CN117887623A - Fender fiber micro-bacterium for producing 2, 6-di-tert-butyl-4-methylphenol and application - Google Patents
Fender fiber micro-bacterium for producing 2, 6-di-tert-butyl-4-methylphenol and application Download PDFInfo
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- methylphenol
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- NLZUEZXRPGMBCV-UHFFFAOYSA-N Butylhydroxytoluene Chemical compound CC1=CC(C(C)(C)C)=C(O)C(C(C)(C)C)=C1 NLZUEZXRPGMBCV-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 235000010354 butylated hydroxytoluene Nutrition 0.000 title claims abstract description 40
- 239000000835 fiber Substances 0.000 title claims abstract description 40
- 241001467578 Microbacterium Species 0.000 title claims abstract description 9
- 238000000855 fermentation Methods 0.000 claims abstract description 15
- 230000004151 fermentation Effects 0.000 claims abstract description 15
- 230000000813 microbial effect Effects 0.000 claims abstract description 13
- 241000079938 Cellulosimicrobium funkei Species 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 22
- 238000004321 preservation Methods 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 8
- 235000013305 food Nutrition 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 7
- 239000003963 antioxidant agent Substances 0.000 claims description 6
- 244000309466 calf Species 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 239000003381 stabilizer Substances 0.000 claims description 6
- 230000003078 antioxidant effect Effects 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 210000004556 brain Anatomy 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 229940121375 antifungal agent Drugs 0.000 claims description 2
- 239000003429 antifungal agent Substances 0.000 claims description 2
- 230000000853 biopesticidal effect Effects 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims 1
- 238000011161 development Methods 0.000 abstract description 4
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- 239000000575 pesticide Substances 0.000 abstract description 3
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- 239000002609 medium Substances 0.000 description 14
- 230000001580 bacterial effect Effects 0.000 description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 244000005700 microbiome Species 0.000 description 9
- 239000000047 product Substances 0.000 description 6
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- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 4
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
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- 230000000694 effects Effects 0.000 description 3
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- 239000010687 lubricating oil Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- 244000141359 Malus pumila Species 0.000 description 2
- 241001125889 Micropterus salmoides Species 0.000 description 2
- 241001448533 Rohdea Species 0.000 description 2
- 241000187747 Streptomyces Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000003674 animal food additive Substances 0.000 description 2
- 235000021016 apples Nutrition 0.000 description 2
- 229940095259 butylated hydroxytoluene Drugs 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000003502 gasoline Substances 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- SJWFXCIHNDVPSH-UHFFFAOYSA-N octan-2-ol Chemical compound CCCCCCC(C)O SJWFXCIHNDVPSH-UHFFFAOYSA-N 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
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- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 244000043261 Hevea brasiliensis Species 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
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- 241001148470 aerobic bacillus Species 0.000 description 1
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- 239000011248 coating agent Substances 0.000 description 1
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- 238000007865 diluting Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000004205 dimethyl polysiloxane Substances 0.000 description 1
- 235000013870 dimethyl polysiloxane Nutrition 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
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- 235000019261 food antioxidant Nutrition 0.000 description 1
- 235000003086 food stabiliser Nutrition 0.000 description 1
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- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
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- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
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- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 238000002470 solid-phase micro-extraction Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Abstract
The invention belongs to the technical field of microbial fermentation, and particularly relates to Fender fiber micro-bacteria for producing 2, 6-di-tert-butyl-4-methylphenol and application thereof. In order to enrich microbial resources capable of producing 2, 6-di-tert-butyl-4-methylphenol (BHT) strain, the invention separates and screens out a Fennella fiber micro-bacterium (Cellulosimicrobium funkei) with BHT producing capability from Luzhou Laojiao medium temperature Daqu, and the strain does not pollute the environment or destroy ecological balance, can be used as a microbial preparation to be applied to feed, can also be used as a biological pesticide to be applied to fruit and vegetable protection, accords with the modern agriculture development trend of improving the environment and protecting ecology, and deserves further deep development.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to Fender fiber micro-bacteria for producing 2, 6-di-tert-butyl-4-methylphenol and application thereof.
