CN103710291A - Bacillus megatherium Z2013513 and method for producing phenyl lactic acid - Google Patents
Bacillus megatherium Z2013513 and method for producing phenyl lactic acid Download PDFInfo
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- NWCHELUCVWSRRS-SECBINFHSA-N (2r)-2-hydroxy-2-phenylpropanoic acid Chemical compound OC(=O)[C@@](O)(C)C1=CC=CC=C1 NWCHELUCVWSRRS-SECBINFHSA-N 0.000 title claims abstract description 51
- 241000194107 Bacillus megaterium Species 0.000 title claims abstract description 33
- 238000004519 manufacturing process Methods 0.000 title description 9
- BTNMPGBKDVTSJY-UHFFFAOYSA-N keto-phenylpyruvic acid Chemical compound OC(=O)C(=O)CC1=CC=CC=C1 BTNMPGBKDVTSJY-UHFFFAOYSA-N 0.000 claims abstract description 43
- 238000000034 method Methods 0.000 claims abstract description 38
- 239000001903 2-oxo-3-phenylpropanoic acid Substances 0.000 claims abstract description 20
- DEDGUGJNLNLJSR-UHFFFAOYSA-N alpha-hydroxycinnamic acid Natural products OC(=O)C(O)=CC1=CC=CC=C1 DEDGUGJNLNLJSR-UHFFFAOYSA-N 0.000 claims abstract description 20
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- 230000009466 transformation Effects 0.000 claims abstract description 8
- 238000006243 chemical reaction Methods 0.000 claims description 24
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 20
- 239000006228 supernatant Substances 0.000 claims description 17
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 16
- 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 15
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
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- 241000193830 Bacillus <bacterium> Species 0.000 claims description 8
- 239000012043 crude product Substances 0.000 claims description 8
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 8
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- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 7
- MQGYVGKMCRDEAF-UHFFFAOYSA-M sodium;2-oxo-3-phenylpropanoate Chemical compound [Na+].[O-]C(=O)C(=O)CC1=CC=CC=C1 MQGYVGKMCRDEAF-UHFFFAOYSA-M 0.000 claims description 7
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- VOXXWSYKYCBWHO-MRVPVSSYSA-N (R)-3-phenyllactic acid Chemical compound OC(=O)[C@H](O)CC1=CC=CC=C1 VOXXWSYKYCBWHO-MRVPVSSYSA-N 0.000 description 5
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Abstract
The invention relates to bacillus megatherium Z2013513(BacillusmegateriumZ2013513) with the preservation number of CCTCCNO: M2013244. The invention also relates to a method for utilizing the bacillus megatherium Z2013513 and adopting phenyl pyruvic acid as a primer to carry out biotransformation to synthesize phenyl lactic acid. The bacillus megatherium Z2013513 is from traditional fermented foods, belongs to general safety strains and can be applied in foods; the strain grows well on beef extract peptone or an LB culture medium, the enlarged culture for collecting thallus is easy, the content of the phenyl pyruvic acid obtained by transformation of growth cells and the content of the phenyl pyruvic acid obtained by transformation of resting cells can reach 5.4g/L and 16.1g/L respectively, and the capability of inhibiting the putrefying bacteria in the foods is stronger.
Description
Technical field
The present invention relates to technical field of microbe application, relate to a strain bacillus megaterium Z2013513(
bacillus megateriumz2013513) and the method for producing phenyl-lactic acid.
Background technology
β-phenyl-lactic acid, claims again PLA or PLA (Phenyllactic acid is called for short PLA), relative molecular mass is 166,121～125 ℃ of fusing points, odorlessness, to acid, thermally-stabilised, 121 ℃ of heating, be not destroyed yet, be present in natural honey.This organic acid is the meta-bolites being produced by multiple-microorganism (especially milk-acid bacteria) metabolism phenylalanine, is one of main active substances of giving the antimycotic and long shelf-life of lactobacillus-fermented product.PLA has been subject to extensive concern as the antibacterial substance of a kind of novel, safety, wide spectrum.This aromatic organic acid being produced by generally recognized as safe milk-acid bacteria metabolism shows that it is to the equal nontoxicity of humans and animals cell after deliberation.In addition, compare with most natural antiseptic agents, PLA has wider antimicrobial spectrum, particularly can pollute by Antifungi, and also its water-soluble and Heat stability is good, action pH wide ranges, these advantages are beneficial to it in the widespread use of foodstuffs industry.Numerous research has confirmed that PLA has wide spectrum and efficient antibacterial activity, and can be used for cattle breeding by substitute antibiotics.Such as, existing data shows that PLA can effectively suppress to comprise food-borne pathogenic bacterium, as
listeria monocytogenes,
enterococcus faecalis,
staphylococcus aureus,
escherichia coli,
providencia stuartii,
klebsiella oxytoca, yeast and mould, as
aspergillus ochraceus,
penicillium roqueforti,
penicillium citrinugrowth Deng multiple-microorganism.
