CN104017853A

CN104017853A - Method for producing gamma-aminobutyric acid by fermentation

Info

Publication number: CN104017853A
Application number: CN201410291576.8A
Authority: CN
Inventors: 江波; 沐万孟; 冯慧杰; 张涛; 缪铭
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2014-06-26
Filing date: 2014-06-26
Publication date: 2014-09-03

Abstract

The invention relates to a method for producing gamma-aminobutyric acid by fermentation, belonging to the field of microbial fermentation engineering. The method comprises the following steps: by using Lactococcus lactis capable of producing gamma-aminobutyric acid as a strain, carrying out mixed microbe culture with Saccharomyces cerevisiae W303-1A in an MRS (Man, Rogosa and Sharpe) culture medium with 15 g/L sodium glutamate, and meanwhile, inoculating, wherein the gamma-aminobutyric acid content in the 36-hour fermentation liquid is 4.7 times of that under single lactobacillus culture conditions. The mixed microbe strain and method have the advantages of high safety and reliability, short production cycle and obviously higher gamma-aminobutyric acid yield.

Description

A kind of method of fermentative production γ-aminobutyric acid

Technical field

The present invention relates to a kind of method of fermentative production γ-aminobutyric acid, belong to microbial fermentation engineering field.

Background technology

γ-aminobutyric acid (γ-aminobutyric acid is called for short GABA) is a kind of naturally occurring, nonprotein composition amino acid, is mainly present in mammal brain, spinal cord.GABA has the brain vigor of raising, hypotensive and cholesterol, stable spirit, prevents obesity, improves the effects such as liver kidney function, thereby it is with a wide range of applications in field of functional food.

At present, the production method of GABA mainly contains two kinds of chemical synthesis and biological processes.Although chemical synthesis purity is higher, cost is higher, and yield is lower, and relates to dangerous solvents in production technique, is difficult to realize in food factories.Biological synthesis process mainly utilizes enzyme catalysis by extract Synthesis before GABA, is both safety of one, cost low method again.Biological method is divided into again two kinds of plants enriched and microorganism fermentations, and plants enriched extraction method is taking plant as raw material, makes through enrichment, extraction, separation, purification, although easy and simple to handle, GABA content is lower, and cost is still higher.And Production by Microorganism Fermentation mild condition, equipment requirements is simple, and security is good, and especially with milk-acid bacteria production security the best, and the GABA producing is difficult for being degraded, and therefore microorganism fermentative production GABA has huge advantage and potentiality in the exploitation of this functional foodstuff.

Milk-acid bacteria and yeast are carried out to mixed fungus fermentation product GABA, and yeast can offer milk-acid bacteria as the nutritive substance such as VITAMIN, amino acid on the one hand, and the inhibition meta-bolites lactic acid of milk-acid bacteria also can be utilized by yeast on the other hand.Know through mixed fungus fermentation experimental result, under same medium culture condition, mixed fungus fermentation produce GABA be under single bacterium culture condition more than 4 times, this phenomenon is up to now there are no report.

Summary of the invention

The object of this invention is to provide a kind of method of fermentative production γ-aminobutyric acid, by mixed fungus fermentation operation simple, that be suitable for conventional application, improve the output of γ-aminobutyric acid.

Technical scheme of the present invention, with one strain produce γ-aminobutyric acid Lactococcus lactis ( lactococcus lactis) SYFS 1.009 and yeast saccharomyces cerevisiae W303-1A( saccharomyces cerevisiae) W303-1A is for mixed bacterium bacterial classification, under triangular flask fermentation condition, use is added with the MRS culture medium culturing of 15 g/L Sodium Glutamates.Total inoculum size is 2%, leaves standstill 30 DEG C of cultivations.Under 30 L fermentor tank conditions, its fermention medium is as follows: glucose 30 g/L, yeast extract paste 40 g/L, potassium primary phosphate 10 g/L, Sodium Glutamate 15 g/L, MgSO ₄0.6 g/L, MnSO ₄h ₂o 0.1 g/L, tween 80 0.5 mL/L.In 30 L fermentor tanks, carry out mixed fungus fermentation, initial pH is controlled at 6.8-7.0, culture temperature is 30 DEG C, naturally be down to 5.0 o'clock until fermented liquid pH, stream adds 25%(w/v) ammoniacal liquor to maintain pH be 5.0 ± 0.5, when 36 h, to the Sodium Glutamate concentrated solution that adds 15 g/L in fermented liquid, in whole fermenting process, add glucose and make its fermented liquid concentration maintain 10 g/L left and right, cultivate 48 h and obtain the high streptococcus acidi lactici fermented solution containing γ-aminobutyric acid.

