Transcript quality-based planning - Department of Food Science and

Winery Sanitation
Molly Kelly
Enology Extension Specialist
Virginia Tech
Cellar Hygiene


Why?
To maintain wine integrity and quality
Sanitation


Anything in contact with the wine is a
potential vector for microbial spoilage
Have appropriate sanitation protocols in
place and implement them
General Principles of
Surface Cleaning and Sanitizing


Remove loose soiling particles from surface
Make bound soiling layer ‘wet’





Allow penetration of layer by solvent and
cleanser
Remove bound soiling layer from surface
Rinse surface
Sanitize surface
If desired, sterilize surface
Purposes of Cleaning, Sanitizing,
and Sterilizing



Get rid of microorganisms
Get rid of nutrients and contaminating
compounds
Prevent entry and build-up of
microorganisms and other contaminants
Some Definitions



Cleaning: Removing dirt, debris and stains
Sanitizing: the reduction of the microbial
population to a safe level (kills 99.9% of
growing bugs)
Sterilizing: Complete Elimination of microbial
Life
Definitions


Cleaning: an attempt to physically and
chemically remove food for
microorganisms and to eliminate
hospitable environments for their growth
Sanitation=Disinfection


Sanitation is an attempt to reduce the number
of spoilage organisms on equipment surfaces
Sterilization is an attempt to kill 100% of
spoilage organisms
QA and QC



Quality Assurance (QA) focuses on attempts to improve
and stabilize production practices in order to prevent
defects
Quality Control (QC) focuses on product testing to
uncover defects
HACCP: means of assuring quality, through the
identification and monitoring of critical control points
from vine to glass
HACCP

Logical system of control based on the
prevention of problems







Look at your process from start to finish
Decide where hazards can occur
Put in controls and monitor them
Write it all down and keep records
Ensure that it continues to work effectively
All wineries would benefit from a basic
outline
http://www.apps.fst.vt.edu/extension/enology/EN/index.html
Was process effective?
Example:
Cleaning Flow
Chart
Soiled Item
Scrape,
brush, shovel,
squegee etc
Water
Temperature
Dependent
on Soil Type
Verify Temperature
Remove Soil
Rinse with water
until water runs
clear
Physical
Methods
Clean
Verify Cleaner
Concentration
Chemical
Methods
Water
Temperature
Dependent
on situation
Water Rinse
No
Verify Rinse Water
neutrality
Was
Looks,
cleaning
Smells,
Feels
Clean?
effective?
Look, smell, touch
No
Yes
ATP Test
Acceptable?
Yes
ATP swabbing test
Item Ready for
Sanittaion
Rule of Thumb



Acid cleaners dissolve alkaline soils
(minerals)
Alkaline cleaners dissolve acid soils and
food wastes
Improper use of detergents can actually
“set” soils, making them more difficult to
remove (e.g. acid cleaners can precipitate
protein)
More is better?



Common misconception: “if a little is
good, more is better”
Using sanitizer concentrations above
recommendations does not sanitize better,
in fact, can be corrosive to equipment and
lead to less cleanable
Follow manufacturer’s label instructions
Chemical and their Use
Action
Chemical
Cleaner
Alkalis, acids, detergents
Sanitizer
Quats, peracetic acid, SO2,
hydrogen peroxide, ozone
Sterilizer
Hot water, steam, alcohol,
UV, ozone
Types of Sanitizers

Heat, w/ water or steam to saturate effect




Very effective anti-microbial, except some
encysted forms
Exposure time critical
Non-corrosive, but energy intensive
Chemical


Effectiveness varies with exposure time,
temperature, concentration, microbial load, soil
load
Can be corrosive
Chemical Sanitizers

Chlorine-containing not recommended
except for out of control microbial
situations


Very effective anti-microbial
Iodine-containing little used for wineries

Effective anti-microbial, except when diluted
Iodine




Iodophores: broad spectrum
Active against bacteria, viruses, yeasts,
molds, fungi
Bottling line (followed by hot water)
Potential for TCA
Quaternary Ammonium
Compounds (QACs)



Positively charged: mode of action is
related to their attraction to negatively
charged materials (bacterial proteins)
Leave a residual antimicrobial film
Floors
Chemical Sanitizers