The method of producing PLA in prior art mainly contains chemical method and biological synthesis process.The problem that chemical process exists is that product optical purity is not high enough, and technical sophistication, contaminate environment is serious, product cost is high and quality product is difficult to control etc.Due to
β-phenyl-lactic acid may be mainly used in food and medicine field future, human consumer, advocate today natural, nutritive food, and the investigator of countries in the world has transferred to biological synthesis process aspect the emphasis of research; Biological synthesis process is realized by the stereoselectivity biocatalysis of complete microorganism cells.Therefore the good microorganism strains that screens highly-solid selectively is the key of the method.As far back as 1986, Kamata etc. just used brevibacterium lactofermentum
brevibacterium Lactofermentumfermentative production R-3-phenyl-lactic acid, output is 1.94 g/L, and has applied for patent; 1998, Die μ Leveux etc. were from geotrichum candidum
geotrichum Candidumnutrient solution in be also separated to R-3-phenyl-lactic acid, this bacterial strain amount that produces R-3-phenyl-lactic acid of fermenting in TSBYE substratum is 0.6 g/L～1 g/L; 2000, the plant lactobacillus that the discoveries such as Lavermicocca are separated to from bread dough
lactobacillus plantarum21B can secrete R-3-phenyl-lactic acid, and they are seeded in plant lactobacillus in wheat-flour hydrolyzed solution, 30 ℃ of cultivation and fermentation 24 h, and the R-3-phenyl-lactic acid amount of generation is 0.009 mol/L.2002, Katrin etc. were also separated to a strain from ensiling grass
l.plantarummiLAB 393, and the S type that it produces and the ratio of R type PLA are 9:1; 1996, Hashimoto etc. screened a pseudomonas from soil
pseudomonassp. BC218, can be converted into phenylacetic aldehyde-cyanalcohol S-3-phenyl-lactic acid.This conversion has higher enantioselectivity, and the e.e. value of the S-3-phenyl-lactic acid of generation is 75%, then passes through preferential crystallization, and e.e. value can reach 99%.This bacterial strain is after mutagenesis, and the output of S-3-phenyl-lactic acid is 12 times of starting strain; It is 99% S-3-phenyl-lactic acid that nitrile enzyme for Diversa company (EC 3.5.5.1) (Nitrilases) can be prepared e.e. value; Recently, the report such as Thierry Fei Shi bacillus (
propionibacteriun freudenreichii)in cheese fermenting process, produced PLA, these PLAs are obtained under hydroxy reductase effect by phenylpyruvic acid.At present
β-phenyl-lactic acid Yi Diversa company has carried out suitability for industrialized production.
Yet these utilize different microorganisms complicated with the process of simple carbon source de novo synthesis PLA, and output is lower, and the related microorganism of most report or desired nutritional require high, or poor growth, and be not suitable for large-scale production application.Phenyl-pyruvic acid, as a kind of comparatively cheap large chemical feedstocks, be take it as substrate, utilizes enzyme catalysis specificity and the high efficiency of microorganism, and one-step synthesis PLA is just becoming current study hotspot.The present invention filters out the safe bacterial strain with the synthetic PLA of efficient catalytic phenyl-pyruvic acid from food, develop on this basis the biosynthesis technology of the synthetic PLA of microorganism Efficient Conversion phenyl-pyruvic acid, effectively solve high, the poky problems of production bacterial strain nutritional requirement such as milk-acid bacteria, promoted significantly output and the production intensity of product.
Summary of the invention
The first object of the present invention is to provide the microorganism strains of a strain source safety, simple, the high synthesis of phenyl lactic acid of cultural method.
Second object of the present invention is to provide a kind of method of utilizing above-mentioned bacterial strains to produce phenyl-lactic acid.
In order to realize the first object of the present invention, the technical solution used in the present invention is:
One, a strain bacillus megaterium Z2013513(
bacillus megateriumz2013513), it was preserved in Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC NO:M 2013244 on June 4th, 2013.
Morphology and the physiological and biochemical property of bacterial strain of the present invention are as follows:
Colony colour: beige;
Aerobic mode: aerobic growth;
Bacterium colony size: 1.2～2.5 * 2.0～10.0 μ m;
Optimum growth temperature: 35-40 ℃;
The optimum initial pH:7 that grows;
Thalli morphology: shaft-like, end circle, single or be short chain and arrange, can move;
Gramstaining: the positive;
Gemma: 1.0～1.2 * 1.5～2.0 microns, ellipse, middle life.
