CN106399195A - Lactobacillus casei and application thereof - Google Patents
Lactobacillus casei and application thereof Download PDFInfo
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- CN106399195A CN106399195A CN201610960989.XA CN201610960989A CN106399195A CN 106399195 A CN106399195 A CN 106399195A CN 201610960989 A CN201610960989 A CN 201610960989A CN 106399195 A CN106399195 A CN 106399195A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/245—Lactobacillus casei
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
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Abstract
The invention provides Lactobacillus casei and an application thereof in transforming and synthesizing microorganisms into phenyllactic acid. The Lactobacillus casei is named Lactobacillus casei HR S67 and is preserved at China Center for Type Culture Collection with a preservation number of CCTCC No. M 2016216. With a bacterial strain of the Lactobacillus casei as an original strain, the phenyllactic acid can be synthesized by fermentation and cultivation, concentration of the fermented liquor can reach 20mg/L; with thallus of the Lactobacillus casei as a whole-cell catalyst, a converted solution containing the phenyllactic acid can be obtained by conducting microbial conversion on a substrate solution containing phenylpyruvic acid, wherein the concentration of the phenyllactic acid can reach 11.2g/L. The bacterial strain of the Lactobacillus casei is separated from pickle food and is a typical probiotics. As a catalyst for fermentation microorganisms or whole-cell microorganisms, the bacterial strain has not only good biosecurity but also the advantages of mild conditions for fermentation and transformation, high product concentration and easy product amplification in the converted solution and good industrial application prospect.
Description
Technical field
The invention belongs to technical field of biochemical industry and in particular to a kind of lactobacillus casei microbial strains and its fermentation
With the application in Synthesis phenyllactic acid.
Background technology
Phenyllactic acid is a kind of food with broad-spectrum antibacterial characteristic and pharmaceutical preservative with no toxicity studied in recent years, is also that synthesis is poly-
The important monomer of phenyllactic acid macromolecular material, can be also used for synthesizing anti-platelet aggregation and myocardial infarction medicine, therefore, benzene breast
Acid all has important application prospect in the many such as chemical industry, material, medicine and food fields.
Traditional chemical synthesiss are prepared phenyllactic acid and are had that raw material sources are more convenient, cost is relatively low, large-scale production is easy
The advantages of realize, but also there is the limitation that some are difficult to overcome, such as using high poison solvent, more serious environmental pollution and product
Middle problem of solvent residual etc..Synthesize phenyllactic acid using fermentable or microorganism conversion, have mild condition, environmental friendliness and
The advantages of synthesis path is easier, becomes the important development direction of the aspect of phenyllactic acid preparation in recent years.
But, when fermentable conjunction or Synthesis phenyllactic acid, there is some crucial problem, the safety of such as bacterial strain
Property, conversion yield be not high, production concentration is very low.Accordingly, it would be desirable to exploring, biological safety is good, high conversion rate and production concentration are high
Phenyllactic acid fermentation synthesis or Synthesis new strains and Synthesis new method.
Content of the invention
For the above-mentioned problems in the prior art, it is an object of the present invention to provide one plant of lactobacterium casei strains, and provide
Its application in phenyllactic acid synthesis.The existing good biological safety of this bacterial strain, has good fermentation synthesis and conversion to close again
Become the ability of phenyllactic acid.
For achieving the above object, taken following technical scheme：
（1）A kind of lactobacterium casei strains are it is characterised in that described Strain Designation is lactobacillus casei（Lactobacillus casei）HRS67, is preserved in China typical culture collection center, and deposit number is：CCTCC No. M 2016216.Preservation
Time：On April 21st, 2016；Preservation address：Wuhan City, Hubei Province Wuchang District Luo Jia Shan Wuhan University Life Science College, postcode
430072.
Further, described lactobacterium casei strains（Lactobacillus casei）HRS67 bacterial strain is Gram-positive bar
Bacterium, 0.8 micron × 1.5 ~ 2.0 microns；Become chain growth, spore of not sprouting；Do not move；Amphimicrobian；Negative catalase；Change
Can heterotrophism.
Further, described lactobacterium casei strains（Lactobacillus casei）HRS67 bacterial strain 16S rDNA sequence
As SEQ ID NO：Shown in 1.