Peroxides







Hydrogen peroxide, ozone (inorganic), peroxyacetic
acid, peroxyborates (organic)
Very effective anti-microbials
Tolerant of mineralized water
Concentration dependent
Not corrosive
Can remove light bio-films
Environmentally friendly
Sanitizers
percarbonates






Per(oxy)carbonate
Release of oxygen radicals via H2O2
Application at room temperature
Effective over wide pH range (1-8)
Alkaline
Degradation to soda ash, water, O2
Sanitizers
Soda Ash Na2CO3





Strong alkaline (pH 11.3 at 1%)
Dissolves proteins, fats, oils,
carbohydrates, tartrates
Neutralizes acidic odors (VA)
Neutral pH best for bacteria
Sanitizing effect?
Evaluating Cleaning
Effectiveness
Evaluating Cleaning
Effectiveness
Biofilms

•
•
•
•
•
A mixed group of microbial
species protected by a
polysaccharide/exopolymer
coating.
Resistant to many chemical cleaners.
Usually need to be removed by physical action
(scrubbing, brushing, high pressure).
Many wine microbes can form or join biofilms.
Commonly found at liquid boundary regions.
Can be invisible to the naked eye.
Winery Recommendations

General Order: Stainless or plastic tanks,
crusher-destemmers, presses, fittings







Cold water, high pressure rinse
Strong inorganic alkaline solution or paste scrub
Cold water, high pressure rinse
Cationic detergent, combined with peroxyacetic
Cold water, high pressure rinse
Hot water, high pressure rinse
Ozone treatment
Winery Recommendations

General Order: Bottling line







Cold water, high pressure rinse
Mild alkaline detergent solution
Cold water, high pressure rinse
Cationic detergent, combined with peroxyacetic
Cold water, high pressure rinse
Hot water, high pressure rinse
Ozone treatment
Winery Recommendations

General Order: Hoses and pumps







Cold water, high pressure rinse w/foam ball
Mild alkaline detergent solution w/foam ball
Cold water, high pressure rinse w/foam ball
Cationic detergent, combined with peroxyacetic,
w/foam ball
Cold water, high pressure rinse, w/foam ball
Hot water, high pressure rinse, no ball
Ozone treatment, no ball
Winery Recommendations

General Order: Barrels free of faulty aromas or tastes





Cold water, high pressure rinse, 1-3 minutes
High pressure steam rinse, 1-3 minutes
Repeat cold and steam rinses twice more
Either refill with clean wine or
Fill with water



add ozone, if available
follow with water + 45 pm SO2/90 ppm citrate
After 1-4 days, empty and refill with wine or empty and burn
sulfur pastille, re-bung and store
Winery Recommendations

General Order: Barrels with faulty aromas or tastes


Option 1 Remove from winery and sell for non-wine uses
Option 2  Clean, sterilize, and re-use, if worth the cost


Rinse cycles done as per barrel without faulty aromas or tastes
Fill with water, put steam wand in water and bring water to 160180F, steam periodically to maintain temperature for 4-6 hours





add ozone, if available
follow with water + 45 pm SO2/90 ppm citrate
After 1-4 days, empty and burn sulfur pastille, re-bung and store
After 1-4 weeks, rinse and fill with clean water, after 1 week, take
samples and then add 90 pm SO2/180 ppm citrate while doing
microbiological assay of samples
If samples are negative for spoilage microorganisms, re-use barrel,
but sample periodically
Winery Recommendations

General Order: Floors and waterproof
walls and ceilings





Cold water, high pressure rinse
Scrub w/mild alkaline detergent
Cold water, high pressure rinse
Hot water, high pressure rinse
Ozone treatment
Winery Recommendations

General Order: Non-waterproof surfaces



Dust w/ damp cloth or sponge
Clean with mild household ammonia-based
product
Wipe with damp sponge or dry towel
Is Sterility a Must?

We don’t Need Sterility


Commercial yeast out-competes wild yeasts,
fungus, mold, and bacteria– especially at wine
pH
We don’t Want Sterility

Sometimes wines may benefit from something
extra…
Sterilizers

Hot water and steam used to sterilize
bottling line




80-90F for 30 min
180F for 20 min
ozone for 20-30 min
Alcohol and UV: lab & for small equipment
Basic Rules

Clean everything BEFORE you use it. And then sanitize.