The acquisition methods of above-mentioned bacillus megaterium Z2013513, through the seed selection of starting strain, LB substratum shaking table is cultivated and phenylpyruvic acid is substrate conversion, then through streptococcus aureus and penicillium citrinum plate screening, obtains respectively simultaneously
Specifically, the screening purification process of described bacillus megaterium Z2013513 comprises the steps:
1, the seed selection of starting strain-
Take commercially available sausage or soy cheese as bacterium source, get appropriate sample and after oscillation treatment 2h, get bacteria suspension and process 10min in 80 ℃ in stroke-physiological saline solution.The bacteria suspension 1mL getting after processing is inoculated in 10mL beef-protein medium in 30-40 ℃ of enrichment culture 12h.Get 0.1mL enrichment culture liquid and be applied in respectively the beef-protein medium containing 2% agar, cultivate 2-3 days, obtain single bacterium colony for 30 ℃.The further line separation on beef-protein medium of several single bacterium colonies of random choose, obtains starting strain 4 ℃ of lower inclined planes and saves backup;
2, thalline activation and conversion fluid preparation
The bacterial strain of institute's seed selection is seeded to respectively to LB substratum, and 37 ℃, 200 revs/min of activation culture 10 hours, then get culture according to 10%(v/v) inoculum size access SYG liquid nutrient medium, 35-40 ℃,, continues to cultivate 8 hours by 200 revs/min.Culture is through 4 ℃, and 4000 revs/min of abandoning supernatant after centrifugal 5 minutes, add appropriate conversion substratum suspension thalline, are placed in 30-40 ℃, and 200 revs/min of shaking tables transform 4-8 hour.Conversion fluid is got supernatant liquor and is carried out bacteriostatic experiment after 10000 revs/min of centrifugal 10 min.
3, streptococcus aureus plate screening
Get one of the slant tube that covers with streptococcus aureus (As1.89), add sterile distilled water 10mL, make bacteria suspension, get 0.1mL and be inoculated in beef-protein medium surface evenly coating, Oxford cup at the equidistant placement high-temperature sterilization of dull and stereotyped different positions, getting above-mentioned steps 2) gained supernatant liquor 200 μ L add in the cup of Oxford, and cultivate 22 hours, choose the bacterial strain that produces inhibition zone in the surrounding of Oxford cup for 37 ℃;
4, penicillium citrinum plate screening
In sterile petri dish, pour PDA solid medium into, penicillium citrinum is made to spore suspension, getting 0.1mL is inoculated on PDA plate culture medium and evenly coating, Oxford cup at the equidistant placement high-temperature sterilization of dull and stereotyped different positions, getting above-mentioned steps 2) gained supernatant liquor 200 μ L add Oxford cup, cultivate 60 hours, choose the bacterial strain that produces inhibition zone in the surrounding of Oxford cup for 28 ℃;
Select and can suppress the corresponding bacterial strain of fermented liquid that above-mentioned streptococcus aureus and Penicillium notatum and inhibition zone are larger, called after bacillus megaterium Z2013513(simultaneously
bacillus megateriumz2013513).
Two, utilize bacillus megaterium Z2013513 to produce a method for phenyl-lactic acid, the method be take phenylpyruvic acid as substrate, utilizes grown cell or the resting cell bio-transformation synthesis of phenyl lactic acid of bacterial strain of the present invention.
Specifically comprise the steps:
1) seed culture: the mono-colony inoculation of bacillus megaterium Z2013513 is activated to LB substratum to 35-40 ℃, 200rpm activation culture 8 hours;
2) enlarged culturing: by above-mentioned steps 1) gained seed culture fluid is by 10% inoculum size access SYD substratum, 35-40 ℃, shaking table 200 turns per minute, cultivate 8 hours to Growth of Cells mid-log phase, by gained nutrient solution at 4 ℃, the centrifugal 5min of 4000rpm, and collect resting cell thalline 2 times with stroke-physiological saline solution washing;
3) conversion production phenyl-lactic acid: by step 2) gained thalline forwards 40 ℃ of conversion 4-18 hour in conversion fluid to.
In described step 3), grown cell transform to be produced the preferred version of phenyl-lactic acid and is: enlarged culturing is after 4 hours, respectively after in 6 hours, the fed-batch medium that adds 0.375mL per hour, transforms 8 hours.
In described step 3), conversion of resting cells is produced the preferred version of phenyl-lactic acid and is: by step 2) thalline be suspended in 10 mL and transform in substratum and in 30 ℃, in 200rpm shaking table, transform.In first 14 hours, within every 2 hours, add 0.5mL fed-batch medium, add altogether approximately 320 mg phenyl-pyruvic acids and 350 mg glucose, 40 ℃ transform 18 hours.
