optimizing cleaning and sanitation sop’s in the winery
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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
UC DAVIS VITICULTURE AND ENOLOGY
Outline
• Introduction
• Winery Spoilage Microbes
• Cleaning and Sanitation Chemicals
• Protocols
• Monitoring Strategies
• Supporting Experimental Data
• Barrel Cleaning and Sanitation
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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
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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
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Winery Cleaning and Sanitation
• Cleaning• Process involving physical removal of organic and inorganic
soils
• Sanitizing• Process involving inactivation of microbes
• Disinfection
• Sanitation
• Sterilization
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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
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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
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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
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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
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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)
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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
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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)
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Sanitizing Chemistries
• 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
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Sanitizing Chemistries
• 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
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Sanitizing Chemistries
• 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.)
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Basic 5‐Step Cleaning/Sanitizing Process
Water Rinse
Water Rinse
Water Rinse
Cleaning Cycle
Sanitizing Cycle
Winery Cleaning and Sanitation
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)
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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
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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
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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
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Winery Microbes
• Research study• 3 yeast
• Saccharomyces cerevisiae, Zygosaccharomyces baili, Brettanomyces bruxellensis
• 4 bacteria• Lactobacillus casei, Acetobacter pasteurianus, Pediococcus parvulus,
Oenococcus oeni
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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
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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
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Biofilm Investigation
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Environmental Swabs: Biofilms on Stainless Steel
• Biofilms formed on 304SS coupons• 3 yeast, 1 mixture of
bacteria
• Two monitoring Methods• ATP bioluminescence
• Plate counts
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ATP Swabbing of Biofilms on Stainless Steel
• Illustrates need for cleaning step
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ATP Swabbing of Biofilms on Stainless Steel
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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
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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
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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
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Time-kill Experiments: S. cerevisiae
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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
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Optimizing Concentrations/Contact Time
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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
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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
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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
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Strategies for Barrel Management
• 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
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Strategies for Barrel Management
• 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
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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.)
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