Screening Of γ-aminobutyric Acid-producing Lactic Acid Bacteria,preparation And Product Development Of Fermented Mulberry Leaf Powder

In this study,a lactic acid bacteria strain with high production ofγ-aminobutyric acid（GABA）was screened from silkworm intestines.GABA production was used as the response value to optimize the medium composition and culture conditions,and a yield of 7.23 mg/m L was obtained.The fermented mulberry leaf powder was prepared with the strain as starting strain.The fermented mulberry leaf powder extract was used to prepare yogurt,and analyze the GABA content and quality characteristics of the yogurt.The specific results were as follows:（1）According to dilution coating plate method and calcium dissolving circle identification,64 strains of lactic acid bacteria were screened from different origins of traditional kimchi,different species of silkworm excrement and intestines.The GABA content in lactic acid bacteria fermentation broth was further analyzed by thin-layer chromatography（TLC）and colorimetric method,and 13 strains were identified having the ability to produce GABA,among which strain JS35 had the highest GABA production.After morphological,physiological-biochemical and molecular biological identification,strain JS35 was identified as Pediococcus pentosaceus.Then the physiological characteristics of JS35 were further analyzed.The results showed that Pediococcus pentosaceus JS35 entered a stable growth stage at 22 h.The survival rate was 84.14%after heating at 60°C for 5 min,and the survival rate was 94.67%after culture at p H 2.5 for 6 h,the survival rate after 24 h of growth at 0.3%bile salt content was 99.40%.That is to say,the strain JS35 had highly tolerant to acid,bile salts and heat.（2）The factors affecting GABA production by Pediococcus pentosaceus JS35 were optimized by single-factor and orthogonal tests.The results showed that the optimal composition of the fermentation medium for GABA production was:glucose 10 mg/m L,yeast powder 12.5 mg/m L,soybean peptone 12.5 mg/m L,L-sodium glutamate 15 mg/m L and Ca Cl₂ 1 mg/m L.The optimal fermentation conditions were obtained as follows:the initial p H was 5.0,fermentation time was 48 h,fermentation temperature was 30°C,inoculation rate with 4%.Under the above conditions,the GABA yield was 7.23 mg/m L.（3）The fermentation was carried out according to the above fermentation conditions with the addition at 5%mulberry leaf powder,using Pediococcus pentosaceus JS35 as the starting strain.During the fermentation process,it was found that mulberry leaf powder promoted the growth of lactic acid bacteria and increased the titratable acidity.The fermented mulberry leaf powder was obtained by freeze-drying the mixed fermentation broth for 48 h.Scanning electron microscopy revealed that the crude fiber structure of mulberry leaf powder was destroyed by the fermentation of Pediococcus pentosaceus JS35.The changes of volatile flavor substances in mulberry leaf powder after fermentation were analyzed by gas chromatography-mass spectrometry analysis.The results showed that that fermentation improved flavor by increasing the contents of benzyl alcohol,phenylethyl alcohol,isospathulenol,acacia alcohol,et al.,while reduced the relative content of geranyl acetone,a major constituent of grass and green aroma in mulberry leaf powder.The effects of the above fermentation on the active ingredients were also explored.The results showed that the contents of GABA,total phenolics and total flavonoids in fermented mulberry leaf powder were increased by 951.3%,16.5%and 22.2%,respectively.The anti-nutritional factor phytic acid was decreased by 20.0%.Additionally,the antioxidant activity of mulberry leaf powder was analyzed,and the results showed that the DPPH radical scavenging capacity and the reducing ability of mulberry leaf extract were significantly improved after fermentation（p<0.01）.（4）The yogurt was prepared by using direct-injection starter,and the effects of unfermented and fermented mulberry leaf powder extract（FME）on the qualities of yogurt were determined.The results showed that yogurt added 0.50%FME contained 20 mg/100m L GABA.Compared with the blank control samples,adding FME promoted the growth of lactic acid bacteria,which reduced the yogurt curdling time by 20%.At 8 h of fermentation,the p H of yogurt without FME was 5.67,while that with 0.50%FME was 4.46,and the titratable acidity of both increased to 3.7 and 9.27,respectively,which further indicated that the addition of FME accelerated curdling by promoting the growth and producing acid of lactic acid bacteria.The effects of FME on the physicochemical properties and textural properties of post-ripened yogurt were investigated.The results showed that the FME gave the yogurt a yellowish color,reduced the brightness（L*）,and increased the red-greenness（a*）and yellow-blueness（b*）values.In the rheological properties,the elastic modulus was increased and the loss angle tangent value was decreased,which indicated that its addition enhanced the gel network strength of the yogurt system.Compared with the control yogurt,the texture results showed that the hardness and gelling properties of yogurt with 0.50%FME were improved significantly,and recovery properties was increased by 4.18 times.However,the effects on cohesiveness and elasticity were not significant（p>0.05）.In addition,the water holding capacity of yogurt with 0.50%FME was improved by 4.06%（p<0.05）.In summary,the results indicated that FME significantly improved the physicochemical and textural properties of yogurt.