In described step 3), to produce the preferred method for transformation of phenyl-lactic acid be that batch fermentation transforms to conversion of resting cells: by step 2) thalline is suspended in 10 mL and transforms in substratum and in 40 ℃, in 200rpm shaking table, transform some hours, every 2h sampling, the centrifugal 10min of 10000rpm, getting supernatant liquor adds the ethyl acetate of 2 times of volumes to extract, reduction vaporization is concentrated, obtain the crude product of phenyl-lactic acid, then with same volume 0.05% trifluoroacetic acid aqueous solution, dissolve, through suitably adopting high performance liquid chromatography (HPLC) method to measure the content of phenyl-pyruvic acid and phenyl-lactic acid after dilution, through 4h, transform, transform in substratum almost approach exhaustion of phenyl-pyruvic acid.
Described LB substratum comprises: yeast extract 5g/L, Tryptones 10g/L, NaCl 10g/L.
Described SYD substratum comprises: glucose 20g/L, Tryptones 10g/L, yeast extract 5g/L, NaCl 5g/L.
Described conversion substratum is: glucose 20g/L, Sodium.beta.-phenylpyruvate 4g/L, the phosphoric acid buffer of pH7.
Described fed-batch medium is: glucose 100g/L, Sodium.beta.-phenylpyruvate 78g/L, the phosphoric acid buffer of pH7.
Further the purification process of phenyl-lactic acid is:
1) by the centrifugal 10min of conversion fluid 10000rpm of gained, reject precipitation, it is 2.0 that supernatant liquor is adjusted PH;
2) get above-mentioned steps 1) gained supernatant liquor adds the ethyl acetate extraction of 2 times of volumes, after get organic phase reduction vaporization to dry, obtain the crude product of phenyl-lactic acid;
3) 0.05% of use same volume the trifluoroacetic acid aqueous solution dissolving step 2) crude product of the phenyl-lactic acid of gained, through suitably adopting high performance liquid chromatography (HPLC) method to measure the content of phenyl-lactic acid after dilution.
Wherein, HPLC condition determination is: chromatographic column is Shimadzu VP-ODSC18, moving phase is the mixed solution of the trifluoroacetic acid/water (B) of trifluoroacetic acid/methyl alcohol (A) of 0.05% and 0.05%, flow velocity 1mL/min, detect wavelength 210nm, 30 ℃ of column temperatures, sample size 10 μ L, use 0.22mm membrane filtration before sample introduction.
Beneficial effect of the present invention is:
Bacillus megaterium Z2013513 of the present invention belongs to and it is generally acknowledged safe bacterial classification, has higher security; This bacterial strain is well-grown on LB and SYG substratum, nutritional requirement is low, growth cycle is short, easily cultivate and preserve, by grown cell, transform and can obtain phenyl-lactic acid 5.4g/L, phenyl-lactic acid content by conversion of resting cells gained can be up to 16.1 g/L, and the phenyl-lactic acid transforming is easy to purify, and the ability that suppresses spoilage organism in food is stronger.
Accompanying drawing explanation
Fig. 1 is the Photomicrograph (1000 *) of bacillus megaterium Z2013513.
Fig. 2 be bacillus megaterium Z2013513 resting cell respectively before transforming phenyl-pyruvic acid, transform 2 hours, the HPLC collection of illustrative plates of 4 hours phenyl-lactic acids that produce.
Fig. 3 is that bacillus megaterium Z2013513 grown cell current adding substrate transforms phenyl-pyruvic acid generation phenyl-lactic acid figure.
Fig. 4 is that bacillus megaterium Z2013513 resting cell current adding substrate transforms phenyl-pyruvic acid generation phenyl-lactic acid figure.
Biomaterial of the present invention, its Classification And Nomenclature is bacillus megaterium Z2013513(
bacillus megateriumz2013513), on June 4th, 2013 be preserved in Chinese Typical Representative culture collection center (be called for short CCTCC, address is: China. Wuhan. Wuhan University), deposit number: CCTCC NO:M 2013244.
embodiment:
enumerate embodiment below the present invention is further described, but therefore do not limit content of the present invention
.
Substratum of the present invention:
Beef-protein medium (g/L): extractum carnis 3g, peptone 10g, NaCl 5g.
Slant preservation substratum (g/L): yeast extract 5g, Tryptones 10 g, NaCl 10 g, agar 20 g.
LB substratum (g/L): yeast extract 5g, Tryptones 10g, NaCl 10g.
Transform substratum (g/L): glucose 20g, Sodium.beta.-phenylpyruvate 4g, 0.2mol/L pH7 phosphoric acid buffer.
Fed-batch medium (g/L): glucose 100g, Sodium.beta.-phenylpyruvate 78g, 0.2mol/L pH7 phosphoric acid buffer.