Background
2, 6-Di-tert-butyl-4-methylphenol (Butylated Hydroxytoluene) is also called butyl hydroxytoluene, butylated hydroxytoluene, 3, 5-di-tert-butyl-4-hydroxytoluene and the like, which are BHT for short, belong to excellent general phenol antioxidants, are nontoxic, nonflammable, non-corrosive and good in storage stability, and are widely used for various lubricating oils, gasoline, paraffin and various raw oil, and can prevent the acid value or viscosity of the lubricating oils and the fuel oil from rising; can be used as food antioxidant and stabilizer to effectively delay rancidity of food; it can also be used as antioxidant and stabilizer for food grade plastics and polymer materials for packaging food, natural rubber and white or light color products of synthetic rubber.
The research also finds that BHT has antifungal activity (Meng Lulu et al, the effect of BHT on gray mold after picking of apples, the influence of defensive enzyme activity and malondialdehyde content, the report of plant protection school 46.3 (2019): 7), has wide application prospect in agriculture, such as disease control after picking of fruits and vegetables, or can be used as a biological pesticide to induce plant disease-resistant active substance (Zhang Ying. Mechanism of Streptomyces rohdea A-1 metabolite to induce anthracnose resistance of apples [ D ] Qingdao agricultural university, 2017.), has the advantages of long resistance duration, wide disease-resistant spectrum, no environmental pollution and the like, and has great research and development potential.
When the BHT is used as a feed additive, the feed additive has certain effect of delaying the rancidity of the feed, for example, experimental results of Li Li (at Li Li. BHA, tolerance research of BHT and different selenium sources in the feed of micropterus salmoides [ D ]. Dai university, 2023-09-21.) show that the aquatic feed added with the BHT has certain fat metabolism promoting effect and oxidation resistance protecting effect on the micropterus salmoides.
The results of experiments of Fender fiber microorganisms (Cellulosimicrobium funkei) belonging to the genus Cellobacter show that the Fender fiber microorganisms can be used as microbial preparations in feeds, improve the quality of the feeds for livestock and poultry, prolong the shelf life and promote the growth and reproduction of livestock and poultry, such as Zhao Ling and the like (Zhao Ling, sun Ranran, zhu Luoyi, and the like; the influence of the Fender fiber microorganisms on the cecal microorganisms of duckling [ J ]. Feed industry, 2016 (13): 4 ]) show that the Fender fiber microorganisms can reduce the quantity of escherichia coli and total aerobic bacteria in the content of the cecal of duckling, have no influence on lactic acid bacteria and bifidobacteria, and improve the cecal microorganism system to a certain extent. The addition of Fender fiber microorganisms was also found in commercial aquaculture composite microbial powders.
Although it has been found that microbial strains such as Bacillus subtilis, saccharomyces cerevisiae, streptomyces rohdea have BHT-producing ability, reports or records on BHT-producing microorganisms such as Fennella fiber have not been made.
Disclosure of Invention
In order to enrich microbial resources capable of producing 2, 6-di-tert-butyl-4-methylphenol (BHT) strain, the invention separates and screens out a Fender fiber micro-bacterium (Cellulosimicrobium funkei) with BHT producing capability from Luzhou Laojiao Daqu, and the strain does not pollute the environment and does not destroy ecological balance.
The Fender fiber micro-bacteria (Cellulosimicrobium funkei) are preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (CGMCC No. 26366) at the 12 th month of 2022, and the preservation address is the microbiological institute of China academy of China, the national institute of sciences No. 3, the Chaoyang area , beijing, and the post code: 100101, entitled: fender fiber micro-bacteria LZLJ-2 (Cellulosimicrobium funkei).
