Download Microorganisms of Juice: Managing Competition in the Tank

Microorganisms of Juice: Managing
Competition in the Tank
Lucy Joseph
U.C. Davis
Department of Viticulture and Enology
Managed Ecosystem
Ecological Succession in Wine
Fermentation
Which Microbes Are Found? –
Vineyard to Must
Factors Affecting Grape Microflora
•
•
•
•
•
•
Moisture/Humidity
Insect Vectors and Damage
Temperature
Vineyard Management Practices
Variety of Grape
Geography
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
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
Bacteria in the Vineyard
Lactobacillus
Lactococcus
Enterococcus
Weissella
Gluconobacter
Pediococcus
Oenococcus
Must
Lactobacillus
Pediococcus
Weissella
Gluconobacter
Acetobacter
Gluconacetobacter
Leuconostoc
Oenococcus
Yeast in the –
Vineyard
Harvested Grapes
Zygosaccharomyces
Hanseniaspora
Issatchenkia
Kluyveromyces
Metschnikowia
Pichia
Candida
Rhodotorula
Sporobolomyces
Sporidiobolus
Hanseniaspora
Metschnikowia
Candida
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
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
Typical Saccharomyces Growth Curve vs Typical
Wine Fermentation Curve
400
30.00
350
25.00
300
20.00
250
Brix
15.00
200
10.00
150
5.00
100
50
0.00
0
(5.00)
250
0.00
0
50
100
150
200
50.00
100.00 150.00 200.00 250.00 300.00 350.00 400.00
Saccharomyces and Non-Saccharomyces Growth
Curves vs Typical Wine Fermentation Curve
400
30.00
350
25.00
300
20.00
250
Brix
15.00
200
10.00
150
5.00
100
50
0.00
0
(5.00)
250
0.00
0
50
100
150
200
50.00
100.00 150.00 200.00 250.00 300.00 350.00 400.00
How Certain Groups are Selected
Natural selection:
•
•
•
•
•
•
Low pH
High sugar concentration – high osmolarity
Other nutrients
High phenolic content
Low oxygen
Alcohol concentration
How Certain Groups are Selected
Artificial Selection:
•
•
•
•
•
Temperature
Potassium metabisulfite
pH - Tartaric acid
Lysozyme
Nutrients
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
Why Do Best Practices Work?
What do they control?
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
Addition of SO2
• Inhibits the growth of spoilage bacteria
• Inhibits growth of wild yeasts including
Brettanomyces
• Destroys thiamin
• Inhibits oxidation
Inoculation
• Directly adds the desirable organism in high
numbers
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
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
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
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
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
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
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.
Grenache Wine
Grenache Filter on Plate
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.
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
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.
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
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.
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