CN101906392B - Lactobacillussp.W2 strain and application thereof - Google Patents
Lactobacillussp.W2 strain and application thereof Download PDFInfo
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- CN101906392B CN101906392B CN2010102073511A CN201010207351A CN101906392B CN 101906392 B CN101906392 B CN 101906392B CN 2010102073511 A CN2010102073511 A CN 2010102073511A CN 201010207351 A CN201010207351 A CN 201010207351A CN 101906392 B CN101906392 B CN 101906392B
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Abstract
The invention relates to Lactobacillus sp.W2. The preservation number of the Lactobacillussp.W2 is CCTCCM209318. The invention also relates to application of the Lactobacillus sp.W2 in biologically transforming phenylpyruvic acid substrate and extracting phenyllactic acid from fermentation solution by using ethyl acetate. The Lactobacillus sp.W2 is from the traditional fermented food, belongs to an acknowledged safe strain and can be applied to food; the strain grows well on an MRS culture medium and is easily cultured and stored; and the content of the phenyllactic acid obtained by fermentation can reach 1.038 grams per liter, and the phenyllactic acid has strong capability of inhibiting putrefactive bacteria in the food.
Description
Technical field
The present invention relates to a strain lactobacillus spp
LactobacillusSp.W2 bacterial strain, and the application in the fermentative prodn phenyl-lactic acid belong to the industrial microorganism art.
Background technology
β-phenyl-lactic acid is claimed 3-phenyl-lactic acid or 2-hydroxyl-3-phenylpropionic acid (Phenyllactic acid is called for short PLA) again; Relative molecular mass is 166,121～125 ℃ of fusing points, odorlessness; To acid, thermally-stabilised; Be not destroyed 121 ℃ of heating yet, natural being present in the honey, nontoxic to human or animal's cell; Its second carbon atom is chiral carbon atom, and two kinds of enantiomer R-(+)-3-phenyl-lactic acid (D-(+)-3-phenyl-lactic acid) and S-(-)-3-phenyl-lactic acid (L-(-)-3-phenyl-lactic acid) are therefore arranged.
β-phenyl-lactic acid is a kind of novel antibacterial substance, and wider antimicrobial spectrum is arranged, and its antagonistic property has received investigator's extensive concern.Particularly its pollution that can suppress fungi can prolong the shelf-lives of food, and it be again a kind of can biosynthetic natural compounds, be expected to as the new type natural aseptic applications in foodstuffs industry or other field.R-3-phenyl-lactic acid and S-3-phenyl-lactic acid all have fungistatic effect, under low concentration, just can effectively suppress most of food spoilage bacterium, and its fungistatic effect will significantly be superior to some common food sanitass, like synthetic preservatives such as Sodium Benzoate, POTASSIUM SORBATE GRANULAR WHITEs.
The method of producing the 3-phenyl-lactic acid in the prior art mainly contains chemical method and biological synthesis process.The problem that chemical process exists is that the 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.Because
β-phenyl-lactic acid is mainly used in the food and medicine field, advocates today natural, nutritive food the human consumer, and the investigator of countries in the world has transferred to the biological synthesis process aspect to the emphasis of research; Biological synthesis process is promptly realized through the stereoselectivity biocatalysis of complete microorganism cells.Therefore the good microorganism strains that screens highly-solid selectively is the key of this method.As far back as 1986, Kamata etc. just used brevibacterium lactofermentum
Brevibacterium LactofermentumFermentative prodn the R-3-phenyl-lactic acid, output is 1.94 g/L, and has applied for patent; 1998, Dieuleveux etc. were from geotrichum candidum
Geotrichum CandidumNutrient solution in also be separated to the R-3-phenyl-lactic acid, this bacterial strain amount that produces the R-3-phenyl-lactic acid of in the TSBYE substratum, fermenting is 0.6 g/L～1 g/L; 2000, the plant lactobacillus that discoveries such as Lavermicocca are separated to from bread dough
Lactobacillus plantarum21B can secrete the R-3-phenyl-lactic acid, and they are seeded in plant lactobacillus in the 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. also were separated to a strain from the ensiling grass
L.plantarumMiLAB 393, and the S type that it produces and the ratio of R type 3-phenyl-lactic acid are 9:1; 1996, Hashimoto etc. screened a pseudomonas from soil
PseudomonasSp. BC218 can be converted into the S-3-phenyl-lactic acid with phenylacetic aldehyde-cyanalcohol.This conversion has higher enantioselectivity, and the e.e. value of the S-3-phenyl-lactic acid of generation is 75%, passes through preferential crystallization again, and the 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 Diversa company (Nitrilases) can prepare the e.e. value with nitrile enzyme (EC 3.5.5.1); Recently, report such as Thierry Fei Shi bacillus
Propionibacteriun freudenreichiiIn the cheese fermenting process, produced the 3-phenyl-lactic acid, these 3-phenyl-lactic acids are obtained under the hydroxy reductase effect by phenylpyruvic acid.At present
β-phenyl-lactic acid has carried out suitability for industrialized production in Diversa company.
