Screening Of Saccharomyces Cerevisiae For High γ-aminobutyric Acid Production And Its Using In Brewing Of Pear Wine

γ-aminobutyric acid(GABA) is a non-protein natural amino acid which is widely existed in plants, animals and microorganisms. GABA has many important physiological functions, such as anti-aging, lowering blood pressure, improving liver function, regulating hormone secretion, improving sleep, enhancing memory ability, preventing and treating obesity. GABA is primarily formed by the decarboxylation reaction of glutamic acid, catalysed by glutamic acid decarboxylase enzyme which is found in lactic acid bacteria, mold, yeast and other microorganisms. As a result, GABA can be produced by extracting glutamic acid decarboxylase or microbial fermentation. With the improvement of people’s living standard and enhancement of health care consciousness, functional foods that are rich in GABA have become a hot spot of research and development. Pear fruits are succulent and crisp, sweet and refreshing. Pear has high sugar content, rich nutrition and contains a variety of proteins, minerals and vitamins, etc., is a good raw material for brewing wine. The aim of this project is to screen out strains of Saccharomyces cerevisiae which not only have high GABA yield, but also a strong ability to produce ethanol, and then use the yeast strain to brew pear wine which will be rich in GABA.The establishment of colorimetry to detect GABA content in the fermented broth was achieved. There was special color reaction between GABA and bromocresol green with the presence of glyoxalic acid and succinic acid. Base on this reaction, a new kind of rapid colorimetric method for determination of GABA content was established. The suitable concentration of bromocresol green, glyoxalic acid and succinic acid were 0.5, 5.0 and 6.0 mg/mL respectively. The appropriate conditions for determination of GABA content were: 4 mL sample solution, adding 2 ml of chromogenic agent, mixing and shakeing under 25℃ water bath for 5 min, determining absorbance value at 617 nm. GABA standard curve regression equation was A=0.1257C-0.0295, and the correlation coefficient R2=0.9958. The precision of test method(repeat measurement sample of RSD) was 0.88%, and the average recovery was 95.33%. This method had good reproducibility and accuracy. The operation was simple, rapid and low cost.Strains of S.cerevisiae with high GABA were screened. After enrichment cultivation, by using the GABA selecting medium, 8 yeast strains were screened from pears, grapes, apples and other fruits’ surface, which could both produce high GABA and had strong ability to produce alcohol. Among these yeast strains, KS45 had the strongest ability to produce GABA, and its GABA yield and ethanol concentration reached 1.146 g/L and 8.0%. Through microbiology and molecular identification, strain KS45 was identified S. cerevisiae. When KS45 was developed in YEPD at 30℃ for 2 d, its colony morphology had larger diameter, and the colony surface was smooth, creamy, neat and easy to be picked. The cells were ovoid under microscopic, and some were elliptic. It was found that some cells had budding phenomenon. When the fermentation medium contained pear juice, KS45 could produce higher GABA under static fermentation, which suggested that pear juice might contain some substances to stimulate S. cerevisiae to synthesize GABA.GABA-rich pear wine was brewed. S. cerevisiae KS45 was inoculated into pear juice for pear wine brewing. The influences of fermentation time, inoculation volume, initial sugar concentration, fermentation temperature, pear juice volume, concentration of SO2 on GABA and alcohol content were investigated. It was found that pear juice volume had a great influence on GABA production. As pear juice volume increased, GABA content in pear wine decreased sharply, which might be due to lack of oxygen supply. The addition of SO2 had certain inhibitory effect on synthesis of GABA. The optimal fermentation conditions for GABA-rich pear wine brewing were that: 10 d static fermentation, 8% inoculum, 20% initial sugar concentration, 30℃, pear juice volume 100 mL/500 mL, SO2 60 mg/L. Under the optimal conditions, the alcohol concentration was 8%, and the GABA content was 1.765 mg/mL. After the examination, methanol content in pear wine meted with the requirements of national standard of wine quality.