GABA Formation By Lactobacillus Pentosus Stains Used In Dairy Industry

γ-Aminobutyric acide(GABA)is a natural amino acids which is composed of non-protein.GABA has very important physiological functions in human,such as the induction of hypotensive,neurotrans mission mediator,tranquilizer effects,the anti-worry anxiously,promoted the sperm conceive,and the improvement liver kidney function etc. GABA is catalyzed and synthesized from by L-glutamate by Glutamate decarboxylase (GAD).Some lactic acid bacteria has the ability that catalyzes the decarboxylation of L-glutamate to GABA.This text got two lactic acid bacteria higher produced GABA. Based on the cell figure,physiology biochemical authenticate and 16S rRNA determined date,substantiate the two lactic acid bacteria were Lactobacillus pentosus.On this basis,we investigate the intrinsic and extrinsic factors affecting the production of GABA by this organism,the separateness and purification of Glutamate decarboxylase(GAD),the character of GAD,manufacture dairy products fermented with GABA-producing strains.1.The HPLC method,Paper chromatography method and Berthelot Spectrophotometry method for qualitative analysis and quantitative analysis GABA was established.â‘ The HPLC condition:The GABA was derivatized to OPA-GABA with OPA;the latter was subsequently separated with C18 separation column(250mm×4.6mm,5μm) and detected with UV absorbance detector at 338 nm.flowed solution:0.1mol/L sodinm phos phate potassinm buter(pH6.0):tetrahydrofuran:methanol= 52:2:46.â‘¡Paper chromatography method:the chromatographic solvent system is:n-butamol: acetic acid glacial:Water=4:2:1(contain the 0.4 g/L ninhydrin);chromatographic resolution at 30℃for 3.5 h,then dry at 90℃for 10 mins,shear the spot descend use liquid to eluent(V75%ethanol absolute:V0.1%CuSO₄ solution=38:2)wash to take off 25 min.The optical density is read at 630 nm.GABA produced was calculatedl using the standard curve..â‘¢The condition of Berthelot Spectrophotometry method:Take 0.6mol/L enzyme reaction solution,plus 0.4mL sodium borate buffer,pH9.0,in order to terminated the reaction then0.8 mL phenol solution and 1.5mL sodium hyochloride were added.Color development was carried out in boiling water for 10 min then immediately put in ice-water bath for 20 min.The optical density is read at 630 nm.GABA produced was calculatedl using the standard curve.2.Two GABA-producing strain of P and Ind-3 was isolated and screened from the traditional fermented food.Based on the cell figure,physiology biochemical authenticate and 16S rRNA determined date,substantiate the two lactic acid bacteria were Lactobacillus pentosus.0.3615mg/mL and 0.1575 mg/mL of GABA was produced by the P and Ind-3 after anaerobic fermentation for 48 hours at 37℃in MRS.3.The optimal conditions for Lactobacillus pentosus P and Lactobacillus pentosus Ind-3 to transform L-Glu into GABA were incubation temperature 37℃in a medium with pH 7.0.The two Lactobacillus pentosus both grown in improved MRS incubated with 20g/L glucose,10g/L tryotone,10g/L yeast extract,10g/L beef extract,MSG concentration of 15 g/L,plus CaCl₂ concentration 0.5mmol/L.A 2%(v/v,quantity was10⁸ cfu/mL) inoculation size(germ age is 12h)was recommended.In this condition,the maximum biosynthesis of GABA by the P incubation for 72h was 1.002 g/L.The maximum biosynthesis of GABA by the Ind-3 incubation for 84h was 0.3262g/L.Tthe GABA concentration continue increased to 1.4064g/Lå’Œ0.4563g/L in store time at 4℃at 30 days.4.Glutamate decarboxvlase(GAD)as one of important enzyme able to convey L-Glu to GABA,was extracted from the cells of Lactobacillus pentosus strains grown in MRS incubated at 37℃for 16h.Its purification and its properties were characterized in the following experiments.The molecular weight of GAD from the two Lactobacillus pentosus was 43 KD.The GAD showed the maximum activity in transformation of L-Glu to GABA if the enzyme was incubated at 37℃for 1 h and maintained in a given pH of 4.8.It was proved that the enzyme was sensitive to heat relatively and never tolerated temperature higher than 60℃.Among metal ions used in the study,Ca²⁺manifested great promoting effects on enzyme activity.the activity of GAD in P and Ind-3 respectively increased by 126%and 223%.The promoting effect of Cu²⁺,Mn²⁺,and Mg²⁺took second place.The activity of GAD in P respectively increased by 63%,109%and 47%, and the activity of GAD in Ind-3 respectively increased60%,12%and 50%.In fact,K⁺, Fe²⁺had an effect of inhibition to GAD.The activity of GAD in P respectively lowered down by 41%and 47%,the GAD activity of Ind-3 respectively declined by 30%and 37%.5.Skim-milk with GABA-producing Lactobacillus pentosus strains were sampled for the determination of GABA concentration after the termination of fermentation and subsequent 21-day refrigerated storage periods.Clearly,addition of MSG to the cultured milks increased the production of GABA by P and Ind-3.An appropriate inclusion of carbon and nitrogen sources in cultured milks also improved GABA formation by Lactobacillus pentosus strains.It was noted that glucose was the best sugar in promoted the two Lactobacillus pentosus grown in milk to produce much more GABA,followed by saccharose and actose.Compared to peptone and beef extract,inclusion of yeast extract in cultured milks was a good way to enhance Lactobacillus pentosus strains to form the most GABA.Addition of 2g/L MSG to the cultured milks was appropriate for the biggest production of GABA by the two Lactobacillus pentosus.Utilization of P and Ind-3 averagely produced as much 3 times GABA as those of plain yogurt products without inoculation of GABA producing strains,up to 0.2647g/L and 0.0677g/L.In addition to the holding of higher GABA,it was observed that GABA concentration in the cultured milks had little changed between the day 0 and day 21 of refrigerated storage,and then slowed increased gently following the 21days.The GABA concentration respectively increased 35%and 45%.