CN111778179B - Arthrobacter protoformiae for degrading lactic acid and application thereof - Google Patents
Arthrobacter protoformiae for degrading lactic acid and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention belongs to the technical field of brewing microorganisms, and particularly relates to arthrobacter protoformis for degrading lactic acid and application thereof. Aiming at the problems that the lactic acid content is high in the late fermentation stage of the Luzhou-flavor liquor and a strain for efficiently degrading the lactic acid needs to be separated, the invention provides the protovitronectin arthrobacter for efficiently degrading the lactic acid. The preservation number of the strain is CGMCC NO.19460, and the 16S rDNA sequence is shown as SEQ ID NO:1 is shown. The Arthrobacter vitrinite has the capacity of efficiently degrading lactic acid, the capacity of degrading the lactic acid reaches 9g/L, new precious strain resources are provided for degrading the lactic acid, and the Arthrobacter vitrinite has important application value.
Description
Technical Field
The invention belongs to the technical field of industrial microorganisms, and particularly relates to a protovitronectici arthrobacterium for degrading lactic acid and application thereof.
Background
The strong aromatic white spirit is one of typical representatives of the traditional white spirit in China, generates wide influence in the world and plays a very important role. The microbial community is a main driving force for forming flavor substances of the liquor, the lactic acid and derivatives thereof are important flavor substances in the Luzhou-flavor liquor, and aiming at the practical problem of overhigh content of the lactic acid in the fermentation process of the Luzhou-flavor liquor, the lactic acid degrading bacteria can be separated and screened from fermented grains by adopting a pure culture technology, so that guidance is provided for realizing the aim of 'reducing the milk'. The lactic acid degrading bacteria screened at present mainly comprise: clostridium (Clostridium), Geobacillus (Terrisporobacter), Bacillus (Bacillus), Clavibacterium (Ilyobacter), Desulfuromicrobium (Desutomaculus).
In order to better degrade lactic acid generated in the brewing process of the Luzhou-flavor liquor, more varieties of lactic acid degrading bacteria are needed.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: in the fermentation process of the Luzhou-flavor liquor, the lactic acid content is high, and a strain capable of efficiently degrading lactic acid needs to be separated to solve the problem.
The technical scheme for solving the technical problems comprises the following steps: provides an arthrobacter protothecoides strain for degrading lactic acid. The preservation number is CGMCC NO. 19460. The preservation time is 3 months and 6 days in 2020, the preservation place is the China general microbiological culture Collection center, and the address is microbial research institute of China academy of sciences No. 3, West Lu No.1 Hospital, North Cheng, the south China area, Beijing, zip code: 100101.
wherein, the 16S rDNA sequence of the arthrobacter vitronectici has the sequence shown in SEQ ID NO: 1.
SEQ ID NO 1 Arthrobacter vitronectii 16S rDNA nucleotide sequence
agtttgatcatggctcaggatgaacgctggcggcgtgcttaacacatgcaagtcgaacgatgaagcccagcttgctgggtggattagtggcgaacgggtgagtaacacgtgagtaacctgcccctgactctgggataagcccgggaaactgggtctaataccggatatgaccatgggacgcatgtcttgtggtggaaagatttatcggttggggatggactcgcggcctatcagcttgttggtgaggtaatggctcaccaaggcgacgacgggtagccggcctgagagggtgaccggccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagcgacgccgcgtgagggatgacggccttcgggttgtaaacctctttcagtagggaagaagcgagagtgacggtacctgcagaagaagcgccggctaactacgtgccagcagccgcggtaatacgtagggcgcaagcgttatccggatttattgggcgtaaagagctcgtaggcggtttgtcgcgtctgccgtgaaagtccgaggctcaacctcggatctgcggtgggtacgggcagactagagtgatgtaggggagactggaattcctggtgtagcggtgaaatgcgcagatatcaggaggaacaccgatggcgaaggcaggtctctgggcatttactgacgctgaggagcgaaagcatggggagcgaacaggattagataccctggtagtccatgccgtaaacgttgggcactaggtgtgggggacattccacgttttccgcgccgtagctaacgcattaagtgccccgcctggggagtacggccgcaaggctaaaactcaaaggaattgacgggggcccgcacaagcggcggagcatgcggattaattcgatgcaacgcgaagaaccttaccaaggcttgacatgttccagaccgcctcagagatggggtttcccttcggggctggttcacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaaccctcgttccatgttgccagcgggttatgccggggactcatgggagactgccggggtcaactcggaggaaggtggggatgacgtcaaatcatcatgccccttatgtcttgggcttcacgcatgctacaatggccggtacaatgggttgcgatactgtgaggtggagctaatccctaaaagccggtctcagttcggattggggtctgcaactcgaccccatgaagtcggagtcgctagtaatcgcagatcagcaacgctgcggtgaatacgttcccgggccttgtacacaccgcccgtcaagtcacgaaagttggtaacacccgaagccgatggcctaaccaccttgtgtggggggagtcgtcggaggtgggactggcgattgggactaagtcgtaacaaggtaacc。
Wherein the capacity of the Arthrobacter protoformiae for degrading lactic acid is less than or equal to 9 g/L.
The cytological characteristics of the Arthrobacter protoformiae for degrading lactic acid are as follows: the thallus is rod-shaped, the bacterial colony is light yellow and round, the surface is smooth and moist, and the edge is neat.
The biological characteristics of the Arthrobacter protoformiae for degrading lactic acid are as follows: at least one of potassium nitrate, pyrazinamide, 2-naphthyl phosphoric acid, 2-naphthyl- α -D-pyranoside, or gelatin (bovine source) can be used. Pyrrolidone-2-naphthylamine, naphthol-ASB 1-beta glucuronate-D, 2-naphthyl-beta-D-galactoside, 1-naphthyl-N-acetyl-beta D glucosaminide, ferric esculetin citrate, urea, glucose, ribose, xylose, mannitol, maltose, lactose (bovine source), sucrose, glycogen cannot be utilized. The hydrogen peroxide test is positive.
The invention also provides an application of the Arthrobacter protoformiae in degrading lactic acid in a wine brewing process.
The invention has the beneficial effects that: the invention firstly separates and identifies the glass fly arthrobacter capable of efficiently degrading lactic acid, the capacity of the glass fly arthrobacter for degrading lactic acid reaches 9g/L, and the glass fly arthrobacter is a novel bacterium capable of degrading lactic acid. The invention provides a new valuable strain resource for effectively degrading lactic acid and has high application value.
The Arthrobacter protoformiae provided by the invention is preserved in China general microbiological culture Collection center (CGMCC) at 3-6.2020, the preservation number is CGMCC NO.19460, and the Arthrobacter protoformiae is named as Arthrobacter protoformiae in a biological classification manner.
Drawings
FIG. 1 shows a morphogram of Arthrobacter protoformiae strain of the present invention;
FIG. 2 shows a diagram of the developing tree of the 16S rDNA system of Arthrobacter protoformiae.
Detailed Description
The invention provides a protovitreous fly arthrobacterium for degrading lactic acid, which has a preservation number of CGMCC NO. 19460. The preservation time is 3 months and 6 days in 2020, the preservation place is the China general microbiological culture Collection center, and the address is microbial research institute of China academy of sciences No. 3, West Lu No.1 Hospital, North Cheng, the south China area, Beijing, zip code: 100101.
the lactic acid degradation capability of the Arthrobacter protoformiae for efficiently degrading lactic acid is less than or equal to 9 g/L.
The strain is obtained by separating fermented grains of Yibin wuliangye Limited company.
The lactic acid degradation capability of the protovitreous fly arthrobacterium capable of efficiently degrading lactic acid is less than or equal to 9g/L, and the protovitreous fly arthrobacterium is a novel protovitreous fly arthrobacterium capable of degrading lactic acid. The invention provides a new valuable strain resource for effectively improving the lactic acid degradation capability, and has very high application value.