The 16S rDNA sequence of the BHT-producing Fender fiber micro-bacteria is shown as SEQ ID NO:1 is shown as follows:
SEQ ID NO1
GTGCTTACCATGCAGTCGAACGATGATGCCCAGCTTGCTGGGCGGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTTGACTTCGGGATAACTCCGGGAAACCGGGGCTAATACCGGATATGAGCCGCCTTCGCATGGGGGTGGTTGGAAAGTTTTTCGGTCAGGGATGGGCTCGCGGCCTATCAGCTTGTTGGTGGGGTGATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGAAGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAAcTACGTGCCAGCAGCCGCGGTAATaCGTAGGGcGCAAGCGTGTCCGGAATTAtTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGGTGTGaAAACTCGAGGCTCAACCTCGAGCTTGCATCGGGTACGGGCAGACTAGAGTGCGGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCCGCAACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGCTCATTCCACGAGTTCCGTGCCGCAGCAAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGCACGAGAAGCCACCAGAGATGGTGGTCTCTTTGGACACTCGTGCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTCCCATGTTGCCAGCGGGTTATGCCGGGGACTCATGGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGCTGCGATACCGTAAGGTGGAGCGAATCCCAAAAAGCCGGTCTC AGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCACGAAAGTCGGTAACACCCGAAGCCCATGGCCCAACCGTTCGCGGGGGGAGTGTC.
The invention also provides a microbial agent, which comprises the Fender fiber micro-bacteria.
The invention also provides application of the Fender fiber micro-bacteria in producing 2, 6-di-tert-butyl-4-methylphenol.
The invention also provides application of the Fender fiber microzyme in preparing an antioxidant, a stabilizer or a food antifungal agent containing 2, 6-di-tert-butyl-4-methylphenol.
Preferably, the antioxidant or stabilizer is an antioxidant or stabilizer added to lubricating oil, gasoline, paraffin, various raw oils, food or food material.
The invention also provides an application of the Fenton fiber micro-bacterium in preparing feed microbial preparations and biopesticides.
Wherein, in the application, the method for producing the 2, 6-di-tert-butyl-4-methylphenol by fermenting the Fender fiber micro-bacteria comprises the following steps: culturing the Fender fiber micro-bacteria in a liquid fermentation culture medium.
Preferably, the liquid fermentation medium comprises 10g of peptone, 12.5g of dehydrated calf brain powder, 5g of dehydrated calf heart powder, 5g of sodium chloride, 2g of glucose and 2.5g of disodium hydrogen phosphate per liter of medium.
Preferably, the inoculum size of the fine fiber micro-bacteria is 5-10v/v%.
Preferably, the fermentation culture temperature is 35-37 and the culture time is 3-5d.
By adopting the fermentation process, the content of 2, 6-di-tert-butyl-4-methylphenol produced by the strain is 0.217mg/L.
The beneficial effects are that: the invention separates and screens out a Fender fiber micro-bacterium (Cellulosimicrobium funkei) with BHT producing capability from Luzhou Laojiao Daqu, the preservation number is CGMCC No.26366, the content of 2, 6-di-tert-butyl-4-methylphenol produced in a liquid fermentation culture medium reaches 0.217mg/L, the strain does not pollute the environment, does not destroy ecological balance, can be used as a microbial preparation to be applied to feed or used as a biological pesticide to be applied to fruit and vegetable protection, accords with the modern agriculture development trend of improving the environment and protecting ecology, and deserves further deep development.
Drawings
FIG. 1 is a colony morphology of the Fender fiber micro-bacteria LZLJ-2 of the present invention;
FIG. 2 is a mass spectrum of 2, 6-di-tert-butyl-4-methylphenol GC-MS molecular fragments produced by the Fender fiber micro-bacterium LZLJ-2 of the present invention;
FIG. 3 is a mass spectrum of GC-MS molecular fragments of 2, 6-di-tert-butyl-4-methylphenol standard.
Preservation description: the Fender fiber micro-bacteria (Cellulosimicrobium funkei) for producing 2, 6-di-tert-butyl-4-methylphenol are preserved in China general microbiological culture collection center (CGMCC) for 12 months and 30 days in 2022, the preservation number is CGMCC No.26366, and the preservation address is the post code of China academy of microorganisms of national institute No. 3 of the national institute of sciences of West 1, in the Chaoyang area of Beijing: 100101, entitled: fender fiber micro-bacteria LZLJ-2 (Cellulosimicrobium funkei).
Detailed Description
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The medium formulation referred to in the examples:
R2A solid medium: yeast extract powder 0.5g/L, peptone 0.5g/L, casein hydrolysate 0.5g/L, glucose 0.5g/L, soluble starch 0.5g/L, dipotassium hydrogen phosphate 0.3g/L, anhydrous magnesium sulfate 0.024g/L, sodium pyruvate 0.3g/L, agar 15.0g/L, pH 7.2+ -0.2, and sterilizing with high pressure steam at 121deg.C for 15 min.
