Screening Of Mannitol-production Strains And The Biosynthesis Of Mannitol

Mannitol is a functional sweetener, which is insulin-independent in human bodymetabolic pathways. It can be made of mouth refreshing agent and prevent dental caries as asweetener. Mannitol is the only one that is not hygroscopic among functional sugar alcohols.With the development of recognition about mannitol and the pursuit of a healthy diet, therewill be a huge demand for mannitol in the future. However, the methods for mannitolproduction nowadays are still stuck in natural extraction and/or chemical synthesis. Mannitolproduction by fermentation has many advantages, such as mild reaction conditions and nobyproduct sorbitol. As a result, mannitol production by fermentation will eventually replacethe conventional production technology in the near future. Thus screening a novel strainproducing mannitol with high yield will be of great importance for commercial process andmarket applications.In this work, strain SK26.001that could produce mannitol was isolated from sugarcanejuice. Based on the morphological observation as well as18S rDNA sequence analysis, strainSK26.001was identified as Candida parapsilosis and named as Candida parapsilosisSK26.001, which was deposited at China Center for Type Culture Collection (CCTCC No. M2012491). The18S rDNA gene sequence includes1446bases and was submitted to GenBankwith the accession number of KF255835.The production of mannitol by the fermentation with C. parapsilosis SK26.001wasstudied from culture medium compositions and fermentation conditions, respectively. Basedon single factor experiment and orthogonal test, the fermentation culture medium wasdetermined. The optimum fermentation culture medium included: glucose200g/L, yeastextract30g/L, CaCl22H2O0.15g/L, FeCl32H2O0.02g/L. The optimum fermentationconditions were: initial pH4, liquid medium volume50mL, inoculum size4%, temperature30oC, rotational speed200r/min. Under the above conditions, the yield of manntiol reached68.5g/L with shaker.Based on the above optimum conditions, the fermentation scale-up experiment wasperformed with strain C. parapsilosis SK26.001in30-liter fermenter. Both batch fermentationand fed-batch fermentation were studied. The results showed that the production of mannitolwas80.3g/L at72h in the30-liter batch fermentation. Mannitol concentration began todecline after72h. In the3-liter fed-batch fermentation the production of mannitol was97.1g/L at120h. The initial glucose concentration was200g/L. Additional glucose was fed forfour times and the total adding amount of glucose was84g/L in the whole process. The totalglucose consumption was284g/L. This experiment laid the foundation for mannitol-industrialized production. The purification method of mannitol from fermentation broth was explored, mainlyincluding the conditions of activated carbon decolorization, ultrafiltration and calciumion-exchange resin. The optimum conditions were: activated carbon decolorizationconcentration3%, temperature40oC, pH5and the reaction time40min. A molecular weight5000-intercept ultrafiltration membrane was chosen for ultrafiltration and the conditions forthe separation of mannitol were studied with calcium ion-exchange resin. The effects ofcolumn temperature, sample size and mobile phase flow rate on separation were studied. Inaddition, the separation curve was drawn to calculate the degree of separation. At last, the bestseparation conditions were determined as temperature60oC, sample size20mL and flow rate1mL/min. Mannitol purify over98.8%and total yield was31.3%under this condition.