CN112342163A

CN112342163A - Burkholderia and application thereof

Info

Publication number: CN112342163A
Application number: CN202011269503.0A
Authority: CN
Inventors: 任婧楠; 彭芷芊; 范刚; 张璐璐; 李晓; 潘思轶
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2020-11-13
Filing date: 2020-11-13
Publication date: 2021-02-09

Abstract

The invention relates to the field of microorganisms, in particular to Burkholderia and application thereof. Firstly, diluting and coating a sample bacterial suspension on a flat plate through primary screening, selecting a single colony, taking valencene as a unique carbon source, carrying out primary screening by drawing a growth curve, and selecting a strain with good growth to carry out the next experiment; secondly, re-screening, analyzing and comparing the yield of the grapefruit ketone generated by bioconversion of valencene by the strains, and selecting strains with high grapefruit ketone yield to perform the next experiment; and (3) carrying out streak separation on the selected strains, and analyzing and measuring the products and the content of the strains in the valencene biotransformation. Compared with the existing physical and chemical synthesis methods, the method has the characteristics of high specificity, mild reaction conditions, high reaction rate, high yield, low cost, few reaction steps and the like, and the produced nootkatone belongs to natural perfume and can be added into foods, cosmetics and other household products.

Description

Burkholderia and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to Burkholderia and application thereof.

Background

Nootkatone is one of important substances in pomelo fragrance components, has a persistent strong citrus-flavor, and is mainly added as a perfume in foods and cosmetics. A certain amount of nootkatone is added into cigarettes, so that the fragrance of the cigarettes can be increased, the fragrance of the cigarettes can be enriched, and the stimulation can be reduced; the nootkatone has sweet pericarp and costus scent, so the nootkatone can also be used for preparing edible essences such as pomelo, orange, tropical fruit and the like.

Natural nootkatone is mainly present in essential oils such as bergamot oil, grapefruit oil, lemon oil, citrus oil, lime oil, and tangerine oil. However, the natural content of the grapefruit seed oil is generally low, so that the grapefruit seed oil used in actual production is mainly derived from chemical synthesis. The most common synthetic methods are oxidation using valencene as a starting material, oxidation of valencene using dichromate, or oxidation using a catalyst in the presence of t-butyl hydroperoxide (TBHP). The chemical synthesis methods use a large amount of heavy metal catalysts, which seriously damage the environment, and other available non-heavy metal catalysts have great potential safety hazards although the environment cannot be damaged, and the production efficiency is too low to meet the production requirements.

European food legislation has specifically established that the nootkatone product obtained by chemical synthesis cannot be sold and used as a natural flavour which can only be prepared by physical processes (extraction from natural raw materials) or by enzymatic/microbiological processes. Microbial conversion is the structural modification of exogenous substrates by microbial cells or enzymes, namely the catalytic reaction of a certain enzyme or a series of enzymes produced in the microbial metabolic process on the substrates, so that the perfumes produced by microbial conversion also belong to natural perfumes. The microbial conversion has the characteristics of high specificity, mild reaction conditions, high reaction rate, high yield, low cost, few reaction steps and the like. At present, the method is widely applied to aspects of organic synthesis, new drug development, in-vitro metabolite prediction, spice production and the like.

Disclosure of Invention

The invention aims to efficiently and accurately screen out strains capable of transforming nootkatone by adopting a screening method taking valencene as a unique carbon source, and transform the valencene which is rich in natural content and low in application value into natural spice nootkatone with stronger fragrance, wider application and higher commercial value.

In order to realize the aim, the invention provides burkholderia, the preservation number is CCTCC NO: m2020510, and the screening method comprises the following steps:

(1) taking 10g of soil sample from research center of orange engineering technology in Hubei province, adding the soil sample into 90g of sterilized normal saline, and mixing uniformly.

(2) 2mL of the mixed liquid is put into 100mL of minimum culture medium, and 1mL of valencene (0.22 μm filter membrane filtration sterilization, 4 ℃ storage) is added, and shake culture is carried out at 37 ℃ and 121r/min for 2-7 days.