SYG substratum (g/L): glucose 20g, Tryptones 10g, yeast extract 5g, NaCl 5g.
PDA solid medium (g/L): potato 200g, glucose 20g, agar 15g, water 1000mL, natural pH.
The HPLC condition determination of product of the present invention is: chromatographic column: VP-ODSC18, moving phase is the mixed solution of the trifluoroacetic acid/water (B) of trifluoroacetic acid/methyl alcohol (A) of 0.05% and 0.05%, flow velocity 1mL/min, detect wavelength 210 nm, 30 ℃ of column temperatures, sample size 10 μ L, use 0.22mm membrane filtration before sample introduction.
embodiment 1: screening and the Purification method of the present embodiment explanation bacillus megaterium Z2013513.
1, the seed selection of starting strain-
Take commercially available sausage or soy cheese as bacterium source, get appropriate sample and after oscillation treatment 2h, get bacteria suspension and process 10min in 80 ℃ in stroke-physiological saline solution.The bacteria suspension 1mL getting after processing is inoculated in 10mL beef-protein medium in 35 ℃ of enrichment culture 12h.Get 0.1mL enrichment culture liquid and be applied in respectively the beef-protein medium containing 2% agar, cultivate 3 days, obtain single bacterium colony for 30 ℃.Several single bacterium colonies of random choose further on beef-protein medium line separated, obtain starting strain and be stored in 4 ℃ standby;
2, thalline activation and conversion fluid preparation
The bacterial strain of institute's seed selection is seeded to respectively to LB substratum, and 37 ℃, 200 revs/min of activation culture 10 hours, then get culture according to 10%(v/v) inoculum size access SYG liquid nutrient medium,, 200 revs/min, continues to cultivate 8 hours by 40 ℃.Culture is through 4 ℃, and 4000 revs/min of abandoning supernatant after centrifugal 5 minutes, add appropriate conversion substratum suspension thalline, are placed in 37 ℃, and 200 revs/min of shaking tables transform 8 hours.Conversion fluid is got supernatant liquor and is carried out bacteriostatic experiment after 10000 revs/min of centrifugal 10 min.
3, streptococcus aureus plate screening
Get and cover with streptococcus aureus (As1.89, purchased from DSMZ of institute of microbiology of Chinese Academy of Sciences country) one of slant tube, add sterile distilled water 10mL, make bacteria suspension, getting 0.1mL and be inoculated in beef-protein medium surface evenly coating, at the Oxford cup of the equidistant placement high-temperature sterilization of dull and stereotyped different positions, get above-mentioned steps 2) gained supernatant liquor 200 μ L add in the cup of Oxford, cultivate 22 hours, choose the bacterial strain that produces inhibition zone in the surrounding of Oxford cup for 37 ℃;
4, penicillium citrinum plate screening
In sterile petri dish, pour PDA solid medium into, penicillium citrinum is made to spore suspension, getting 0.1mL is inoculated on PDA plate culture medium and evenly coating, Oxford cup at the equidistant placement high-temperature sterilization of dull and stereotyped different positions, getting above-mentioned steps 2) gained supernatant liquor 200 μ L add Oxford cup, cultivate 60 hours, choose the bacterial strain that produces inhibition zone in the surrounding of Oxford cup for 28 ℃;
Select and can suppress the corresponding bacterial strain of fermented liquid that above-mentioned streptococcus aureus and Penicillium notatum and inhibition zone are larger, called after bacillus megaterium Z2013513(simultaneously
bacillus megateriumz2013513).
Morphology and the physiological and biochemical property of this bacterial strain are as follows:
Colony colour: beige;
Aerobic mode: aerobic growth;
Bacterium colony size: 1.2～2.5 * 2.0～10.0 μ m;
Optimum growth temperature: 35-40 degree Celsius;
The optimum initial pH:7 that grows;
Thalli morphology: shaft-like, end circle, single or be short chain and arrange, can move;
Gramstaining: the positive;
Gemma: 1.0～1.2 * 1.5～2.0 microns, ellipse, middle life.
embodiment 2: the authentication method of the present embodiment explanation bacillus megaterium Z2013513.
1, the evaluation of gemma bacterial strain:
Mensuration and compare of analysis to the sequence of the 16SrDNA part of this bacterium, be accredited as bacillus megaterium, through the preservation of microbial preservation program, by its Classification And Nomenclature, is bacillus megaterium Z2013513(
bacillus megateriumz2013513), its preservation registration number is: M2013244.
2, the feature of bacillus megaterium Z2013513:
Morphological feature:
Bacillus megaterium Z2013513 bacterial strain, on LB solid medium, is cultivated 8 hours for 37 ℃, formed obvious bacterium colony, diameter is between 1.0～2.0 mm, circle, is round shape in substratum, neat in edge, oyster white, opaque, surface wettability is smooth, not chromogenesis.Examine under a microscope, Gram-positive, motion, cell rod-short, 0.29～0.45 mm * 1.58～4.20 mm, produces spore.
