The Study On Constructing A High-producing Erythritol Strain By The Treatment Of UV And Electrofusion

Erythritol is a natural existed four-carbon sugar polyols ,chemical name is 1,2,3,4-butanetetrol, molecular formula is C₄H₁₀O₄ and molecular weight is 122.22, borling point is 329-331℃,melting point is 126℃,its appearance is white and crystal powder .It can absorb more energy when it is soluted in water, dissolution is -97.4J/g, erythritol has a positive enthalpy of solubilization , thus providing a strong cooling effect in ingestion . It can give a better tasty.Erythritol occurs widely in nature and has been found naturally in several foods including mushroom , watermelon, pear, grape, wine, sake ,beer ,soy sause and fermented foods. It has a 60%-70% sweetness relative to sucrose and it is a good low-calorie sweetener .It can not be used by mutation streptococcus. Erythritol has been used safely as a noncariogenic sweetener in many countries, owing to inability of erythritol metabolism in cariogenic organisms. More than 90% of ingested erythritol is not metabolized by the human body and is excreted unchanged in the urine without changing blood glucose and insulin levels. Therefore,it could be used advantageously in special foods for people with diabetes and fatness. It is a non-caloric and non-cariogenic sweetner, Erythritol has great potential for application in food.The nature determination of high erythritol-producers was obtained from erythritol-producing strains that were isolated in lab by Ultraviolet radiation treatment .Different strains were fused by means of protoplast fusion .High-producing engineering microorganisms and the growth condition of the strain were studied deeply in the experiment.Examinations were compared under various cultivation condition in the while of activating, collection and screening on the erythritol-producing strains. Plobing a method with a special coloring-matter of TCL , that is, made a piece of normal TCL through the normal sample spoting samples with gradient and certain quantity. In the investigation , just requiring a result of TCL with samples that comparing with normal-board. So we can analized the erythritol that came from the sample with the qualitative and quantitive methods.We designed a infinite highway to select the high-producing erythritol strains in the mutable strains and selecting aiming-fusants among the plentiful inosculate strains in the experiment. Fixing on the condition of high-producing erythritol strains in thetreatment of UV. The growth-condition of the high-producing erythritolstrains achieved from the protoplast fusion were investigated on the temperature, cultural-time and pH. At last ,making a certain on the cultivate-condition of the high-producing erythritol strains of Candida RH-UV-L4-F9 and its cultivate-temperature is 32°C,cultivate-time:6 days, the pH of initial-culture is 5 6 .The optimal concentration of glucose is 20%,Nitrogen-source: 0.5% yeast extract and 0.1% carbamide. In conclusion, the culture condition of microzyme is studied, erythritol content in the cultivated supernatant is 120.6mg/g,which increased 3.61 times than the initial strain.In the experiment, we make some breakthrough in the factors of effect formation of protoplast, regeneration and parameters of electrofusion. The outcome proves that enzyme's chroma, the time of disposal by enzyme, the optimal temperature. pH osmotic pressure, the choice of steady matter have a great effect in the formation of protoplasts and regeneration. On the base of every effect factors, we do single-factor experiment, and emply orthogonal test to ensure the optimal condition in the formation of protoplasts regeneration. At the same time, this study employs the method of electrofusion to complete protoplast fusion, and this avoids employing chemical method of fusion produce poison cell, and increase the control of reaction and the rate of fusion., this make a solid ground in further study and high-production erythritol. However, if we want get more erythritol from it , it must be study further.In a word, this study make a steady basis for constructing gene-enginerring erythritol which can produce plenty of erythritol.