CN101440382A - Preparation of biological preservative 4-hydroxyphenyl lactic acid - Google Patents
Preparation of biological preservative 4-hydroxyphenyl lactic acid Download PDFInfo
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- CN101440382A CN101440382A CNA2008102440532A CN200810244053A CN101440382A CN 101440382 A CN101440382 A CN 101440382A CN A2008102440532 A CNA2008102440532 A CN A2008102440532A CN 200810244053 A CN200810244053 A CN 200810244053A CN 101440382 A CN101440382 A CN 101440382A
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Abstract
The invention provides a method for a biological antiseptic 4-hydroxyl phenyllactic acid, which relates to a method for preparing 4-hydroxyl phenyllactic acid by fermentation bioconversion of microbe, and belongs to the field of food biotechnology. The preparation method comprises that lactic acid bacteria are used as fungi, 4-hydroxyl phenylypruvic acid or 4-hydroxyl phenylypruvic acid salt is used as a conversion substrate; and by adding a fermentation medium formed by carbon sources, nitrogen sources and inorganic slat, the 4-hydroxyl phenyllactic acid is prepared by fermentation bioconversion. The lactic acid bacteria are cultured at a temperature of between 25 and 40 DEG C in an MRS culture medium, grow in good conditions and are used as a seed culture solution; the fermentation medium carbon source is glucose, maltose, lactose or sucrose, and the nitrogen source is a mixture of one or more than one of corn steep liquor, yeast extract, peptone and soy bean pulp; and after fermentation, detections show that the concentration of the 4-hydroxyl phenyllactic acid in the fermentation liquid is between 1.0 and 20.0mmol/L. The product prepared by the method has the advantages of safety, reliability and strong bacteria inhibiting function.
Description
Technical field
The present invention relates to a kind of preparation method of biological preservative 4-hydroxyphenyl lactic acid, be specifically related to a kind of method that the microbial fermentation bio-transformation prepares the 4-hydroxyphenyl lactic acid, belong to technical field of food biotechnology.
Background technology
The 4-hydroxyphenyl lactic acid is a kind of novel antibacterial substance that milk-acid bacteria produces.Studies show that the 4-hydroxyphenyl lactic acid has wide spectrum and suppresses the fungi function.
The 4-hydroxyphenyl lactic acid is a kind of natural antiseptic agent.It is present in the natural honey, and is nontoxic to the humans and animals cell.Milk-acid bacteria is to be known as safety (Generally recognized as safety, microorganism GRAS) is used for food preservation over the past thousands of years.Therefore, 4-hydroxyphenyl lactic acid be considered to continue first-generation antibacterial substance that milk-acid bacteria produces such as lactic acid, acetic acid beyond s-generation antibacterial substance such as the Nisin bacteriocins such as (nisins), can be used for the novel antibacterial substance of food system.Compare with Nisin, the antibacterial spectrum width of 4-hydroxyphenyl lactic acid, stability is high, wetting ability is strong.
The preparation of 4-hydroxyphenyl lactic acid at present has the synthetic and bioconversion method of chemical catalysis.Chemical synthesis has many adverse factors, and for example severe reaction conditions produces multiple by product and chemical pollutant, and numerous by products of formation make separation, purification step become complicated again.Recent study personnel prepare the 4-hydroxyphenyl lactic acid in the method for seeking bio-transformation, bioconversion method, and mild condition is polluted and is lacked, and the reaction specificity is strong.Because milk-acid bacteria is the microorganism of GRAS (being known as safety), producing the 4-hydroxyphenyl lactic acid with safe bacterial strain bio-transformation becomes current focus.Recently some milk-acid bacteria of report can utilize the fermentation of MRS substratum to generate the 4-hydroxyphenyl lactic acid, but yield poorly, the bacterial classification individual difference is big, production peak is 0.29mmol/L.