Summary of the invention
The purpose of this invention is to provide a brace has the microorganism strains of high synthesis of phenyl lactic acid ability, make this bacterium source safety, cultural method simple, and the productive rate of phenyl-lactic acid is high with respect to the prior art bacterial strain; Another technical purpose of the present invention is to provide the application of this microorganism strains in the fermentative prodn phenyl-lactic acid.
In order to realize technical purpose of the present invention, technical scheme of the present invention is:
One, the present invention's separation screening, purifying from the classical acid milk-product of the Inner Mongol obtains a strain lactic bacilli strains, its classification called after lactobacillus spp
LactobacillusSp.W2, preservation registration number are CCTCC NO:M 209318.
Lactobacillus spp of the present invention
LactobacillusThe biological property of sp.W2 is following:
Morphological feature: on the MRS substratum, form tangible bacterium colony, diameter between 0.5～2.0 mm, circle, neat in edge, oyster white, opaque, surface wettability is smooth, not chromogenesis.Gram-positive is not moved, the cell thin rod shape, and 0.29～0.45 mm * 1.58～4.20 mm does not give birth to spore.
Cultural characteristic: 30～37 ℃ of optimum growth temperatures, amphimicrobian, it is poor to grow during aerobic, and the initial pH of the righttest growth is 6.5.
Two, lactobacillus spp of the present invention
LactobacillusThe application of sp.W2 in the fermentative prodn phenyl-lactic acid is with lactobacillus spp
LactobacillusSp.W2 is a bacterial classification, is conversion of substrate with the phenylpyruvic acid, and the fermentation method bio-transformation prepares phenyl-lactic acid.
Concrete application method: with lactobacillus spp
LactobacillusSp.W2 is seeded to the MRS liquid nutrient medium and carries out activation, then seed culture fluid is inserted in the fermention medium by 5% inoculum size, cultivates 20～24 hours for 35～37 ℃.
Further the purification process of phenyl-lactic acid is: with lactobacillus spp
LactobacillusThe sp.W2 fermented liquid carries out centrifugal, and the reject deposition is got the ethyl acetate extraction that supernatant adds 2 times of volumes; Reduction vaporization concentrates; Obtain the bullion of phenyl-lactic acid, then with the dissolving of 10 mL, 0.05% trifluoroacetic acid aqueous solution, the content of high effective liquid chromatography for measuring phenyl-lactic acid.
Beneficial effect of the present invention is:
Lactobacillus spp of the present invention
LactobacillusSp.W2 derives from traditional leavened food, belongs to the generally recognized as safe bacterial classification, can in food, use; This bacterial strain is well-grown on the MRS substratum, cultivates easily and preserves, can be through the phenyl-lactic acid content of fermentation gained up to 1.038 g/L, and the ability that suppresses spoilage organism in the food is stronger.
Description of drawings
Fig. 1 is a lactobacillus spp
LactobacillusThe thalli morphology of sp.W2 (1000 *).
Fig. 2 is a lactobacillus spp
LactobacillusThe HPLC collection of illustrative plates of the phenyl-lactic acid that liquid state fermentation is produced of sp.W2.
Mikrobe lactobacillus spp of the present invention
LactobacillusThe preservation date of sp.W2 is on December 28th, 2009, and depositary institution's full name is Chinese typical culture collection center, is called for short CCTCC, and its preservation registration number is CCTCC NO:M 209318.
Embodiment
The substratum that the present invention adopted:
Enrichment medium:The MRS liquid nutrient medium.
Isolation medium(%): yeast extract paste 0.75%, peptone 0.75%, glucose 1%, 100 mL of Tomato juice, tween 80 0.05%, agar 2%, water 900 mL, pH6.8～7.0.
Differential medium(BCP substratum) (%): peptone 0.5%, and Carnis Bovis seu Bubali cream 0.5%, yeast extract paste 0.5%, tween-80 0.05%, glucose 1%, agar 2%, water 1000mL adds 1.6% purpurum bromocresolis solution, 1.4 mL, pH6.8～7.0.
