CN103756932B - Myroides spp strain and application thereof in preparation of allyl methyl sulfide - Google Patents
Myroides spp strain and application thereof in preparation of allyl methyl sulfide Download PDFInfo
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Abstract
The invention discloses a Myroides spp strain, the strain name is WTC05-2, the category name is Myroides sp., the Myroides spp strain is collected in China General Microbiological Culture Collection Center (CGMCC) on January 9, 2013, and the collection number is CGMCC No. 7113. The strain disclosed by the invention has strong capability of producing allyl methyl sulfide and can be used for producing and preparing allyl methyl sulfide, and allyl methyl sulfide is produced by adopting a microbial fermentation method. Compared with a chemical production method, the microbial fermentation method has the advantages of mild reactions, green and natural raw materials, simple process, low energy consumption and the like, is conductive to environmental protection and easy to popularize and use, and further lays a technical foundation for production of allyl methyl sulfide by utilizing microbial fermentation.
Description
Technical field
The present invention relates to a strain fragrance Pseudomonas bacterial strain and preparing the application in allyl methyl thioether, belong to biological technical field.
Background technology
Allyl methyl thioether (Allyl methyl sulfide), Chinese another name: propenylmethyl thioether, allyl methyl sulfide; No. CAS: 10152-76-8; Molecular formula: C
4h
8s; Molecular weight: 88.17; Molecular structure:
boiling point: 88.6 DEG C of at 760mmHg; Density 0.85g/mL
[1].
Natural diallyl sulfide is present in the vegetables such as garlic, onion more.Wherein, allyl methyl thioether is a kind of foodstuff additive generally used; medicine intermediate; it has important biological activity; as follows: (1) anti-oxidant activity, can scavenging free radicals, the generation of nitrosamine in barrier bodies; anti-lipid peroxidation thing to the damage of membrane structure, thus plays liver-protective effect
[2,3].The people such as keemun soldier analyze antioxidant component in bacillus natto rice bran fermented product, find that the anti-oxidant vigor of ethyl acetate phase and ether phase is the strongest, be respectively 168 and 163kU/g, wherein ethyl acetate phase oxidation-resistant active ingredient mainly allyl methyl thioether
[4].(2) Zhao Huaiqing, the permanent hero of difficult ripple have studied For-carrying green onion (Allium victorialis) medicinal extract and compound thereof to the effect of cultured myocardial, experimental result shows that volatile extract has obvious enhancement to cultured myocardial heart rate and amplitude, and then carry out composition analysis to having bioactive volatile extract, show that Methyl disulfide, allyl methyl trisulfide, allyl methyl thioether and diallyl disulfide all have enhancement in various degree to cultured myocardial heart rate and amplitude
[5].(3) diallyl sulfide or the main component of hypoglycemic drug, the function that can improve Regular Insulin or the redoxomorphism improved under the mercaptan compound such as gsh, halfcystine participates in organism.It should be noted that diallyl sulfide only reduces abnormal hyperglycemia, and on blood glucose level normal without impact
[3,6].Liu Hao, Cui Meizhi, Li Chunyan have studied the impact of garlicin on blood glucose in diabetic rats, and the chemistry of garlicin is called diallyl trisulfide, are isolated a kind of compounds from garlic bulbs
[7].Result display garlicin can reduce the blood sugar of diabetes rat, and hypoglycemic effect becomes positive correlation with dosage.Have studied the impact of garlicin on normal rat blood sugar simultaneously, before and after experiment, comparing difference is without significant (P>0.05), with negative control group comparing difference without significant (P>0.05).The blood sugar of known garlicin on normal rat does not affect
[8].(4) with the Bulbus Allii quintessence oil that allyl methyl thioether is main component, in anti-tumor aspect growth capable of inhibiting cell, cell cycle regulation relies on plain kinases, participate in tumorigenic signal transduction, inducing tumor cell differentiation and apoptosis, reduce oncogene, increase expression of tumor suppressor gene, regulate bioenzyme activity
[9].
