaggcgacg atggatagcc ggcctgagag ggtgaacggc 240

cacaagggga ctgagacacg gcccttactc ctacgggagg cagcagtggg gaatattgga 300

caatgggcca caagcctgat ccagcaattc tgtgtgcacg atgaaggtct tcggattgta 360

aagtgctttc agttgggaag aagaaagtga cggtaccaac agaagaagcg acggctaaat 420

acgtgccagc agccgcggta atacgtatgt cgcaagcgtt atccggattt attgggcgta 480

aagcgcgtct aggcggaaaa ataagtctga tgttaaaatg cggggctcaa ctccgtattg 540

cgttggaaac tgtttttcta gagtactgga gaggtgggcg gaactacaag tgtagaggtg 600

aaattcgtag atatttgtag gaatgccgat ggagaagtca gctcactgga cagatactga 660

cgctgaagcg cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt 720

aaacgatgat cactaggtgt tggggggtcg aacctcagcg cccaagctaa cgcgataagt 780

gatccgcctg gggagtacgc acgcaagtgt gaaactcaaa ggaattgacg gggacccgca 840

caagcggtgg agcatgtggt ttaattcgac gcaacgcgag aaaccttacc agcgtttgac 900

atcctaagaa gtttccagag atagattcgt gccggcttgc cggaacttag tgacaggtgg 960

tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1020

cccctattgt atgttgctac cattaagttg agcactcatg cgatactgcc tgcgatgagc 1080

aggaggaagg tggggatgac gtcaagtcat catgcccctt atacgctggg ctacacacgt 1140

gctacaatgg gcagtacaga gagttgccaa cccgcgaggg tgagctaatc tcttaaagct 1200

gttcttagtt cggattgtac tctgcaactc gagtacatga agttggaatc gctagtaatc 1260

gcaaatcagc atgttgcggt gaatacgttc tcgggtcttg tacacaccgc ccgtcacacc 1320

acgagagttg gttgcacctg aagtagcagg cctaaccgta agg 1363

Claims

1. Whale bacillus NK01, deposited in Guangdong province microbial strain collection center with the deposit number GDMCC No. 61502.

2. Use of cetacean NK01 as an amino acid producing bacterium for the production of valine, leucine, isoleucine, glycine, alanine, phenylalanine and/or proline.

3. The use as claimed in claim 2, wherein the whale bacillus NK01 can use gelatin and starch.