Yeast saccharomyces cerevisiae W303-1A is the common genetically engineered Host Strains of a strain, and the king of Southern Yangtze University in 2008 pays doctorate paper " structure of flocculence Saccharomyces cerevisiae gene engineering bacteria and fermentation " turning etc. all report; Lactococcus lactis Lactococcus lactis SYFS 1.009, at volume the 3rd phase 79-85 page " Lactococcus lactis in May, 2004 " Wuxi Light Industry Univ.'s journal " the 23rd ?the separation and purification of L-Glutamic decarboxylase and part zymologic property ", and " foodstuffs industry science and technology " Vol.35, No.14, has report in 2014 197-201 pages " the high density fermentation research of Lactococcus lactis ".Above bacterial strain is Southern Yangtze University's Food science and the preservation of technology National Key Laboratory, and ensures to provide to the public in 20 years.

Beneficial effect of the present invention: the present invention is owing to having adopted such scheme, and described substratum is safe and reliable, with short production cycle, and γ-aminobutyric acid output is high.Simple to operate in fermenting process, greatly improve the output of γ-aminobutyric acid in fermented liquid.

Embodiment

Embodiment 1

Substratum preparation:

(1) take respectively each component, with distilled water dissolving, wherein glucose dissolves separately, and initial substratum pH is adjusted to 6.8-7.0;

(2) 115 DEG C of independent sterilizings of glucose 20 minutes, other components pack Autoclave or 121 DEG C of sterilizings of fermentor tank into 20 minutes;

(3) before inoculation, by aseptic technique, glucose is added in triangular flask or fermentor tank, stirring and evenly mixing, to obtain final product.

Get ordinary method and activate Lactococcus lactis and the S. cervisiae after two generations, be 2% by total inoculum size, inoculative proportion is 1:1, be inoculated in (glucose 20 g/L, peptone 10 g/L, yeast extract paste 5 g/L in the MRS substratum that is added with 15 g/L Sodium Glutamates simultaneously, extractum carnis 10 g/L, diammonium hydrogen citrate 2 g/L, sodium acetate 5 g/L, K ₂hPO ₄2 g/L, Sodium Glutamate 15 g/L, MnSO ₄4H ₂o 0.25 g/L, MgSO ₄7H ₂o 0.58 g/L, tween 80 l mL, pH 6.8 ~ 7.0), 30 DEG C of triangular flasks leave standstill cultivates 36 h, and in fermented liquid, GABA content is 3.8 g/L, is 4.7 times under the single bacterium culture condition of milk-acid bacteria.

Embodiment 2: get ordinary method and activate Lactococcus lactis and the S. cervisiae after two generations, with total inoculum size inoculum size of 2%, be inoculated in by inoculative proportion 1:1 in the 30 L fermentor tanks that defined medium is housed simultaneously, fermentation tank culture medium consists of: glucose 30 g/L, yeast extract paste 40 g/L, potassium primary phosphate 10 g/L, Sodium Glutamate 15 g/L, MgSO ₄0.6 g/L, MnSO ₄h ₂o 0.1 g/L, tween 80 0.5 mL/L.Initial pH is controlled at 6.8 ~ 7.0, in culturing process, dissolved oxygen amount is 5%, in the time that fermented liquid pH is down to 5.0 naturally, stream adds 25%(w/v) ammoniacal liquor to maintain pH be 5.0 ± 0.5, when 36 h to the Sodium Glutamate concentrated solution that adds 15 g/L in fermented liquid, in whole fermenting process, add glucose and make its fermented liquid concentration maintain 10 g/L left and right, in 48 h secondary fermentation liquid, alpha-aminobutyric acid content is 7.5 g/L.

Claims

1. a method for fermentative production γ-aminobutyric acid, is characterized in that step is: extracting lactic acid galactococcus ( lactococcus lactis) SYFS 1.009 and yeast saccharomyces cerevisiae W303-1A( saccharomyces cerevisiae) W303-1A is mixed fungus fermentation bacterial classification, be inoculated in triangle shaking flask in the ratio of viable count 1 ︰ 1, inoculation simultaneously, substratum is MRS substratum, total inoculum size is 2%, leaves standstill 30 DEG C of cultivations;

When cultivation, initial control pH is 6.8 ~ 7.0, in culturing process, dissolved oxygen amount is 5%, in the time that fermented liquid pH is down to 5.0 naturally, it is that to maintain pH be 4.5 ~ 5.5 for 25% ammoniacal liquor that stream adds mass volume ratio, when 36 h to the Sodium Glutamate concentrated solution that adds 15g/L in fermented liquid, in whole fermenting process, add glucose and make its fermented liquid concentration maintain 10 g/L, after 48 h, obtain the fermented liquid of γ-aminobutyric acid.

2. the method for fermentative production γ-aminobutyric acid according to claim 1, is characterized in that: when fermentation culture, substratum is MRS substratum, specifically consists of: glucose 30g/L, yeast extract paste 40g/L, potassium primary phosphate 10g/L, Sodium Glutamate 15g/L, MgSO ₄0.6g/L, MnSO ₄h ₂o 0.1g/L, tween 80 0.5mL/L.