Research On Isolation, Identification, Metabolism And High Cell Density Cultivation Of Lactobacillus Bacterias From Tofu Acid Whey

Tofu whey would become tofu acid whey in the process of storage with naturally fermentation. Naturally fermented tofu acid whey was widely used as coagulant in the process of making tofu, due to its good capacity of coagulation. Studies have shown that the security of naturely fermented tofu acid whey was not high, because lots of unknown microorganisms grew in it. In this thesis, two strains of lactobacillus bacterias were isolated from naturally fermented tofu acid whey by using modified MRS medium. And then, their 16 S r DNA sequences and fermentation characteristics in tofu whey were further analyzed. A suitable strain, which had predominant capacity of utilizing tofu whey to produce acids, was obtained. Based on this study, the metabolism and high-density culture of this strain were further evaluated. A safe, efficient way of preparing tofu coagulant was obtained. Main conclusions were as follows:(1) Identification and fermentation performance of isolated strains were studied. A suitable strain, with the name of Lactobacillus amylolyticus L6, which had predominant capacity of utilizing tofu whey to produce acids, was obtained. Two strains of bacteria, which named L5 and L6 respectively, were isolated from naturally fermented tofu acid whey by using modified MRS medium. And then, they were identified through their 16 S r DNA sequences. The results showed that both of them were Lactobacillus amylolyticus. All of them were active in the range of 37~47 ℃. Among of them, the high temperature resistance of strain L5 was better. Strains showed strong ability of utilizing tofu whey to produce acids. The titratable acidity of tofu whey fermented by strain L6 for 48 hours at the temperature of 42 ℃ reached 110 oT. In addition, the titratable acidity of tofu whey fermented by strain L5 for 48 hours at the temperature of 47 ℃ reached 90 oT.(2) The carbohydrate metabolism of Lactobacillus amylolyticus L6 in tofu whey was studied. And also, the biological conversion of isoflavones, antioxidant activities of fermented tofu whey and the correlation between them were researched. The content of fructose, glucose, sucrose, raffinose, stachyose, was 0.36 g/L, 0.46 g/L, 5.16 g/L, 1.80 g/L and 5.48 g/L respectively. All of Fructose, glucose and sucrose were consumed in tofu whey, when fermented by strain L6 for 36 hours under the temperature of 42 ℃. After fermentation for 36 hours at the temperature of 42 ℃, the content and availability of raffinose and stachyose were 0.95 g/L, 2.73 g/L and 52.78%, 49.82% resprctively. The amount of lactic acid increased 18.6 times, up to 5.21 g/L, with the most rapid increase. Tofu whey was rich in soy isoflavones, with the content of 238.16 mg/L and 90% of the total content of five kinds of isoflavone was in the form of glycoside. The percentage of isoflavone aglycones increased to 37%, with the content of daidzein increased 4.4 times; the content of genistein increased 35.3 folds, after fermentation by strain L6 at the temperature of 42 ℃ for 36 hours. Antioxidant activities of tofu whey and fermented tofu whey were measured using FRAP, DPPH·, ABTS+ and Iron(II)-chelating assaies. The antioxidant activities of fermented tofu whey were found to be higher than that of tofu whey. In addition, the antioxidant activities of tofu whey increased with the extension of fermented time. The Pearson’s correlation analyses(P<0.05) showed that the content of isoflavone aglycones was positively related to the abilities of reducing Fe3+, scavenging DPPH· and ABTS+. However, there was no influence on the ability of Iron(II)-chelating.(3)The proliferation medium and the conditions of proliferation of Lactobacillus amylolyticus L6 were researched. Tofu whey was used as based medium, the effects of seven additives, which include peptone, yeast extract, corn starch, glucose, sodium acetate, ammonium citrate dibasic and tomato juice, were evaluated by using a two-level partial factorial design(27-22). Results showed that peptone, yeast extract and corn starch were the main affecting factors, while the other additives had no significant effect on the proliferation of strain L6(OD600 value). Then, the steepest ascent experiment, the rotatable central composite design and response surface analysis were used to determine the optimal levels of the main factors. The optimized conditions were: tofu whey supplemented with 0.82%(w/v) peptone, 1.07%(w/v) yeast extract and 2.2%(w/v) corn starch. In the optimized medium, the growth curve of strain L6 and suitable temperature for proliferation was studied. It resulted in cell production of strain L6 to be increased to 4.5×109 cfu/ml at the temperature of 42 ℃ for 12 hours.Finally, a suitable strain named L6, with prebiotic characteristic, which had predominant capacity of utilizing tofu whey to produce acids, was obtained. It was identified through its 16 S r DNA sequences. The results showed that it was Lactobacillus amylolyticus. Fructose, glucose and sucrose were priority used by strain L6, in the tofu whey, with large amount production of lactic acid. The fermentation of Lactobacillus amylolyticus L6 could promote isoflavones to convert from the form of glycoside to aglycones(Daidzein and Genistein). The proliferation medium and the conditions of proliferation of Lactobacillus amylolyticus L6 wree further researched for the application of the foundation.