1. Types of Native Fermentations Linda F. Bisson Wine Flavor 101 January 2016

2. Native Fermentations: Definitions The term “native” with respect to wine production has several meanings: –Not deliberately inoculated –No commercial yeast strains present –Only vineyard yeast present –Vineyard/winery resident yeast only present –No manipulation of native microbiota –Only autochthonous yeast present

3. Source of “Native” Flora  Vineyard  Winery  Early in season grape microbiota may be most important but as microbes become established on winery surfaces, winery microbiota become more important  Genetic data suggests winery microbes likely over- winter in the winery and build up from winery, not vineyard, biota  And it all depends upon sanitation practices! Both are sources of microbes

4. Native Fermentations: Goals  Complexity, complexity, complexity  Non-Saccharomyces yeast and bacteria able to contribute to aroma, flavor and mouthfeel properties of the wine  Slower fermentation of Saccharomyces contributes to aroma and flavor profile of wine  Wines are more unique and appealing than they would be using a “generic” strain  Enhanced varietal character due to hydrolases and other enzymes produced by a diverse microbiota or to production of aroma-enhancing compounds (matrix effects)

5. Not Deliberately Inoculated  No commercial inoculum  No tank-to-tank inoculum  May be “inoculated” by winery biota –Tank/barrel residents –Hoses/equipment residents  Depending on sanitation practices may be conducted by vineyard biota

6. No Commercial Yeast Strains Present  Winery has never used commercial starters (ever)  Winery has not introduced materials (bulk wines; used equipment) that has seen commercial starter cultures  Winery may take steps to not be accidentally “inoculated” by natural starters from adjacent wineries  Yeast lees returned to vineyard?

7. Only Vineyard Yeast Present  Challenging: requires intense sanitation program to eliminate winery residents each year  Vineyard yeast variability from year to year both in numbers and types of strains should be evident  Winery lees may or may not be put back in the vineyard

8. Vineyard/Winery Resident Yeast Only Present  Recognizes challenges of not developing winery “house” strains  House strain lees may be added back to vineyard so origins the next season may be confounded

9. No Manipulation of Native Microbiota  No use of sulfur dioxide or other antimicrobial  No additions of nutrients that would alter microbial dynamics  No oxygen addition except that occurring during transfers  No temperature control that would alter microbial dynamics

10. Only Autochthonous Yeast Present  Autochthonous: “originating in the place found”  Strains present are not just found in the wild but originated in that region  Challenging with wine yeast as yeast DNA analyses have shown “migration patterns” for specific strains  Issues: –If one generates a “house autochthonous” strain and uses it as an inoculum, is it still “native”? –Should “heritage yeast” be its own category of fermentation type?

11. The Negative Impacts of Native Microbiota  Arrest of alcoholic/ML fermentations  Spoilage character formation –From the non-Saccharomyces microbes –From Saccharomyces due to enhanced competition for nutrients and growth factors  Loss of varietal character –Degradation of varietal aroma/flavor compounds –Modification of varietal aroma/flavor compounds –Masking of varietal impact compounds

12. When Are Negative Impacts Likely?  Condition of fruit: high incidence of rot/damage in vineyard  High insect presence  Lack of a robust native strain of Saccharomyces ( a trade-off between sanitation practices and use of “house” strains  Deficiencies in grape composition: nutritional stress  Undesirable vineyard residents: the dreaded “bad lactics”

13. Condition of the Fruit  Damaged Clusters –Rot amplifies acetic acid bacteria –Mold metabolites may be present  Uneven Ripening/Raisining –Leads to differences in flora on the surfaces of the fruit  Presence of Material Other than Grape –Soil, leaves, bark: all contain microbes –Some of these microbes can persist early in fermentation

14. Timing of Harvest  During ripening the berry surface flora change –More seepage from the berry providing nutrients –Yeast population continues to increase in relative numbers post- veraison  Aerobic basidiomycetes are replaced by the fermentative ascomycetes –Consume available oxygen rapidly –Create localized anaerobic zones –Produce toxic waste products  Aerobic bacilli and pseudomonads replaced by acetic and lactic acid bacteria –Produce toxins and toxic waste products

15. Factors Impacting Biota and Persistence  pH  Temperatures of holding or processing: everything is a selection  Oxygen exposure  Nutrient level and diversity of nutritional components  The starting biota and strains present

16. Native Fermentations: Styles  100% native: no additions of any microbes ever  Native yeast but with ML inoculation  Native Saccharomyces only or native non- Saccharomyces? (manipulate biota using sulfur dioxide)  Partial natives: inoculated at some point (the insurance policy method)  Manipulated microbiota “natives”

17. Manipulated Microbiota  Sulfur dioxide or other antimicrobial agent used  Temperature of pre-incubation/fermentation  pH adjustment or selection  Level of aeration  Nutrient addition practices  Inoculation with non-Saccharomyces yeasts and bacteria

18. Partial Natives  Temporal inoculation: after x days  Inoculation at a specific Brix/ethanol level  Inoculation at “first sign of trouble”  Addition of SO 2 to arrest non-Saccharomyces biota  Selective feeding of Saccharomyces population

19. The VEN124L Trial:  Test the impact of timing of inoculation of a commercial strain of Saccharomyces  Used Albariño  No sulfur dioxide  No nutrient additions  Used a neutral yeast: EC1118

20. Treatments ● Control: inoculated with EC1118 at 0 hours ● 24hrs: inoculated with EC1118 at 24 hours ● 48hrs: inoculated with EC1118 at 48 hours ● 72hrs: inoculated with EC1118 at 72 hours ● 96hrs: inoculated with EC1118 at 96 hours ● Native: no inoculation, left to ferment with native yeast

21. Fermentation Data

22. Inoculation Timing Tasting:  Glass 1: Inoculated Time 0  Glass 2: Inoculated 24 hrs.  Glass 3: Inoculated 48 hrs.  Glass 4: Inoculated 72 hrs.  Glass 5: Inoculated 96 hrs.  Glass 6: Uninoculated