At present, the preparation method of allyl methyl thioether mainly chemosynthesis or natural extract.Chemosynthesis complex manufacturing, comprises the technological processs such as backflow, distillation, freezing, acidifying, alkalization, press filtration, often has high temperature, high pressure, negative pressure, and uses inflammable and explosive organic reagent in a large number, reaction intermediate or byproduct more; And often can produce large quantity of exhaust gas, waste water, waste residue in chemosynthesis, thus contaminate environment; Thus it applies the restriction of high risk, Product Safety and environmental contamination in being produced.Along with the raising of living standards of the people, to the requirement increasingly stringent of Product Safety, food that is green, pure natural becomes everybody common pursuit.But the compound of natural extract is usually because content low, resource-constrained, complex structure, can not adopt the shortcomings such as chemical process synthesis and be difficult to be developed to product innovation.Biological synthesis process is by enzymatic chemical reaction, there is regioselectivity and the feature such as stereoselectivity is good, catalytic efficiency is high, reaction conditions is gentle, reaction type is many, environmental friendliness, therefore, utilize biological synthesis process to prepare allyl methyl thioether and will become the research direction in the natural manufacture of allyl methyl thioether with great potential.But through the retrieval of document and patent, utilize the correlation technique of this thinking and method both at home and abroad so far there are no document or patent report.
Fragrance bacterium (Myroides) is a class gram negative bacillus, has not yet to see the patent information or the scientific documents report that produce allyl methyl thioether about fragrance bacterium.Strictly aerobic on plain agar nutritional medium, not haemolysis on blood agar
[10], there is certain biological safety.Patent application of the present invention screens the fragrance Pseudomonas bacterial strain CGMCC 7113 that allyl methyl thioether is produced in a strain from earth, and the reported first method of its fermentative production allyl methyl thioether.
Reference:
[1] allyl methyl thioether. China Chemical Industry manufactures net (http://www.chemmade.com/).
[2] Liu Shucheng. Bulbus Allii quintessence oil is to the anti-oxidation characteristics [J] of food oils. Food science, 2001 (6): 128-131.
[3] Sun Junshe, Gao Kongrong. the chemistry of garlic and drug effect and utilization [J]. Foods in Guangzhou science and technology, 1994 (S1): 8-11.
[4] keemun soldier, Song Junxia, Chen Jun. Analysis of antioxidant ingredients [J] in bacillus natto rice bran fermented product. Agriculture of Anhui science, 2012,40 (12): 7414-7416,741.
[5] Zhao Huaiqing, difficult ripple is permanent male. and For-carrying green onion medicinal extract and compound thereof are to the effect [J] of cultured myocardial. Shenyang Pharmaceutical University's journal, 1999,16 (4): 274-277.
[6]Yin MC,Hwang SW,Chan KC.Nonenzymatic antioxidant activity of fourorganosulfur compounds derived from garlic.J Agric Food Chem，2002，50(21)：6143-6147。
[7] Ma Fengying, Wang Wenying. the Pharmacological Advancement [J] of garlicin. herbal medicine, 1997,28 (11): 697-702.
[8] Liu Hao, Cui Meizhi, Li Chunyan. garlicin is on the impact [J] of blood glucose in diabetic rats. Chinese Clinical rehabilitation, 2004,8 (21): 4264-4265.
[9] Xiang Meilin, Su Qi. the antitumor progress of diallyl sulfide [J]. foreign medical science: oncology fascicle, 2004,31 (10): 736-738.
[10]M.VANCANNEYT,P.SEGERS,U.TORCK,et al.Reclassification ofFlavobacterium odoraturn(Stutzer 1929)Strains to a New Genus,Myroides,as Myroidesodoratu scomb.nov.and Myroides odoratimimus sp.nov.IJSEM[J],1996,46(4):926-932。
Summary of the invention
For above-mentioned prior art, for the heavy demand of people to safety and Health natural product, the invention provides the fragrance Pseudomonas bacterial strain that allyl methyl thioether is produced in a strain, and provide it and preparing the application in allyl methyl thioether, and preparation method.