At the low problem of 4-hydroxyphenyl lactic acid fermentation yield, this patent research and utilization 4-hydroxyphenylphruvic acid or 4-hydroxypropiophenonepreparation hydrochlorate are added in the fermention medium as substrate, can increase substantially the output of 4-hydroxyphenyl lactic acid.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of biological preservative 4-hydroxyphenyl lactic acid, particular content relates to a kind of method of utilizing the lactobacillus-fermented bio-transformation to prepare the 4-hydroxyphenyl lactic acid.Milk-acid bacteria can effectively utilize substrate 4-hydroxyphenylphruvic acid or 4-hydroxypropiophenonepreparation hydrochlorate among the present invention, fermentation transforms and generates the 4-hydroxyphenyl lactic acid, transformation efficiency height, specificity are strong, mild condition, by product are few, for the bio-transformation of 4-hydroxyphenyl lactic acid provides a new approach.
Technical scheme of the present invention: as bacterial classification, is conversion of substrate with 4-hydroxyphenylphruvic acid or 4-hydroxypropiophenonepreparation hydrochlorate with milk-acid bacteria, adopts carbon source, nitrogenous source and inorganic salt to form fermention medium, and the fermentation method bio-transformation prepares the 4-hydroxyphenyl lactic acid.
(1) bacterial classification: adopt milk-acid bacteria, Lactobacillus sp.SK007, Lactobacillus plantarumAS1.550, Lactobacillus plantarum SK002, Lactobacillus pentose BCRC15317 or Lactococcus lactis 1.009.
When transforming as strain bio, select for use Lactobacillus sp.SK007 effect good especially with milk-acid bacteria.
(2) substratum, in g/L:
Storage medium: glucose 15, yeast extract paste 10, lime carbonate 15, agar 15, pH 6.5-7.0;
Seed culture medium: glucose 10, peptone 10, yeast extract paste 10, anhydrous sodium acetate 10, potassiumphosphate 0.2, sal epsom 0.2, manganous sulfate 0.05, pH 6.5-7.0;
(3) preparation technology:
Culture presevation: used culture presevation in storage medium, 4 ℃ of preservations, two week switchings once.
Seed culture: change seed culture medium over to from storage medium and need activate 2 times, cultivate 12h for each 30 ℃.
Fermentation: seed culture fluid is with the inoculum size of 1%-3% (V/V), insert and add different carbon sources, in the fermention medium that nitrogenous source and inorganic salt are formed, fermention medium consists of: 4-hydroxyphenylphruvic acid or 4-hydroxypropiophenonepreparation hydrochlorate 4-40mmol/L, carbon source 5-30g/L, nitrogenous source 5-30g/L, anhydrous sodium acetate 10g/L, potassiumphosphate 0.2g/L, sal epsom 0.2g/L, manganous sulfate 0.05g/L, pH 6.2-6.8,25-40 ℃ leaves standstill cultivation, and 12-36h obtains containing the fermented liquid of 4-hydroxyphenyl lactic acid, used carbon source is a glucose, maltose, lactose or sucrose, used nitrogenous source are corn steep liquor, yeast extract paste, peptone, in the soybean meal one or more are used with.
Preferred fermention medium is: 4-hydroxyphenylphruvic acid or 4-hydroxypropiophenonepreparation hydrochlorate 4mmol/L, glucose 20g/L, corn steep liquor 10g/L, yeast extract paste 10g/L, anhydrous sodium acetate 10g/L, potassiumphosphate 0.2g/L, sal epsom 0.2g/L, manganous sulfate 0.05g/L, pH 6.2-6.4.
The method that detects the 4-hydroxyphenyl lactic acid is as follows:
It is centrifugal to get fermented liquid, and supernatant liquor is through filtering with microporous membrane (0.22 μ m), and HPLC analyzes on the filtrate.HPLC condition: Agilent 1100; Chromatographic column: ZORBAX 300SB C18 (5 μ m, 4.6mm * 250mm), moving phase is the mixed solution of the trifluoroacetic acid/water (B) of trifluoroacetic acid/methyl alcohol (A) of 0.05% and 0.05%, the gradient elution program is: 0～20min is by 10%A linear change to 100%, 20～23min keeps 100% A, and 23～25min is by 100% A linear change to 10%.Flow velocity 1mL/min detects wavelength 210nm, 30 ℃ of column temperatures, sampling volume 10 μ L.
