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Winery Sanitation Molly Kelly Enology Extension Specialist Virginia Tech.

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1 Winery Sanitation Molly Kelly Enology Extension Specialist Virginia Tech

2 Cellar Hygiene Why? Why? To maintain wine integrity and quality To maintain wine integrity and quality

3 Sanitation Anything in contact with the wine is a potential vector for microbial spoilage Anything in contact with the wine is a potential vector for microbial spoilage Have appropriate sanitation protocols in place and implement them Have appropriate sanitation protocols in place and implement them

4 General Principles of Surface Cleaning and Sanitizing Remove loose soiling particles from surface Remove loose soiling particles from surface Make bound soiling layer ‘wet’ Make bound soiling layer ‘wet’ Allow penetration of layer by solvent and cleanser Allow penetration of layer by solvent and cleanser Remove bound soiling layer from surface Remove bound soiling layer from surface Rinse surface Rinse surface Sanitize surface Sanitize surface If desired, sterilize surface If desired, sterilize surface

5 Purposes of Cleaning, Sanitizing, and Sterilizing Get rid of microorganisms Get rid of microorganisms Get rid of nutrients and contaminating compounds Get rid of nutrients and contaminating compounds Prevent entry and build-up of microorganisms and other contaminants Prevent entry and build-up of microorganisms and other contaminants

6 Some Definitions Cleaning: Removing dirt, debris and stains Cleaning: Removing dirt, debris and stains Sanitizing: the reduction of the microbial population to a safe level (kills 99.9% of growing bugs) Sanitizing: the reduction of the microbial population to a safe level (kills 99.9% of growing bugs) Sterilizing: Complete Elimination of microbial Life Sterilizing: Complete Elimination of microbial Life

7 Definitions Cleaning: an attempt to physically and chemically remove food for microorganisms and to eliminate hospitable environments for their growth Cleaning: an attempt to physically and chemically remove food for microorganisms and to eliminate hospitable environments for their growth Sanitation=Disinfection Sanitation=Disinfection Sanitation is an attempt to reduce the number of spoilage organisms on equipment surfaces Sanitation is an attempt to reduce the number of spoilage organisms on equipment surfaces Sterilization is an attempt to kill 100% of spoilage organisms Sterilization is an attempt to kill 100% of spoilage organisms

8 QA and QC Quality Assurance (QA) focuses on attempts to improve and stabilize production practices in order to prevent defects 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 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: means of assuring quality, through the identification and monitoring of critical control points from vine to glass

9 HACCP Logical system of control based on the prevention of problems Logical system of control based on the prevention of problems Look at your process from start to finish Look at your process from start to finish Decide where hazards can occur Decide where hazards can occur Put in controls and monitor them Put in controls and monitor them Write it all down and keep records Write it all down and keep records Ensure that it continues to work effectively Ensure that it continues to work effectively All wineries would benefit from a basic outline All wineries would benefit from a basic outline

10 Example: Cleaning Flow Chart Was cleaning effective?

11 Rule of Thumb Acid cleaners dissolve alkaline soils (minerals) Acid cleaners dissolve alkaline soils (minerals) Alkaline cleaners dissolve acid soils and food wastes 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) Improper use of detergents can actually “set” soils, making them more difficult to remove (e.g. acid cleaners can precipitate protein)

12 More is better? Common misconception: “if a little is good, 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 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 Follow manufacturer’s label instructions

13 Chemical and their Use ActionChemical Cleaner Alkalis, acids, detergents Sanitizer Quats, peracetic acid, SO2, hydrogen peroxide, ozone Sterilizer Hot water, steam, alcohol, UV, ozone

14 Types of Sanitizers Heat, w/ water or steam to saturate effect Heat, w/ water or steam to saturate effect Very effective anti-microbial, except some encysted forms Very effective anti-microbial, except some encysted forms Exposure time critical Exposure time critical Non-corrosive, but energy intensive Non-corrosive, but energy intensive Chemical Chemical Effectiveness varies with exposure time, temperature, concentration, microbial load, soil load Effectiveness varies with exposure time, temperature, concentration, microbial load, soil load Can be corrosive Can be corrosive

