Microorganisms of Juice: Managing Competition in the Tank

1. Microorganisms of Juice: Managing Competition in the Tank Lucy Joseph U.C. Davis Department of Viticulture and Enology

2. Managed Ecosystem

3. Ecological Succession in Wine Fermentation

4. Which Microbes Are Found? –Vineyard to Must

5. Factors Affecting Grape Microflora • Moisture/Humidity • Insect Vectors and Damage • Temperature • Vineyard Management Practices • Variety of Grape • Geography

6. Environmental Conditions that Affect the Microflora of Must • pH - Low • Nutrients - high sugar, variable nitrogen • Temperature - fermentation creates heat • Oxygen - fermentation is low oxygen • Inhibitors - pesticides, sulfur, alcohol, acetic acid, etc. • Winery Practices - additions, sanitation, stylistic considerations

7. Fungi found on Grapes • Botrytis cinerea – bunch rot • Plasmopara viticola – downy mildew • Erysiphe necator – powdery mildew • Penicillium – green mold • Aspergillus – black mold • Alternaria – black smut • Cladosporium – post-harvest • Rhizopus – soft rot

8. Bacteria in the - Vineyard Must Lactobacillus Lactobacillus Lactococcus Pediococcus Enterococcus Weissella Weissella Gluconobacter Gluconobacter Acetobacter Pediococcus Gluconacetobacter Oenococcus Leuconostoc Oenococcus

9. Yeast in the – Vineyard Harvested Grapes Zygosaccharomyces Hanseniaspora Hanseniaspora Metschnikowia Issatchenkia Candida Kluyveromyces Metschnikowia Pichia Candida Rhodotorula Sporobolomyces Sporidiobolus

10. Yeast Found in Must • Hanseniaspora uvarum, guilliermondii, opuntiae • Metschnikowia pulcherrima • Candida zemplinina, glabrata, diversa • Pichia anomala, fermentans, guilliermondii • Clavispora lusitaniae • Zygosaccharomyces bailii • Kluveromyces thermotolerans • Kazachstania species • Issatchenkia occidentalis, orientalis, terricola • Saccharomyces cerevisiae

11. Saccharomyces in the Vineyard • Saccharomyces occurs in only about 1 sound berry in 1000 tested • In damaged berries that increases to about 1 in 4 berries tested • The number of Saccharomyces cells on damaged berries is about 104 to 105 cfu/ml • The total microbial counts in damaged berries is 106 to 107 cfu/ml

12. Typical Saccharomyces Growth Curve vs Typical Wine Fermentation Curve

13. Saccharomyces and Non-Saccharomyces Growth Curves vs Typical Wine Fermentation Curve

14. How Certain Groups are Selected Natural selection: • Low pH • High sugar concentration – high osmolarity • Other nutrients • High phenolic content • Low oxygen • Alcohol concentration

15. How Certain Groups are Selected Artificial Selection: • Temperature • Potassium metabisulfite • pH - Tartaric acid • Lysozyme • Nutrients

16. What Are Some “Best Practices”? • Harvest brix less than 27o brix • Harvest acid content – pH 3.2 to 3.6, TA 0.6 to 0.8 • SO2 added at the crusher • Temperatures less than 30oC • Pump overs to manage temperature oxygen levels • Addition of nutrients only as needed

17. Why Do Best Practices Work?What do they control?

18. Acid Adjustment • High acid favors growth of yeasts early in fermentation • Most microbes, especially bacteria, are not acid tolerant • pH often increases during fermentation which favors the ML fermentation • pH greater than 3.6 encourages spoilage lactics • Brettanomyces is more tolerant to low pH

19. Addition of SO2 • Inhibits the growth of spoilage bacteria • Inhibits growth of wild yeasts including Brettanomyces • Destroys thiamin • Inhibits oxidation

20. Inoculation • Directly adds the desirable organism in high numbers

21. Engineering Practices • Adjusting temperature • Cold soaks • Tank temperature, jacketed • Pump overs • Adjusting oxygen – Cap Management • Pump overs – Rack and return • Punch down • Stirring or aeration • Micro-ox

22. Temperature • Cold soaks (15-20oC) encourage growth of non-Saccharomyces yeast early in fermentation • Cool temperatures during fermentation inhibit growth of spoilage bacteria and some yeasts • Warm temperatures can favor ML bacteria • Cool storage temperatures discourage spoilage organisms during storage

23. Low Oxygen • Strict aerobic organisms cannot compete under low oxygen conditions • Filamentous fungi • Acetic acid bacteria • Anaerobic and facultative anaerobes grow under low oxygen • Saccharomyces • ML bacteria

24. Risks and Rewards • Long hang times of fruit allow for development of desirable flavor and color and adds complexity • Over doing it results in higher sugars that can produce too much alcohol that can arrest fermentation, leave residual sugars, and leave nutrients for spoilage organisms • Acidity may also suffer and be too low allowing spoilage organism to flourish

25. Risks and Rewards • Cold soaks can allow growth of wild yeasts that produce desired esters and other flavor compounds that add complexity • Typically yeasts like Hanseniaspora, Pichia, and Candida occur • Wild yeasts can also produce acetic acid and ethyl acetate in large amounts and this can be particularly risky with damaged fruit

26. Risks and Rewards • Low oxygen prevents the growth of strict aerobic organisms like acetic acid bacteria and filamentous fungi • Too much oxygen allows a bloom of aerobic organisms that often occur as a film on the surface of the wine • Micro-ox done incorrectly in the presence of microbes, for example wood, results in the bloom of atypical microbes in wine

27. Case Study 1 • A winemaker approached Dr. Bisson with a wine that had a mousey taste and low bacteria numbers by microscopic observation. No bacteria were found by plating or QPCR. • The wine was filtered and filters plated on both bacterial (MLAB) and yeast (YM and WL) media.

28. Grenache Wine

29. Grenache Filter on Plate

30. What Is Causing the Contamination? • There are many sources of bacterial contamination including: air, dust or soil, grapes, wood, water, and corks. • The wines were typical except that the SO2 level was low to none. • The wines showed contamination with many species of bacteria. • All of these wines were treated by micro-oxidation in the presence of wood.

31. What Is Causing the Bacillus Contamination? In bottling lines, Bacillus spores can survive heat treatments that have replaced chemical sanitation measures. Bacillus is a strict aerobe but is typically acid sensitive Ethanol is effective at inducing sporulation in Bacillus

32. Case Study #2 • Wine was returned from the distributor due to high turbidity. Wine had no aroma or flavor defect, only high turbidity in some bottles. • Bacteria were visible under the microscope but did not grow on plates and QPCR came back negative.

33. What was in the wine? • Initial plating indicated Bacillus but the Bacillus didn’t look like what we saw under the microscope • It might be Bacillus spores • Looking further we also found moderately high levels of Staphylococcus pasteuri

34. The wine • The wine was sweetened with added juice. I don’t know if the juice was filtered. • Synthetic corks were used. • The pH of the wine was high. • The bottling line was cleaned with only hot water, bleach was no longer being used.

35. Best Practices • Developed over time because they work • How and why they work has not always been understood • Significant deviation from the best practices rarely goes well