The present invention is achieved by the following technical solutions:
One strain fragrance Pseudomonas bacterial strain, bacterial strain is called WTC05-2, and Classification And Nomenclature is Myroides sp., and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 9th, 2013, deposit number is CGMCC No.7113.
Bacterial strain of the present invention is that applicant (Wutong Aroma Chemicals Co., Ltd.) screens and obtains from gathered soil sample, its biological property meets the fragrance Pseudomonas feature reported: Gram-negative rods, diameter 0.5 μm, length 1 ~ 2 μm (as shown in Figure 1B), atrichia, without coasting ability, non-agglomerated grows, can produce yellow pigment (as shown in Figure 1A), culture has fruit aroma, and during fermentation culture 48h, fragrance is the denseest.
Bacterial strain of the present invention, after measured, the gene order length of its 16S rRNA is 1481bp, as shown in SEQ ID NO.1.By using U.S.'s Biotechnology Information center (National Center for Biotechnology Information, NCBI) BLASTN program comparison, the gene order of fragrance bacterium reference culture (Myroides) the 16S rRNA that the gene order of the 16S rRNA of bacterial strain of the present invention and NCBI register has higher homology (98 ~ 99%), and further phylogenetic tree construction (as shown in Figure 2), the bacterial strain that the present invention screens and Myroides odoratimimus, Myroides profundi sibship is nearer.
In the present invention, the substratum observed for thalli morphology is TSB substratum (Tryptones 17g/L, phytone 3g/L, sodium-chlor 5g/L, potassium primary phosphate 2.5g/L, glucose 2.5g/L, PH7.2).
In the present invention, observation of morphological method is the observation of common fluorescent inverted microscope.
In the present invention, 16SrRNA Phylogenetic Tree construction process: the Clustal W in application MEGA4.0 carries out Multiple Sequence Alignment together to the similar sequences in the sequence recorded and gene pool, with Neighbor-Joining method phylogenetic tree construction, and carry out 1000 Bootstraps inspections.
The separation and purification of bacterial strain of the present invention and the basic skills of 16S rRNA gene sequencing are: the pedotheque for separating of microorganism is gathered by applicant (Wutong Aroma Chemicals Co., Ltd.) and provided.By pedotheque enrichment culture in LB substratum, after 30 DEG C, 200 revs/min cultivation 12h, get different dilution bacterium liquid coating LB agar plate, choose single bacterium colony and carry out line separation, cultivate 24h for 30 DEG C; Choose single bacterium colony in LB liquid, cultivate 12h for 30 DEG C, 200 revs/min, bacterial classification preserved by Ultralow Temperature Freezer, utilizing bacterial genomes to extract test kit (sky root) simultaneously and extract strain gene group DNA, take strain gene group DNA as template, uses bacterial 16 S rDNA universal primer to increase, test kit (sky root) purifying pcr amplification product is reclaimed with glue, electrophoresis is verified, is connected to PMD19-T carrier, is transformed into E.coli DH5 α.Through ammonia benzyl resistance screening, obtain positive colony.16S rDNA order-checking is completed by Shandong Academy of Agricultural Sciences, is compared by the DNA sequence dna that sequence and American National Bioinformatics Institute (NCBI) are included.According to this flow process, obtain a strain and produce the strong bacterial strain of allyl methyl thioether ability, be numbered WTC05-2, called after Myroides sp., preservation has been carried out on January 9th, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.7113.
The above-mentioned LB substratum for strains separation purifying consists of: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, pH7.2.
The above-mentioned universal primer for bacterial strain 16S rDNA amplification is:
Forward primer is 27f:5 ’ – AGAGTTTGATCCTG GCT CAG-3 ', as shown in SEQ ID NO.2;
Reverse primer is 1492r:5 '-GGTTACCTTGTTACGACTT-3 ', as shown in SEQ ID NO.3.