15 Chemical Sanitizers Chlorine-containing not recommended except for out of control microbial situations Chlorine-containing not recommended except for out of control microbial situations Very effective anti-microbial Very effective anti-microbial Iodine-containing little used for wineries Iodine-containing little used for wineries Effective anti-microbial, except when diluted Effective anti-microbial, except when diluted

16 Iodine Iodophores: broad spectrum Iodophores: broad spectrum Active against bacteria, viruses, yeasts, molds, fungi Active against bacteria, viruses, yeasts, molds, fungi Bottling line (followed by hot water) Bottling line (followed by hot water) Potential for TCA Potential for TCA

17 Quaternary Ammonium Compounds (QACs) Positively charged: mode of action is related to their attraction to negatively charged materials (bacterial proteins) Positively charged: mode of action is related to their attraction to negatively charged materials (bacterial proteins) Leave a residual antimicrobial film Leave a residual antimicrobial film Floors Floors

18 Chemical Sanitizers Peroxides Peroxides Hydrogen peroxide, ozone (inorganic), peroxyacetic acid, peroxyborates (organic) Hydrogen peroxide, ozone (inorganic), peroxyacetic acid, peroxyborates (organic) Very effective anti-microbials Very effective anti-microbials Tolerant of mineralized water Tolerant of mineralized water Concentration dependent Concentration dependent Not corrosive Not corrosive Can remove light bio-films Can remove light bio-films Environmentally friendly Environmentally friendly

19 Sanitizers percarbonates Per(oxy)carbonate Per(oxy)carbonate Release of oxygen radicals via H2O2 Release of oxygen radicals via H2O2 Application at room temperature Application at room temperature Effective over wide pH range (1-8) Effective over wide pH range (1-8) Alkaline Alkaline Degradation to soda ash, water, O2 Degradation to soda ash, water, O2

20 Sanitizers Soda Ash Na 2 CO 3 Strong alkaline (pH 11.3 at 1%) Strong alkaline (pH 11.3 at 1%) Dissolves proteins, fats, oils, carbohydrates, tartrates Dissolves proteins, fats, oils, carbohydrates, tartrates Neutralizes acidic odors (VA) Neutralizes acidic odors (VA) Neutral pH best for bacteria Neutral pH best for bacteria Sanitizing effect? Sanitizing effect?

21 Evaluating Cleaning Effectiveness

22

23 Biofilms A mixed group of microbial species protected by a polysaccharide/exopolymer coating. 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.

24 Winery Recommendations General Order: Stainless or plastic tanks, crusher-destemmers, presses, fittings General Order: Stainless or plastic tanks, crusher-destemmers, presses, fittings Cold water, high pressure rinse Cold water, high pressure rinse Strong inorganic alkaline solution or paste scrub Strong inorganic alkaline solution or paste scrub Cold water, high pressure rinse Cold water, high pressure rinse Cationic detergent, combined with peroxyacetic Cationic detergent, combined with peroxyacetic Cold water, high pressure rinse Cold water, high pressure rinse Hot water, high pressure rinse Hot water, high pressure rinse Ozone treatment Ozone treatment

25 Winery Recommendations General Order: Bottling line General Order: Bottling line Cold water, high pressure rinse Cold water, high pressure rinse Mild alkaline detergent solution Mild alkaline detergent solution Cold water, high pressure rinse Cold water, high pressure rinse Cationic detergent, combined with peroxyacetic Cationic detergent, combined with peroxyacetic Cold water, high pressure rinse Cold water, high pressure rinse Hot water, high pressure rinse Hot water, high pressure rinse Ozone treatment Ozone treatment

26 Winery Recommendations General Order: Hoses and pumps General Order: Hoses and pumps Cold water, high pressure rinse w/foam ball Cold water, high pressure rinse w/foam ball Mild alkaline detergent solution w/foam ball Mild alkaline detergent solution w/foam ball Cold water, high pressure rinse w/foam ball Cold water, high pressure rinse w/foam ball Cationic detergent, combined with peroxyacetic, w/foam ball Cationic detergent, combined with peroxyacetic, w/foam ball Cold water, high pressure rinse, w/foam ball Cold water, high pressure rinse, w/foam ball Hot water, high pressure rinse, no ball Hot water, high pressure rinse, no ball Ozone treatment, no ball Ozone treatment, no ball

