Isolation Of ?-aminobutyric Acid(GABA)-producing Lactobacillus Plantarum Identification And Its Its Application In Banana Fermented Beverage

Gamma aminobutyric acid(GABA)is a kind of natural non protein amino acid,which has many physiological functions such as reducing blood pressure,treating epilepsy,improving brain cells,enhancing memory and promoting sleep.Hainan is rich in banana resources.Banana is a kind of fruit with good medicine and food.Regular consumption of banana can not only prevent nerve fatigue,but also moisten the lungs and stop coughing and prevent constipation.The production of banana lactobacillus beverage rich in GABA is a good way of banana deep processing,which can not only alleviate the high cost of banana transportation in Hainan,but also create good economic and social benefits,and lay the foundation for the creation of Hainan featured products.Banana is also a kind of fruit with high GAD activity.It can enrich GABA with its endogenous GAD.At present,there is no report to isolate and screen the Lactobacillus strains producing GABA from banana plants.Therefore,this study focuses on the screening of GABA producing lactic acid bacteria strains from the flower,fruit,stem,leaf and root soil of banana,and taking this strain as the fermentation strain,mixing banana juice as the natural fermentation medium,combining with the health characteristics of lactic acid bacteria,to produce GABA rich banana lactic acid bacteria beverage.In order to provide theoretical basis and reference for the industrialization and standardization of banana lactobacillus beverage which is rich in GABA,and to open up a new way for the further processing industry of Hainan banana,the effects of various factors on GABA and sensory quality in the fermentation process were studied,and the fermentation conditions were optimized.The main results are as follows:(1)Firstly,the detection method of GABA in the fermentation broth was studied.The legal analysis of GABA by thin layer chromatography,and the quantitative analysis of GABA by high performance liquid chromatography-ultra high performance liquid chromatography.The developing agent components in the thin layer chromatography analysis method were determined.The optimal developing agent ratio was when n-butanol:glacial acetic acid:water(V/V)=4:1:1:1.The Rf values were significantly different.The color development conditions were 90°C for 10 min.Comparison of Ultra-High Performance Liquid Chromatography(UPLC)and High-Performance Liquid Chromatography(HPLC)Pre-column Ophthalaldehyde(OPA)Derivative-UV Detection Method Linear regression equation of concentration and absorbance.The GABA measured by the two methods had a good linear relationship in the range of mass concentration of100.0?1000.0?g/m L(correlation coefficient R~2>0.998).The relative standard deviation(RSD)of the precision test results of the UPLC method and the HPLC method are both less than 1.5%.The average relative standard deviation(RSD)of the UPLC method is0.529%,and the average recovery is 99.59%.The average relative standard deviation of the HPLC method(RSD)Was 1.175%,and the average recovery was 98.06%.It shows that both detection methods can accurately and quantitatively analyze the GABA content in lactic acid bacteria fermentation broth.Compared with the two,UPLC method is faster and more suitable for the detection of a large number of samples,which lays the foundation for subsequent research.(2)Then,thin-layer chromatography qualitative analysis was used to isolate 19 strains of GABA-producing lactic acid bacteria from 49 strains of lactic acid bacteria isolated from banana plants in Hainan,of which 5 strains had strong GABA-producing ability.Then,through ultra-high performance liquid chromatography quantitative analysis method,a strain G4-1 with high GABA production was finally obtained.The content of GABA in the fermentation product of lactic acid bacteria reached 0.928 g/L,which is a strain with high potential for high GABA production.Subsequently,the morphological characteristics and physiological and biochemical characteristics of strain CT4-1 were studied.The physiological and biochemical identification results of this strain were consistent with those of Lactobacillus plantarum in the Berger Bacterial Identification Manual.The 16 S r DNA gene sequence It was identified as L.plantarum and was initially named L.plantarum CT4-1.The antibacterial results showed that the strain was against Escherichia coli,Salmonella,Staphylococcus aureus,and Listeria monocytogenes and Bacillus subtilis all have bacteriostatic effects,indicating that the lactic acid bacteria have antibacterial activity,so they are intended to be used in fermented foods to inhibit the growth of spoilage bacteria in foods and improve the Quality and flavor to extend shelf life.(3)Then optimize the process of banana lactic acid bacteria drink rich in GABA.The single-factor and Box-Behnken response surface method were used to optimize the fermentation conditions of banana lactic acid bacteria beverages.The results showed that the optimal fermentation conditions for banana lactic acid bacteria beverages were:banana juice addition amount of 8%,sucrose addition amount of 5%,and whole milk powder addition.The amount is 10%and the inoculation amount is 5%.Fermented banana lactic acid bacteria beverage under this condition is refreshing,delicate,moderately sweet and sour,fresh milk flavor,and has banana-specific flavor.The sensory score is 95 points.(4)In order to enhance the stability of the beverage,three stabilizers,xanthan gum,CMC-Na and sodium alginate,were selected,and the stabilizers were selected by single factor and 3 factor 3 level orthogonal experiments,and the precipitation rate and sensory evaluation were used.Evaluation of stability effects.The results show that the single stabilizer has a stabilizing effect on the banana lactic acid bacteria beverage.When the amount of xanthan gum is 0.10%,the precipitation rate is 7.86%,which is the best among the three stabilizers.Orthogonal experiments determine that the optimal blending ratio of stabilizers is xanthan gum,CMC-Na,and sodium alginate in the amounts of 0.075%,0.10%,and 0.05%,respectively.At this time,the sensory score of the beverage is 93 points,stabilizing effect.optimal.(5)Finally,in order to explore the effect of cold storage on the quality of banana lactobacillus beverage,the p H,the number of viable Lactobacillus and the sensory quality of banana lactobacillus beverage were measured within 28 days at 0-4°C.The results showed that:under the condition of cold storage,the viable bacteria number of banana lactobacillus beverage was higher than the national standard(1×10~6CFU/ml),the p H value decreased from 3.83 to 3.78,and the sensory quality stability of the beverage was good within 21 days.