Cultural characteristic:
Optimum growth temperature 35-40 ℃, aerobic growth, the initial pH of the most suitable growth is 7.0.
embodiment 3: the method in the grown cell fermentative production phenyl-lactic acid of the present embodiment explanation bacterial strain of the present invention.
1) seed culture: to 35 ℃ of LB liquid nutrient mediums, 200 turn per minute activation culture 8 hours by the mono-colony inoculation of bacillus megaterium Z2013513;
2) enlarged culturing: by above-mentioned steps 1) gained seed culture fluid is by 10% inoculum size access SYD substratum, and 40 ℃, shaking table 200 turns per minute and cultivates 4h acquisition grown cell;
3) transform and produce phenyl-lactic acid
After enlarged culturing 4 hours, respectively after in 6 hours, the fed-batch medium of adding 0.375mL per hour, transforms 8 hours.
Get step 3) conversion fluid, through 10000rpm centrifugal 5 minutes, obtain supernatant liquor, the ethyl acetate extraction that adds 2 times of volumes in supernatant liquor, reduction vaporization is concentrated, obtains the crude product of phenyl-lactic acid, then with 0.05% trifluoroacetic acid aqueous solution, dissolve, adopt high performance liquid chromatography (HPLC) method to measure the content of phenyl-lactic acid, the final content of phenyl-lactic acid reaches 5.4g/L, and transformation efficiency reaches 45%(Fig. 3).
embodiment 4: the resting cell batch fermentation of the present embodiment explanation bacterial strain of the present invention is produced the method in phenyl-lactic acid.
1) seed culture is supported: by the mono-colony inoculation of bacillus megaterium Z2013513 to LB liquid nutrient medium 35-40 ℃, 200rpm activation culture 8 hours;
2) enlarged culturing: by above-mentioned steps 1) gained seed culture fluid is by 10% inoculum size access SYD substratum, 35 ℃, 200rpm cultivates 8 hours to Growth of Cells mid-log phase, and gained nutrient solution is in 4 ℃, the centrifugal 5min of 4000rpm collects thalline, and washs 2 times by stroke-physiological saline solution;
3) resting cell is criticized formula and is transformed production phenyl-lactic acid
By step 2) thalline is suspended in 10 mL and transforms in substratum and in 40 ℃, in 200rpm shaking table, transform some hours, every 2h sampling, the centrifugal 10min of 10000rpm, get supernatant liquor and add the ethyl acetate of 2 times of volumes to extract, reduction vaporization is concentrated, obtains the crude product of phenyl-lactic acid, then with same volume 0.05% trifluoroacetic acid aqueous solution, dissolve, through suitably adopting high performance liquid chromatography (HPLC) method to measure the content of phenyl-pyruvic acid and phenyl-lactic acid after dilution.Through 4h, transform, transform in substratum almost approach exhaustion of phenyl-pyruvic acid, generate phenyl-lactic acid 2.7g/L simultaneously, phenyl-pyruvic acid transformation efficiency reaches 67.5% as shown in Figure 2.
embodiment 5: the method that the resting cell Fed-batch Fermentation Process of the present embodiment explanation bacterial strain of the present invention is produced phenyl-lactic acid.
1) seed culture: by the mono-colony inoculation of bacillus megaterium Z2013513 to 35 ℃ of LB liquid nutrient mediums, 200rpm activation culture 8 hours;
2) enlarged culturing: by above-mentioned steps 1) gained seed culture fluid is by 10% inoculum size access SYD substratum, 40 ℃, 200rpm cultivates 8 hours to Growth of Cells mid-log phase, and gained nutrient solution is in 4 ℃, the centrifugal 5min of 4000rpm collects thalline, and washs 2 times by stroke-physiological saline solution;
3) flow addition in batches transform production phenyl-lactic acid
By step 2) thalline is suspended in 10 mL and transforms in substratum and in 40 ℃, start to transform in 200rpm shaking table.In first 14 hours of 2h, within every 2 hours, in conversion fluid, add 0.5mL fed-batch medium, amount to and add approximately 320 mg phenyl-pyruvic acids and 350 mg glucose, cotransformation finishes for 18 hours at 40 ℃.Final phenyl-lactic acid content can reach 16.1g/L, and transformation efficiency reaches 50%(Fig. 4).
Result shows, the resting cell of this bacterium, the conversion strategy that adopts stream to add can effectively transform phenyl-pyruvic acid and generate phenyl-lactic acid, and output, transformation efficiency and productivity that the technique that present method adopts and bacterial strain transform phenyl-pyruvic acid synthesis of phenyl lactic acid are significantly better than take milk-acid bacteria as basic bio-transformation index.
