1. Molly Kelly Enology Extension Specialist Virginia Tech
Winery Sanitation
Molly Kelly
Enology Extension Specialist
Virginia Tech

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

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

4. 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

5. 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

6. 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

7. 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

8. 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 8

9. 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 9 9

10. Example: Cleaning Flow Chart
A flow chart of the cleaning process might look like this.
Was cleaning effective?

11. 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)

12. 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

13. 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

14. 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

15. 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

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

17. 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

18. 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

19. 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

20. 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?

21. Evaluating Cleaning Effectiveness

22. Evaluating Cleaning Effectiveness
Check rinse water to ensure that the cleaner has been rinsed (pH, color, aroma of rinse water)
Visually inspect the equipment for residual stains, tartrates, etc.
Look most carefully at the “dead” zones and in crevices
Notice the sheeting action of water…even sheeting implies a clean surface, while “beading” implies a dirty surface.
Use your nose and check if the equipment smells clean Check for VA (volatile acidity/acetic acid), wine aroma, aroma of residual cleaning agents, other off aromas
Feel the surface of the equipment for stickiness, roughness, etc to determine if the surface is clean and smooth
Does it look, smell, and feel clean? Do you see stains, or smell aromas, or feel solids or stickiness. Look at boundary layers, blind spots, and crevices
Use microbial methods (ATP testing or plating)

23. 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.
Biofilims are very important in the wine industry. They are likely to be present in many situations, especially at boundary layers between liquid and air (rings around tops of tanks, and especially in hoses and in place lines). An odd effect of this is that a bioluminescence or other swabbing test might show a higher amount of contamination AFTER cleaning than before cleaning, as the detergent may have damaged the protective layers and exposed the cells. Swab testing must be consistently done with a good degree of force to ensure that the swab breaks through this layer and picks up the underlying cells.
Heat will kill the cells in the biofilm but the cells may persist, giving the confusing possibility of a negative growth test and a positive ATP test.

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

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

26. Winery Recommendations
General Order: Hoses and pumps
Cold water, high pressure rinse w/foam ball
Mild alkaline detergent solution 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

27. 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

28. 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 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

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

30. 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

31. Is Sterility a Must? We don’t Need Sterility We don’t Want 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…

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

33. 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.

34. 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
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
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

36. 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

37. 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).

38. Cleaning and sanitation steps
Debris removal without water
Rinse with water
Wash with cleaning agent
Rinse with acid
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
Check MSDS sheets when in doubt
Wear protective gear if needed

40. 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.

41. Verifying Concentration of Chemical SanitizerspH
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

42. Verifying Temperature
Digital Thermometers
Infrared thermometers
Steam test strips and tape (for autoclaves)
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
Many opportunities for problems
People with different responsibilities
Multiple wines to bottle
Operation and maintenance of multiple equipment stations 44

45. 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 45

46. 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 46

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

48. 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 48

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

50. Brettanomyces
Practical winery and vineyard
Wikipedia.com

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

52. 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 52

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

54. Acetobacter Gram negative rods or cocci Strictly aerobic
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 54

55. 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 55

56. 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

57. Lactic acid bacteria spoilage
Typical spoilage times:
During “stuck” fermentations
Finished wines with low SO2, residual malic acid or sugar 57

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

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

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

61. 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) 61

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

63. 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

64. 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 64

65. Bacteria prevention/control
Grapes
Minimize damage to skins
Pick when cool
Sorting
Add SO2 to picking bins
Minimize transport distance
Adequate hygiene 65

66. Bacteria prevention/control
Winery equipment
Regular cellar hygiene
Wines
Short or no skin contact
Adequate SO2
Exclusion of air
Filtration
Acid addition 66

67. 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. 67

68. 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. 68

69. 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, microns for yeasts and molds

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

71. Swab Test Method Semi-quantitative analysis
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

72. Airborne Microorganisms
Sartorius stedim biotech
Sedimentation plates
Leave plates open for 30 min-2 hrs
Incubate

73. Culture Media Yeasts and molds
Wort 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

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

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

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

77. 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 77

78. Contact Information
Molly Kelly
Enology Extension Specialist
Food Science Dept., Virginia Tech
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.
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, 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 79