（2）A kind of lactobacillus casei（Lactobacillus casei）Application in fermentation synthesis phenyllactic acid for the HRS67.
Further, this application is with described lactobacillus casei（Lactobacillus casei）HRS67 bacterial strain is the bacterium that sets out
Strain, is fermented in liquid medium within, is obtained the fermentation liquid containing phenyllactic acid.
Further, described fluid medium consists of：Described fluid medium consists of：Glucose 20 g, albumen
Peptone 10 g, citric acid hydrogen diamine 2 g, anhydrous sodium acetate 5 g, manganese sulfate 0.25 g, magnesium sulfate 0.58 g, dipotassium hydrogen phosphate 2 g,
Yeast extract 5 g, Carnis Bovis seu Bubali cream 10 g, Tween 80 1 g, deionized water 1 L, pH value is natural.
Further, described fermentation condition is：25 ~ 35 DEG C of temperature, fermentation time 24 ~ 72h, standing for fermentation.
Further, in the described fermentation liquid containing phenyllactic acid, final concentration of 5.3 ~ 20 mg/L of phenyllactic acid.
（3）A kind of lactobacillus casei（Lactobacillus casei）HRS67 is prepared in phenyllactic acid in microorganism conversion
Application.
Further, this is used for described lactobacillus casei（Lactobacillus casei）HRS67 strain fermentation gained
Thalline be whole-cell biocatalyst, microorganism conversion is carried out to the substrate solution containing phenylpyruvic acid, obtains containing phenyllactic acid
Conversional solution.
Further, the described substrate solution phenylpyruvic acid containing phenylpyruvic acid, glucose and phosphate buffered saline
Form.
Further, the consisting of of the described substrate solution containing phenylpyruvic acid：Glucose 14 g, phenylpyruvic acid 15 g, 100
The phosphate buffer 1 L of mM pH 8.
Further, described microorganism conversion condition is：Lactobacterium casei strains（Lactobacillus casei）HRS67
Bacterial strain cell concentration 100 g/L, conversion temperature is 35 DEG C, and transformation time is 4 h.
Further, in the described conversional solution containing phenyllactic acid, the final concentration of phenyllactic acid is up to 11.2 g/L, conversion ratio
74%.
By using above-mentioned technology, compared with prior art, the present invention has the advantages that：
（1）The strains separation that the present invention provides, from kimchi foodstuffs, is typical probiotic bacteria, and its fermentation synthesis phenyllactic acid has very well
Biological safety.
（2）With the bacterial strain of present invention offer for full cell microorganism catalyst, during Synthesis phenyllactic acid, there is conversion bar
Part is gentle, conversion is rapid, production concentration is high in conversional solution, the advantages of amplify easy, in conversional solution the concentration of phenyllactic acid up to
11.2 g/L, have good prospects for commercial application.
Specific embodiment
In order to better illustrate the object, technical solutions and advantages of the present invention, with reference to specific embodiment to the present invention
It is further described, but protection scope of the present invention is not limited to that：
Embodiment 1
The present invention carries out Isolation and screening of bacterial strain from conventional Kimchi.Take a certain amount of Pickles juice, be diluted in sterilized water,
Solid medium containing Calcium Carbonate（Composition：Glucose 20 g, peptone 10 g, citric acid hydrogen diamine 2 g, anhydrous sodium acetate 5
G, manganese sulfate 0.25 g, magnesium sulfate 0.58 g, dipotassium hydrogen phosphate 2 g, yeast extract 5 g, Carnis Bovis seu Bubali cream 10 g, Tween 80 1 g, fine jade
Fat 20 g, Calcium Carbonate 30 g, deionized water 1 L, pH value is natural）On carry out primary dcreening operation, 30 DEG C of temperature, quiescent culture 24 ~ 72 h.
Choose the negative single bacterium colony of hydrogen peroxide, be inoculated in fluid medium（Composition：Glucose 20 g, peptone 10
G, citric acid hydrogen diamine 2 g, anhydrous sodium acetate 5 g, manganese sulfate 0.25 g, magnesium sulfate 0.58 g, dipotassium hydrogen phosphate 2 g, yeast
Cream 5 g, Carnis Bovis seu Bubali cream 10 g, Tween 80 1 g, deionized water 1 L, pH value is natural）, 30 DEG C of quiescent culture 24 h；Secondary screening is multiple,
Obtain one plant of lactobacillus strain HRS67.
