Winery Sanitation

Molly KellyEnology 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

Soiled Item

Rinse with water until water runs

clear

Clean

Water Rinse

ATP Test Acceptable?

Was process effective?

Verify Temperature

Verify Cleaner Concentration

Verify Rinse Water neutrality

Cleaning

Remove Soil

Look, smell, touch

Looks, Smells, Feels

Clean?

Yes

ATP swabbing testYes

No

No

Scrape, brush, shovel, squegee etc

Chemical Methods

Physical Methods

Water Temperature Dependent on situation

Water Temperature Dependent

on Soil Type

Item Ready for Sanittaion

Example:Cleaning Flow

Chart

Was cleaning

effective?

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

Sanitizerspercarbonates

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

SanitizersSoda 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 160-180F, 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 NotesCitric 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 NotesStraight-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 (min)

Optimum pH

Temp range

Peracetic acid

5-20 >9 70-100

Ozone 1-30 - Cold

Quats 15m-24hr 6-10 70-120

Sulfur dioxide

15s-20min <3 cold

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) define equipment, concentration of sanitizer,

time, temperature, surface type, cleaning state, bioload, etc.

Validate that the SOP was effective 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.

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 Concentration of Chemical Sanitizers

Verifying Temperature Digital Thermometers Infrared thermometers Steam test strips and tape (for autoclaves) Temperature indicating labels

Verifying TimeHeat 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

BrettanomycesBrettanomyces• 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

B. bruxellensis on WL agar

Brettanomyces.wordpress.com

Brettanomyces

Wikipedia.com

Practical winery and vineyard

Brett Smells like:

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

• Gram negative rods or cocci

• Strictly aerobic

• 0.6 to 0.9 microns by 1 to 3 microns

• Formation of acetic acid from ethanol

angeladouglaslab.com, Cornell University

AAB controlAAB 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

Lactobacillus fermentumFermentacionvegetales.wikispaces.com

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) define equipment, concentration of sanitizer,

time, temperature, surface type, cleaning state, bioload, etc.

Validate that the SOP was effective 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

• 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

Sartorius stedim biotech

Surface Testing

• For smooth surfaces

• Place membrane on surface to be tested and place on agar plate

• Use sterile technique

Sartorius stedim biotech

Swab Test Method

• Semi-quantitative analysis

• Moist sterile cotton swabs for dry areas (sterile physiological saline)

• Dry sterile cotton swabs for moist areas

Sartorius stedim biotech

Sartorius stedim biotech

Airborne Microorganisms

Sartorius stedim biotech

• Sedimentation plates

• Leave plates open for 30 min-2 hrs

• Incubate

Culture Media

Wort Malt Extract Wallerstein Lysine (wild yeasts)

Orange Serum (AAB, LAB) Tomato Juice (O. oeni)Sartorius stedim biotech

Yeasts and molds

Addition of Actidione will allow only Brettanomyces and some Kloeckera to grow

Bacteria

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: mkkelly@vt.edu Cell phone: 540-315-6920

ReferencesLansing, 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.