CN102634563A - Screening method of strain capable of producing acetoin and acetoin production method based on strain fermenting method - Google Patents

Abstract

The invention describes a screening method of a strain capable of producing acetoin and an acetoin production method based on a strain fermenting method, belonging to the technical field of the biological engineering. A soil sample of the strain is dried, suspended, diluted, coated, and then qualitatively analyzed and screened by a Voges-Proskauer(V-P) experiment, and then the screened strain is subject to physiological and biochemical and molecular biological identification, and a Bacillus amyloliquefaciens capable of producing the acetoin with high yield is obtained by using a high-performance liquid chromatography method. The invention also discloses an acetoin production method based on the strain, wherein the yield of the acetoin is 35g/L after fermenting lasts for 72 hours.

Description

A kind ofly produce the screening method of acetoin bacterial strain and with this strain fermentation method production acetoin

Technical field

A kind ofly produce the screening method of acetoin bacterial strain and use this bacterial strain production acetoin, belong to bioengineering field.

Background technology

3-oxobutanol (3-hydroxybutanone) has another name called acetoin; Has pleasant fragrance; Can be used as a kind of flavouring agent is widely used in the foodstuff production; As be used for the flavor potentiator of cream, coffee and prepare dairy products, strawberry type essence etc., also can be used as 2 of synthetic important industrial chemicals of mikrobe and liquid fuel, the precursor substance of 3-butyleneglycol and di-acetyl.In addition, the 3-oxobutanol also can be used as a kind of hardware and software platform compound, is widely used in other numerous industries, and USDOE classified it as one of hardware and software platform compound of 30 kinds of preferential developments utilizations in 2004.Therefore, the 3-oxobutanol is widely used in fields such as food, pharmacy, chemical industry as a kind of important chemosynthesis midbody and multifunctional material.Its working method has: chemical synthesis, enzyme transforming process and microbe fermentation method.Wherein, chemical synthesis has realized suitability for industrialized production, and operation is simple for this method, but pollution is heavy, cost is high, raw material receives oil restriction, quality product to be difficult to reach the requirement of flavouring agent.Microbe fermentation method has low production cost, environmentally friendly, advantages such as product purity is high, reaction conditions gentleness, and the problems such as environment that chemical synthesis and enzyme transforming process face, resource, quality product that can solve receive increasing concern.

Summary of the invention

The purpose of this invention is to provide that a kind of acetoin is produced bacterium and screening method thereof and with this strain fermentation method production acetoin.

Technical scheme of the present invention:

1, a kind of screening method that produces the acetoin bacterial strain:

Near from the system wine workshop soil soil sampling takes by weighing about 5.0g, in 80 ℃ baking oven, toasts 48h, joins in the sterilized water of 200ml and processes suspension.This bacteria suspension is diluted to 10 as mother solution gradient ^-6, get 10 ^-4-10 ^-6Dilution bacterium liquid is coated on the enrichment medium, is inverted for 37 ℃ and cultivates 24h, and single bacterium colony of the different colonial morphologies of picking carries out the V-P The effects, and with the V-P positive strain fermention medium of transferring one by one, the line number of going forward side by side is preserved; Respectively get 2mL after cultivating end, the centrifugal 10min of 10000r/min detects the acetoin content in the supernatant with the HPLC method, filters out the bacterial strain that acetoin is produced in tens strains, and wherein the ability of bacterial strain FMME044 product acetoin is the strongest.

2, according to the said a kind of screening method that produces the acetoin bacterial strain of claim 1, it is following that its screening obtains producing the authentication method of acetoin bacterial strain:

1) Physiology and biochemistry is identified

Catalase test, V.P. and M.R. test, gelatine liquefication and starch hydrolysis, salt tolerant test, nitrate reduction test;

2) 16S rDNA molecular biology identification

Extract test kit with bacterial genomes and extract bacterial genomes DNA; Adopt subtilis 16S rDNA universal primer to carry out pcr amplification; Amplified production reclaims test kit with glue and reclaims purifying, and is connected to cloning vector pMD 18 T Vector, is transformed in the e. coli jm109; Utilize the penicillin resistance of cloning vector to screen positive transformant, and with universal primer transformant is carried out plasmid PCR and identify.