The slant preservation substratum(%): yeast extract paste 1%, lime carbonate 2%, glucose 1%, agar 2%, water 1000 mL, pH7.0.
Liquid seed culture medium: the MRS liquid nutrient medium.
Fermention medium(%): peptone 1.0%, Carnis Bovis seu Bubali cream 1.0%, yeast extract paste 0.5%, glucose 2.0%, tween-80 0.1%; Potassium hydrogenphosphate 0.2%, sodium-acetate 0.5%, dibasic ammonium citrate 0.2%, sal epsom 0.05%; Manganous sulfate 0.025%, phenylpyruvic acid 0.5%, water 1000 mL, pH7.5.
Embodiment 1
Present embodiment explanation lactobacillus spp
LactobacillusThe screening of sp.W2 and purifying authentication method.
1, the seed selection of bacterial strain:
Accurately take by weighing sour milk slag (purchasing the pearl Mu Qin flag in Xilin Hot, the Inner Mongol) 25 g, under the aseptic condition, adding fills in the triangular flask of 225 mL MRS liquid nutrient mediums 35 ℃ of enrichment culture 24 h.Draw 1 mL sour milk slag nutrient solution and carry out gradient dilution; Drawing different dilution enrichment culture liquid 1 mL places on the aseptic isolation medium that has solidified; Be coated on whole planar surface to bacterium liquid equably with aseptic spreading rod, 35 ℃ of heat insulating culture 2～3 days, the macroscopic bacterium colony that grows in the picking substratum; The separation and Culture of on the BCP plate culture medium, ruling respectively; Substratum is the xanchromatic bacterium colony and is further purified around the picking colony, until obtaining single bacterium colony, is transferred on the slant preservation substratum.
2, fermented liquid preparation:
The bacterial strain of institute's seed selection is seeded to the MRS liquid nutrient medium respectively, 37 ℃, cultivated 24 hours, get an amount of fermented liquid then and descend the step bacteriostatic experiment.
3, the screening of antibacterial bacterial strain:
Get and cover with streptococcus aureus (As1.89; Purchase in Chinese common micro-organisms culture presevation administrative center) one of slant tube, add zero(ppm) water 10 mL that bacterium is crossed in death of monks or nuns, process bacteria suspension; Get 0.1 mL and be inoculated in the nutrient agar plate medium surface, evenly with the glass rod coating.At the Oxford cup of the equidistant placement high-temperature sterilization of dull and stereotyped different positions, fermented liquid 200 mL that get step 2 preparation again add the Oxford cup, cultivate observations 20～24 hours down for 37 ℃.Select the bacterial strain that produces inhibition zone on every side of cup in the Oxford.
In sterile petri dish, pour the PDA solid medium into; The penicillium citrinum (3.4039 that will be separated to from bread; Purchase in Chinese common micro-organisms culture presevation administrative center) process spore suspension, get 0.1 mL and be inoculated on the PDA plate culture medium, be coated with equably with spreading rod.At the Oxford cup of the equidistant placement high-temperature sterilization of dull and stereotyped different positions, get fermented liquid 200 mL adding Oxford cup in the step 2 again, cultivated observations 48～72 hours down for 28 ℃.Select the bacterial strain that produces inhibition zone on every side of cup in the Oxford.
Select and to suppress the pairing bacterial strain of the bigger fermented liquid of above-mentioned streptococcus aureus and penicillium spp and inhibition zone, called after W2 simultaneously.
Embodiment 2
Present embodiment explanation lactobacillus spp
LactobacillusThe authentication method of sp.W2.
1, the evaluation of bacterial strain W2:
Mensuration and compare of analysis to the sequence of the 16SrDNA of W2 part are accredited as lactobacillus spp
LactobacillusSp., through the evaluation and the preservation of microbial preservation program, with its classification called after lactobacillus spp
LactobacillusSp.W2, its preservation registration number are CCTCC NO:M 209318.
2, lactobacillus spp
LactobacillusThe characteristic of sp.W2:
Morphological feature:
With lactobacillus spp
LactobacillusThe sp.W2 bacterial strain was cultivated 48 hours for 35 ℃ on the MRS solid medium, formed tangible bacterium colony, and diameter is between 0.5～2.0 mm, and circle is needle-like in the substratum, neat in edge, and oyster white, opaque, surface wettability is smooth, not chromogenesis.Examine under a microscope, Gram-positive is not moved, the cell rod-short, and 0.29～0.45 mm * 1.58～4.20 mm does not give birth to spore.