The above-mentioned system for bacterial strain 16S rDNA amplification is: add each 1.0 μ L of 27f and 1492r that concentration is 10mmol/L in every 30 μ L reaction systems, the dNTP 2.4 μ L of 2mmol/L, 2 × GC Buffer I 15 μ L, the LA-Taq archaeal dna polymerase 0.2 μ L of 5U/ μ L, template 3uL, 18.2M Ω .cm ultrapure water 8.0 μ L.Gene amplification reagent used is purchased from the precious Bioisystech Co., Ltd in Dalian.
The above-mentioned condition for bacterial strain 16S rDNA amplification is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30S, 55 DEG C of annealing 30S, 72 DEG C extend 90S, totally 35 circulations; 72 DEG C extend 15min; Be cooled to 4 DEG C and be incubated 15min.
The fermentation condition optimization method of bacterial strain of the present invention is: after being activated on LB slant medium by strains tested, be inoculated in the 300ml triangular flask that 50ml LB substratum is housed, 30 DEG C, 200 revs/min, 12h cultivated by shaking table, is then inoculated in LB, TB, SOB, TSB substratum according to 1.5% inoculum size respectively, 30 DEG C, 200 revs/min of shaking tables are cultivated, and respectively when 3h, 6h, 9h, 12h, 18h, 24h, 30h, 42h, measure cell concentration (OD in 600nm wavelength
600).
The above-mentioned composition of the substratum for strain fermentation is respectively:
TB: peptone 12g/L, yeast leaching powder 24g/L, glycerine 4ml/L, KH
2pO
42.31g/L, K2
hpO
412.54g/L, pH7.2.
2 × YT: peptone 16g/L, yeast leaching powder 10g/L, NaCl5g/L, pH7.0.
SOB: peptone 20g/L, yeast leaching powder 5g/L, NaCl0.5g/L, KCl0.186g/L, MgCl
20.95g/L, pH7.0.
TSB: Tryptones 17g/L, phytone 3g/L, sodium-chlor 5g/L, potassium primary phosphate 2.5g/L, glucose 2.5g/L, pH7.2.
The above-mentioned basis for estimation for strain fermentation condition optimizing is: (1) directly judges by sense of smell the change that in strain fermentation process, fragrance is deep or light; (2) output is estimated by the purifying of product, the semi-quantitative analysis of gas chromatography mass spectrometry.
Bacterial strain of the present invention, produces allyl methyl thioether ability by force, may be used for manufacture allyl methyl thioether.During application, concrete mode is as follows:
(1) bacterial classification is selected: select bacterial strain Myroides sp.WTC05-2 of the present invention, its culture presevation is numbered CGMCCNo.7113;
(2) actication of culture: above-mentioned bacterial classification is lined solid medium, 30 DEG C of quiescent culture 18 hours, for subsequent use;
(3) seed culture: the bacterial strain transfering loop picking list bacterium colony that above-mentioned steps (2) is cultivated, be inoculated in and be equipped with in the 300mL triangular flask of 50mL liquid seed culture medium, put concussion in shaking table and cultivate, its rotating speed is 200 revs/min, cultivate 12 hours, obtain seed liquor for 30 DEG C;
(4) fermentation culture: be the inoculum size of 1.0% with volume ratio, continues to be inoculated in by step (3) gained seed liquor and is equipped with in the 500mL shaking flask of 100mL fermention medium, 30 DEG C, rotating speed be the condition of 200 revs/min under shake-flask culture 42h, obtain fermented liquid;
(5) product enrichment and purifying: add D101 macroporous adsorbent resin in above-mentioned fermented liquid, extracts 24h, filters, then carry out wash-out: first use water wash resin, then use organic reagent drip washing resin, collect organic reagent elutriant, steam except organic solvent, obtain allyl methyl thioether.
Further, can also comprise the following steps (6): the detection of above-mentioned organic reagent elutriant: adopt gas chromatography mass spectrometry (GC-MS) whether to identify in organic reagent elutriant containing allyl methyl thioether and concentration thereof.
