Download Juice Nutrient Analysis and Additions: Methods and

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
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temperatures than Saccharomyces and less tolerant of
higher temperatures
Saccharomyces more tolerant of sulfur dioxide
Equivalent tolerances to low pH across nonSaccharomyces 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
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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
Molecular Forms of SO2 as a Function of pH
100
90
Percentage in form (%)
80
70
60
Molecular SO2
50
Bisulfite (HSO3)
40
Sulfite (SO3)
30
20
10
0
0
1
2
3
4
5
pH
6
7
8
9
10
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).
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
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
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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 recontamination
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