OPTIMIZING CLEANING AND SANITATION SOP’S IN THE WINERY

UC DAVIS VITICULTURE AND ENOLOGY

Cory Marx, PhD Student, Oberholster Group

OPTIMIZING CLEANING AND SANITATION SOP’S IN THE WINERY

FERMENTATION READINESS: HARVEST DECISIONS FROM THE VINEYARD TO THE WINERY

SENSORY THEATER, UC DAVIS

JULY 29TH, 2019

ANITA OBERHOLSTER, CORY MARX, PAUL VAN DER MERWE


Outline

• Introduction

• Winery Spoilage Microbes

• Cleaning and Sanitation Chemicals

• Protocols

• Monitoring Strategies

• Supporting Experimental Data

• Barrel Cleaning and Sanitation


Winery Cleaning and Sanitation• Cleaning and sanitizing is a preventative process, not a

corrective one• Minor time investment compared to total winemaking process,

often overlooked• Established, written protocols ensure product quality and

worker safety

Benefits:• Improved product quality• Reduced operating cost• Longer equipment shelf Life• Safe working environment

Costs/Consequences:• Spoiled/unsaleable product• Damaged reputation• Damaged equipment• Hazardous conditions


Winery Cleaning and Sanitation

• Involves all aspects of winery• FDA-regulated food processing facilities

• Numerous materials, wide variation in equipment• Stainless steel, plastic, concrete, rubber• Tanks, hoses, barrels, bottling lines, drains



• Cleaning• Process involving physical removal of organic and inorganic

soils

• Sanitizing• Process involving inactivation of microbes

• Disinfection

• Sanitation

• Sterilization


Definitions• Disinfection – Reduction in

harmful/pathogenic cells (log 3, 99.9%)

• Sanitation – Effective elimination of potential spoilage microbes (log 6+)

• Sterilization – Elimination of all viable cells (log 12+)

• PPE – Personal Protective Equipment

• PEL – Permissible Exposure Limit

• OSHA defined, TWA and ceiling values

• SDS (MSDS) – Safety Data Sheet


Winery Microbes

• Wineries have high microbial load, especially during harvest

• Must and wine properties place ‘selective pressure’ on microbial communities• Low pH, ethanol major population drivers• Spoilage microbes only group of concern

• All heat labile

A. pasteurianusS. cerevisiae


Winery Microbes

• Spoilage microorganisms require a vector to travel through the winery (except for fruit flies)• Vectors include workers, HVAC system, improperly sanitized

equipment or tools

• 0.45 μm filtration remove commercial yeast (~5-10 μm) but not all bacteria or some wild yeast• Should not be used as only remedy for bacteria


Winery Microbes

• Yeasts, molds, lactic and acetic acid bacteria main sources of wine spoilage• Common spoilage yeast genera

• Saccharomyces, Brettanomyces, Zygosaccharomyces• Common bacteria genera

• Lactobacillus, Acetobacter, Oenococcus, Pediococcus

• Planktonic (suspended in medium) • Sessile (surface-associated) – biofilm

• Biofilms more difficult to inactivate/remove• Inactivation is not enough – should be removed


Assessing Microbial Spoilage: The basics

• Sensory• Plating and/or microscopy• Molecular methods ex. Scorpion• Have ID guides on hand

Illustrated Guide to Microbes and Sediments in Wine, Beer, and Juice, by Charles G. Edwards (Winebugs LLC and GusmerEnterprises inc. 2005)


Common Cleaning Chemistries

• Caustics• NaOH, KOH• Capable of dissolving soils• Have biocidal activities (at typical 1-2% concentration)

• Non-caustic Alkaline Products• Often sodium carbonate/potassium percarbonate-based• Trisodium phosphate (TSP), hydrogen peroxide, sodium

metasilicate common in formulations

• Acid Cleaners• Phosphoric/Nitric acid-based cleaners


Sanitizing Chemistries

• Cl- - based compounds• Broad-spectrum activity• No evidence of TCA issues with chlorine dioxide• Important to maintain pH < 7 (must thoroughly rinse alkaline

cleaners)• Hazardous to health - requires PPE

• I- - based compounds• No-rinse formulations available• Can stain equipment• Temperature sensitive (can’t be used with >120oF water)



• Quaternary Ammonium Compounds (QUATs)• Has residual activity, can be left on surfaces that won’t be used

immediately• Affected by water quality• Relatively narrow-spectrum

• SO2 (acidified to pH ~3)• Inexpensive• Frequently already stocked in winery BUT corrosive to metals• Hazardous to health• Not generally recommended as a primary sanitizing agent



• Peracetic Acid (PAA) • Breaks down to acetic acid, oxygen, water• Can use as no-rinse sanitizer• Effective at low temperatures• Less effective against some yeast and molds• Store cold• Expensive



• Ozone• Broad spectrum, strong oxidizer• Breaks down to molecular oxygen (O2)• Half-life important (24hrs as a gas, but seconds dissolved in water)• Dedicated equipment required

• Heat/Steam• Temperatures > 185oF sufficient to inactivate winery spoilage

microorganisms• Heat is a full-spectrum technique, but requires dedicated equipment

and can have high energy costs• Others (UV, ultrasonic, etc.)


