| Oxidative stress and damage cause systemic and metabolic diseases among various organisms,large amount of researches focusing on intervention against Oxidative stress have arouse attentions in various fields of science, including life sciences, medicine and nutrition science. A huge number of studies on Lactic Acid Bacteria (LAB) have demonstrated that LAB physiologically functioned as stimulator to immune system, and participated in reduction of serum cholesterol and incidence of cardiovascular disease, as well as in alleviation of oxidative stress, regulation of intestinal flora and restraint of pathogenic colonization. Therefore, this paper was embarked upon screen and identification of specific LAB strains isolated from traditional fermented foods, with potential for antioxidation capacity. The prebiotics and processing properties of the isolates were analyzed according to the capacity in relieving oxidative stress.Subsequently, analyses of the intervening effect on the oxidation stress induced by D-galactose were performed. Finally, the potential mechanisms and effects of antioxidation were tested in vivo,and the applications of isolated LAB in functional food fermentation were discussed. This study possesses great value both in theoretical research and in practical application, and results were as follows.1. Screening and identification of lactic acid bacteria with excellent antioxidant activities in vitro.A total of 83 LAB strains were isolated from traditional fermented food. Lactobacillus rhamnosus GG (ATTCC 53103) was used as the reference strain with positive antioxidation capacity. Antioxidant effects of isolated LAB strains were evaluated in vitro according to capacities of free radical scavenging action, indicators including DPPH, hydroxyl free radical.Lipid peroxidation and potassium ferricyanide were also used in the present experiment. The best candidate strains according to the results were M9-1, LP-4 and P13. The DPPH and hydroxyl free radical scavenging action, lipid peroxidation and potassium ferricyanide reduction activity of strain M9-1 were 65.12%?62.26%?61.02% and 299.26?mol/L cysteine equivalent, respectively,those of strain LP-4 were 79.37%?64.55%?59.88% and 369.55 ?mol/L cysteine equivalent,respectively,and those of strain of P13 were 73.02%?59.02%?68.88% and 284.01 ?mol/L cysteine equivalent, respectively. Physiological and biochemical characteristics analysis and phylogenetic analysis methods were performed to identify candidate strains, the strains LP-4 and M9-4 were identified as Lactobacillus paracasei while strain P13 was Lactobacillus Fermentum.2. Probiotic properties and application base of the isolated strains M9-1, LP-4 and P13A series of methods were performed to evaluate the probiotic and application potential of strains M9-1, LP-4 and P13, including antagonistic activity against foodborne pathogens, survival rates under gastrointestinal tract conditions (acidity, bile and pancreatin), decarboxylase activity,bile salt hydrolase activity, hemolytic activity, cell surface hydrophobicity, aggregation capacity and adhesion properties to human cell lines. Compared with the control strain L. rhamnosus GG ATCC53103, strains M9-1 and LP-4 showed great probiotic potential and promising application value, Specifically, strain LP-4 showed higher adherence to Caco-2 cells, cell surface hydrophobicity and aggregation capacity than those of ATCC53103 (p < 0.05) while strain M9-1 showed similar capacity as ATCC53103 (p > 0.05). However, the adherence to Caco-2 cells, cell surface hydrophobicity and aggregation capacity of strain P13 were lower than ATCC53103 (p<0.05). The results of enzyme assays revealed that strain P13 have histidine decarboxylase activity and tyrosine decarboxylase activity.3. Repair of D-galactose induced oxidative stress by M9-1 and LP-4 strains in mice model.The ability, of the two strains M9-1 and LP-4, to reduce the oxidative stress in vivo was assessed by mice suffering oxidative stress induced by D-galactose, and vitamin C was used as a positive drug control. The results showed that strain M9-1 and LP-4 had the potential to alleviate oxidative stress in blood, kidney and liver of mice. The superoxide dismutase activity and the glutathione peroxidase activity of plasma, liver and kidney increased in different degree, while the level of malondialdehyde and protein carbonyl in liver and kidney decreased in different degree, the level of endotoxin in serum decreased significantly (p < 0.05). At the transcriptional level, RT-PCR analysis suggested that the gene expression of SOD, GSH-Px, HO-1 NRF2, and TRX increased in various degree in vivo. Interestingly, there was a dose-response relationship between the relieving effect and administration dose. Compared with the repair effects of positive antioxidants in high dose volume, strain LP-4 showed significant advance in oxidative damage repair function in rat.4. Studies on optimization of culture conditions for high cell density cultivation of strain LP-4Based on MRS broth, optimizations of medium composition and culture conditions for strain LP-4 were studied by means of single factor and the orthogonal design. The optimized medium was determined with modifications of glucose 30 g/L, tryptone 35 g/L and tomato juice 60 g/L in original MRS, and optimized cultivating conditions were achieved with the inoculation amount of 3%, pH 6.5, at 34?. After being cultivated in optimized medium and condition for 18 h, the optical density at 600 nm of LP-4 increased by 18.12% compared with the cell density of before optimization.5. Preliminary study of strain LP-4 in fermented functional foodCompared with the yogurt fermented by Streptococcus thermophilus and treptococcus thermophilus, additional strain LP-4 during fermentation significantly increased the antioxidant capacity and acetaldehyde content in yogurt (p < 0.05), as well as the viscosity and the water holding of yogurt (p < 0.05). Compared with naturally fermented vegetable, the antioxidant capacity and sensory score of vegetables fermented by extra strain LP-4 increased significantly (p< 0.05), while the nitrite content and fermentation time decreased significantly (p < 0.05). During the shelf life of yogurt, the survival bacterial number stayed above 1.04×107 CFU/g during a 30-day storage at 4?. The results confirmed the promising potential and application of strain LP-4 in fermented functional food. |
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