Clean everything AFTER you use it.

Clean the winery premises, not just the equipment, on a regular
basis.

Keep the winery free of clutter.

Watch for pests (bacteria, mold, wild yeast, rodents, etc.

Deal with pomace IMMEDIATELY.
Generic Chemicals for Cleaning and
Sanitation
Type
Dose
Notes
Citric Acid
3 Tbsp/gal
Neutralizes alkalinity from other cleansers
and helps to activate MBS
Trisodium Phosphate
1 Tbsp/gal
Effective against organic soils but can cause
minerals to precipitate as scale
Soda Ash
0.25c/gal
Effective no-frills cleaning compound
Sodium Metabisulfite
3 Tbsp/gal
Very good must treatment; questionable
product when used alone
Sodium Hydroxide
1 tsp/5 gal
Very effective; very caustic; very hazardous
and difficult to rinse
Ammonia
1.5 Tbsp/gal
Difficult to rinse, but very effective on
removing labels; stinks
Formulated Chemicals for Cleaning
and Sanitation
Type
Dose
Notes
Straight-A, B-Brite
1-2 Tbsp/gal
Alkaline products containing
percarbonate
Iodophor
1 Tbsp/5 gal
Sanitizer only; color is not as good an
indicator of strength as is thought
Dishwashing Detergent
1 Tbsp/gal
Effective, but only use unscented
versions
MBS/Citric Acid
8oz/1oz/gal
Very effective sanitizer with some
cleaning effects; strong SO2 release
may be hazardous to some people
Note: B. Zoecklein et al. Wine Analysis and Production: 20
g/hL KMS and 300 g/hL citric acid at 140F
Sanitizers
Sanitizer
Time of use Optimum
(min)
pH
Peracetic acid 5-20
>9
Temp range
Ozone
1-30
Quats
15m-24hr
Sulfur dioxide 15s-20min
Cold
70-120
cold
6-10
<3
70-100
Special Cases: Barrels

There’s no good way to deal with old barrels
that have “gone off…”



Chemicals will either taint the wood or extract
essence.
But here are some ideas.
Treat barrels right




Don’t let them dry out.
Store with a MBS/citric acid solution (2 oz/2 oz/5 gal water)
Clean the outside as well as the inside
Recover with a percarbonate based cleaner (1
Tbsp/gal), let sit 24 hours, rinse, then rinse with citric
acid (0.5 tsp/gal).
Cleaning and sanitation steps







Debris removal without water
Rinse with water
Wash with cleaning agent
Rinse with water
Rinse with acid
Rinse with water
Use sanitizer & repeat rinsing if cleaner is
not anti-microbial
Precautions



Cleaners and sanitizers are corrosive and
can be harmful to skin, respiratory system
and eyes
Check MSDS sheets when in doubt
Wear protective gear if needed
Validating Winery Sanitation

Perform the sanitation operation using a
standard operating procedure (SOP)


Validate that the SOP was effective



define equipment, concentration of sanitizer, time,
temperature, surface type, cleaning state, bioload,
etc.
Use swabbing or soak/rinse water to get sample
Use microscope scans, plating, or PCR methods
Changing any parameter in the SOP would
indicate the need for additional validation.
Verifying Concentration
of Chemical Sanitizers

pH


pH test strips for acid sanitizers
Concentration (critical to get the right range and
sensitivity)






Ozone test strips
Iodine test strips
Peracetic acid test strips
Quaternary Ammonia strips
Peroxide test strips
Sulfite test strips
Verifying Temperature




Digital Thermometers
Infrared thermometers
Steam test strips and tape (for autoclaves)
Temperature indicating labels
Verifying Time
Heat sanitizing time starts
when the equipment reaches
the desired temperature
The time to get equipment up
to temperature can be
significant, especially with
large equipment such as filler
bowls
Time/Temperature strips may
be available that meet
specific needs
Bottling and Packaging


One of the most critical steps in wine
production
Many opportunities for problems



People with different responsibilities
Multiple wines to bottle
Operation and maintenance of multiple
equipment stations
Sterile bottling rooms?