The raw glass fly arthrobacter obtained by screening of the invention has high speed and high efficiency for degrading lactic acid, can be completely degraded after 40 hours, 72 hours and 80 hours when the concentration of the lactic acid is respectively 3 g/L, 6 g/L and 9g/L, and has important value.
The following examples are intended to illustrate specific embodiments of the present invention without limiting the scope of the invention to the examples.
Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.
The enrichment medium used in the invention comprises the following components: NaCl 1g, K2HPO4 1.0g、NH4Cl 1g、MgCl20.5g、NaNO3Dissolving 1.0g and 2g of glucose in 500ml of distilled water, dissolving 20g of vinasse, 10ml of yellow water, 6g of yeast powder and 20ml of feints in 500ml of distilled water, stirring for half an hour, filtering by using 8 layers of gauze, mixing, and adjusting the pH value to 6.5-7.0 by using NaOH.
The composition of the LB culture medium used by the invention comprises: 10g of beef extract, 5g of yeast extract powder, 10g of NaCl, 1000ml of distilled water and NaOH for adjusting the pH value to 7.0.
The slant and the plate are corresponding culture media, and 2% agar powder is added.
EXAMPLE 1 Strain isolation
1. Enrichment culture
A pit mud sample taken from a brewing workshop of Yibin wuliangye Co., Ltd is inoculated into an enrichment medium containing 4g/L of lactic acid in an inoculation amount of 4%, and is subjected to static culture at 30 ℃ for 15 days.
2. Screening
Taking 10mL of enrichment culture solution in 90mL of sterile physiological saline, shaking and uniformly mixing, taking supernatant for gradient dilution, taking 10-2-10-5Coating the diluent on an enrichment medium plate containing 4g/L of lactic acid, standing and culturing for 15 days at 30 ℃, selecting bacterial colonies on the plate, streaking, culturing, repeating for three times until purified strains are obtained by separation, and storing on a slant. The strain was named WLY-B-L3.
Example 2 Strain identification
1. Morphological characteristics of the Strain
The WLY-B-L3 strain obtained by separation is gram-positive bacteria and rod-shaped, and the bacterial colony on the MRS culture medium is light yellow, the surface is wet and smooth, the edge is neat, and the middle is convex.
2. Physiological and biochemical characteristics
The WLY-B-L3 strain obtained by separation can utilize at least one of potassium nitrate, pyrazinamide, 2-naphthyl phosphoric acid, 2-naphthyl-alpha-D-pyranoside and gelatin (bovine source). Pyrrolidone-2-naphthylamine, naphthol-ASB 1-beta glucuronate-D, 2-naphthyl-beta-D-galactoside, 1-naphthyl-N-acetyl-beta D glucosaminide, ferric esculetin citrate, urea, glucose, ribose, xylose, mannitol, maltose, lactose (bovine source), sucrose, glycogen cannot be utilized. The hydrogen peroxide test is positive.
3. 16S rDNA identification
Inoculating the strain WLY-B-L3 into LB culture medium, standing and culturing at 30 ℃ for 7 days, taking 1mL of bacterial liquid, centrifugally collecting thalli, extracting genomic DNA by using a bacterial genome extraction kit, and preserving at-20 ℃ for later use.
The bacterial genome 16S rDNA is amplified by using primers 27F and 1492R, wherein the nucleotide sequence of 27F is shown as SEQ ID NO. 2, and the nucleotide sequence of 1492R is shown as SEQ ID NO. 3.
227F nucleotide sequence of SEQ ID NO
agtttgatcmtggctcag。
31492R nucleotide sequence
ggttaccttgttacgactt。
Amplification system (25. mu.L total): 10 XPCR buffer 2.5. mu.L, dNTP (2.5mmol/L each) 1. mu.L, DNA 10ng, primer 27F (10. mu. mol/L) 0.5. mu.L, primer 1492R (10. mu. mol/L) 0.5. mu.L, and double distilled water to 25. mu.L. The amplification procedure is as follows: 4min at 94 ℃; 30 cycles of 94 ℃ for 45s, 55 ℃ for 45s and 72 ℃ for 60 s; the reaction was stopped at 72 ℃ for 10min and 4 ℃.