27 Winery Recommendations General Order: Barrels free of faulty aromas or tastes General Order: Barrels free of faulty aromas or tastes Cold water, high pressure rinse, 1-3 minutes Cold water, high pressure rinse, 1-3 minutes High pressure steam rinse, 1-3 minutes High pressure steam rinse, 1-3 minutes Repeat cold and steam rinses twice more Repeat cold and steam rinses twice more Either refill with clean wine or Either refill with clean wine or Fill with water Fill with water add ozone, if available add ozone, if available follow with water + 45 pm SO 2 /90 ppm citrate follow with water + 45 pm SO 2 /90 ppm citrate After 1-4 days, empty and refill with wine or empty and burn sulfur pastille, re-bung and store After 1-4 days, empty and refill with wine or empty and burn sulfur pastille, re-bung and store

28 Winery Recommendations General Order: Barrels with faulty aromas or tastes General Order: Barrels with faulty aromas or tastes Option 1  Remove from winery and sell for non-wine uses Option 1  Remove from winery and sell for non-wine uses Option 2  Clean, sterilize, and re-use, if worth the cost Option 2  Clean, sterilize, and re-use, if worth the cost Rinse cycles done as per barrel without faulty aromas or tastes Rinse cycles done as per barrel without faulty aromas or tastes Fill with water, put steam wand in water and bring water to  F, steam periodically to maintain temperature for 4-6 hours Fill with water, put steam wand in water and bring water to  F, steam periodically to maintain temperature for 4-6 hours add ozone, if available add ozone, if available follow with water + 45 pm SO 2 /90 ppm citrate follow with water + 45 pm SO 2 /90 ppm citrate After 1-4 days, empty and burn sulfur pastille, re-bung and store 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 SO 2 /180 ppm citrate while doing microbiological assay of samples After 1-4 weeks, rinse and fill with clean water, after 1 week, take samples and then add 90 pm SO 2 /180 ppm citrate while doing microbiological assay of samples If samples are negative for spoilage microorganisms, re-use barrel, but sample periodically If samples are negative for spoilage microorganisms, re-use barrel, but sample periodically

29 Winery Recommendations General Order: Floors and waterproof walls and ceilings General Order: Floors and waterproof walls and ceilings Cold water, high pressure rinse Cold water, high pressure rinse Scrub w/mild alkaline detergent Scrub w/mild alkaline detergent Cold water, high pressure rinse Cold water, high pressure rinse Hot water, high pressure rinse Hot water, high pressure rinse Ozone treatment Ozone treatment

30 Winery Recommendations General Order: Non-waterproof surfaces General Order: Non-waterproof surfaces Dust w/ damp cloth or sponge Dust w/ damp cloth or sponge Clean with mild household ammonia-based product Clean with mild household ammonia-based product Wipe with damp sponge or dry towel Wipe with damp sponge or dry towel

31 Is Sterility a Must? We don’t Need Sterility We don’t Need Sterility Commercial yeast out-competes wild yeasts, fungus, mold, and bacteria– especially at wine pH Commercial yeast out-competes wild yeasts, fungus, mold, and bacteria– especially at wine pH We don’t Want Sterility We don’t Want Sterility Sometimes wines may benefit from something extra… Sometimes wines may benefit from something extra…

32 Sterilizers Hot water and steam used to sterilize bottling line Hot water and steam used to sterilize bottling line 80-90F for 30 min 80-90F for 30 min 180F for 20 min 180F for 20 min ozone for min ozone for min Alcohol and UV: lab & for small equipment Alcohol and UV: lab & for small equipment

33 Basic Rules Clean everything BEFORE you use it. And then sanitize. Clean everything BEFORE you use it. And then sanitize. Clean everything AFTER you use it. Clean everything AFTER you use it. Clean the winery premises, not just the equipment, on a regular basis. Clean the winery premises, not just the equipment, on a regular basis. Keep the winery free of clutter. Keep the winery free of clutter. Watch for pests (bacteria, mold, wild yeast, rodents, etc. Watch for pests (bacteria, mold, wild yeast, rodents, etc. Deal with pomace IMMEDIATELY. Deal with pomace IMMEDIATELY.

