The Dynamic Changes Of Yeast Populations During Icewine Fermentation And Screening Of The Yeast Strains

Icewine is the best in all wines,it has the characteristics of taste lubrication,sweet and mellow,rich aroma and so on.The growth of ice grapes as an ingredient in producing icewine has strict requirements on climate and latitude.Icewine is produced in only a few countries such as Canada,Germany,Austria,and China.At present,Vidal blanc is widely used for producing icewine,as it has high resistance to harsh climates,high rate of juice and gives relatively stable yields.Vidal blanc icewine is considered the highest quality icewine because of its appealing aroma and taste(pineapple,apricot,honey,et al.).Due to the high osmotic pressure in ice grape juice,in general,yeast cannot be growth,reproduction and fermentation.Therefore,the yeasts used in icewine production are imported from other countries,and icewine production enterprises in China don't have the indigenous yeasts which are core technology for producing icewine.A lot of money is spent every year to buy commercial yeasts to produce icewine,which are seriously restricted the development of China's icewine industry and products quality improvement.In this study,Vidal blanc ice grapes(Wunvshan&Sun Valley)from Liaoning province were used as experimental materials,the yeast diversity and dynamics associated with the fermentation of Vidal blanc icewine was investigated.The tolerance,the fermentation performance and ?-glucosidase activity of yeast strain was researched,which is in order to screen excellent Saccharomyces and non-Saccharomyces yeast strains.In this study,the diversity of yeasts in the icewine fermentation process was clarified,and the succession of yeast populations was revealed.The screening of the indigenous yeast strains with excellent performance,which is a theoretical foundation and technical support for icewine brewing industry in China.The main research results are as follow.First of all,the yeast diversity and dynamics associated with the spontaneous fermentation of Vidal blanc icewine was investigated by morphological identification method.Samples were collected from the different stages of the icewine fermentation and analyzed using culture-dependent(Wallerstein laboratory nutrient agar)method,and then the isolates were identified by sequencing the internal transcribed spacer(ITS)region and the 26S rDNA D1/D2 domain genes.Aureobasidium,Candida,Cryptococcus,Curvibasidium,Debaryomyces,Filobasidium,Hanseniaspora,Metschnikowia,Pichia,Rhodotorula,Saccharomyces,Sporidiobolus,Sporobolomyces,Torulaspora,Zygosaccharomyces,Zygoascus were detected in samples of "Wunvshan" and "Sun Valley".During the spontaneous fermentation of "Wunvshan" icewine,Candida,Hanseniaspora,Metschnikowia,and Rhodotorula were predominant in the initial stages,Candida,Metschnikowia,and Saccharomyces dominated in the middle stages,and Saccharomyces was predominant in the final stages of the fermentation.During the spontaneous fermentation of "Sun Valley"icewine,Candida,Cryptococcus,Filobasidium were predominant in the initial stages,Candida,Pichia,and Saccharomyces dominated in the middle stages,and Candida and Saccharomyces were predominant in the final stages of the fermentation.During the inoculated fermentation,Saccharomyces was predominant all the time.The second,the samples were also analyzed using culture-independent method(high-throughput sequencing,HTS).There were sixty-eight genera in "Wunvshan" samples:twelve genera of wine yeasts(Saccharomyces,Candida,Zygosaccharomyces,et al.),and Aureobasidium,Mrakiella,Mrakia,and so on.while there were thirty-six genera in "Sun Valley" samples:eight genera of wine yeasts(Saccharomyces,Candida,Cryptococcus,et al.),and Aureobasidium,Davidiella,Alternaria,and so on.During the spontaneous fermentation of "Wunvshan" icewine,Cryptococcus and were predominant in the initial stages,Candida,Cryptococcus,and Rhodotorula dominated in the middle stages,Metschnikowia and Saccharomyces was predominant in the final stages of the fermentation.During the spontaneous fermentation of "Sun Valley" icewine,Cryptococcus and Filobasidium were predominant in the initial stages,Candida and Saccharomyces dominated in the middle stages,and Candida and Saccharomyces were also predominant in the final stages of the fermentation.During the inoculated fermentation,Saccharomyces was predominant.The yeast dynamic changes are directly related to the comsuption of sugar and the production of acohol in ice grape juice.In the initial stages,non-Saccharomyces yeasts with high tolerance of sugar were predominant;then,Saccharomyces yeast was gradually dominated,non-Saccharomyces yeasts were declined;in the final stages,Saccharomyces with high tolerance of acohol was predominant.The third,the tolerance of non-Saccharomyces yeasts strains(Debaryomyces?Metschnikowia,Hanseniaspora,Pichia,Torulaspora and Candida)and Saccharomyces cerevisiae were studied.The tolerances of Saccharomyces cerevisiae strains isolated were well.Moreover,the fermentation performances of S.cerevisiae strains were studied.An excellent S.cerevisiae strain with fast fermentation speed and low volatile acid content was screened.Moreover,?-glucosidase activities of non-Saccharomyces yeasts strains were studied.As a result,C.railenensis was better than the other non-Saccharomyces yeasts.The application of C.railenensis in icewine fermentation has not been reported,but its?-glucosidase activitie was high,indicating that it has practical application significance.Finally,the mixed culture fermentation(C.railenensis and S.cerevisiae)and the single culture fermentation(S.cerevisiae)were carried out;the dynamic changes of yeast populations during the icewine fermentation,and the difference of metabolites and aroma of icewine were studied.During the single culture fermentation,S.cerevisiae was increased firstly and then went down.During the mixed culture fermentation,C.railenensis didn't change much in the first 48 hours;after S.cerevisiae joined,C.was increased slightly,but C.railenensis wasn't found after 14 d.The difference of metabolites and aroma of icewine was researched by HS-SPME-GC-TOFMS(HeadSpace Solid Phase MicroExtraction Gas Chromatography Tandem Time-of-Flight Mass Spectrometry)and the electronic nose technologies.There were 600 metabolites in all,and there are 248 different metabolites in between the mixed culture fermentation and the single culture fermentation.The 53 important metabolites related to ice wine aroma were selected in the 248 different metabolites.The relative contents of 13 substances,such as benzaldehyde,acetoin and beta-damascenone,these in the mixed culture fermentation were significantly higher than that in the single culture fermentation;on the contary,the relative contents of 40 substances(1-butanol,ethyl acetate,et al.)in the single culture fermentation were higher than that in the mixed culture fermentation.The two groups showed significant differences in aroma performance.C.railenensis participating in the fermentation of S.cerevisiae inoculated also reduced alcohol and volatile acid contents of icewine.The icewine aroma of the mixed culture fermentation and the single culture fermentation were detected by the PEN3 electronic nose and sensory evaluation.The results showed that the aroma substances of the mixed culture fermentation icewine were much richer than those of the single culture fermentation icewine.The mixed culture fermentation icewine has a much stronger aroma and taste than the single culture fermentation icewine in the aroma of roasted caramel and nuts.The single culture fermentation icewine has a stronger aroma than the mixed culture fermentation icewine in the aroma of tropical fruits.In this study,C.railenensis and S.cerevisiae strains with good performances of tolerances and fermentation were screened,and they might be able to use for icewine fermentation.