In above-mentioned application, solid medium described in step (2) (3) (4), liquid seed culture medium, fermention medium are pancreas peptone soybean broth substratum, its formula consists of: Tryptones 17g/L, phytone 3g/L, sodium-chlor 5g/L, potassium primary phosphate 2.5g/L, glucose 2.5g/L, pH7.2.Difference is only: also add the agar that concentration is 2% in solid medium.
In above-mentioned application, step (5) is specially: in above-mentioned fermented liquid, add D101 macroporous adsorbent resin according to 20g/L addition, extract 24h, filter with 100 object silks, resin is added in glass column (internal diameter 20mm), resin loading height 10mm, according to 2ml/min flow velocity, first use 4 volumes of deionized water drip washing resins, use the organic reagent drip washing resin of 3 volumes again, collect the organic reagent elutriant (first of the 3rd column volume, because impurity is more in the organic reagent elutriant of two column volumes, be unfavorable for the separation and purification of target product, therefore impurity is only collected few, the organic reagent elutriant of the 3rd column volume that purity is high) be elutriant containing allyl methyl thioether, steam except organic solvent, obtain allyl methyl thioether.
In above-mentioned application, the D101 macroporous adsorbent resin in step (5), needs before using to anticipate, processing mode is: with the saturated aqueous common salt of 2 times of resin volumes, soak 18 ~ 20h, then drain salt solution, clean with clear water rinsing, make the water not displaing yellow of discharge, be the sodium hydroxide solution (its consumption is 2 times of resin volume) of 2% ~ 4% again with mass percent, soak 2 ~ 4h, after draining alkali lye, rinse resin until water is neutral, stand-by.
In above-mentioned application, the organic reagent in step (5) is ether.
In above-mentioned application, the GC-MS analysis condition described in step (6) is:
(1) GC conditions: RTX-1 chromatographic column, column temperature 80 DEG C, carrier gas He, carrier gas flux 3.0ml/min;
(2) mass spectrometric detection condition is: detector temperature 250 DEG C, starts to detect after 4.5min, quality of scanning scope 45 ~ 700.
Confirm through experiment: utilize strain fermentation of the present invention, 500ml triangular flask carries out shake flask fermentation, and through 42 hours, the output of allyl methyl thioether can reach 70mg/L.
Bacterial strain of the present invention; produce allyl methyl thioether ability strong; adopt Production by Microorganism Fermentation allyl methyl thioether; than chemical production method have reaction temperature and, simple, the low power consumption and other advantages of raw material green natural, technique; and be conducive to environment protection; be easy to promote the use of, produce allyl methyl thioether for utilizing fermentable and established technical foundation.
Accompanying drawing explanation
Fragrance Pseudomonas bacterial strain of the present invention, bacterial strain is called WTC05-2, Classification And Nomenclature is Myroides sp., be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 9th, 2013, deposit number is CGMCC No.7113, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode: 100101.
Fig. 1: the grown form feature of bacterial strain of the present invention, wherein, A illustrate bacterial strain of the present invention on TSB substratum, grow 24h after form; B illustrates the cellular form of bacterial strain of the present invention.
Fig. 2: the evolutionary tree that bacterial strain of the present invention is drawn based on 16S rRNA gene order.
Fig. 3: the fermentation condition optimization figure of bacterial strain of the present invention, depict the growth curve of WTC05-2 respectively in LB, TB, 2 × YT, SOB, TSB substratum in figure, and judged by sense of smell, choose the substratum of applicable fermentative production allyl methyl thioether.
Fig. 4: bacterial strain of the present invention ferments after 42h in pancreas peptone soybean broth, and the gas chromatogram of extracted with diethyl ether fermented liquid, shows in figure, the appearance time of allyl methyl thioether is 9.858min.
Fig. 5: bacterial strain of the present invention produce the mass spectrum of allyl methyl thioether.
