Screening And Mutagenesis Of Glucan-producing Lactic Acid Bacteria And Optimization Of Fermentation Conditions

Lactic acid bacteria can utilize exogenous simple carbohydrates and secrete polysaccharides, which could affect the products’ flavor and quality, or achieve prebiotic effect so that the polysaccharides were widely studied and applied. Leuconostoc mesenteroides is a kind of lactic acid bacteria which can produce extracellular polysaccharides and take advantage of sucrose to synthesis glucan under the catalytic of dextransucrase. The glucan from Leuconostoc mesenteroides were widely used in medicine, cosmetic, food and biochemical reagent industry.The main content of this research includes: One high-yield glucan strain of lactic acid bacteria with independent intellectual property rights was obtained through isolation, screening and mutagenesis. Further the enzymatic characteristics of dextransucrase from lactic acid bacteria were studied. Finally, the fermentation medium and parameters for producing glucan were optimized. The main results are as follows:(1) One lactic acid bacteria strain named 2-17 isolated from kimchi juice sample could produce glucan for(5.24±0.14) g/L. The strain was identified as Leuconostoc mesenteroides ssp. mesenteroides by a series of physiological and biochemical tests and analysing the 16 S r DNA sequence.(2) The strain isolated was disposed by NTG and ultraviolet to improve the production of glucan. The optimal composite mutagenic condition was etermined as the following: it was processed under 0.5 mg/m L NTG for 80 min, and then exposured to 45 cm 30 W UV lamp for 30 s, after the strain was cultured in the MRS medium for 9 h. One mutant strain UN2-18 produced glucan for(7.54±0.08) g/L was obtained by being treated the same composite mutagenesis again, the production of which was increased compared with the original strain by 43.89%. After the strain were obtained by continuous subculturing for ten generations, the production of glucan remained at about 7.5 g/L.(3) Dextransucrase from mutant strain UN2-18 exhibited optimum catalytic activity for temperatures 33 ℃and a p H of 5.5. Besides, enzyme activity was stable during 20-30 ℃. Mg2+, Ca2+, Mn2+ enhanced the dextransucrase activity. The low concentration of Co2 + behaved activative effect, while the high concentration inhibited inhibitory effect. Fe2 + showed inhibitory on enzyme activity. EDTA and urea had a stronger inhibition to the enzyme activity. Kinetic parameters of Km was 31 m M, and the maximum reaction rate was 4.5 U/ml. 178 k Da molecular size appeared on SDS-PAGE after reaction in situ and PAS staining.The necessary materials of mutant strain for growth and dextransucrase synthesis included: ① Amino acid: isoleucine, glutamine, methionine, threonine, valine, leucine, phenylalanine, tryptophan, arginine, tyrosine, cysteine, serine, histidine. ② Bases: adenine, uracil. ③ B vitamins including pantothenic acid, lipoic acid, nicotinic acid, folic acid, pyridoxal, riboflavin, whey acid, inosine, biotin. ④ Minerals: K2HPO4, Mg Cl2, Mn SO4.(4) Different degrees of hydrolysis, skimmed milk, soybean meal, whey powder and unhydrolyzed wheat bran and corn starch were chosen as the nitrogen source, and were compared their influence on the glucan production. When the soybean meal was hydrolyzed 10%, the production of glucan was the highest, which was 7.37±0.12g/L. Further single factor was applied to optimize medium composition for producing glucan followed by response surface method. The optimum condition were 9.41% sucrose, 10% degree of hydrolysis of soybean meal 2.09%, 2.09% K2HPO4, when the strain gaven highest conversion rate of sucrose, which was 33.81±0.67% and the glucan production was 31.8±0.63g/L.(5) Using the culture medium optimized, the optimal fermentation conditions in 10 L fermentor were: fermentation temperature was 28 ℃, inoculation size was 4%, rotating speed was 150 r/min, initial p H was 6.0 and was controled for 8 h, and then fermented naturally until 20 h, when glucan production reached 34.15±0.27g/L and the conversion of sucrose was 36.29%.This study obtained one high-yield glucan strain of Leuconostoc mesenteroides subsp. mesenteroides with independent intellectual property rights. Glucan production reached 7.54±0.08g/L after the strain was mutanted. Glucan production reached 34.15±0.27g/L under the optimization of fermentation medium and fermentation parameters and the conversion of sucrose was 36.29%.