Not an absolute necessity
Bottling area should be screened-off from
fermentation areas and excessive air
movement
Easily sanitized floors, walls and ceilings
Sources of contamination at
bottling




Filter pad drip trays: drain often during runs
Fill bowls: Mist filler spouts with 70% ethanol to
inhibit microbial growth
Corker: likely to have spilled wine. Ethanol misting
of corker jaws during bottling
Activity: Limit number of people around
filling/corking area
Spoilage Microorganisms on Grapes

Non-Saccharomyces yeasts

Kloeckera apiculata

Acetic acid bacteria

Lactic acid bacteria

Lysozyme
Brettanomyces
• Most serious of spoilage yeasts
• Red wine in barrels
• Bottled wines
• Transmitted by fruit flies
• Grows on cellobiose, toasted barrels
• Tolerance to sulfur dioxide
Brettanomyces growing in culture
Brettanomyces.wordpress.com
B. bruxellensis on WL agar
Brettanomyces
Practical winery and vineyard
Wikipedia.com

Smells like:









Brett
Barnyard
Horse (blanket, sweat, saddle)
Wet dog
Tar
Tobacco
Creosote
Plastic
Leather
Pharmaceutical (band aid)
Yeast prevention/control



Grapes
 Minimize damage to skins
 Pick when cool
 Sorting
 Add SO2 to picking bins
 Minimize transport distance
 Adequate hygiene
Winery equipment
 Regular cellar hygiene
Wines
 Adequate SO2
 Absence of air/oxygen
 Filtration
Microorganisms on Grapes

Non-Saccharomyces yeasts

Kloeckera apiculata

Acetic acid bacteria

Lactic acid bacteria

Lysozyme
Acetobacter
angeladouglaslab.com, Cornell
University
•Gram negative rods or cocci
•Strictly aerobic
•0.6 to 0.9 microns by 1 to 3 microns
•Formation of acetic acid from ethanol
AAB control
•Low pH (acid)
•Minimize oxygen incorporation
•Maintain cool temperatures (<50F)
•Free sulfur dioxide levels according to pH
•High VA wines can be blended with unaffected wine or
treated by RO
Microbes during fermentation



Non-Saccharomyces yeasts
Lactic acid bacteria (LAB)
What to watch for:





Ethyl acetate
Spontaneous or sluggish fermentation
Spontaneous MLF
VA
Off odors
Lactic acid bacteria spoilage

Typical spoilage times:


During “stuck” fermentations
Finished wines with low SO2, residual malic
acid or sugar
Lactobacillus spp.
Enologyaccess.org
Fermentacionvegetales.wikispaces.com
Lactobacillus fermentum
Lactobacillus brevis
LAB

Volatile acidity-metabolism of citric acid and
glucose to acetic acid

Mousiness

Geranium taint-metabolism of sorbic acid

Ropiness –production of extracellular
polysaccharides
Pediococcus
Practicalwinery.com
Pediococcus spp. and one Lactobacillus chain
Mousey taint

Aftertaste: not volatile at wine pH;mix
with neutral pH of saliva become apparent




Mouse urine
Rancid nuts
Can also smell like acetaldehyde, corn chips
(when rub in palms)
Comes from Brett (rarely) and LAB
(usually)
Stuck Ferments

Lactobacillus
Pediococcus
Brettanomyces
Acetobacter
Film yeasts (Candida and Pichia)

Watch for:








VA increase
Film yeast
Spontaneous MLF
CO2 production
Microbes during MLF

Acetobacter
Lactobacillus
Pediococcus
Brettanomyces

Film yeasts

Oenococcus

Watch for:






VA increase >0.15 g/L (from citric acid)
Film
Off-odors and flavors
VA post fermentation sources



Headspace in barrels
Oxidation of wine
“acetic aroma” not exclusively a result of
acetic acid

Also due to ethyl acetate (ester)

Esters are volatile compounds formed by
combination of an alcohol and an acid
Bacteria prevention/control

Grapes






Minimize damage to skins
Pick when cool
Sorting
Add SO2 to picking bins
Minimize transport distance
Adequate hygiene
Bacteria prevention/control