16S rDNA sequencing: purifying PCR amplification products by using a gel recovery kit; the sequencing work of the purified product is completed by Shanghai Biotechnology Limited, and the 16S rDNA sequence of the strain WLY-B-L3 is obtained as shown in SEQ ID NO. 1.
And (3) construction of a phylogenetic tree: the similarity analysis of BLASTn and RDP Classifier is carried out on the determined 16S rDNA gene sequence of the bacterium and a GenBank database respectively, a standard strain which is relatively close to the genetic relationship of the experimental strain is selected to carry out sequence comparison by Clustalw software, and the MEGA5 software is adopted to carry out phylogenetic analysis to construct a phylogenetic tree, as shown in figure 2.
The strain WLY-B-L3 is identified as Arthrobacter protoformis (Arthrobacter protophorae) by integrating the thallus morphology, physiological and biochemical characteristics and 16S rDNA gene sequence. The bacterial strain WLY-B-L3 is preserved in China general microbiological culture Collection center (CGMCC for short, address: No. 3 of West Lu No.1 of Beijing city Kogyo-Yang district, microbiological research institute of Chinese academy of sciences, postal code 100101) in 3.6.2020 with the preservation number of CGMCC NO. 19460.
Example 3 lactic acid degradation experiment
Inoculating seed liquid of the strain WLY-B-L3 into LB culture solution containing 3, 6 and 9g/L of lactic acid (pH 7.0) according to the inoculation amount of 5.0% (v/v), culturing at 30 ℃ and 120r/min, taking the bacterial liquid at different time, measuring the residual quantity of the lactic acid, and inoculating sterile water with the same volume as a blank control in a control test. Each sample was repeated 3 times and averaged.
The method for measuring the lactic acid comprises the following steps: taking 0.1mL of the fermentation liquid, diluting the fermentation liquid to 1mL by using 0.1% phosphoric acid aqueous solution, uniformly oscillating the fermentation liquid in a 2mL centrifuge tube, and filtering the fermentation liquid by using a 0.2-micrometer filter membrane to obtain a sample to be detected. The measurement was carried out by high performance liquid chromatography. A chromatographic column: venusil ASB C184.6250mm, 5 μm, by Agela. Column temperature: at 30 ℃. Mobile phase: a: methanol; b: 0.1% phosphoric acid in water. Gradient elution: 0 min: 100B; 8 min: 100B; 25 min: 10B; 28.5 min: 10B; 29 min: 100B; 35 min: 100B; stopping: 35 min; flow rate: 1.0 mL/min.
The results of lactic acid degradation are shown in table 1 below.
TABLE 1 degradation rates of the strain WLY-B-L3 in lactic acid medium of different initial concentrations
| Initial concentration (lactic acid, g/L) | Incubation time (h) | Final concentration (g/L) | Degradation Rate (%) |
| 3 | 40 | - | 100 |
| 6 | 72 | - | 100 |
| 9 | 80 | - | 100 |
Note: "-" indicates no detection.
