The Discrepancy Analysis On Y-aminobutyric Acid Production Ability Among Lactobacillus And Its Fermented Milk Used In Sleep Improvement

Nowadays,many people are suffering from different kinds of sleep disorders,especially insomnia due to various metal pressure and physiological diseases.Even though traditional insomnia-treating drugs may be effective in soothing symptoms,there will be different kinds of side effects.Therefore,dietary therapy to help improve sleep has broad application prospects.Previous studies reported the sleep-improvement effect of some Lactobacillus strains and ?-aminobutyric acid(GABA),a kind of their metabolite.However,few studies had explored the sleep-improvement effect of Lactobacillus strains which produce GABA.In the present study,68 Lactobacillus strains were screened to determine their production ability of GABA.Correlation analysis was performed to investigate the association between the expression levels of key genes in the glutamate decarboxylase(GAD)system and the output of GABA.GABA-rich milk product fermented by selected Lactobacillus was then developed and the fermentation parameters were optimized.Functional evaluation of the sedative and hypnotic effects of Lactobacillus strains and GABA fermented milk was performed in animal experiments.The main results are as follows:Firstly,the GABA producing ability of 68 strains distributed in 8 Lactobacillus species was determined by high-performance liquid chromatography(HPLC).14 strains of GABAproducing Lactobacillus were screened out.The Lactobacillus brevis DL1-11 strain had the highest GABA yield for 5072±280 mg/L.The genome information of the eight above-tested species of Lactobacillus was searched and downloaded from the Genebank and the genome data of 812 strains were obtained.The distribution of the GAD system genes in these genomes was investigated.Similar to HPLC result,the relative complete GAD system occurred frequently in the genome of Lactobacillus brevis and Lactobacillus plantarum,indicating the evolutional conservation of GABA-producing ability in these two species.The transcription of all GAD-related genes including gadA,gadB,gadC and gadR in L.brevis or L.plantarum at different growth stages were studied by qRT-PCR.The results showed that the expression levels of gadB and gadC in L.brevis were similar and peaked in the late logarithm or in the early stage of stabilization.The high-yield GABA of L.brevis in the study may be related to the high expression of gadC and gadB.The expression of gadB in L.plantarum was similar to that of L.brevis,but the correlation between GABA production and gadB expression was weak.GABA-producing Lactobacillus strains,mixing with commercially available L.bulgaricus and Streptococcus thermophilu,were used to ferment milk.Again,the GABA yields in fermented milk were determined,and L.brevis DL1-11 also possessed highest one.After that,the fermentation parameters,including monosodium glutamate addition,fermentation temperature and fermentation time were optimized.Both the yields of GABA and the sensory evaluation results were used as indexes.The optimal fermentation parameters were 0.10% monosodium glutamate,37°C and 9 h.Under this condition,the sensory score of the fermented milk was 7.67±1.00 and the GABA content was 84.10±2.68 mg/100 g.The effects of L.brevis DL1-11 and its fermented milk on the improvement of anxiety and sleep quality were evaluated in animal experiment.The open field experiment,elevated plus maze test as well as pentobarbital-induced sleep test were adopted.In the open field test,mice in both L.brevis DL1-11 group and its fermented milk with high-dose GABA group had a significantly increased frequency on entering the central region and a prolonged time period staying in the central region of the open field.The effect of fermented milk with high-dose GABA on the elevated plus maze experiment was relatively small.However,mice in L.brevis DL1-11 group stayed longer in the open arm and had a more frequent entrance to the open arm when compared with the control group.All of these results indicated the anxiety-relieving potentials of both L.brevis DL1-11 and its fermented milk with high-dose GABA.As for pentobarbital-induced sleep test,mice in both L.brevis DL1-11 group and fermented milk with high-dose GABA group had a significantly prolonged sleep time induced by sodium pentobarbital and a shortened the sleep latency induced by sodium barbital,indicating the sleep improvement effect of both L.brevis DL1-11 and its fermented milk.Besides,gut microbiota composition analysis showed that in both L.brevis DL1-11 and fermented milk with high-dose GABA group,the composition of the mouse flora changed significantly.The total content of short-chain fatty acid SCFAs in fermented milk with high-dose GABA group increased significantly.The content of butyric acid,isobutyric acid and valeric acid in the milk group was significantly higher when compared to the control group.All of these suggests their potentials in modulating the gut microenvironment.