34 Generic Chemicals for Cleaning and Sanitation TypeDoseNotes 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

35 Formulated Chemicals for Cleaning and Sanitation TypeDoseNotes 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 SO 2 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

36 Sanitizers Sanitizer Time of use (min) Optimum pH Temp range Peracetic acid 5-20> Ozone1-30-Cold Quats15m-24hr Sulfur dioxide 15s-20min<3cold

37 Special Cases: Barrels There’s no good way to deal with old barrels that have “gone off…” There’s no good way to deal with old barrels that have “gone off…” Chemicals will either taint the wood or extract essence. Chemicals will either taint the wood or extract essence. But here are some ideas. But here are some ideas. Treat barrels right Treat barrels right Don’t let them dry out. Don’t let them dry out. Store with a MBS/citric acid solution (2 oz/2 oz/5 gal water) Store with a MBS/citric acid solution (2 oz/2 oz/5 gal water) Clean the outside as well as the inside 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). Recover with a percarbonate based cleaner (1 Tbsp/gal), let sit 24 hours, rinse, then rinse with citric acid (0.5 tsp/gal).

38 Cleaning and sanitation steps Debris removal without water Debris removal without water Rinse with water Rinse with water Wash with cleaning agent Wash with cleaning agent Rinse with water Rinse with water Rinse with acid Rinse with acid Rinse with water Rinse with water Use sanitizer & repeat rinsing if cleaner is not anti-microbial Use sanitizer & repeat rinsing if cleaner is not anti-microbial

39 Precautions Cleaners and sanitizers are corrosive and can be harmful to skin, respiratory system and eyes Cleaners and sanitizers are corrosive and can be harmful to skin, respiratory system and eyes Check MSDS sheets when in doubt Check MSDS sheets when in doubt Wear protective gear if needed Wear protective gear if needed

40 Validating Winery Sanitation Perform the sanitation operation using a standard operating procedure (SOP) Perform the sanitation operation using a standard operating procedure (SOP) define equipment, concentration of sanitizer, time, temperature, surface type, cleaning state, bioload, etc. define equipment, concentration of sanitizer, time, temperature, surface type, cleaning state, bioload, etc. Validate that the SOP was effective Validate that the SOP was effective Use swabbing or soak/rinse water to get sample Use swabbing or soak/rinse water to get sample Use microscope scans, plating, or PCR methods Use microscope scans, plating, or PCR methods Changing any parameter in the SOP would indicate the need for additional validation. Changing any parameter in the SOP would indicate the need for additional validation.

41 pH pH pH test strips for acid sanitizers pH test strips for acid sanitizers Concentration (critical to get the right range and sensitivity) Concentration (critical to get the right range and sensitivity) Ozone test strips Ozone test strips Iodine test strips Iodine test strips Peracetic acid test strips Peracetic acid test strips Quaternary Ammonia strips Quaternary Ammonia strips Peroxide test strips Peroxide test strips Sulfite test strips Sulfite test strips Verifying Concentration of Chemical Sanitizers

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

43 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

44 Bottling and Packaging One of the most critical steps in wine production One of the most critical steps in wine production Many opportunities for problems Many opportunities for problems People with different responsibilities People with different responsibilities Multiple wines to bottle Multiple wines to bottle Operation and maintenance of multiple equipment stations Operation and maintenance of multiple equipment stations

45 Sterile bottling rooms? Not an absolute necessity Not an absolute necessity Bottling area should be screened-off from fermentation areas and excessive air movement Bottling area should be screened-off from fermentation areas and excessive air movement Easily sanitized floors, walls and ceilings Easily sanitized floors, walls and ceilings

46 Sources of contamination at bottling Filter pad drip trays: drain often during runs Filter pad drip trays: drain often during runs Fill bowls: Mist filler spouts with 70% ethanol to inhibit microbial growth 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 Corker: likely to have spilled wine. Ethanol misting of corker jaws during bottling Activity: Limit number of people around filling/corking area Activity: Limit number of people around filling/corking area

47 Spoilage Microorganisms on Grapes Non-Saccharomyces yeasts Non-Saccharomyces yeasts Kloeckera apiculata Kloeckera apiculata Acetic acid bacteria Acetic acid bacteria Lactic acid bacteria Lactic acid bacteria Lysozyme Lysozyme