Fig. 6: allyl methyl thioether canonical plotting.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
The screening of embodiment 1 bacterial strain obtains and 16S rRNA gene sequencing
Pedotheque for separating of microorganism is gathered by applicant (Wutong Aroma Chemicals Co., Ltd.) and is provided.By pedotheque enrichment culture in LB substratum, after 30 DEG C, 200 revs/min cultivation 12h, get different dilution bacterium liquid coating LB agar plate, choose single bacterium colony and carry out line separation, cultivate 24h for 30 DEG C; Choose single bacterium colony in LB liquid, cultivate 12h for 30 DEG C, 200 revs/min, bacterial classification preserved by Ultralow Temperature Freezer, utilizing bacterial genomes to extract test kit (sky root) simultaneously and extract strain gene group DNA, take strain gene group DNA as template, uses bacterial 16 S rDNA universal primer to increase, test kit (sky root) purifying pcr amplification product is reclaimed with glue, electrophoresis is verified, is connected to PMD19-T carrier, is transformed into E.coli DH5 α.Through ammonia benzyl resistance screening, obtain positive colony.16S rDNA order-checking is completed by Shandong Academy of Agricultural Sciences, is compared by the DNA sequence dna that sequence and American National Bioinformatics Institute (NCBI) are included.According to this flow process, obtain a strain and produce the strong bacterial strain of allyl methyl thioether ability, be numbered WTC05-2, called after Myroides sp., preservation has been carried out on January 9th, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.7113.
The above-mentioned LB substratum for strains separation purifying consists of: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, pH7.2.
The above-mentioned universal primer for bacterial strain 16SrDNA amplification is:
Forward primer is 27f:5 ’ – AGAGTTTGATCCTG GCT CAG-3 ';
Reverse primer is 1492r:5 '-GGTTACCTTGTTACGACTT-3 '.
The above-mentioned system for bacterial strain 16S rDNA amplification is: add each 1.0 μ L of 27f and 1492r that concentration is 10mmol/L in every 30 μ L reaction systems, the dNTP 2.4 μ L of 2mmol/L, 2 × GC Buffer I 15 μ L, the LA-Taq archaeal dna polymerase 0.2 μ L of 5U/ μ L, template 3uL, 18.2M Ω .cm ultrapure water 8.0 μ L.Gene amplification reagent used is purchased from the precious Bioisystech Co., Ltd in Dalian.
The above-mentioned condition for bacterial strain 16S rDNA amplification is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30S, 55 DEG C of annealing 30S, 72 DEG C extend 90S, totally 35 circulations; 72 DEG C extend 15min; Be cooled to 4 DEG C and be incubated 15min.
The optimization of embodiment 2 strain fermentation condition
After strains tested (WTC05-2) is activated on LB slant medium, be inoculated in the 300ml triangular flask that 50ml LB substratum is housed, 30 DEG C, 200 revs/min, 12h cultivated by shaking table, is then inoculated in LB, TB, SOB, TSB substratum according to 1.5% inoculum size respectively, 30 DEG C, 200 revs/min of shaking tables are cultivated, and respectively when 3h, 6h, 9h, 12h, 18h, 24h, 30h, 42h, measure cell concentration (OD in 600nm wavelength
600), as shown in Figure 3, as seen in Figure 3, WTC05-2 is other four kinds of substratum poor growths relative in TSB substratum, better can accumulate secondary metabolite for result, therefore select TSB substratum as follow-up fermention medium.
The above-mentioned composition of the substratum for strain fermentation is respectively:
TB: peptone 12g/L, yeast leaching powder 24g/L, glycerine 4ml/L, KH
2pO
42.31g/L, K
2hPO
412.54g/L, pH7.2.
2 × YT: peptone 16g/L, yeast leaching powder 10g/L, NaCl 5g/L, pH7.0.
SOB: peptone 20g/L, yeast leaching powder 5g/L, NaCl 0.5g/L, KCl 0.186g/L, MgCl
20.95g/L, pH7.0.
TSB: Tryptones 17g/L, phytone 3g/L, sodium-chlor 5g/L, potassium primary phosphate 2.5g/L, glucose 2.5g/L, pH7.2.
The above-mentioned basis for estimation for strain fermentation condition optimizing is: (1) directly judges by sense of smell the change that in strain fermentation process, fragrance is deep or light; (2) output is estimated by the purifying of product, the semi-quantitative analysis of gas chromatography mass spectrometry.
