Fermentative Production Of Glutathione By Candida Utilis Using The Hydrolysate Of Banana Peels As The Substrate

Glutathione, as a tripetide of glutamate, cysteine and glycine, is one of antioxidants abundant in organism. It plays an important role in maintaining the normal cellular redox environment. So it is widely used in medical, cosmetic, food and health products. Lignocellulosic materials, such as banana peels, are low price renewable resources.It cuts the cost of fermentation using the hydrolyzate of banana peels as the carbon source, which also benefits to the reasonable treatment on banana peels. Based on the fact that the Candida utilis has the ability to utilize hexose and pentose degraded from lignocellulosic biomass, the GSH fermentation on the banana peels by Candida utilis was carried out in this thesis. The conditions of GSH fermentation on xylose were optimized by statistical methods and we drew the conclusion that the fermentable sugars in hydrolyzate of banana peels were an ideal carbon source. Then the production conditions of fermentable sugars were optimized by the uniform design and the high efficiency fermentable sugars were obtained. Further studies on the cell growth and GSH production by the different dissolved oxygen were carried out in the last chapter of this thesis. The main conclusions were as follows:(1) The analysis on banana peels showed that it contained massive carbohydrates and little crude protein and fat. Glucose, mannose, xylose and arabinose were found in the hydrolyzate of banana peels by the HPLC. Based on the single factor experiments, response surface methodology was applied for optimization the GSH fermentation condition on the xylose, and the optimal conditions were as follows: xylose 20 g/L, (NH₄)₂SO₄ 9.59 g/L, KH₂PO₄ 3 g/L, L-methionine 5.72 g/L , MgSO₄ 0.20 g/L, temperature 30℃, volume of medium 50 mL. The GSH yield on the optimized conditions was improved by 80.09% compared to the yield on the starting medium.(2) Glucose, mannose, arabinose and mixed sugars, were further investigated as the sole carbon source based on the medium compositions obtained with xylose. It was shown that C. utilis SZU 07-01 could also grow well on the medium with the sole carbon source of glucose and mannose at 20 g/L. However, arabinose cannot be well utilized by the yeast for GSH production, and the fermentation results of the mixed sugars were similar to the mannose. Then the hydrolyzate of banana peels was investigated in the further study and we found that DCW and GSH could be achieved at 7.92 g/L and 116.9 mg/L, respectively, which were improved by 41.4% and 126.5% compared to the results of mixed sugars. Based on the results above, it was shown that the hydrolyzate of banana peels was a good novel substrate for the GSH production by C. utilis SZU 07-01 for both the high DCW and GSH yield.(3) In order to investigate the process of fermentable sugars based on the yield of reducing sugars and the yield of GSH, the single factor experiments were applied for investigating the effects of substrate loading rate, hydrolysis time, H2SO4 concentration and temperature. Then uniform design was applied to optimize the key parameters influencing the hydrolysis of banana peels by dilute sulfuric acid. The optimized conditions of hydrolysis were obtained by the analysis of regression respectively: (a) based on the yield of reducing sugars: substrate loading rate 17.4%(w/v), hydrolysis time 26.6 min, H2SO4 concentration 1%(v/v), temperature 120℃; (b) based on the yield of GSH: substrate loading rate 14%(w/v), hydrolysis time 45 min, H2SO4 concentration 1%(v/v), temperature 118℃. The application of banana waste for the production of glutathione will also give a good reference for other useful chemical compounds using lignocellulosic material as substrate.(4) The effects of dissolved oxygen on the cell growth and GSH yield in flasks under the different agitation rates and medium volumes were investigated by C. utilis SZU 07-01 on the hydrolyzate of banana peels. Then the further studies in batch fermentation were carried out. All the above results showed that DCW and GSH were improved by the elevation of dissolved oxygen.(5) The consumption of fermentable sugars were investigated in the batch fermentation and it was found that glucose was the preferential carbon source, the xylose and mannose were inferior to glucose, however, the efficiency of arabinose was the lowest. When medium contained glucose, it would inhibit the utilization of the other sugars. Furthermore, glucose and xylose could be consumed quickly under lower dissolved oxygen, but the DCW and GSH yield were lower than the higher dissolved oxygen. Then analysis of DCW and GSH under different dissolve oxygen together with the fermentation kinetic models from different aspects was applied to explain the results.