(3) Diluting the bacterial liquid obtained in the step (2) to 10 degrees with sterilized normal saline^-1、10^-2、10^-3、10^-4、 10^-5And 10^-6And 6 dilution gradients in total, coating the gradient bacterial liquid on a plate to separate strains, and culturing at 37 ℃ until bacterial colonies completely grow.

(4) After the bacterial colony grows out in the step (3), selecting a plate with proper concentration to pick out a single bacterial colony, and inoculating the single bacterial colony in an LB culture medium for activated culture.

(5) Primary screening: taking appropriate amount of activated bacteria liquid, adding 1% valencene in minimum culture medium (0.22 μm filter membrane filtration sterilization, 4 deg.C storage), culturing at 37 deg.C under 210r/min, and measuring bacteria liquid OD every 12h₆₀₀Values, growth curves of the strains were plotted.

(6) And (5) selecting strains with good growth according to the growth curve drawn in the step (5), and transferring the strains to an LB culture medium for activated culture.

(7) Taking 1mL of bacterial liquid to be cultured in 100mLLB culture medium at 37 ℃ at 210r/min, and obtaining the OD of bacterial liquid₆₀₀When the value reaches 0.6-0.7, 0.1% valencene is added (0.22 μm filter membrane filtration sterilization,stored at 4 ℃), and cultured for further 72 h.

(8) Re-screening: and respectively taking fermentation liquor cultured for 24h, 48h and 72h for SPME-GC-MS detection analysis, and detecting the yield of the nootkatone generated by strain conversion.

(9) Selecting strains with high yield of nootkatone, performing plate streaking separation, and continuously separating for 3 times. And picking single colonies on the plate to perform activated culture in an LB culture medium.

(10) Transferring 1mL of bacterial solution into 100mLLB culture medium, culturing at 37 deg.C and 210r/min until bacterial solution OD₆₀₀When the value reaches 0.6-0.7, 0.1% valencene is added (0.22 μm filter membrane filtration sterilization, 4 deg.C storage), and culture is continued for 72 h.

(11) SPME-GC-MS detection is carried out on fermentation liquor cultured for 24h, 48h and 72h respectively, determination and analysis are carried out on transformation products, and the analysis results of the obtained bacterial strain biotransformation products are shown in Table 1:

TABLE 1

(12) The colony morphology of the strain on a solid culture medium is observed morphologically according to a manual of identifying common bacteria systems, and a plurality of physiological and biochemical indexes of the strain are measured at the same time, and the analysis of the physiological and biochemical characteristics of the obtained strain is shown in table 2:

TABLE 2

(13) Extracting DNA of the strain, carrying out PCR amplification and sequencing of 16SrDNA, and carrying out strain homology analysis.

Preferably, the minimum medium components in step (2) are as follows: 6g/LNa₂HPO₄、0.50 g/LNaCl、3g/LKH₂PO₄、1g/LNH₄Cl, dissolved in 0.85% physiological saline.

Preferably, the composition of the LB medium in step (4) is as follows: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl, dissolved in distilled water.

Preferably, all of the operations described are performed in a sterile operating station, maintaining a sterile environment.

The invention also provides a spice, wherein the nootkatone in the spice is prepared by the following steps of preserving in the formula of CCTCC NO: m2020510 by bioconversion of Burkholderia.

Biological preservation Instructions

And (3) classification and naming: burkholderia plantarii PZQ14, burkholderia sp. Is preserved in China center for type culture Collection (CMGWU university, Wuhan, China) in 2020 on 9/17 th month with the preservation number of CCTCCNO: M2020510.

The invention has the beneficial effects that:

(1) the nootkatone produced by the present invention belongs to natural flavors. Compared with the existing production technology of nootkatone synthesized by a chemical synthesis method, the method for producing nootkatone by a microbiological method has the characteristics of high specificity, mild reaction conditions, high reaction rate, high yield, low cost, few reaction steps and the like.

(2) The invention provides a screening method of a bacterial strain which can use valencene as a unique carbon source and has high conversion rate of nootkatone. The screening method is efficient and accurate, and has the advantages of reducing workload, improving efficiency and the like.