The application of embodiment 3 bacterial strain in manufacture allyl methyl thioether
Concrete mode is as follows:
(1) bacterial classification is selected: select bacterial strain Myroides sp.WTC05-2 of the present invention, its culture presevation is numbered CGMCCNo.7113;
(2) actication of culture: above-mentioned bacterial classification is lined solid medium, 30 DEG C of quiescent culture 18 hours, for subsequent use;
(3) seed culture: the bacterial strain transfering loop picking list bacterium colony that above-mentioned steps (2) is cultivated, be inoculated in and be equipped with in the 300mL triangular flask of 50mL liquid seed culture medium, put concussion in shaking table and cultivate, its rotating speed is 200 revs/min, cultivate 12 hours, obtain seed liquor for 30 DEG C;
(4) fermentation culture: be the inoculum size of 1.0% with volume ratio, continues to be inoculated in by step (3) gained seed liquor and is equipped with in the 500mL shaking flask of 100mL fermention medium, 30 DEG C, rotating speed be the condition of 200 revs/min under shake-flask culture 42h, obtain fermented liquid;
(5) product enrichment and purifying: add the D101 macroporous adsorbent resin of anticipating according to 20g/L addition in fermented liquid, extract 24h, filter with 100 object silks, resin is added in glass column (internal diameter 20mm), resin loading height 10mm, according to 2ml/min flow velocity, first use 4 volumes of deionized water drip washing resins, use the organic reagent drip washing resin of 3 volumes again, collect the organic reagent elutriant of the 3rd column volume, be the elutriant containing allyl methyl thioether, steam except organic solvent, obtain allyl methyl thioether;
In above-mentioned application, the D101 macroporous adsorbent resin in step (5), needs before using to anticipate, processing mode is: with the saturated aqueous common salt of 2 times of resin volumes, soak 20h, then drain salt solution, clean with clear water rinsing, make the water not displaing yellow of discharge, be the sodium hydroxide solution (its consumption is 2 times of resin volume) of 3% again with mass percent, soak 3h, after draining alkali lye, rinse resin until water is neutral, stand-by.
(6) detection of eluted product crude extract: adopt gas chromatography mass spectrometry (GC-MS) to identify whether containing allyl methyl thioether in above-mentioned organic reagent elutriant, and its concentration.
In above-mentioned application, solid medium described in step (2) (3) (4), liquid seed culture medium, fermention medium are pancreas peptone soybean broth substratum, its formula consists of: Tryptones 17g/L, phytone 3g/L, sodium-chlor 5g/L, potassium primary phosphate 2.5g/L, glucose 2.5g/L, pH7.2.Difference is only: also add the agar that concentration is 2% in solid medium.
In above-mentioned application, the organic reagent in step (5) is ether.
In above-mentioned application, the GC-MS analysis condition described in step (6) and result thereof are:
(1) GC conditions: RTX-1 chromatographic column, column temperature 80 DEG C, carrier gas He, carrier gas flux 3.0ml/min;
(2) mass spectrometric detection condition is: detector temperature 250 DEG C, starts to detect after 4.5min, quality of scanning scope 45 ~ 700;
(3) in the gas chromatogram obtained, allyl methyl thioether appearance time is 9.858min, as shown in Figure 4, can be found out by this collection of illustrative plates, and allyl methyl thioether content in extract is maximum, and peak area is 1429067.
(4) mass spectral results of allyl methyl thioether as shown in Figure 5, can be found out by this collection of illustrative plates, and the molecular weight of this material is 88, meets the molecular size range of allyl methyl thioether.In addition, compare with the standard mass spectrum of allyl methyl thioether in database, similarity is greater than 99%, therefore judges that this material is allyl methyl thioether.
(5) by drawing allyl methyl thioether typical curve (Fig. 6), the concentration that the calculated by peak area according to allyl methyl thioether obtains allyl methyl thioether in fermented liquid is 70mg/L.