48 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

49 Brettanomyces growing in culture B. bruxellensis on WL agar Brettanomyces.wordpress.com

50 Brettanomyces Wikipedia.com Practical winery and vineyard

51 Brett Smells like: Barnyard Horse (blanket, sweat, saddle) Wet dog Tar Tobacco Creosote Plastic Leather Pharmaceutical (band aid) Smells like: Barnyard Horse (blanket, sweat, saddle) Wet dog Tar Tobacco Creosote Plastic Leather Pharmaceutical (band aid)

52 Yeast prevention/control Grapes Minimize damage to skins Pick when cool Sorting Add SO 2 to picking bins Minimize transport distance Adequate hygiene Winery equipment Regular cellar hygiene Wines Adequate SO 2 Absence of air/oxygen Filtration Grapes Minimize damage to skins Pick when cool Sorting Add SO 2 to picking bins Minimize transport distance Adequate hygiene Winery equipment Regular cellar hygiene Wines Adequate SO 2 Absence of air/oxygen Filtration

53 Microorganisms on Grapes Non-Saccharomyces yeasts Non-Saccharomyces yeasts Kloeckera apiculata Kloeckera apiculata Acetic acid bacteria Acetic acid bacteria Lactic acid bacteria Lactic acid bacteria Lysozyme Lysozyme

54 Acetobacter Gram negative rods or cocciGram negative rods or cocci Strictly aerobicStrictly aerobic 0.6 to 0.9 microns by 1 to 3 microns0.6 to 0.9 microns by 1 to 3 microns Formation of acetic acid from ethanolFormation of acetic acid from ethanol angeladouglaslab.com, Cornell University

55 AAB control Low pH (acid)Low pH (acid) Minimize oxygen incorporationMinimize oxygen incorporation Maintain cool temperatures (<50F)Maintain cool temperatures (<50F) Free sulfur dioxide levels according to pHFree sulfur dioxide levels according to pH High VA wines can be blended with unaffected wine or treated by ROHigh VA wines can be blended with unaffected wine or treated by RO

56 Microbes during fermentation Non-Saccharomyces yeasts Non-Saccharomyces yeasts Lactic acid bacteria (LAB) Lactic acid bacteria (LAB) What to watch for: What to watch for: Ethyl acetate Ethyl acetate Spontaneous or sluggish fermentation Spontaneous or sluggish fermentation Spontaneous MLF Spontaneous MLF VA VA Off odors Off odors

57 Lactic acid bacteria spoilage Typical spoilage times: Typical spoilage times: During “stuck” fermentations During “stuck” fermentations Finished wines with low SO 2, residual malic acid or sugar Finished wines with low SO 2, residual malic acid or sugar Typical spoilage times: Typical spoilage times: During “stuck” fermentations During “stuck” fermentations Finished wines with low SO 2, residual malic acid or sugar Finished wines with low SO 2, residual malic acid or sugar

58 Lactobacillus spp. Enologyaccess.org Lactobacillus fermentum Fermentacionvegetales.wikispaces.com Lactobacillus brevis

59 LAB Volatile acidity-metabolism of citric acid and glucose to acetic acid Volatile acidity-metabolism of citric acid and glucose to acetic acid Mousiness Mousiness Geranium taint-metabolism of sorbic acid Geranium taint-metabolism of sorbic acid Ropiness –production of extracellular polysaccharides Ropiness –production of extracellular polysaccharides Volatile acidity-metabolism of citric acid and glucose to acetic acid Volatile acidity-metabolism of citric acid and glucose to acetic acid Mousiness Mousiness Geranium taint-metabolism of sorbic acid Geranium taint-metabolism of sorbic acid Ropiness –production of extracellular polysaccharides Ropiness –production of extracellular polysaccharides

60 Pediococcus Practicalwinery.com Pediococcus spp. and one Lactobacillus chain

61 Mousey taint Aftertaste: not volatile at wine pH;mix with neutral pH of saliva become apparent Aftertaste: not volatile at wine pH;mix with neutral pH of saliva become apparent Mouse urine Mouse urine Rancid nuts Rancid nuts Can also smell like acetaldehyde, corn chips (when rub in palms) Can also smell like acetaldehyde, corn chips (when rub in palms) Comes from Brett (rarely) and LAB (usually) Comes from Brett (rarely) and LAB (usually) Aftertaste: not volatile at wine pH;mix with neutral pH of saliva become apparent Aftertaste: not volatile at wine pH;mix with neutral pH of saliva become apparent Mouse urine Mouse urine Rancid nuts Rancid nuts Can also smell like acetaldehyde, corn chips (when rub in palms) Can also smell like acetaldehyde, corn chips (when rub in palms) Comes from Brett (rarely) and LAB (usually) Comes from Brett (rarely) and LAB (usually)