Beneficial effect of the present invention: the invention provides a kind of 4-of utilization hydroxyphenylphruvic acid or 4-hydroxypropiophenonepreparation hydrochlorate is substrate, the lactobacillus-fermented bio-transformation prepares the method for 4-hydroxyphenyl lactic acid, and the output of 12h 4-hydroxyphenyl lactic acid can reach 1.0-20.0mmol/L.
Embodiment
Embodiment 1 different strain is to the influence of 4-hydroxyphenyl lactic acid output
In fermention medium, add the 4-hydroxyphenylphruvic acid of 4mmol/L, the above-mentioned preferred substratum of by specification carries out fermenting experiment to some milk-acid bacterias of primary election, behind the fermentation 12h fermented liquid is detected, the result is as shown in table 1, various milk-acid bacterias can both transform the 4-hydroxyphenylphruvic acid and generate the 4-hydroxyphenyl lactic acid, wherein the 4-hydroxyphenyl lactic acid fermentation yield of Lactobacillus sp.SK007 is higher, and different strain has difference comparatively significantly to the generation of 4-hydroxyphenyl lactic acid.
Table 1 different strain is to producing the influence of 4-hydroxyphenyl lactic acid
Embodiment 2 fermentation times are to producing the influence of 4-hydroxyphenyl lactic acid
The described fermentation condition of by specification, add the 4-hydroxyphenylphruvic acid of 4mmol/L, produce the detection of 4-hydroxyphenyl lactic acid in the different fermentations time by Lactobacillus sp.SK007, the content of 4-hydroxyphenyl lactic acid is the highest when finding fermentation 18h, along with fermentation time prolongs, the content of 4-hydroxyphenyl lactic acid has decline slightly.So determine that fermentation time is 18h, fermentation time is as shown in table 2 to the influence of producing the 4-hydroxyphenyl lactic acid.
Table 2 fermentation time is to producing the influence of 4-hydroxyphenyl lactic acid
Embodiment 3 different substrates are to producing the influence of 4-hydroxyphenyl lactic acid
The described fermentation condition of by specification, add different substrate 4-hydroxyphenylphruvic acid or the 4-hydroxyphenylphruvic acid sodium of 4mmol/L respectively, by Lactobacillus sp.SK007 is produced the detection of 4-hydroxyphenylphruvic acid behind fermentation 18h, 4-hydroxyphenylphruvic acid and 4-hydroxyphenylphruvic acid sodium are that the 4-hydroxyphenyl lactic acid fermentation yield of substrate is close, and prove the effective substrate that is the fermentative preparation of 4-hydroxyphenyl lactic acid with 4-hydroxyphenylphruvic acid and 4-hydroxyphenylphruvic acid sodium.