Nucleotide sequence in positive colony order-checking gained 16S rDNA sequence and the GenBank DB carries out homology analysis, obtains the nearest bacterial classification Bacillus amyloliquifaciens of sibship with it, and homology reaches 99%.

Confirm that through Physiology and biochemistry and 16S rDNA molecular biology identification this bacterial strain is a bacillus amyloliquefaciens, and called after Bacillus amyloliquifaciens FMME044.

3, the working method of a kind of acetoin disclosed by the invention, it is characterized in that adopting Bacillus amyloliquifaciens FMME044 is starting strain, with seed culture and liquid fermenting production acetoin;

1) seed culture:

Seed culture medium is in g/L: glucose 10, Carnis Bovis seu Bubali cream 5, peptone 5, initial pH 7.0;

Culture condition: under 37 ℃ of temperature, the shaking speed 200rpm condition, cultivate 10-12h;

2) liquid fermentation and culture:

Fermention medium is in g/L: glucose 120, and peptone 10, yeast powder 10, NaCl 5, K ₂HPO ₄3, MgSO ₄0.4, initial pH 7.0;

Fermentation condition: inoculum size 10% (v/v), under 37 ℃ of temperature, the shaking speed 200rpm condition, fermentation 60-72h.

3) the 7L ferment tank is cultivated:

Fermention medium is in g/L: glucose 120, and peptone 10, yeast powder 10, NaCl 5, K ₂HPO ₄3, MgSO ₄0.4, initial pH 7.0;

7L fermentation cylinder for fermentation substratum liquid amount is 4L, and inoculum size 10% (v/v), temperature are 37 ℃, and mixing speed is 400r/min, and air flow is 1.0vvm, does not control pH, incubation time 32-48h in the process.

4, the mensuration of acetoin output

Get the centrifugal 10min of fermented liquid 10000rpm, collect supernatant, and with acetoin as standard substance, the standardized solution of preparation 1,2,3,4,5g/L.With supernatant and standardized solution behind 0.45 μ m filtering with microporous membrane, with the content of high effective liquid chromatography for measuring acetoin.

Chromatographic condition:

Chromatographic column: Aminex HPX-87H;

Moving phase: the 5mmol/L dilute sulphuric acid, with 0.45 μ m membrane filtration;

Column temperature: 60 ℃;

Detect wavelength: 290nm;

Sample size: 20 μ L

Flow velocity: 0.6ml/min.

Typical curve presents good linear relationship (Fig. 1), regression equation: y=0.0027, R between 1-5g/L ²=0.9999.The output that obtains the 72h acetoin in view of the above is 35g/L, like Fig. 1.

Description of drawings

Fig. 1 acetoin typical curve

Fig. 2 chromatogram detected result, A 5g/L acetoin standard specimen, B 32h fermented liquid

Embodiment

Below be the embodiment of bacillus amyloliquefaciens (Bacillus amyloliquifaciens FMME044) screening, evaluation and fermentative prodn acetoin.

Embodiment 1

From near the face of land soil sampling system wine workshop, take by weighing about 5.0g, in 80 ℃ baking oven, toast 48h, join in the sterilized water of 200mL and process suspension.This bacteria suspension is diluted to 10 as mother solution gradient ^-6, get 10 ^-4-10 ^-6Dilution bacterium liquid is coated on the enrichment medium, is inverted for 37 ℃ and cultivates 24h, and single bacterium colony of the different colonial morphologies of picking carries out the V-P The effects, and with the V-P positive strain fermention medium of transferring one by one, the line number of going forward side by side is preserved; Respectively get 2mL after cultivating end, the centrifugal 10min of 10000r/min detects the acetoin content in the supernatant with the HPLC method, filters out the bacterial strain that acetoin is produced in tens strains, and wherein the ability of bacterial strain FMME044 product acetoin is the strongest.