Basic 5‐Step Cleaning/Sanitizing Process

Water Rinse

Water Rinse

Water Rinse

Cleaning Cycle

Sanitizing Cycle


Water Rinse 1 – Room temperature water pre-rinse to remove gross soil. Can be followed by warm water rinse. Hot water may ‘cook’ on debris, and should be avoided at this stage

Water Rinse 2 – Remove cleaner residue, loosened debris, neutralize. Using room temperature water facilitates application of some sanitizers

Water Rinse 3 – Remove sanitizer residue (if necessary)


Cleaning and Sanitizing Strategies

• Cleaning Choices• Manual (hose, buckets, brushes)• Semi-Automatic (spray ball, mobile sprayer)• CIP (Clean-in-place)• COP (Clean-out-of-place)• Immersion


Protocols and Monitoring Strategies

• Written protocols• Employee checklists • Monitoring strategy to validate

protocol• ATP and environmental swabbing • pH meters and strips • Temperature tape and thermometers


HACCP, ISO, and formalized SOP’s

• HACCP (Hazard Analysis and Critical Control Point) defined in Codex Alimentarius• Established methods for critically

assessing and developing product stream and protocols

• Write down SOP’s • Use checklists

Understanding Wine Technology 3rd Edition by David Bird (DBQA publishing, Britain, 2010)

Example CCP Flow


Winery Microbes

• Research study• 3 yeast

• Saccharomyces cerevisiae, Zygosaccharomyces baili, Brettanomyces bruxellensis

• 4 bacteria• Lactobacillus casei, Acetobacter pasteurianus, Pediococcus parvulus,

Oenococcus oeni


Initial Chemical Screening

• Planktonic cells• Microbes exposed to chemical treatment in 96 well plate,

sampled out to petri dishes at 5 min intervals• 7 microbes

• 20 treatments• 9 cleansers, 10 sanitizers, H2O

Organism S. cerevisae B. bruxellensis Z. bailii A. pasteurianus L. casei P. parvulus O. oeni

Treatment 2% NaOH

Cleaners

1% KOH 2% KOH 0.75% Inventek 2% Cleanskin‐K 1% Destainex 2% 231‐Xtra 2% 440‐K 17% Sterilex 1% H2O2

Sanitizers

100 ppm PAA 200 ppm PAA 100 ppm PAA + 1% H2O2 20 mM KHSO4 20 mM KHSO4+ 1% H2O2 40 mM KHSO4+ 1% H2O2 20 mM KHSO4 + 2% citric acid 0.2% Quat Sanitizer II 0.15% BTF Iodophor Sanitizer 100 ppm ClO2


Initial Chemical Screening• Biofilms

• Biofilms developed by incubating

microbes in 50% grape juice

medium for 10 days at 30˚C in

96-well plates

• Biofilms exposed to chemical treatment, rinsed at 5 min intervals to stop exposure

• Capacity to remove biofilm measured via crystal violet staining of well plates

• 20 Chemical treatments

• 4 trials with 3 yeast each, 1 mixture of all 4 bacteria


Biofilm Investigation


Environmental Swabs: Biofilms on Stainless Steel

• Biofilms formed on 304SS coupons• 3 yeast, 1 mixture of

bacteria

• Two monitoring Methods• ATP bioluminescence

• Plate counts


ATP Swabbing of Biofilms on Stainless Steel

• Illustrates need for cleaning step


ATP Swabbing of Biofilms on Stainless Steel


Minimum Inhibitory/Biocidal Concentration

• Widely-used approach to determine minimum concentration of antimicrobial agents

• Microbes exposed to serial dilution of antimicrobial agents• Incubated overnight in 96-well plates

• After incubation, absorbance data acquire

• Wells sampled out to petri dishes• No growth in well plate = inhibition

• No growth in petri dish = biocidal


Experimental – MIC/MBC/Biofilm

• 3 Yeast• Saccharomyces cerevisiae, Zygosaccharomyces baili, Brettanomyces

bruxellensis

• 10 Cleaning/Sanitizing Formulations• NaOH*, KOH*, peracetic acid (PAA), Mixture (percarbonate,