Winery equipment


Regular cellar hygiene
Wines





Short or no skin contact
Adequate SO2
Exclusion of air
Filtration
Acid addition
Validating Winery Sanitation

Perform the sanitation operation using a
standard operating procedure (SOP)


Validate that the SOP was effective




define equipment, concentration of sanitizer, time,
temperature, surface type, cleaning state, bioload,
etc.
Use swabbing or soak/rinse water to get sample
Use microscope scans, plating, or PCR methods
Scorpions™ testing can provide fast results and target
specific wine spoilage organisms.
Changing any parameter in the SOP would
indicate the need for additional validation.
Swab or Soak Sampling


Specify the details of the sampling technique in the SOP
Swabbing area and location







Use good sterile technique
Rub hard to break up biofilms
Place swab in labeled container of sterile saline
Sterile water soak/rinse sampling may be appropriate for
some situations.
A concentration step may be helpful if low cell numbers are
anticipated.
A negative result (no cells found) does NOT indicate
sterility.
Ongoing testing and historical trends important in
developing and implementing effective programs.
Membrane Filter Method
Sartorius stedim biotech
•Isolate small number of microbes from liquid sample
•Demonstrate their presence as colonies on culture medium
•Cellulose nitrate membrane: 0.45 micron for bacteria, 0.65-8.0 microns for
yeasts and molds
Surface Testing
Sartorius stedim biotech
•For smooth surfaces
•Place membrane on surface to be tested and place on agar plate
•Use sterile technique
Swab Test Method
Sartorius stedim
biotech
Sartorius stedim
biotech
•Semi-quantitative analysis
•Moist sterile cotton swabs for dry areas (sterile physiological saline)
•Dry sterile cotton swabs for moist areas
Airborne Microorganisms
Sartorius stedim biotech
•Sedimentation plates
•Leave plates open for 30 min-2 hrs
•Incubate
Yeasts and molds
Wort
Culture Media
Malt Extract
Wallerstein
Lysine (wild yeasts)
Addition of Actidione will allow only Brettanomyces and some Kloeckera to grow
Bacteria
Orange Serum (AAB, LAB)
Tomato Juice (O. oeni)
Sartorius stedim biotech
Cellar Hygiene



Why?
To maintain wine integrity and quality
Poor wine quality is usually due to poor
sanitation practices!
Sources/areas of build up





Vineyard
Diluted pools of juice
Second-hand barrels
Imported bulk wine
Areas of winery that are difficult to reach
General guidelines


Proactive and not reactive
Constant and immediate attention


Clean up immediately
Save time at end of day
Conclusions

Prevent and control microbial
contamination

Reducing population during winemaking
So2
 Temp
 Filtration


Quality control plan
SOPs based on cGMPs
 HACCP


Sample wines/taste for faults
Contact Information





Molly Kelly
Enology Extension Specialist
Food Science Dept., Virginia Tech
Email: [email protected]
Cell phone: 540-315-6920
References
Lansing, R. May 2011. Managing Bottling Operations. Wine Business Monthly.
Neradt, F. 1982. Sources of reinfections during cold-sterile bottling of wine. Am. J. Enol. Vitic. Vol. 33.
no. 3.
Pregler, B. Dec 2009. What’s Cool: Accurate Membrane Filter Integrity Testing.
Wine Business Monthly.
Tracy, R. and Skaalen, B. Jan/Feb 2009. Bottling-last line of microbial defense. Practical Winery and
Vineyard.
Ritchie, G., Napa Valley College, VWT 280, Cellar Hygiene, 2007.
Menke, S., Cleansers and Sanitizers, Penn State Enology Extension, 2007.
Butzke, C., Barrel Maintenance, Dept. of Food Science, Purdue University, 2007.
Howe, P., ETS Laboratories, SOWI “Current Issues” Workshops March 2011.
Crowe, A. Avoiding Stuck Ferments, Wine Business Monthly, August 2007.
Zoecklein, B. et al, Wine Analysis and Production, Aspen Publishers, 1999.
Van de Water, L., Practical Winery and Vineyard Journal, Sept/Oct 2009.
Just, E. and H. Regnery. Microbiology and Wine Preventive care and monitoring in the wine industry.
Sartorius Stedim Biotech. 2008.