LB liquid medium: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, and steam sterilization at 121for 15 minutes.
LB solid medium: 15g/L agar was added to the LB liquid medium, and the mixture was autoclaved at 121for 15 minutes.
Liquid fermentation medium: 10.0g/L peptone, 12.5g/L dehydrated calf brain powder, 5.0g/L dehydrated calf heart powder, 5.0g/L sodium chloride, 2.0g/L glucose, 2.5g/L disodium hydrogen phosphate, pH 7.4+ -0.2, and autoclaving at 121deg.C for 20min.
Example 1: isolation and purification of strains
(1) The separation method comprises the following steps: taking 20g of luzhou-temperature Daqu sample in a luzhou-state wine, putting the luzhou-temperature Daqu sample into a conical flask filled with 180mL of sterile distilled water, and oscillating for 10min by a constant-temperature shaking table to fully scatter and mix the sample uniformly. Taking 1mL of sample suspension, diluting to 10 -210-7 by adopting a double-ratio dilution method, sucking 100 mu L of the diluted solution of each gradient, uniformly coating the diluted solution on an R2A solid culture medium plate, preparing two plates in parallel, inverting, culturing in a culture box at 37 for 36-48h, and observing in time.
(2) And (3) scribing and purifying: and taking out the plate with the colonies, picking single colonies with different colony morphologies, and carrying out secondary streaking until all the single colonies are purified.
(3) And (3) strain preservation: the single colony of each strain after purification is picked into 5mL LB liquid medium, placed at 37 for 24h, 1mL of bacterial liquid is sucked into a bacteria-preserving tube, 0.5mL of 60% sterile glycerol solution is added, resuspended and placed at-80 for preservation.
Example 2: detection of ability to produce 2, 6-di-tert-butyl-4-methylphenol
(1) Preparing a bacterial liquid to be tested:
After the glycerol preservation tube of the strain obtained by screening is dissolved, the strain is respectively inoculated into LB liquid culture medium, and is cultured for 24 hours at 37 , and after three generations of activation, the strain is inoculated into 50mL of LB liquid culture medium according to the inoculum size of 2% (volume ratio) for 24 hours, so as to obtain the bacterial liquid to be tested.
(2) HS-SPME/GC-MS method for detecting 2, 6-di-tert-butyl-4-methylphenol:
The headspace solid-phase microextraction/gas chromatography-mass spectrometer technique is applied: taking supernatant, adding the supernatant into a headspace bottle, adding saturated NaCl solution, keeping the temperature of the prepared sample at 60 for 5min, extracting the sample by using a 50/30 mu mDVB/CAR/PDMS extraction head at 60 for 50min, and desorbing the sample at 250 for 5min at a GC sample inlet after the extraction is finished. And (5) matching the compound search result with an NIST standard spectrum library, and confirming that the similarity reaches more than 80% as a target compound.
GC-MS detection chromatographic conditions:
Gas chromatography conditions: HP-INNOWAX column (60 m. Times.0.25 mm. Times.0.25 m); heating program: the initial temperature is 40 for 5min, the temperature is increased to 100 at 4 /min, the temperature is increased to 230 at 6 /min, the temperature is maintained for 10min, and the carrier gas is high-purity helium (1.0 mL/min); the temperature of the sample inlet is 250 , and the flow is not split.
Mass spectrometry conditions: electron ionization source with electron energy of 70eV; electron multiplier voltage 350V; the ion source temperature is 230 ; the temperature of the transmission line is 250 ; the mass range is 40-450 m/z.
By the detection method, as shown in figures 1, 2 and 3, a strain with the ability of producing 2, 6-di-tert-butyl-4-methylphenol is screened from the separated strains, and the biological characteristics are as follows: culturing on LB solid medium at 37deg.C for 1 day, as shown in FIG. 1, the colony is yellow, wet and convex, round and opaque, and has neat edge and thick texture. The strain was selected for further investigation.
