Identification Of A Mannitol-Producing Strain And Optimization Of Its High Yield Conditions

The main function of mannitol from previous studies is as a functional sweetener.It does not have any effect on insulin in the body after digestion in the human body.There are many types of functional sugar alcohols,and mannitol is advantageous for storage due to its non-hygroscopic character and therefore has a significant advantage over other functional sugar alcohols.Modern people have begun to pay more attention to the health of the diet,and mannitol has been widely favored because of its good flavor and harmlessness to the human body.Mannitol production so far has continued to rise.However,the current methods for extracting mannitol are mainly two methods:direct extraction from nature and synthesis of mannitol by chemical means.Both methods are relatively backward.The latest proposed microbial fermentation method due to the fermentation process of mild production conditions and the absence of by-product sorbitol and other advantages,in the future can replace these old production methods.Therefore,in order to adapt to the further research of mannitol's microbial fermentation method and the future industrialization development,it is of great significance to select a strain with high mannitol yield from the laboratory.In this paper,a mannitol-producing strain(cp.20164071)was screened out from the yeast producing biodiesel for production of lipids.The strain was identified as Candida parapsilosis by microbial morphology observation and 18 S rDNA sequence analysis.Through the preliminary fermentation culture of the strain,the biomass,pH,sugar metabolism,nitrogen metabolism and mannitol yield were detected during the fermentation.These would lay foundation for the subsequent optimization of the fermentation conditions.The medium composition for Candida parapsilosis cp.20164071 to produce mannitol was studied.A single factor experiment was used to determine the approximate range of the optimal content of each fermentation medium component.Then the Plackett-Burman experiment was used to find out the significant factors affecting the mannitol production.Then the optimum fermentation medium formula was: glucose 83g/L,yeast extract 30g/L,malt extract 22g/ L et al.,the predicted maximum mannitol production was 51.27 g/L,and the average mannitol production in the validation test was 51.15 g/L,which was similar to the predicted value.After optimization,the yield of mannitol increased 32.24% compared with the previous yield(38.68 g/L).Fermentation conditions for the production of mannitol from Candida parapsilosis cp.20164071 were investigated.Single factor experiments were used to screen out the three main influencing factors of inoculum size,liquid volume and initial p H value.Then the optimum fermentation conditions were as follows:inoculation volume 5%,pH 5.8 and liquid volume 118 mL/500 mL,the maximum mannitol production was 51.20 g/L.Three groups of validation tests were performed under optimal conditions.The average mannitol yield was 51.11 g/L,similar to the predicted value.The optimized mannitol production was 32.37% higher than that before optimization(38.68 g/L).The effects of sugar supplemental and nitrogen metabolism on mannitol production were explored.Through the detection of sugar,nitrogen metabolism and biomass changes in Candida parapsilosis,it was concluded that the first 48 h was an important period for the growth and development of Candida parapsilosis,and the biomass increased significantly.The mannitol production reached a maximum of50.31g/L at 72 h.The sugar-supplemented fermentation was carried out,and sugar was added at 15 g/L at 72 h.The mannitol production increased by up to 60.28 g/L.Under the optimal time of sugar supplementation,the effects of different sugar supplements on mannitol production were further studied.It was found that when the sugar supplement amount was 45 g/L,the mannitol production reached a maximum of65.49 g/L.Compared with unsupplemented sugar fermentation production increased by 30.17%.The influence of amino acids on the production of mannitol by Candida smoothaceans was investigated one by one.Three main influencing factors,glutamic acid,alanine and proline,were selected and optimized by response surface methodology to obtain the optimal culture conditions: alanine 0.042 g/L,proline0.021 g/L,and glutamate at 0.082 g/L,the maximum mannitol yield was 56.281 g/L.Under the optimal conditions,three groups of verification tests were performed.The average mannitol yield was 56.143 g/L,similar to the predicted value,indicating that the regression equation can reflect the impact of various factors on mannitol production more realistically and accurately.The model is in good agreement with the actual situation.