Sequence listing
<110> Yibin wuliangye GmbH
<120> lactic acid-degrading Arthrobacter protoformiae and use thereof
<130> A200382K (preface)
<141> 2020-06-04
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1485
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agtttgatca tggctcagga tgaacgctgg cggcgtgctt aacacatgca agtcgaacga 60
tgaagcccag cttgctgggt ggattagtgg cgaacgggtg agtaacacgt gagtaacctg 120
cccctgactc tgggataagc ccgggaaact gggtctaata ccggatatga ccatgggacg 180
catgtcttgt ggtggaaaga tttatcggtt ggggatggac tcgcggccta tcagcttgtt 240
ggtgaggtaa tggctcacca aggcgacgac gggtagccgg cctgagaggg tgaccggcca 300
cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga atattgcaca 360
atgggcgcaa gcctgatgca gcgacgccgc gtgagggatg acggccttcg ggttgtaaac 420
ctctttcagt agggaagaag cgagagtgac ggtacctgca gaagaagcgc cggctaacta 480
cgtgccagca gccgcggtaa tacgtagggc gcaagcgtta tccggattta ttgggcgtaa 540
agagctcgta ggcggtttgt cgcgtctgcc gtgaaagtcc gaggctcaac ctcggatctg 600
cggtgggtac gggcagacta gagtgatgta ggggagactg gaattcctgg tgtagcggtg 660
aaatgcgcag atatcaggag gaacaccgat ggcgaaggca ggtctctggg catttactga 720
cgctgaggag cgaaagcatg gggagcgaac aggattagat accctggtag tccatgccgt 780
aaacgttggg cactaggtgt gggggacatt ccacgttttc cgcgccgtag ctaacgcatt 840
aagtgccccg cctggggagt acggccgcaa ggctaaaact caaaggaatt gacgggggcc 900
cgcacaagcg gcggagcatg cggattaatt cgatgcaacg cgaagaacct taccaaggct 960
tgacatgttc cagaccgcct cagagatggg gtttcccttc ggggctggtt cacaggtggt 1020
gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080
cctcgttcca tgttgccagc gggttatgcc ggggactcat gggagactgc cggggtcaac 1140
tcggaggaag gtggggatga cgtcaaatca tcatgcccct tatgtcttgg gcttcacgca 1200
tgctacaatg gccggtacaa tgggttgcga tactgtgagg tggagctaat ccctaaaagc 1260
cggtctcagt tcggattggg gtctgcaact cgaccccatg aagtcggagt cgctagtaat 1320
cgcagatcag caacgctgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaag 1380
tcacgaaagt tggtaacacc cgaagccgat ggcctaacca ccttgtgtgg ggggagtcgt 1440
cggaggtggg actggcgatt gggactaagt cgtaacaagg taacc 1485
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agtttgatcm tggctcag 18
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ggttaccttg ttacgactt 19
Claims (4)
1. Arthrobacter protoformiae (Arthrobacter protophorae) which degrades lactic acid is characterized in that: the preservation number of the Arthrobacter protoformiae is CGMCC NO. 19460.
2. The Arthrobacter protoformiae of claim 1, characterized in that: the 16S rDNA sequence of the arthrobacter vitronectici has the sequence shown in SEQ ID NO: 1.
3. The Arthrobacter protoformiae of claim 1, characterized in that: the capacity of degrading lactic acid of the arthrobacter vitronectici is less than or equal to 9 g/L.
4. Use of Arthrobacter protoformiae according to any of claims 1-3 for degrading lactic acid in a brewing process.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200902721A (en) * | 2007-03-19 | 2009-01-16 | Sumitomo Chemical Co | Method for producing lactic acid |
| CN101892172A (en) * | 2009-12-31 | 2010-11-24 | 青岛康地恩生物科技有限公司 | Bare-glass fly arthrobacter strain and method for producing feeding proteins by using bare-glass fly arthrobacter strain to decompose gossypol |
| CN104328068A (en) * | 2014-09-30 | 2015-02-04 | 中南民族大学 | Arthrobacter protophormiae strain, screening method and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200902721A (en) * | 2007-03-19 | 2009-01-16 | Sumitomo Chemical Co | Method for producing lactic acid |
| CN101892172A (en) * | 2009-12-31 | 2010-11-24 | 青岛康地恩生物科技有限公司 | Bare-glass fly arthrobacter strain and method for producing feeding proteins by using bare-glass fly arthrobacter strain to decompose gossypol |
| CN104328068A (en) * | 2014-09-30 | 2015-02-04 | 中南民族大学 | Arthrobacter protophormiae strain, screening method and uses thereof |
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