After testing, the physiological and biochemical property of bacterial strain HRS67 is：Gram-positive bacillus, 0.8 micron × 1.5 ~ 2.0 microns；
Become chain growth, spore of not sprouting；Do not move；Amphimicrobian；Negative catalase；Chemoheterotrophy.
The 16S rDNA sequence such as SEQ ID NO of bacterial strain HRS67：Shown in 1.
In conjunction with bacterial strain physiological and biochemical property and 16S rDNA sequence, enter with GreenGenes16S rRNA gene database
Row compares, and bacterial strain GBS3 is accredited as lactobacillus casei（Lactobacillus casei）, it is named as lactobacillus casei
（Lactobacillus casei）HRS67, is preserved in China typical culture collection center, preservation on April 21st, 2016
Numbering is：CCTCC No. M 2016216.
Embodiment 2
With lactobacillus casei of the present invention（Lactobacillus casei）HRS67 bacterial strain is starting strain, in 1 L liquid
Body culture medium（Composition：Glucose 20 g, peptone 10 g, citric acid hydrogen diamine 2 g, anhydrous sodium acetate 5 g, manganese sulfate 0.25
G, magnesium sulfate 0.58 g, dipotassium hydrogen phosphate 2 g, yeast extract 5 g, Carnis Bovis seu Bubali cream 10 g, Tween 80 1 g, deionized water 1 L, pH value
Natural）In carry out standing for fermentation, temperature control at 25 ~ 35 DEG C, ferment 24 hours, obtain the fermentation containing lactic acid and phenyllactic acid
Liquid；Tested and analyzed with high performance liquid chromatography, final concentration of 5.3 mg/L of phenyllactic acid in gained fermentation liquid.
Embodiment 3
With lactobacillus casei of the present invention（Lactobacillus casei）HRS67 bacterial strain is starting strain, in 1 L liquid
Body culture medium（Composition：With embodiment 2）In carry out standing for fermentation, temperature control at 25 ~ 35 DEG C, ferment 72 hours, obtain containing breast
Acid and the fermentation liquid of phenyllactic acid；Tested and analyzed with high performance liquid chromatography, phenyllactic acid concentration 20 mg/L in gained fermentation liquid.
Embodiment 4
With lactobacillus casei of the present invention（Lactobacillus casei）HRS67 bacterial strain is starting strain, in 1.5 L
Fluid medium（Composition：With embodiment 2）In carry out standing for fermentation, temperature control at 25 ~ 35 DEG C, ferment 72 hours, take wherein
3.1 g thalline of centrifugation gained, as whole-cell catalyst, take 10 mL to contain 0.14 g glucose and 0.15 g phenylpyruvic acid
Solution（100 mM pH 8 phosphate buffer）For substrate, convert 4 hours, 35 DEG C of temperature, obtain conversional solution；Use efficient liquid phase
Chromatograph is tested and analyzed, and in gained conversional solution, phenyllactic acid concentration is 11.2 g/L, molar yield 74%.
SEQUENCE LISTING
<110>Zhejiang Polytechnical University
<120>A kind of lactobacillus casei and its application
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 950
<212> DNA
<213>Bacterial strain HRS67
<400> 1
gcggcggctg ctataatgca gtcgaacgag ttctcgttga tgatcggtgc ttgcaccgag 60
attcaacatg gaacgagtgg cggacgggtg agtaacacgt gggtaacctg cccttaagtg 120
ggggataaca tttggaaaca gatgctaata ccgcatagat ccaagaaccg catggttctt 180
ggctgaaaga tggcgtaagc tatcgctttt ggatggaccc gcggcgtatt agctagttgg 240
tgaggtaatg gctcaccaag gcgatgatac gtagccgaac tgagaggttg atcggccaca 300
ttgggactga gacacggccc aaactcctac gggaggcagc agtagggaat cttccacaat 360
ggacgcaagt ctgatggagc aacgccgcgt gagtgaagaa ggctttcggg tcgtaaaact 420
ctgttgttgg agaagaatgg tcggcagagt aactgttgtc ggcgtgacgg tatccaacca 480
gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttatc 540
cggatttatt gggcgtaaag cgagcgcagg cggtttttta agtctgatgt gaaagccctc 600
ggcttaaccg aggaagcgca tcggaaactg ggaaacttga gtgcagaaga ggacagtgga 660
actccatgtg tagcggtgaa atgcgtagat atatggaaga acaccagtgg cgaaggcggc 720
tgtctggtct gtaactgacg ctgaggctcg aaagcatggg tagcgaacag gattagatac 780
cctggtagtc catgccgtaa acgatgaatg ctaggtgttg gagggtttcc gcccttcagt 840
gccgcagcta acgcattaag cattccgcct ggggagtacg accgcaaggt tgaaactcaa 900
aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 950
Claims (7)
1. a kind of lactobacterium casei strains（Lactobacillus casei）HRS67, deposit number is：CCTCC No. M
2016216.
