SELECTION OF ANTIMICROBIALS

Linda F. Bisson

Department of Viticulture and Enology

Quality Control Management during Crush and Fermentation

August 7, 2014

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

Types of Microorganisms• Yeast• Gram positive bacteria• Gram negative bacteria

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

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

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

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

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

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

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

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

Microbial Inhibitors Approved for Use in Wine

• Lysozyme• DMDC(DiMethylDiCarbonate)/Velcorin• SO2

• Heat treatments

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

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 SO2 concentrations

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)

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

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 SO2 to control bacteria so there is

some impact

Sulfur Dioxide

• Microbes in wine vary in sensitivity to SO2

• Yeast in general less sensitive than bacteria• Lactic acid bacteria appear more sensitive than acetic

acid bacteria• Bacteria generally inhibited at

0 1 2 3 4 5 6 7 8 9 100

10

20

30

40

50

60

70

80

90

100

Molecular SO2

Bisulfite (HSO3)

Sulfite (SO3)

pH

Per

cen

tag

e in

fo

rm (

%)

Molecular Forms of SO2 as a Function of pH

pH Molecular SO2 (%) Bisulfite ion(%)

Free SO2 (mg/L) for 0.825 mg/L molecular*

3.0 5.56 94.4 14.8

3.1 4.47 95.5 18.5

3.2 3.58 96.4 23.1

3.3 2.87 97.1 28.8

3.4 2.29 97.7 36.0

3.5 1.83 98.2 45.1

3.6 1.46 98.5 56.5

3.7 1.15 98.8 71.1

3.8 0.924 99.0 89.3

3.9 0.736 99.2 112.0

4.0 0.585 99.4 141.0

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).

Inhibitory Levels of SO2

• Saccharomyces: 0.825 mg/L molecular SO2

• Acetobacter: 0.05 to 0.6 mg/L molecular SO2

• Lactic Acid Bacteria: 0.01 to 0.2 mg/L molecular SO2

• Brettanomyces and non-Saccharomyces yeast: 0.1 to 0.6 mg/L molecular SO2

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

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

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?

Impact of SO2 on Microbial Populations

Data courtesy of Nick Bokulich