62 Stuck Ferments Lactobacillus Lactobacillus Pediococcus Pediococcus Brettanomyces Brettanomyces Acetobacter Acetobacter Film yeasts (Candida and Pichia) Film yeasts (Candida and Pichia) Watch for: Watch for: VA increase VA increase Film yeast Film yeast Spontaneous MLF Spontaneous MLF CO2 production CO2 production

63 Microbes during MLF Acetobacter Acetobacter Lactobacillus Lactobacillus Pediococcus Pediococcus Brettanomyces Brettanomyces Film yeasts Film yeasts Oenococcus Oenococcus Watch for: Watch for: VA increase >0.15 g/L (from citric acid) VA increase >0.15 g/L (from citric acid) Film Film Off-odors and flavors Off-odors and flavors

64 VA post fermentation sources Headspace in barrels Headspace in barrels Oxidation of wine Oxidation of wine “acetic aroma” not exclusively a result of acetic acid “acetic aroma” not exclusively a result of acetic acid Also due to ethyl acetate (ester) Also due to ethyl acetate (ester) Esters are volatile compounds formed by combination of an alcohol and an acid Esters are volatile compounds formed by combination of an alcohol and an acid Headspace in barrels Headspace in barrels Oxidation of wine Oxidation of wine “acetic aroma” not exclusively a result of acetic acid “acetic aroma” not exclusively a result of acetic acid Also due to ethyl acetate (ester) Also due to ethyl acetate (ester) Esters are volatile compounds formed by combination of an alcohol and an acid Esters are volatile compounds formed by combination of an alcohol and an acid

65 Bacteria prevention/control Grapes Grapes Minimize damage to skins Minimize damage to skins Pick when cool Pick when cool Sorting Sorting Add SO 2 to picking bins Add SO 2 to picking bins Minimize transport distance Minimize transport distance Adequate hygiene Adequate hygiene Grapes Grapes Minimize damage to skins Minimize damage to skins Pick when cool Pick when cool Sorting Sorting Add SO 2 to picking bins Add SO 2 to picking bins Minimize transport distance Minimize transport distance Adequate hygiene Adequate hygiene

66 Bacteria prevention/control Winery equipment Winery equipment Regular cellar hygiene Regular cellar hygiene Wines Wines Short or no skin contact Short or no skin contact Adequate SO 2 Adequate SO 2 Exclusion of air Exclusion of air Filtration Filtration Acid addition Acid addition Winery equipment Winery equipment Regular cellar hygiene Regular cellar hygiene Wines Wines Short or no skin contact Short or no skin contact Adequate SO 2 Adequate SO 2 Exclusion of air Exclusion of air Filtration Filtration Acid addition Acid addition

67 Validating Winery Sanitation Perform the sanitation operation using a standard operating procedure (SOP) Perform the sanitation operation using a standard operating procedure (SOP) define equipment, concentration of sanitizer, time, temperature, surface type, cleaning state, bioload, etc. define equipment, concentration of sanitizer, time, temperature, surface type, cleaning state, bioload, etc. Validate that the SOP was effective Validate that the SOP was effective Use swabbing or soak/rinse water to get sample Use swabbing or soak/rinse water to get sample Use microscope scans, plating, or PCR methods Use microscope scans, plating, or PCR methods Scorpions™ testing can provide fast results and target specific wine spoilage organisms. 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. Changing any parameter in the SOP would indicate the need for additional validation.