2. a kind of lactobacillus casei as claimed in claim 1（Lactobacillus casei）HRS67 is in fermentation synthesis benzene breast
Application in acid.
3. application as claimed in claim 2 is it is characterised in that with described lactobacterium casei strains（Lactobacillus casei）HRS67 bacterial strain is starting strain, is fermented, obtain the fermentation liquid containing phenyllactic acid in liquid medium within.
4. application as claimed in claim 3 is it is characterised in that described fluid medium forms：Glucose 20 g, peptone
10 g, citric acid hydrogen diamine 2 g, anhydrous sodium acetate 5 g, manganese sulfate 0.25 g, magnesium sulfate 0.58 g, dipotassium hydrogen phosphate 2 g, ferment
Female cream 5 g, Carnis Bovis seu Bubali cream 10 g, Tween 80 1 g, deionized water 1 L, pH value is natural, and the condition of described fermentation is：Temperature 25 ~
35 DEG C, fermentation time 24 ~ 72h, standing for fermentation.
5. a kind of lactobacillus casei as claimed in claim 1（Lactobacillus casei）HRS67 closes in microorganism conversion
Become the application in phenyllactic acid.
6. application as claimed in claim 5 is it is characterised in that with described lactobacterium casei strains（Lactobacillus casei）HRS67 bacterial strain is whole-cell biocatalyst for the described thalline of starting strain fermentation, with phenylpyruvic acid as precursor, enters
Row microorganism conversion, obtains the conversional solution containing phenyllactic acid.
7. application as claimed in claim 6 is it is characterised in that described microorganism conversion condition is：Glucose 14 g/L, benzene
Acetone acid 15 g/L, solvent is the phosphate buffer of 100 mM pH 8, lactobacterium casei strains（Lactobacillus casei）HRS67 bacterial strain cell concentration 100 g/L, 35 DEG C of conversion temperature, transformation time 4 h, mixing speed 80 rpm.
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Cited By (5)
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| CN108384730A (en) * | 2018-01-15 | 2018-08-10 | 浙江工业大学 | A kind of Lactobacillus paracasei and its application in Synthesis phenyllactic acid |
| CN108441436A (en) * | 2018-01-15 | 2018-08-24 | 浙江工业大学 | A kind of Lactobacillus paracasei and its application |
| CN112251383A (en) * | 2020-10-28 | 2021-01-22 | 四川农业大学 | Lactobacillus casei capable of producing phenyllactic acid and application thereof |
| CN114686526A (en) * | 2021-12-30 | 2022-07-01 | 广州君研生物科技有限公司 | Lactobacillus casei fermentation product and skin care product containing lactobacillus casei fermentation product |
| CN115074290A (en) * | 2022-07-06 | 2022-09-20 | 福建生物工程职业技术学院 | Lactobacillus casei for co-production of phenyllactic acid and gamma-aminobutyric acid and application thereof |
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Cited By (6)
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| CN112251383B (en) * | 2020-10-28 | 2023-03-28 | 四川农业大学 | Lactobacillus casei capable of producing phenyllactic acid and application thereof |
| CN114686526A (en) * | 2021-12-30 | 2022-07-01 | 广州君研生物科技有限公司 | Lactobacillus casei fermentation product and skin care product containing lactobacillus casei fermentation product |
| CN115074290A (en) * | 2022-07-06 | 2022-09-20 | 福建生物工程职业技术学院 | Lactobacillus casei for co-production of phenyllactic acid and gamma-aminobutyric acid and application thereof |
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