Drawings

FIG. 1 is a graph showing the growth profile of the strain obtained in step (5);

FIG. 2 is a graph showing a comparative analysis of the yield of nootkatone obtained in step (8);

FIG. 3 is a cross-sectional drawing of a plate of four strains obtained by the final screening;

FIG. 4 is a graph showing the growth of the strain in LB medium;

Detailed Description

Example one

The screening steps of the strain which takes valencene as a unique carbon source and has high conversion rate of nootkatone are as follows:

(1) 10g of soil sample is taken from research center of orange engineering technology in Hubei province. The soil sample was added to 90g of sterilized normal saline and mixed well. Taking 2mL of uniformly mixed liquid into 100mL of minimum culture medium, adding 1mL of valencene (0.22 mu m filter membrane filtration sterilization, preservation at 4 ℃), performing shake culture at 37 ℃ at 121r/min for 2-7 days, separating strains by using a gradient dilution coating method, performing culture at 37 ℃ until the strains completely grow, and picking out single colonies.

(2) Primary screening: and inoculating the picked single colony in LB culture medium for activating culture. Taking appropriate amount of bacterial liquid, adding 1% valencene (0.22 μm filter membrane filtration sterilization, 4 deg.C preservation), culturing at 37 deg.C under 210r/min, and measuring bacterial liquid OD every 12h₆₀₀Values, growth curves of the bacteria were plotted. And selecting a strain with good growth, and transferring the strain to an LB culture medium for enrichment culture.

(3) Re-screening: taking 1mL of bacterial liquid to be cultured in 100mLLB culture medium at 37 ℃ at 210r/min, and obtaining the OD of bacterial liquid₆₀₀And when the value reaches 0.6-0.7, adding 0.1% valencene in valencene (0.22 mu m filter membrane filtration sterilization, preservation at 4 ℃), continuously culturing for 72h, respectively taking fermentation liquor cultured for 24h, 48h and 72h, carrying out SPME-GC-MS detection analysis, detecting the yield of the grapefruit ketone generated by strain conversion, and selecting a strain with high yield of the grapefruit ketone for the next experiment.

(4) Analytical determination of the bioconversion products of the strains: the selected strains were subjected to plate streaking separation and separated 3 times in succession. Selecting single colony on the plate, performing activated culture in LB culture medium, transferring to 100mLLB culture medium, culturing at 37 deg.C under 210r/min, and culturing as bacterial liquid OD₆₀₀When the value reaches 0.6-0.7, adding 0.1% valencene myrcene (0.22 mu m filter membrane filtration sterilization, preservation at 4 ℃), continuously culturing for 72h, respectively taking fermentation liquor cultured for 24h, 48h and 72h, carrying out SPME-GC-MS detection, and carrying out determination analysis on the conversion product.

(5) GC-MS analysis of the conversion products:

accurately weighing 5mL of the centrifuged fermentation liquid, transferring the centrifuged fermentation liquid into a 20mL jaw sample bottle, adding 1.8g of NaCl to promote volatilization of volatile components, sealing the fermentation liquid by using a polytetrafluoroethylene spacer, balancing the fermentation liquid on a magnetic stirrer at a constant temperature of 40 ℃ for 15min, inserting an activated 50/30 mu m DVB/CAR/PDMS extraction head (activated at 270 ℃ for 1h) through the spacer, pushing out a fiber head, adsorbing the fermentation liquid in a headspace for 40min, and then inserting a GC-MS sample inlet for desorption for 5 min.

Gas chromatography conditions: 6890N type gas chromatograph has capillary column HP-5(30m × 250 μm × 0.25 μm), temperature programming, initial temperature of 40 deg.C, holding for 3min, heating to 160 deg.C at 3 deg.C/min, holding for 2min, heating to 220 deg.C at 8 deg.C/min, and holding for 3 min. The injection port temperature was 250 ℃. Mass spectrum conditions: 5975B mass spectrometer with ion source temperature 230 deg.C, quadrupole rod temperature 150 deg.C, ionization mode EI, electron energy 70eV, and mass range of 45-550 AMU/sec¹⁶。

And (3) qualitative analysis: analyzing and identifying by GC-MS combined instrument and utilizing C₆～C₃₀Retention time of normal paraffins the retention index of each chromatographic peak was calculated. The analysis result is subjected to preliminary retrieval and data analysis by using a computer spectrum (NIST05/WILEY7.0), and then is compared and subjected to manual spectrogram analysis by combining with retention indexes of documents to confirm each chemical composition of the volatile substances.

