Construct and Aroma-producing Yeast for Kiwi Wine by Protoplast Fusion
|School||Northwest University of Science and Technology|
|Course||Of Food Science|
|Keywords||protoplast fusion kiwi wine aroma-productive yeast single-inactivation|
China is a major country producing kiwi fruit, but only a small fraction of kiwi is processed, which causes farmers to gain little benefit from kiwifruit. Kiwi wine, as a fine processed product, proved to be nutritious and healthy, can provide the kiwi industry high extra value. Therefore, making good use of kiwi resource to produce kiwi wine can alleviate the crisis between supply and demand and so help the kiwi industry lead a healthy way. However, as a result of absence of good yeasts, kiwi wine was evaluated as poor banquet, less fruity aroma, weak typicality, which gav.j kiwi wine bad quality. So, construct good yeasts became a key step to solve the kiwi wine problems. Protoplast fusion had long been proved to be an effective way to industry yeasts breeding because of its high frequency genes integration and recombination. In this paper, protoplast fusion was carried out between a yeast with strong fermentation vigor and a high aroma productive yeast to construct good yeasts for quality kiwi wine. heresults showed that,1.Y1 and Y3 were selected as parental strains in term of ermentation vigor, sugar utilization velocity during fermentation, ysio-chemical analysis of fresh wine and organleiptical evaluation of end product.2.The genetic maker (his") of Yl was obtained by UV-induced mutation. 3.Protoplast eparation procedure:?Culture: grown in liquid YEPD medium (50ml) for 5h at 28 °C on a rotary shaker (150 rpm) according to the growth curve.②Pretreatment: 108 cells were suspended in 3ml pretreatment buffer at 30°C for 15min.③Optimal cells wall enzymatic lysis condition: suspended 20mg/ml snailase in PBS pH 6.0, incubated at 37"C (Yl for 45 min, Y3 for 60 min) with interval gentle shaking. The ratio of protoplasts formation reached to 98.2% and frequency of protoplasts regeneration 20.3% for Yl. 95.3% and 21.3% for Y3. 4. Inactivation condition of Y3 protoplasts was obtained: 80°C for SOmin. S.PEG-induced protoplast fusion was carried out between live Yl(his’) protoplasts and Y3 heat-inactivated protoplasts. Fusants were recovered on YNB minimal medium. The fusion frequency was 10-5.6.Sixty-nine clones obtained were screened for the larger colony, only 10 clones: Fl, F12, F13, F17, F25, F30, F38, F41, F52 and F65 had good fermentation vigor.Further screen work was carried out by kiwi wine fermentation using these fusants. Results showed that F25 and F41 had strong vigor and good aroma producing characteristics.T.Aromatic components GC-MS determination showed that volatile compounds in kiwi wines fermented <WP=7>with parental and fusants varied in kinds and amount. Further principle components analysis showed that difference lied in some esters and phenyl ethyl alcohol.8.Cluster analysis of aromatic compounds in kiwi wine showed that Y3 was donor of aroma.9.Identification results showed that 10 fusants were real hybrids between parental strains and stable during subculture.