EDTA, QUAT), KHSO4, H2O2, ClO2

• Serial dilutions of antimicrobials (50% series)• 24-hr contact time

• Sampled to nutrient agar, microplate reader


Minimum Inhibitory/Biocidal Concentration

Experimental – Fluorescence

• Saccharomyces cerevisiae• Peracetic acid – 100, 150 mg/L• Dual-Channel Fluorescence• EtOH linearization (proxy validation)• Time-Kill Trials

30‐60 minutes

Cells stained with RFP/GFP

Live cells able to expel RFP from membrane/cytoplasm

Staining Principle

• Linear EtOH kill curve demonstrates utility of propidium iodide as viability proxy

• Assay suggests effective concentrations of peracetic acid work in 5 minutes or less

Results – Fluorescence


Time-kill Experiments: S. cerevisiae


In-tank Fermentation Trials

• 2B Mill stainless steel coupons (3.5 cm x 8.125 cm) mounted on custom device

• Inoculated Cab Sauvfermentation trials to simulate tank-dirty soils

• Three trials• Cleaners only• Cleaners followed by sanitizers

(5-step)• Optimization of protocols

• ATP swabbing and plate counts obtained


Optimizing Concentrations/Contact Time


Major Areas of Microbial Contamination

• Winery tank surfaces and bottling lines - cleanest

• Drains and area around the bung of barrels - high levels of microbial contamination

• Pumps, hoses, thieves, winery process water – re-contaminate


Strategies for Fermentation-Soil Protocols

• Pay attention to critical areas• Valves, gaskets, ports• Places in the shadow of spray balls or mechanical agitation• Take care in cleaning floors, especially with hose water

• Develop a strategy for monitoring the success of a protocol• Visual clean is not good enough

• Written protocols and checklists


Strategies for Barrel Management

• Brettanomyces identified to penetrate barrel staves up to 9 mm• For thermal treatments: Inactivated at 55C with D-value of up to 80 sec.• Water treatment were determined as 9∙7 min at 55°C, 5∙4 min at 57∙5°C,

3∙0 min at 60∙0°C and 1∙7 min at 62∙5°C

• Barrel thermal water treatment at 60˚C for 19 min (include filling time of 6 in, heat propagation into the wood, 10 min) – affective 8 log reduction

• Appear to penetrate deeper into French barrels, especially with higher toast levels

• Hot water• Normal protocol: Fill barrels with 50-70C water, soak for variable time

Cartwright, Z.M., Glawe, D., and Edwards, C.G. (2018) Reduction of Brettanomyces bruxellensis Populations from Oak Barrel Staves Using Steam, Am. J. Enol. Vitic. 69:4; De Lourdes Alejandra Aguilar Solis, M., Gerling, C., and Worobo, R. (2013) Sanitation of Wine Cooperage using Five Different Treatment Methods: and In Vivo study, Appellation Corness Research Focus 2013‐3



• Steam• 12 min required to produce no culturable cells at 9 mm depth.

• Takes approximately 4 min for barrel to reach 55C at 9 mm depth

• Ozone• 1 mg/L for 30 min

• Hindered by flow diffusion into staves, reacts easily with organics in barrel

• Chemical Control, Ultrasonic, SO2

Cartwright, Z.M., Glawe, D., and Edwards, C.G. (2018) Reduction of Brettanomyces bruxellensis Populations from Oak Barrel Staves Using Steam, Am. J. Enol. Vitic. 69:4; De Lourdes Alejandra Aguilar Solis, M., Gerling, C., and Worobo, R. (2013) Sanitation of Wine Cooperage using Five Different Treatment Methods: and In Vivo study, Appellation Corness Research Focus 2013‐3; Photo, Grapevine Magazine



• Cross contamination• Barrel sampling and topping • Sanitize wine thief during sampling• Critical that topping wine is microbially stable

• Use filtered wine• Or do plating before use for topping

• Carry 70% EtOH to spray outer/inner bung


Summary• A proper cleaning regimen is the most important aspect of a

cleaning and sanitation program• The basic five-step cleaning and sanitizing protocol provides a

foundation for developing a successful program for a winery of any size

• There is no right or wrong choice for cleaning and sanitizing chemicals

• Choice should depend on budget, the application (surface, soil load), regulations or personal preference, but should be validated by a monitoring program

• Even when visually clean, a cleaning step cannot be omitted or the risk of contamination from biofilms will occur

• Pay attention to critical surfaces (gaskets, hoses, drains, etc.)

Documents

OPTIMIZING CLEANING AND SANITATION SOP’S IN THE WINERY