Claims (9)
1. a strain bacillus megaterium, is characterized in that, its Classification And Nomenclature is bacillus megaterium Z2013513(
bacillus megateriumz2013513), Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC NO:M 2013244 on June 4th, 2013, have been preserved in.
2. utilize the method that bacillus megaterium Z2013513 produces phenyl-lactic acid described in claim 1, it is characterized in that, through seed culture, after enlarged culturing, by grown cell or conversion of resting cells, produce phenyl-lactic acid.
3. method according to claim 2, is characterized in that, the step that grown cell is produced phenyl-lactic acid comprises:
1) seed culture: by the mono-colony inoculation of bacillus megaterium Z2013513 to LB liquid nutrient medium, 35-40 ℃, 200rpm activation culture 8 hours;
2) enlarged culturing: by above-mentioned steps 1) gained seed culture fluid accesses in SYD substratum by 10% inoculum size, 35-40 ℃, and shaking table 200 turns per minute and cultivates 4h acquisition grown cell;
3) transform and produce phenyl-lactic acid
After enlarged culturing 4 hours, respectively after in 6 hours, the fed-batch medium that adds 0.375mL per hour, transforms 8 hours.
4. method according to claim 3, is characterized in that:
Described LB substratum comprises: yeast extract 5g/L, Tryptones 10g/L, NaCl 10g/L;
Described SYD substratum comprises: glucose 20g/L, Tryptones 10g/L, yeast extract 5g/L, NaCl 5g/L;
Described fed-batch medium is: glucose 100g/L, Sodium.beta.-phenylpyruvate 78g/L, the phosphoric acid buffer of pH7.
5. method according to claim 2, is characterized in that, the step that described resting cell is produced phenyl-lactic acid comprises:
1) seed culture is supported: by the mono-colony inoculation of bacillus megaterium Z2013513 to LB liquid nutrient medium, 35-40 ℃, 200rpm activation culture 8 hours;
2) enlarged culturing: by above-mentioned steps 1) gained seed culture fluid is by 10% inoculum size access SYD substratum, 35-40 ℃, shaking table 200 turns per minute, cultivate 8 hours to Growth of Cells mid-log phase, by gained nutrient solution at 4 ℃, the centrifugal 5min of 4000rpm, and collect resting cell thalline 2 times with stroke-physiological saline solution washing;
3) transform and produce phenyl-lactic acid
By above-mentioned steps 2) gained thalline forwards in conversion fluid, and 40 ℃ transform 4-18 hour.
6. method according to claim 5, is characterized in that, by step 2) thalline be suspended in 10 ml and transform in substratum and in 30 ℃, in 200rpm shaking table, transform;
In first 14 hours, within every 2 hours, add 0.5ml fed-batch medium, add altogether approximately 320 mg phenyl-pyruvic acids and 350 mg glucose, 40 ℃ transform 18 hours.
7. method according to claim 5, it is characterized in that, the method for transformation of step 3) is that batch fermentation transforms: by step 2) thalline is suspended in 10 mL and transforms in substratum and in 40 ℃, in 200rpm shaking table, transform some hours, every 2h sampling, the centrifugal 10min of 10000rpm, getting supernatant liquor adds the ethyl acetate of 2 times of volumes to extract, reduction vaporization is concentrated, obtain the crude product of phenyl-lactic acid, then with same volume 0.05% trifluoroacetic acid aqueous solution, dissolve, through suitably adopting high performance liquid chromatography (HPLC) method to measure the content of phenyl-pyruvic acid and phenyl-lactic acid after dilution.
8. according to the either method described in claim 5,6 or 7, it is characterized in that:
Described LB substratum comprises: yeast extract 5g/L, Tryptones 10g/L, NaCl 10g/L;
Described SYD substratum comprises: glucose 20g/L, Tryptones 10g/L, yeast extract 5g/L, NaCl 5g/L;
Described conversion substratum is: glucose 20g/L, Sodium.beta.-phenylpyruvate 4g/L, the phosphoric acid buffer of pH7;
Described fed-batch medium is: glucose 100g/L, Sodium.beta.-phenylpyruvate 78g/L, the phosphoric acid buffer of pH7.