68 Swab or Soak Sampling Specify the details of the sampling technique in the SOP Specify the details of the sampling technique in the SOP Swabbing area and location Swabbing area and location Use good sterile technique Use good sterile technique Rub hard to break up biofilms Rub hard to break up biofilms Place swab in labeled container of sterile saline Place swab in labeled container of sterile saline Sterile water soak/rinse sampling may be appropriate for some situations. Sterile water soak/rinse sampling may be appropriate for some situations. A concentration step may be helpful if low cell numbers are anticipated. A concentration step may be helpful if low cell numbers are anticipated. A negative result (no cells found) does NOT indicate sterility. A negative result (no cells found) does NOT indicate sterility. Ongoing testing and historical trends important in developing and implementing effective programs. Ongoing testing and historical trends important in developing and implementing effective programs.

69 Membrane Filter Method Isolate small number of microbes from liquid sampleIsolate small number of microbes from liquid sample Demonstrate their presence as colonies on culture mediumDemonstrate their presence as colonies on culture medium Cellulose nitrate membrane: 0.45 micron for bacteria, microns for yeasts and moldsCellulose nitrate membrane: 0.45 micron for bacteria, microns for yeasts and molds Sartorius stedim biotech

70 Surface Testing For smooth surfacesFor smooth surfaces Place membrane on surface to be tested and place on agar platePlace membrane on surface to be tested and place on agar plate Use sterile techniqueUse sterile technique Sartorius stedim biotech

71 Swab Test Method Semi-quantitative analysisSemi-quantitative analysis Moist sterile cotton swabs for dry areas (sterile physiological saline)Moist sterile cotton swabs for dry areas (sterile physiological saline) Dry sterile cotton swabs for moist areasDry sterile cotton swabs for moist areas Sartorius stedim biotech

72 Airborne Microorganisms Sartorius stedim biotech Sedimentation platesSedimentation plates Leave plates open for 30 min-2 hrsLeave plates open for 30 min-2 hrs IncubateIncubate

73 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

74 Cellar Hygiene Why? Why? To maintain wine integrity and quality To maintain wine integrity and quality Poor wine quality is usually due to poor sanitation practices! Poor wine quality is usually due to poor sanitation practices!

75 Sources/areas of build up Vineyard Vineyard Diluted pools of juice Diluted pools of juice Second-hand barrels Second-hand barrels Imported bulk wine Imported bulk wine Areas of winery that are difficult to reach Areas of winery that are difficult to reach Vineyard Vineyard Diluted pools of juice Diluted pools of juice Second-hand barrels Second-hand barrels Imported bulk wine Imported bulk wine Areas of winery that are difficult to reach Areas of winery that are difficult to reach

76 General guidelines Proactive and not reactive Proactive and not reactive Constant and immediate attention Constant and immediate attention Clean up immediately Clean up immediately Save time at end of day Save time at end of day

77 Conclusions Prevent and control microbial contamination Prevent and control microbial contamination Reducing population during winemaking Reducing population during winemaking So2 So2 Temp Temp Filtration Filtration Quality control plan Quality control plan SOPs based on cGMPs SOPs based on cGMPs HACCP HACCP Sample wines/taste for faults Sample wines/taste for faults

78 Contact Information Molly Kelly Molly Kelly Enology Extension Specialist Enology Extension Specialist Food Science Dept., Virginia Tech Food Science Dept., Virginia Tech Cell phone: Cell phone:

79 References Lansing, R. May Managing Bottling Operations. Wine Business Monthly. Neradt, F Sources of reinfections during cold-sterile bottling of wine. Am. J. Enol. Vitic. Vol. 33. no. 3. Pregler, B. Dec What’s Cool: Accurate Membrane Filter Integrity Testing. Wine Business Monthly. Wine Business Monthly. Tracy, R. and Skaalen, B. Jan/Feb Bottling-last line of microbial defense. Practical Winery and Vineyard. Ritchie, G., Napa Valley College, VWT 280, Cellar Hygiene, Menke, S., Cleansers and Sanitizers, Penn State Enology Extension, Butzke, C., Barrel Maintenance, Dept. of Food Science, Purdue University, Howe, P., ETS Laboratories, SOWI “Current Issues” Workshops March Crowe, A. Avoiding Stuck Ferments, Wine Business Monthly, August Zoecklein, B. et al, Wine Analysis and Production, Aspen Publishers, Van de Water, L., Practical Winery and Vineyard Journal, Sept/Oct Just, E. and H. Regnery. Microbiology and Wine Preventive care and monitoring in the wine industry. Sartorius Stedim Biotech


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