Embodiment 2

FMME044 bacterial strain to screening carries out physio-biochemical characteristics evaluations (seeing table 1) by " microbial taxonomy ", and extracts the test kit process for extracting by bacterial genomes and extract genomic dna, is forward and reverse primer with P1 and P2:

P1：5′-CAGATGGGAGCTTGCTCCCTG-3′，

P2：5′-CGACTTCACCCCAATCATCTG-3′。

With pcr amplification 16S rDNA gene, entrust the big cara gene of China to carry out 16S rDNA order-checking, obtain this bacterial strain part 16S rDNA sequence after, on the NCBI website, carry out the homology compare of analysis with the BLAST gopher.Identify based on 16SrDNA sequential analysis and physio-biochemical characteristics,, think bacillus amyloliquefaciens, called after Bacillus amyloliquifaciens FMME044 in conjunction with growth and fermentation character research to this bacterium.

Bacterial strain FMME044 part 16S rDNA sequence:

1 gccaagagca?tgacggcaag?tggacgattc?gacttcaccc?caatcatctg?tcccaccttc

61 ggcggctggc?tccaaaaagg?ttacctcacc?gacttcgggt?gttacaaact?ctcgtggtgt

121?gacgggcggt?gtgtacaagg?cccgggaacg?tattcaccgc?ggcatgctga?tccgcgatta

181?ctagcgattc?cagcttcacg?cagtcgagtt?gcagactgcg?atccgaactg?agaacagatt

241?tgtgggattg?gcttaacctc?gcggtttcgc?tgccctttgt?tctgcccatt?gtagcacgtg

301 tgtagcccag?gtcataaggg?gcatgatgat?ttgacgtcat?ccccaccttc?ctccggtttg

361 tcaccggcag?tcaccttaga?gtgcccaact?gaatgctggc?aactaagatc?aagggttgcg

421 ctcgttgcgg?gacttaaccc?aacatctcac?gacacgagct?gacgacaacc?atgcaccacc

481 tgtcactctg?cccccgaagg?ggacgtccta?tctctaggat?tgtcagagga?tgtcaagacc

541 tggtaaggtt?cttcgcgttg?cttcgaatta?aaccacatgc?tccaccgctt?gtgcgggccc

601 ccgtcaattc?ctttgagttt?cagtcttgcg?accgtactcc?ccaggcggag?tgcttaatgc

661 gttagctgca?gcactaaggg?gcggaaaccc?cctaacactt?agcactcatc?gtttacggcg

721 tggactacca?gggtatctaa?tcctgttcgc?tccccacgct?ttcgctcctc?agcgtcagtt

781 acagaccaga?gagtcgccct?tcgccactgg?tgttcctcca?catctctacg?catttcaccg

841 ctacacgtgg?aattccactc?tcctcttctg?cactcaagtt?ccccagtttc?caatgaccct

901 ccccggttga?gccgggggct?ttcacatcag?acttaagaaa?ccgcctgcga?gccctttacg

961 cccaataatt?ccggacaacg?cttgccacct?acgtattacc?gcggctgctg?gcacgtagtt

1021?agccgtggct?ttctggttag?gtaccgtcaa?ggtgccgccc?tatttgaacg?gcacttgttc

1081?ttccctaaca?acagagcttt?acgatccgaa?aaccttcatc?actcacgcgg?cgttgctccg

1141?tcagactttc?gtccattgcg?gaagattccc?tactgctgcc?tcccgtagga?gtctgggccg

1201?tgtctcagtc?ccagtgtggc?cgatcaccct?ctcaggtcgg?ctacgcatcg?tcgccttggt

1261?gagccgttac?ctcaccaact?agctaatgcg?ccgcgggtcc?atctgtaagt?ggtagccgaa

1321?gccacctttt?atgtctgaac?catgcggttc?aaacaagcat?ccggtattag?ccccggtttc

1381?ccggagttat?cccagtctta?caggcaggtt?acccacgtgt?tactcacccg?tccgccgcta

1441?acatcaggga?gcaagctccc?atctgaatct?ccagaggatc?gccgggaacc?gaggacgag

Table 1 bacterial strain (FMME044) Physiology and biochemistry qualification result