Claims (9)
1. a strain fragrance Pseudomonas bacterial strain (
myroides sp.) WTC05-2, its deposit number is CGMCC No.7113.
2. fragrance Pseudomonas bacterial strain according to claim 1 is preparing the application in allyl methyl thioether.
3. application according to claim 2, is characterized in that: during application, comprises the following steps:
(1) bacterial classification is selected: the bacterial strain selecting claim 1;
(2) actication of culture: above-mentioned bacterial classification is lined solid medium, 30 DEG C of quiescent culture 18 hours, for subsequent use;
(3) seed culture: the bacterial strain transfering loop picking list bacterium colony that above-mentioned steps (2) is cultivated, be inoculated in and be equipped with in the 300mL triangular flask of 50mL liquid seed culture medium, put concussion in shaking table and cultivate, its rotating speed is 200 revs/min, cultivate 12 hours, obtain seed liquor for 30 DEG C;
(4) fermentation culture: be the inoculum size of 1.0% with volume ratio, step (3) gained seed liquor being continued to be inoculated in is equipped with in the 500mL shaking flask of 100mL fermention medium, 30 DEG C, rotating speed be the condition of 200 revs/min under shake-flask culture 42h, obtain fermented liquid;
(5) product enrichment and purifying: add D101 macroporous adsorbent resin in above-mentioned fermented liquid, extracts 24h, filters, then carry out wash-out: first use water wash resin, then use organic reagent drip washing resin, collect organic reagent elutriant, steam except organic solvent, obtain allyl methyl thioether.
4. application according to claim 3, it is characterized in that: further comprising the steps of (6): the detection of above-mentioned organic reagent elutriant: adopt gas chromatography mass spectrometry (GC-MS) to identify whether containing allyl methyl thioether in organic reagent elutriant, and its concentration.
5. application according to claim 3, it is characterized in that: the solid medium in described step (2) (3) (4), liquid seed culture medium, fermention medium are pancreas peptone soybean broth substratum, its formula consists of: Tryptones 17g/L, phytone 3g/L, sodium-chlor 5g/L, potassium primary phosphate 2.5g/L, glucose 2.5g/L, pH7.2; The agar that concentration is 2% is also added in solid medium.
6. application according to claim 3, is characterized in that: the organic reagent in described step (5) is ether.
7. application according to claim 3, it is characterized in that: described step (5) is specially: in above-mentioned fermented liquid, add D101 macroporous adsorbent resin according to 20g/L addition, extract 24h, filter with 100 object silks, resin is added in glass column, resin loading height 10mm, according to 2ml/min flow velocity, first use 4 volumes of deionized water drip washing resins, use the organic reagent drip washing resin of 3 volumes again, collect the organic reagent elutriant of the 3rd column volume, be the elutriant containing allyl methyl thioether, steam except organic solvent, obtain allyl methyl thioether.
8. the application according to claim 3 or 7, it is characterized in that: the D101 macroporous adsorbent resin in described step (5), need before using to anticipate, processing mode is: with saturated common salt water soaking 18 ~ 20h, then drain salt solution, clean with water rinse, make the water not displaing yellow of discharge, soak 2 ~ 4h with the sodium hydroxide solution that mass percent is 2% ~ 4% again, after draining alkali lye, rinse resin until water is neutral.
9. application according to claim 4, is characterized in that: in described step (6), GC-MS analysis condition is:
(1) GC conditions: RTX-1 chromatographic column, column temperature 80 DEG C, carrier gas He, carrier gas flux 3.0ml/min;
(2) mass spectrometric detection condition is: detector temperature 250 DEG C, starts to detect after 4.5min, quality of scanning scope 45 ~ 700.
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| Reclassification of Flavobacterium odoraturn(Stutzer 1929)Strains to a New Genus,Myroides,as Myroides odoratu scomb.nov.and Myroides odoratimimus sp.nov.;M.VANCANNEYT et al.;《IJSEM》;19961231;926-932 * |
| 茖葱浸膏及其化合物对培养心肌细胞的作用;赵怀清等;《沈阳药科大学学报》;19991031;274-277 * |
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