9. method according to claim 2, is characterized in that, also comprises further purifying phenyl-lactic acid:
1) by the centrifugal 10min of conversion fluid 10000rpm of the claims 1-7 any one gained, reject precipitation, it is 2.0 that supernatant liquor is adjusted PH;
2) get above-mentioned steps 1) gained supernatant liquor adds the ethyl acetate extraction of 2 times of volumes, after get organic phase reduction vaporization to dry, obtain the crude product of phenyl-lactic acid;
3) with 0.05% trifluoroacetic acid aqueous solution dissolving step 2 of same volume) crude product of the phenyl-lactic acid of gained, after suitably diluting, adopt the content of high effective liquid chromatography for measuring phenyl-lactic acid.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108004277A (en) * | 2018-01-24 | 2018-05-08 | 贵州大学 | One kind prepares linoleic method using bacillus megaterium |
| CN108410756A (en) * | 2018-02-11 | 2018-08-17 | 浙江工业大学 | Thermophilic pyridine Rhodococcus sp and its application in degradable organic pollutant |
| CN112553119A (en) * | 2020-12-25 | 2021-03-26 | 宁夏启元药业有限公司 | Bacillus megaterium strain and application thereof |
| CN116135965A (en) * | 2023-02-09 | 2023-05-19 | 大连工业大学 | Bacillus megaterium PH3 for biosynthesis of resveratrol and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831394A (en) * | 2010-02-05 | 2010-09-15 | 山东大学 | Heat-resistant bacillus and application thereof in preparing phenyl lactic acid |
| CN102115777A (en) * | 2010-11-29 | 2011-07-06 | 昆明理工大学 | Screening method of lactic acid bacteria strains of high-yield DL-3-phenyllactic acid |
| CN102382783A (en) * | 2011-10-17 | 2012-03-21 | 天津科技大学 | Bacillus coagulans and fermentation liquor and fermentation method thereof and applications of fermentation liquor taken as biopesticide |
| CN102604858A (en) * | 2012-01-10 | 2012-07-25 | 江南大学 | Bacterial strain for preparing phenyllactic acid and method for preparing phenyllactic acid through bacterial strain fermentation |
| WO2012105495A1 (en) * | 2011-01-31 | 2012-08-09 | 旭化成ケミカルズ株式会社 | Phenylpyruvate reductase and method for manufacturing optically-active phenyllactic acid and 4-hydroxyl-phenyllactic acid using same enzyme |
-
2014
- 2014-01-02 CN CN201410000615.4A patent/CN103710291B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831394A (en) * | 2010-02-05 | 2010-09-15 | 山东大学 | Heat-resistant bacillus and application thereof in preparing phenyl lactic acid |
| CN102115777A (en) * | 2010-11-29 | 2011-07-06 | 昆明理工大学 | Screening method of lactic acid bacteria strains of high-yield DL-3-phenyllactic acid |
| WO2012105495A1 (en) * | 2011-01-31 | 2012-08-09 | 旭化成ケミカルズ株式会社 | Phenylpyruvate reductase and method for manufacturing optically-active phenyllactic acid and 4-hydroxyl-phenyllactic acid using same enzyme |
| CN102382783A (en) * | 2011-10-17 | 2012-03-21 | 天津科技大学 | Bacillus coagulans and fermentation liquor and fermentation method thereof and applications of fermentation liquor taken as biopesticide |
| CN102604858A (en) * | 2012-01-10 | 2012-07-25 | 江南大学 | Bacterial strain for preparing phenyllactic acid and method for preparing phenyllactic acid through bacterial strain fermentation |
Non-Patent Citations (2)
| Title |
|---|
| A.THIERRY ET AL: "PRODUCTION OF CHEESE FLAVOUR COMPOUNDS DERIVED FROM AMINO ACID CATABOLISM BY PROPIONIBACTERIUM FREUDENREICHII", 《LAIT》 * |
| 龚春燕 等: "多粘类芽孢杆菌HY96-2发酵液化学成分研究", 《天然产物研究与开发》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108004277A (en) * | 2018-01-24 | 2018-05-08 | 贵州大学 | One kind prepares linoleic method using bacillus megaterium |
| CN108004277B (en) * | 2018-01-24 | 2021-04-02 | 贵州大学 | Method for preparing linoleic acid by using bacillus megatherium |
| CN108410756A (en) * | 2018-02-11 | 2018-08-17 | 浙江工业大学 | Thermophilic pyridine Rhodococcus sp and its application in degradable organic pollutant |
| CN108410756B (en) * | 2018-02-11 | 2020-12-08 | 浙江工业大学 | Rhodococcus pyridinivorans and application thereof in degradation of organic pollutants |
| CN112553119A (en) * | 2020-12-25 | 2021-03-26 | 宁夏启元药业有限公司 | Bacillus megaterium strain and application thereof |
| CN116135965A (en) * | 2023-02-09 | 2023-05-19 | 大连工业大学 | Bacillus megaterium PH3 for biosynthesis of resveratrol and application thereof |
| CN116135965B (en) * | 2023-02-09 | 2024-02-27 | 大连工业大学 | Bacillus megaterium PH3 for biosynthesis of resveratrol and application thereof |
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