The different substrates of table 3 are to producing the influence of 4-hydroxyphenyl lactic acid
Claims (3)
1. the preparation method of a 4-hydroxyphenyl lactic acid, it is characterized in that with the milk-acid bacteria being bacterial classification, with 4-hydroxyphenylphruvic acid or 4-hydroxypropiophenonepreparation hydrochlorate is conversion of substrate, adopts carbon source, nitrogenous source and inorganic salt to form fermention medium, and the fermentation method bio-transformation prepares phenyllactic acid;
(1) bacterial classification: milk-acid bacteria;
(2) substratum, in g/L:
Storage medium: glucose 15, yeast extract paste 10, lime carbonate 15, agar 15, pH 6.5-7.0;
Seed culture medium: glucose 10, peptone 10, yeast extract paste 10, anhydrous sodium acetate 10, potassiumphosphate 0.2, sal epsom 0.2, manganous sulfate 0.05, pH 6.5-7.0;
Fermention medium: 4-hydroxyphenylphruvic acid or 4-hydroxypropiophenonepreparation hydrochlorate 4-40mmol/L, carbon source 5-30, nitrogenous source 5-30, anhydrous sodium acetate 10, potassiumphosphate 0.2, sal epsom 0.2, manganous sulfate 0.05, pH 6.2-6.8;
(3) preparation technology:
Culture presevation: used culture presevation in storage medium, 4 ℃ of preservations, two week switchings once;
Seed culture: change seed culture medium over to from storage medium and need activate 2 times, cultivate 12h for each 30 ℃;
Fermentation: seed culture fluid is with the inoculum size of volume ratio 1%-3%, insert in the fermention medium, 25-40 ℃ leaves standstill cultivation 12-36h, obtain containing the fermented liquid of 4-hydroxyphenyl lactic acid, used carbon source is glucose, maltose, lactose or sucrose, and used nitrogenous source is that in corn steep liquor, yeast extract paste, peptone, the soybean meal one or more are used with.
2. the preparation method of 4-hydroxyphenyl lactic acid according to claim 1, it is characterized in that fermention medium is: 4-hydroxyphenylphruvic acid or 4-hydroxypropiophenonepreparation hydrochlorate 4mmol/L, glucose 20g/L, corn steep liquor 10g/L, yeast extract paste 10g/L, anhydrous sodium acetate 10g/L, potassiumphosphate 0.2g/L, sal epsom 0.2g/L, manganous sulfate 0.05g/L, pH 6.2-6.4.
3. the preparation method of 4-hydroxyphenyl lactic acid according to claim 1 is characterized in that bacterial classification selects Lactobacillus sp.SK007, Lactobacillus plantarum AS 1.550, Lactobacillusplantarum SK002, Lactobacillus pentose BCRC15317 or Lactococcus lactis 1.009 for use.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906392A (en) * | 2010-06-23 | 2010-12-08 | 常熟理工学院 | Lactobacillussp.W2 strain and application thereof |
| CN102703530A (en) * | 2012-06-08 | 2012-10-03 | 沈阳科纳提克生物科技有限公司 | Process for producing propionate |
| CN103540541A (en) * | 2013-07-16 | 2014-01-29 | 湖南泰谷生物科技股份有限公司 | Fermentation process of lactic streptococci |
| CN107805620A (en) * | 2017-10-12 | 2018-03-16 | 昆明理工大学 | One strain gene engineering bacterium Lactococcus lactis and its application |
| 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 |
| CN108624533A (en) * | 2018-05-10 | 2018-10-09 | 浙江工商大学 | A kind of method that phenyl-lactic acid is isolated and purified in lactobacillus plantarum |
-
2008
- 2008-12-19 CN CNA2008102440532A patent/CN101440382A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906392A (en) * | 2010-06-23 | 2010-12-08 | 常熟理工学院 | Lactobacillussp.W2 strain and application thereof |
| CN101906392B (en) * | 2010-06-23 | 2012-02-01 | 常熟理工学院 | Lactobacillussp.W2 strain and application thereof |
| CN102703530A (en) * | 2012-06-08 | 2012-10-03 | 沈阳科纳提克生物科技有限公司 | Process for producing propionate |
| CN103540541A (en) * | 2013-07-16 | 2014-01-29 | 湖南泰谷生物科技股份有限公司 | Fermentation process of lactic streptococci |
| CN103540541B (en) * | 2013-07-16 | 2015-09-16 | 湖南泰谷生物科技股份有限公司 | The zymotechnique of streptococcus acidi lactici |
| CN107805620A (en) * | 2017-10-12 | 2018-03-16 | 昆明理工大学 | One strain gene engineering bacterium Lactococcus lactis and its application |
| 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 |
| CN108624533A (en) * | 2018-05-10 | 2018-10-09 | 浙江工商大学 | A kind of method that phenyl-lactic acid is isolated and purified in lactobacillus plantarum |
| 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 |
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Open date: 20090527 |