Quantitative analysis: and quantifying the nootkatone by adopting an external standard method. Other products are quantified by using cyclohexanone as an internal standard and comparing and calculating the peak area of the cyclohexanone with the peak area of the aroma substances. The aroma concentration and conversion are calculated according to the following formula:

example two

Strain identification

(1) Morphological and physiological biochemical assays: the colony morphology of the strain on a solid culture medium is observed morphologically according to a 'handbook for identifying common bacteria systems', and a plurality of physiological and biochemical indexes of the strain are measured at the same time.

(2) Extracting bacterial DNA by using a DNA extraction kit, and amplifying 16SrDNA by using a PCR technology, wherein the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃, 30sec, annealing at 58 ℃, 30sec, extension at 72 ℃, 45sec, 35 cycles; terminal extension at 72 ℃ for 5 min. And (3) detecting the PCR amplification product by agarose gel electrophoresis, sending the PCR amplification product to Wuhantianyihuiyuan biotechnology limited company for sequencing, and performing homology analysis and comparison on a sequencing result and a known 16SrDNA sequence in GenBank.

EXAMPLE III

The growth curve of the strain in LB medium is drawn:

inoculating strain seed solution into LB culture medium at 1%, culturing at 37 deg.C under 210r/min, and measuring OD every four hours with sterile LB culture medium as blank control₆₀₀Value, triplicate, in OD₆₀₀The average is the ordinate and the time is the abscissa, and the growth curve of the strain is plotted.

The above description is only a preferred embodiment of the present invention, and does not limit the present invention, and those skilled in the art can make various modifications and variations. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> Burkholderia and application thereof

<141> 2020-11-13

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1426

<212> DNA

<213> Burkholderia plantarii sp. PZQ14)

