| Probiotic resources are one of the important biological resources in the health era.Although China is rich in resources,there are few commercial strains from local sources,and many starter strains still rely on imports.This study selected probiotic resources that could tolerate the gastrointestinal environment and have potential commercial value from the feces of local babies in Guangzhou,which enriched the probiotic resource database of China's independent property rights.Taking the selected strains as the research object,the safety evaluation was carried out from the aspects of in vitro safety experiments and animal experiments.Several probiotic properties of the strain were studied:the ability to inhibit common intestinal pathogenic bacteria,intestinal adhesion,hydrophobicity and self-cohesion,and anti-cancer ability.The antioxidant capacity of the strain was evaluated from two aspects of in vitro antioxidant and cellular antioxidant.The high-density culture of the strain was optimized.The specific research content is divided into the following parts:(1)60 strains of bacteria were screened from the intestines of healthy infants one-month-old infants in Guangzhou.After preliminary physiological and biochemical identification,7strains of suspected lactic acid bacteria were selected;the strains were exposed to artificial simulated gastrointestinal environment to determine the strains Tolerance of gastrointestinal tract,and using LGG(Lactobacillus rhammosus GG)as a control,finally selected strain B1107-01;B1107-01 was more tolerant than LGG in the simulated gastric fluid of p H 2.0 and the level of tolerance to simulated intestinal fluid equivalent to LGG.Identified by molecular biology,B1107-01 is Lactobacillus rhamnosus.(2)The safety evaluation of LR-ZB1107-01 showed that the strain had no hemolytic activity,no nitroreductase and amino acid decarboxylase activity,and was sensitive to four common antibiotics.Animal oral acute toxicity test results show that LR-ZB1107-01 is safe and non-toxic.The probiotic performance study of LR-ZB1107-01 showed that it has a certain inhibitory capacity against four common intestinal pathogenic bacteria.The inhibitory effect is equivalent to the level of LGG and has a certain intestinal adhesion capacity(13.3±3.2 Cells/cell),and hydrophobicity(48.6%±0.4)>LGG(27.2%±1.2),self-aggregation(86.7%±2.0)is equivalent to LGG(87.5%±1.0).The fermentation supernatant and bacterial suspension of the strain have certain inhibitory ability on the growth and reproduction of Hep G2 liver cancer cells.(3)The antioxidant properties of LR-ZB1107-01 were studied.Antioxidant in vitro indicators include DPPH free radical scavenging rate,hydroxyl free radical scavenging rate and superoxide anion free radical scavenging rate.The results showed that at a cell density of 2×10~8 CFU/m L,the DPPH free radical scavenging rate was:fermentation supernatant(98.71%±1.0)>bacterial suspension(98.13%±1.3)>cell lysate(44.16%±1.7),hydroxyl radical scavenging rate:fermentation supernatant(93.33±4.3)>bacterial suspension(75.70%±0.6)>cell lysate(37.85%±3.0),superoxide anion free radical scavenging rate:fermentation Supernatant(50.53%±1.9)>cell lysate(23.23%±2.2)>bacterial suspension(18.44%±1.1).The results of cell antioxidant experiments showed that the fermentation supernatant and bacterial suspension of the strain also had a certain protective effect on Caco-2 cells against oxygen damage.(4)The high-density culture method of LR-ZB1107-01 was optimized.Optimal concentration of various nutritional factors:4%glucose,3.5%yeast extract powder,4%bacteriological peptone,0.025%manganese sulfate,0.012%magnesium sulfate,0.03%D-isoascorbic acid,0.06%vitamin B,0.03%vitamin C,and 0.04%folic acid(m/v).In the Plackett-Burmen experiment,D-isoascorbic acid,magnesium sulfate,and bacteriological peptone contributed the most to the growth of LR-ZB1107-01,at 40.963%,21.449%,and13.138%,respectively.In the Box-Behnken experiment,a model was established based on three factors:D-isoascorbic acid,magnesium sulfate,and bacteriological peptone.The fitting degree of the equation is R~2=0.9586,which has good credibility degree.When D-isoascorbic acid is0.02%,magnesium sulfate is 0.14%,and bacteriological peptone is 4%,the maximum response value Y(live bacteria)can be obtained.Verification experiments were carried out on the culture medium obtained by this formula,and the results showed that the actual bacterial cell concentration was 9.08×10~8 CFU/m L,which was about 11 times higher than that in the basic MRS medium. |
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