1. SELECTION OF ANTIMICROBIALS Linda F. Bisson Department of Viticulture and Enology Quality Control Management during Crush and Fermentation August 7, 2014

2. Use of Antimicrobials in Wine Production Control of unwanted populations during processing Control of timing of wanted populations Elimination of microbial activity prior to bottling

3. Types of Microorganisms Yeast Gram positive bacteria Gram negative bacteria

4. Yeast in Juice Similar tolerances to high sugar concentrations Non-Saccharomyces yeasts more tolerant of low temperatures than Saccharomyces and less tolerant of higher temperatures Saccharomyces more tolerant of sulfur dioxide Equivalent tolerances to low pH across non- Saccharomyces and Saccharomyces yeast Differences in ability to consume nutrients and oxygen from the fermentation Similar nutritional needs

5. Bacteria in Juice Gram positive Thick peptidoglycan layer outside cell plasma membrane Gram negative Thin peptidoglycan layer Peptidoglycan sandwiched between two lipid bilayers: inner and outer membranes Gram positive and gram negative bacteria are sensitive to different inhibitors that attack the cell surface Lactic acid bacteria are gram positive Acetic acid bacteria are gram negative

6. Factors Controlling Levels of Microbial Populations pH Temperature Oxygen Nutrients Other organisms: Natural bio-control

7. pH Juice pH value range (2.8 to 4.0+) support growth of yeast with growth higher and higher pH values within this range Juice pH below pH 3.5 favors acetic acid bacteria and Oenococcus (of the lactic acid bacteria) Juice pH above 3.6 enables growth of lactic acid bacteria with the higher the pH in this range permitting a larger diversity of lactic acid bacteria to proliferate

8. Temperature Saccharomyces grows and metabolizes over the range of 12 to 42°C ( 53 to 107°F) Temperature tolerance is reduced at higher ethanol levels Strains vary in tolerance of temperature shifts: depends upon ability to adapt to the new temperature Lactic acid bacteria typically grow at temperatures ranging from 18 to 48°C (64 to 118°F) but varies by strain

9. Oxygen Yeast and bacteria both grow better in the presence of low levels of oxygen Oxygen restriction best mechanism of controlling acetic acid bacteria Saccharomyces very efficient at oxygen stripping from juice if metabolically active due to proton motive force created across their plasma membrane If Saccharomyces metabolism is inhibited, membrane proton motive force may be reduced allowing bacterial populations to proliferate

10. Nutrients Yeast and bacteria use the same nutrients Yeast are focused on generating energy from sugars Lactic acid bacteria are focused on generating energy from proton movements (organic acids and amino acids serve as energy sources) Acetic acid bacteria are focused on generating energy from partial oxidation reactions (ethanol to acetic acid) Populations that dominate early will consume available nutrients and may not re-release them to the environment Can use differential feeding to control populations and microbial interactions

11. Other Organisms Competition for nutrients Production of inhibitors Acetic acid for yeast Ethanol for non-tolerant organisms Bacteriocins Short chain fatty acids Inhibitory peptides: killer factors Cell number inhibition: “crowding” high populations inhibit other organisms perhaps by changing redox conditions of juice

12. Microbial Inhibitors Approved for Use in Wine Lysozyme DMDC(DiMethylDiCarbonate)/Velcorin SO 2 Heat treatments

13. Lysozyme Effective against gram positive bacteria Hydrolytic enzyme attacking cell wall Grape tannin can interact with lysozyme and inactivate it Loss of lysozyme and tannin can impact color stability in reds Effective concentration of lysozyme varies from 250 to 1000 PPM Due to levels of microbes present Due to complexing/binding components in juice/wine Source is egg whites but found in a variety of mammalian tissues and secretions like tears

14. Lysozyme Is an allergen and certain countries require labeling Residual lysozyme leads to haze formation particularly in white wines Ineffective against acetic acid bacteria and yeast Some lactic acid bacteria strains are resistant/tolerant of lysozyme Enables use of lower SO 2 concentrations

15. Lysozyme: Uses Delay malolactic fermentation: inoculate with more resistant strain later Prevent wild malolactic fermentation: inoculate with more resistant strain later or remove with bentonite treatment Prevent any malolactic fermentation Stabilize wine post malolactic fermentation In juice inhibit “bad” lactic acid bacteria Treat a stuck fermentation (remember that if populations are high a high concentration of lysozyme might be needed)

16. Lysozyme Inactivation Removed by bentonite Complexes with tannin: can be inactivated by tannin additions Removed by silica sol Removed by oak chips Removed by carbon fining

17. DMDC/Velcorin Effective mostly against yeast, bacteria are more resistant Crossed plasma membrane and reacts with and inactivates proteins and nucleic acids Hydrolyzes rapidly in juice and wine producing methanol and carbon dioxide Use requires training and use of specialized dosing equipment: explosion hazard, toxic to humans Enables use of lower SO 2 to control bacteria so there is some impact

18. Sulfur Dioxide Microbes in wine vary in sensitivity to SO 2 Yeast in general less sensitive than bacteria Lactic acid bacteria appear more sensitive than acetic acid bacteria Bacteria generally inhibited at

19. Molecular Forms of SO 2 as a Function of pH

20. pHMolecular SO 2 (%)Bisulfite ion (%) Free SO 2 (mg/L) for 0.825 mg/L molecular* 3.05.5694.414.8 3.14.4795.518.5 3.23.5896.423.1 3.32.8797.128.8 3.42.2997.736.0 3.51.8398.245.1 3.61.4698.556.5 3.71.1598.871.1 3.80.92499.089.3 3.90.73699.2112.0 4.00.58599.4141.0 Percentage of sulfur dioxide forms in molecular and bisulfite states in a 14% v/v ethanol solution with 80 mM ion strength at various pH values, adapted from Boulton et al (1996).

21. Inhibitory Levels of SO 2 Saccharomyces: 0.825 mg/L molecular SO 2 Acetobacter: 0.05 to 0.6 mg/L molecular SO 2 Lactic Acid Bacteria: 0.01 to 0.2 mg/L molecular SO 2 Brettanomyces and non-Saccharomyces yeast: 0.1 to 0.6 mg/L molecular SO 2

22. Heat Treatments HTST (high temperature short time) treatments can be used to eliminate grape surface or juice populations of microbes Both yeast and bacteria are sensitive to high heat Enzymes will also be inactivated potentially impacting aroma profile

23. Factors Impacting Effectiveness of Antimicrobial Additions Bioload: higher population numbers require higher doses of inhibitor Type of vessel used for fermentation Gunk build-up on equipment provides a protective layer reducing access of agent Presence of detoxifying microorganisms Removal of synergistic effects: bacteriocins for example Insect populations in winery and opportunities for re- contamination Adjacencies of vineyard/winery Level of rot of fruit at harvest Sanitation practices

24. Choice of Inhibitor What are you trying to inhibit? Is a short term acting inhibitor sufficient? Is recontamination an issue? Presence of factors reducing efficacy of inhibitor? Presence of resistant strains? Presence of inactivating organisms?

25. Impact of SO 2 on Microbial Populations Data courtesy of Nick Bokulich