Isolation,Identification And Probiotic Characteristics Of Lactic Acid Bacteria From Soy Sauce Residue

Objective: Soy sauce is a condiment made by natural fermentation.Many microorganisms are involved in the fermentation process.Soy sauce residue,a by-product of soy sauce production,was rich in nutrition,which was mainly used as feed or fertilizer.Because of its high salt content,its application was limited.The development of lactic acid bacteria resources in soy sauce residue can provide microbial resources for improving the physicochemical properties and fermentation quality of soy sauce residue,and has positive significance for improving the comprehensive utilization rate of soy sauce residue.Methods: A sample of soy sauce residue was collected from a condiment company in Guangdong Province.Lactic acid bacteria were isolated by MRS medium.The phylogenetic status of lactic acid bacteria in soy sauce residue was studied by morphological observation,physiological and biochemical experiments and 16 S rRNA gene sequence analysis.The growth and probiotic characteristics of lactic acid bacteria were also studied.Conclusion: 1.18 suspected lactic acid bacteria strains were isolated from soy sauce residue samples by MRS medium.The results of colony and morphological observation showed that the colony of 12 suspected strains of lactic acid bacteria was white with a diameter between 0.5 mm and 1.5 mm.The morphology of these strains was medium-long rod and in short chain distribution.The colony of 6 suspected lactic acid bacteria strains was grey-white with a diameter between 0.3 mm and 1.0 mm.The morphology of these strains was spherical and mostly in short chains distribution.2.Intergeneric identification of suspected strains of lactic acid bacteria was carried out by microbial biochemical identification tube.The results showed that the enzyme contact,nitrate reduction and gelatin liquefaction tests of 12 suspected strains of rod-shaped lactic acid bacteria were negative and their preliminary identification were Lactobacillus.Six suspected strains of spherical lactic acid bacteria were negative in enzyme contact,nitrate reduction and glucose gas production tests,and were preliminarily identified as Lactococcus.The 16 S rRNA gene sequence analysis was used to identify lactic acid bacteria.Interspecific identification of lactic acid bacteria was carried out by 16 S rRNA gene sequence analysis.The results showed that,eighteen strains of isolates from soy sauce residue belong to two genera: Lactococcus(33.3%)and Lactobacillus(66.7%).They belong to two species: Lactococcus lactis(33.3%)and Lactobacillus paracasei(66.7%).And they belong to four subspecies: Lactococcus lactis subsp.horiadne,Lactococcus lactis subsp.lactis,Lactobacillus paracasei subsp.tolerans,Lactobacillus paracasei subsp.paracasei.It was concluded that Lactobacillus and Lactococcus were widely distributed in soy sauce residues at sampling stage.The results showed that Lactobacillus was the dominant strain in soy sauce residue during the sampling stage,accounting for 66.7% of the total lactic acid bacteria isolated.3.The growth characteristics and probiotic characteristics of lactic acid bacteria were studied.The results of the growth test of lactic acid bacteria showed that:(1)18 strains of lactic acid bacteria grew well in at 37°C in MRS medium.(2)Lactic acid bacteria grew well under the conditions of salt concentration less than 6%,the temperature between 30°C?37°C,and p H 4.0?p H 9.0.The probiotic characteristics of lactic acid bacteria showed that:(1)Lactic acid bacteria grew well in the environment without bile salt.(2)The results showed that the survival rate of strains HT4,HT31,HT51,HT90,HT102,HT111,HT122,HT127,HT155,HT158,HT159,HT160,HT223,HT227,HT253 and HT256 was more than 30% after 3 hours of tolerance to artificial gastric juice.The survival rate of strains HT4,HT31,HT51,HT90,HT102,HT111,HT122,HT125,HT127,HT155,HT158,HT159,HT197,HT223,HT253,HT256 was more than 30% after 4 hours of tolerance to artificial gastric juice.The survival rate of lactic acid bacteria HT4,HT31,HT51,HT90,HT102,HT111,HT122,HT127,HT155,HT158 and HT223 was over 30% after their inoculation in artificial intestinal fluid for 8 hours.(3)The hydrophobicity test of lactic acid bacteria showed that strains HT4,HT111 and HT155 had better hydrophobicity,which was up to 20%.The hydrophobicity of strains HT31 and HT256 was slightly lower,18.52% and 17.59% respectively.(4)The antioxidant tests of strains HT4,HT31,HT111 and HT155 showed that the DPPH and hydroxyl radical scavenging ability of the cell suspension and cell-free extract group of strains HT4,HT31 were better,proving that they had good antioxidant properties.In summary,the probiotic characteristics of strains HT4 and HT31 were good,which can be used as potential lactic acid bacteria starter for further study.It also proved that lactic acid bacteria in soy sauce residue have great development value.