Embodiment 3

Step 1: medium preparation

Seed culture medium (g/L): glucose 10, Carnis Bovis seu Bubali cream 5, peptone 5, NaCl 5, and initial p H 7.0;

Fermention medium (g/L): glucose 120, peptone 10, yeast powder 10, NaCl 5, K ₂HPO ₄3, MgSO ₄7H ₂O 0.4, initial pH 7.0;

Step 2: seed preparation

Seed culture medium is 50mL in the 500mL triangular flask, 121 ℃ of sterilization 15min.It is in 15% the glycerine pipe that bacterial strain is preserved in final concentration, gets 200 μ L preservation bacterium liquid and inserts in the 50ml seed culture medium and carry out seed culture, and 37 ℃, 200rpm cultivates 12h.

Step 3: shake flask fermentation is cultivated

Seed culture medium carries out fermentation culture with 10% inoculum size access 50mL fermention medium.At 37 ℃, 200rpm condition bottom fermentation 60-72h, it is centrifugal to get fermented liquid, collects supernatant is measured the acetoin in the fermented liquid with HPLC content.Output is 35g/L.

Embodiment 4

Step 1: with embodiment 3

Step 2: with embodiment 3

Step 3:7L ferment tank is cultivated

7L fermentation cylinder for fermentation substratum liquid amount is 4L; Inoculum size is 10% (v/v), and culture temperature is 37 ℃, and mixing speed is 400r/min; Air flow is 1.0vvm; Do not control pH, it is centrifugal that incubation time 32-48h (exhausting in glucose) gets fermented liquid, collects supernatant is measured the acetoin in the fermented liquid with HPLC content.Output is 35g/L.

Claims (3)

1. screening method that produces the acetoin bacterial strain is characterized by:

The soil sampling from the face of land, vegetable plot, countryside, Wuxi takes by weighing about 5.0g, in 80 ℃ baking oven, toasts 48h, joins in the sterilized water of 200ml and processes suspension.This bacteria suspension is diluted to 10 as mother solution gradient ^-6, get 10 ^-4-10 ^-6Dilution bacterium liquid is coated on the enrichment medium, is inverted for 37 ℃ and cultivates 24h, and single bacterium colony of the different colonial morphologies of picking carries out the V-P The effects, and with the V-P positive strain fermention medium of transferring one by one, the line number of going forward side by side is preserved; Respectively get 2ml after cultivating end, the centrifugal 10min of 10000r/min detects the acetoin content in the supernatant with the HPLC method, filters out the bacterial strain that acetoin is produced in tens strains, and wherein the ability of bacterial strain FMME044 product acetoin is the strongest.

2. according to the said a kind of screening method that produces the acetoin bacterial strain of claim 1; It is bacillus amyloliquefaciens through Physiology and biochemistry and 16S rDNA molecular biology identification that its screening obtains producing the acetoin bacterial strain, and called after Bacillus amyloliquifaciensFMME044.

3. the working method of an acetoin, it is characterized in that adopting Bacillus amyloliquifaciens FMME044 is starting strain, with seed culture and liquid fermenting production acetoin;

1) seed culture:

Seed culture medium is in g/L: glucose 10, and Carnis Bovis seu Bubali cream 5, peptone 5, initial p H 7.0;

Culture condition: under 37 ℃ of temperature, the shaking speed 200rpm condition, cultivate 10-12h;

2) liquid fermentation and culture:

Fermention medium is in g/L: glucose 120, and peptone 10, yeast powder 10, NaCl 5, K ₂HPO ₄3, MgSO ₄0.4, initial pH 7.0;

Fermentation condition: inoculum size 10% (v/v), under 37 ℃ of temperature, the shaking speed 200rpm condition, fermentation 60-72h;

3) the 7L ferment tank is cultivated:

Fermention medium is in g/L: glucose 120, and peptone 10, yeast powder 10, NaCl 5, K ₂HPO ₄3, MgSO ₄0.4, initial pH 7.0;

7L fermentation cylinder for fermentation substratum liquid amount is 4L, and inoculum size 10% (v/v), temperature are 37 ℃, and mixing speed is 400r/min, and air flow is 1.0vvm, does not control pH, incubation time 32-48h in the process.

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