Example 3: molecular characterization of bacteria
And (3) amplifying and culturing the target strain, taking fresh bacterial liquid in the logarithmic growth phase, centrifuging and collecting bacterial cells, and extracting genome DNA by utilizing Ezup column type bacterial genome DNA extraction kit of Shanghai biological technology company. The full-length sequence of the 16S rDNA is amplified by adopting a bacterial universal primer 27F/1492R, and the method is concretely as follows:
SEQ ID NO227F(5-AGAGTTTGATCCTGGCTCAG-3)
SEQ ID NO31492R(5-GGTTACCTTGTTACGACTT-3)
Reaction system (25. Mu.L)
TABLE 1PCR reaction system
Reaction procedure
TABLE 2PCR reaction procedure
The PCR reaction was performed in 30 cycles of denaturation-annealing-extension according to the above procedure, and the PCR product was checked by electrophoresis on a 1.0% agarose gel at a voltage of about 11V/cm for 20min.
The purification of PCR products was performed as described in the Shanghai Biotechnology Co small amount of gel recovery PCR product purification kit, and sequencing was performed by Shanghai Biotechnology Co.
The gene sequence of the 16S rDNA fragment obtained by sequencing is subjected to BLAST comparison by NCBI, the bacterial colony morphology is combined, the bacterial strain species information is determined, the bacterial strain is identified as Fender fiber micro-bacteria (Cellulosimicrobium funkei), and the bacterial strain is defined as Fender fiber micro-bacteria LZLJ-2 and is preserved in China general microbiological culture collection center (CGMCC) at 12-30 of 2022, and the preservation number is CGMCC No.26366.
Example 4: fermentation culture of Fender fiber micro-bacteria LZLJ-2
After the obtained Fender fiber micro-bacteria LZLJ-2 glycerol storage tube was dissolved, the obtained solution was inoculated into 10mL of a liquid-filled LB liquid medium at an inoculum size of 1%, and 1% of the liquid-filled LB liquid medium was inoculated into 150mL of the liquid-filled LB liquid medium at an inoculum size of 1d based on shaking culture at 37and 120r/min to prepare a seed liquid. Inoculating into liquid fermentation culture medium according to 5% inoculum size, and shake culturing at 37deg.C for 3d at 120 r/min. The sample after fermentation was taken, and the content was calculated by the same method as in example 2, using 0.822mg/mL of 2-octanol as an internal standard and a liquid fermentation medium as a blank. The test result shows that the Fender fiber micro-bacteria LZLJ-2 produce 0.217mg/L of 2, 6-di-tert-butyl-4-methylphenol under the process.
Claims (9)
1. Fender fiber micro-bacteria (Cellulosimicrobium funkei) for producing 2, 6-di-tert-butyl-4-methylphenol, characterized in that: the preservation number is CGMCC No.26366.
2. A microbial agent is characterized in that: a micro-bacterium comprising the Fender fiber of claim 1.
3. Use of a micro-bacterium of the Fender fiber of claim 1 for producing 2, 6-di-tert-butyl-4-methylphenol.
4. Use of a fine fiber of fens' fiber as claimed in claim 1 for the preparation of an antioxidant, stabilizer or food antifungal agent comprising 2, 6-di-tert-butyl-4-methylphenol.
5. Use of the fine fimbriae of claim 1 in the preparation of feed microbial preparations and biopesticides.
6. Use according to any one of claims 3-5, characterized in that: the method for producing 2, 6-di-tert-butyl-4-methylphenol by fermenting the Fender fiber micro-bacteria comprises the following steps: culturing the Fender fiber micro-bacteria in a liquid fermentation culture medium.
7. The use according to claim 6, characterized in that: the liquid fermentation culture medium comprises 10g of peptone, 12.5g of dehydrated calf brain powder, 5g of dehydrated calf heart powder, 5g of sodium chloride, 2g of glucose and 2.5g of disodium hydrogen phosphate per liter of culture medium.
8. The use according to claim 6, characterized in that: the inoculation amount of the micro-bacteria of the Fender fiber is 5-10v/v%.
9. The use according to claim 6, characterized in that: the fermentation culture temperature is 35-37 and the culture time is 3-5d.
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