<400> 1

tgtgcggctg ccttaccatg caagtcgaac ggcagcacgg gtgcttgcac ctggtggcga 60

gtggcgaacg ggtgagtaat acatcggaac atgtcctgta gtgggggata gcccggcgaa 120

agccggatta ataccgcata cgatctacgg atgaaagcgg gggaccttcg ggcctcgcgc 180

tatagggttg gccgatggct gattagctag ttggtggggt aaaggcctac caaggcgacg 240

atcagtagct ggtctgagag gacgaccagc cacactggga ctgagacacg gcccagactc 300

ctacgggagg cagcagtggg gaattttgga caatgggcga aagcctgatc cagcaatgcc 360

gcgtgtgtga agaaggcctt cgggttgtaa agcacttttg tccggaaaga aatccttggt 420

tctaatatag ccgggggatg acggtaccgg aagaataagc accggctaac tacgtgccag 480

cagccgcggt aatacgtagg gtgcaagcgt taatcggaat tactgggcgt aaagcgtgcg 540

caggcggttt gttaagaccg atgtgaaatc cccgggctca acctgggaac tgcattggtg 600

actggcaagc tagagtatgg cagagggggg tagaattcca cgtgtagcag tgaaatgcgt 660

agagatgtgg aggaataccg atggcgaagg cagccccctg ggccaatact gacgctcatg 720

cacgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc ctaaacgatg 780

tcaactagtt gttggggatt catttcctta gtaacgtagc taacgcgtga agttgaccgc 840

ctggggagta cggtcgcaag attaaaactc aaaggaattg acggggaccc gcacaagcgg 900

tggatgatgt ggattaattc gatgcaacgc gaaaaacctt acctaccctt gacatggtcg 960

gaatcccgct gagaggtggg agtgctcgaa agagaaccgg cgcacaggtg ctgcatggct 1020

gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgtcc 1080

ttagttgcta cgcaagagca ctctaaggag actgccggtg acaaaccgga ggaaggtggg 1140

gatgacgtca agtcctcatg gcccttatgg gtagggcttc acacgtcata caatggtcgg 1200

aacagagggt tgccaacccg cgagggggag ctaatcccag aaaaccgatc gtagtccgga 1260

ttgcactctg caactcgagt gcatgaagct ggaatcgcta gtaatcgcgg atcagcatgc 1320

cgcggtgaat acgttcccgg gtcttgtaca caccgcccgt cacaccatgg gagtgggttt 1380

taccagaagt ggctagtcta accgcaagga ggacggtcac cacggt 1426

Claims

1. Burkholderia, the preservation number is CCTCC NO: m2020510.

2. Burkholderia according to claim 1, characterized in that 16S rDNA is as indicated in the SIPO sequence listing.

3. Use of burkholderia according to claim 1 for the production of the natural flavor nootkatone.

4. A preservation number is CCTCC NO: the screening method of M2020510 Burkholderia is characterized by comprising the following steps:

(1) taking 10g of soil sample from research center of orange engineering technology in Hubei province, adding the soil sample into 90g of sterilized normal saline, and mixing uniformly;

(2) taking 2mL of uniformly mixed liquid, adding 1mL of valencene into 100mL of minimum culture medium, and performing shake culture at 37 ℃ for 2-7 days;

(3) diluting the bacterial liquid obtained in the step (2) to 10 degrees with sterilized normal saline^-1、10^-2、10^-3、10^-4、10^-5And 10^-66 dilution gradients in total, coating each gradient bacterial liquid on a plate separation strain, and culturing at 37 ℃ until bacterial colonies completely grow;

(4) after the bacterial colony grows out in the step (3), selecting a flat plate with proper concentration to pick out a single bacterial colony, and inoculating the single bacterial colony in an LB culture medium for activated culture;

(5) primary screening: taking a proper amount of activated and cultured bacterial liquid in a minimum culture medium, adding 1% valencene and myrcene, performing shake culture at 37 ℃, and measuring the OD of the bacterial liquid every 12h₆₀₀Value, plotting the growth curve of the strain；

(6) Selecting strains with good growth according to the growth curve drawn in the step (5), and transferring the strains to an LB culture medium for activated culture;

(7) taking 1mL of bacterial liquid into 100mL of LB culture medium, performing shake culture at 37 ℃, and obtaining bacterial liquid OD₆₀₀Adding 0.1% valencene to 0.6-0.7, and culturing for 72 hr;

(8) re-screening: carrying out SPME-GC-MS detection analysis on fermentation liquor cultured for 24h, 48h and 72h respectively, and detecting the yield of the grapefruit ketone generated by strain conversion;

(9) selecting strains with high yield of nootkatone, performing plate streaking separation, and continuously separating for 3 times. Picking single colony on the plate to be activated and cultured in an LB culture medium;

(10) transferring 1mL of the bacterial solution into 100mL of LB culture medium, performing shake culture at 37 ℃, and obtaining OD of the bacterial solution₆₀₀Adding 0.1% valencene to 0.6-0.7, and culturing for 72 hr;

(11) carrying out SPME-GC-MS detection on fermentation liquor cultured for 24h, 48h and 72h respectively, and determining and analyzing a conversion product;

(12) the colony morphology of the strain on a solid culture medium is observed morphologically according to a 'handbook for identifying common bacteria systems', and a plurality of physiological and biochemical indexes of the strain are measured at the same time.

(13) Extracting DNA of the strain, carrying out PCR amplification and sequencing of 16S rDNA, and carrying out homology analysis of the strain.

5. A flavor containing nootkatone, characterized in that nootkatone is prepared from nootkatone having a preservation number of CCTCC NO: m2020510 by bioconversion of Burkholderia.

6. The method for screening Burkholderia according to claim 4, wherein the minimum medium in the step (2) comprises: 6g/L Na₂HPO₄、0.50g/L NaCl、3g/L KH₂PO₄、1g/L NH₄Cl, and dissolved in 0.85% physiological saline.

7. The method for producing natural flavor nootkatone by biotransformation according to claim 4, wherein the LB medium composition in step (4) comprises: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl, dissolved in distilled water.

8. The method for producing nootkatone, a natural flavor, by bioconversion of claim 4, wherein all of said steps are performed in a sterile processing station.