Research On Fermentation Production Of γ-poly-glutamic Acid By Bacillus Subtilis

y-poly-glutamic acid (y-PGA) is a kind of amino acid polymers which is made of glutamic acid linked by y-amide linkage. It is water soluble, biodegradable, non-poisonous to human and environmentally friendly. Because of its excellent properties, y-PGA shows a promising development and application in the field of chemical engineering, medicine, food, agriculture, environment, commodities and so on. Currently, y-PGA is mainly produced using microbial fermentation.In the paper, the fermentation conditions of y-PGA produced by Bacillus subtilis is thoroughly studied in both liquid-state fermentation and solid-state fermentation. Fermentation conditions and extraction methods were optimized and the characterization of y-PGA was investigated. Main results are as follows.Firstly, in the liquid-state fermentation, single factor experiments and response surface methodology (RSM) were used to optimize the medium composition and the fermentation conditions. The results showed the optimum medium was as follows: sucrose 31.5 g/L, soy peptone 4 g/L, L-glutamic acid 44.3 g/L, potassium dihydrogen phosphate 0.3 g/L, calcium chloride anhydrous 0.2 g/L and NaCl 3 g/L. The optimized cultivation conditions were decided as follows:initial pH value 7.5, medium volume 30%, inoculums size 7% and temperature 37℃. The maximum yield of y-PGA measured by dry weight method was 18.7 g/L after 48 h under the optimum conditions.Secondly, to improve the y-PGA production ability of Bacillus subtilis furthermore, solid-state fermentation was studied on the basis of liquid-state fermentation using soybean meal as the main fermentation substrate. The composition of the optimal substrates investigated by single factor experiments were as follows: every 100 g soybean meal with 10 g perlite and 150 ml nutrient solution. The nutrient solution contains:sucrose 30 g/L, L-glutamic 40 g/L, potassium dihydrogen phosphate 0.3 g/L and calcium chloride anhydrous 0.2 g/L. The solid-state fermentation got a maximum yied under following conditions:initial pH value 7, inoculums size 5% and temperature 37℃ in 4 days. Under these conditons. the yield of y-PGA measured by dry weight method reached 228.02 g/kg (soybean meal).Thirdly, the method of organic solvent precipitation was used to prepare y-PGA sample. Effects of different factors on the yield of y-PGA were discussed, including volume ratio of ethanol to broth. pH value and y-PGA concentration of broth. To get highest y-PGA recovery from the broth, the extraction procedure should be performed as follows. The y-PGA concentration needed to be kept under 30 g/L without extra pH adjustment. Mixed the broth with ethanol (Vethanol/Vbroth=2.5), and then store the mixture at 4℃ in refrigerator for 24 h. Redissolved the gained precipitation and repeated the abovementioned procedure again until the y-PGA sample is obtained. y-PGA accounted for 78.7% in the sample and the ultraviolet absorption peak of sample y-PGA arises at 207nm.Finally, viscosity, moisture absorption and moisture retention ability were discussed in the paper. Temperature, pH value and ultrasound affected viscosity of y-PGA in different level. Among those factors, high temperature, strong acid and ultrasound influenced the viscosity of y-PGA the most. The three factors decreased the viscosity of y-PGA in relatively short time, in which, ultrasound was a faster and more convenient method. Lower viscosity created by temperature, pH value or ultrasound can solve the problem taken by high viscosity of y-PGA in application. The y-PGA showed a superior moisture absorption-retention performance between glycerol and hyaluronic acid according to the moisture absorption-retention activity test, which was able to keep the moisture in a dynamic equilibrium, thus showed a promising application in cosmetics and commodities.In this paper, the preparation of y-PGA by Bacillus subtilis in liquid-state and solid-state fermentation was studied in detail. The y-PGA production ability of Bacillus subtilis was increased to 18.7 g/L in liquid-state fermentation and 228.02 g/kg soybean meal in solid-state fermentation, compared to the original 5.5 g/L. Soybean meal was applied as main substrate in solid-state fermentation, which improved the yield of y-PGA and provided a new way for the high value utilization of soybean meal as well. The results indicated that the production of y-PGA by solid-state fermentation using soybean meal as the substrate showed great advantage and potential in industrial application.