Cultural characteristic:
30～37 ℃ of optimum growth temperatures, amphimicrobian, it is poor to grow during aerobic, and the initial pH of the righttest growth is 6.5.
Embodiment 3
Present embodiment explanation lactobacillus spp
LactobacillusThe application of sp.W2 in the fermentative prodn phenyl-lactic acid.
Will
LactobacillusSp.W2 is seeded to the MRS liquid nutrient medium and ferments, and 37 ℃ of culture temperature were cultivated 17 hours, then seed culture fluid is inserted in the fermention medium by 5% inoculum size, cultivates 20～24 hours for 35～37 ℃.The gained fermented liquid carries out centrifugal; The reject deposition is got the ethyl acetate extraction that supernatant adds 2 times of volumes, and reduction vaporization concentrates; Obtain the bullion of phenyl-lactic acid; With the dissolving of 10 mL, 0.05% trifluoroacetic acid aqueous solution, adopt performance liquid chromatography (HPLC) method to measure the content of phenyl-lactic acid then, the phenyl-lactic acid content of gained can be up to 1.038 g/L.
The HPLC condition determination is: moving phase is the mixed solution of the trifluoroacetic acid/water (B) of trifluoroacetic acid/methyl alcohol (A) of 0.05% and 0.05%, and flow velocity 1 mL/min detects wavelength 210 nm, 30 ℃ of column temperatures, before the sample size 25 μ L, sample introduction with 0.22 mm membrane filtration.It detects collection of illustrative plates shown in accompanying drawing 2.
Claims (3)
- One strain lactobacillus spp ( LactobacillusSp.) W2, its preservation registration number are CCTCC No:M 209318.
- Lactobacillus spp 2. according to claim 1 ( LactobacillusSp.) application of W2 in the fermentative prodn phenyl-lactic acid, it is characterized in that with lactobacillus spp ( LactobacillusSp.) W2 is a bacterial classification, is conversion of substrate with the phenylpyruvic acid, and the fermentation method bio-transformation prepares phenyl-lactic acid.
- Lactobacillus spp 3. according to claim 2 ( LactobacillusSp.) application of W2 in the fermentative prodn phenyl-lactic acid is characterized in that concrete grammar is: will ( LactobacillusSp.) W2 is seeded to the MRS liquid nutrient medium and ferments, and 37 ℃ of culture temperature were cultivated 17 hours, then seed culture fluid is inserted in the fermention medium by 5% inoculum size, cultivates 20～24 hours for 35～37 ℃; Wherein said fermention medium is: peptone 1.0%, Carnis Bovis seu Bubali cream 1.0%, yeast extract paste 0.5%, glucose 2.0%, tween-80 0.1%; Potassium hydrogenphosphate 0.2%, sodium-acetate 0.5%, dibasic ammonium citrate 0.2%, sal epsom 0.05%; Manganous sulfate 0.025%, phenylpyruvic acid 0.5%, water 1000 mL, pH7.5.
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| CN108624533B (en) * | 2018-05-10 | 2021-03-09 | 浙江工商大学 | Method for separating and purifying phenyl lactic acid from lactobacillus plantarum |
| CN110618206B (en) * | 2019-08-22 | 2022-06-14 | 重庆科技学院 | Method for simultaneously detecting benzoic acid, sorbic acid, saccharin sodium, phenyllactic acid and phenylalanine in fermented food |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1940078A (en) * | 2006-08-23 | 2007-04-04 | 江南大学 | Production of biological antiseptic agent phenyllactic acid |
| CN101440382A (en) * | 2008-12-19 | 2009-05-27 | 江南大学 | Preparation of biological preservative 4-hydroxyphenyl lactic acid |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1940078A (en) * | 2006-08-23 | 2007-04-04 | 江南大学 | Production of biological antiseptic agent phenyllactic acid |
| CN101440382A (en) * | 2008-12-19 | 2009-05-27 | 江南大学 | Preparation of biological preservative 4-hydroxyphenyl lactic acid |
Non-Patent Citations (2)
| Title |
|---|
| 李兴峰.乳酸生物合成苯乳酸的研究.《中国博士学位论文全文数据（工程科技I辑）》.2009, * |
| 李兴峰等.苯丙氨酸及苯丙酮酸对Lactobacillus sp. SK007 合成苯乳酸的影响.《过程工程学报》.2007,第7卷(第6期), * |
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