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Microbiology and WinePreventive care and monitoring in the wine industry

Contents 1. Project Objective

1. Project Objective 4

2. Which Microbes 5are found in the Winery?

3. Testing Methods 7

4. Materials for Routine 16 Microbiological Testing

5. Flow Chart of Winemaking 20and Checklists for Testing

6. Cleaning and Checklists for Cleaning 26

7. Plant Hygiene Training 31

8. Risk Assessment and HACCP 36

This brochure is intended to help winemak-ers and wineries test their products and thehygiene of their facilities and to assessthese under microbiological aspects.The brochure begins with a description ofthe particular microbes most frequentlyencountered in wineries and it outlines theproperties of the must, wine, or variousadditives that influence the growth ofmicroorganisms. The various wine diseasesof microbial origin are not dealt with in this brochure, as there is considerableliterature available on this subject.

The third chapter addresses test methodsthat should be used in the various areasand for different samples. Following thischapter is a description of the equipmentand culture media that can be used for this purpose. Further methods for microbialdifferentiation and colony identificationare also discussed.

A flow chart illustrates the various stagesof the winemaking process, ranging fromdelivery of grapes to bottling, and a riskassessment is carried out for each stage.

Finally, based on this flow chart, a test planis provided that can be used for routinetesting in wineries. The test plan includes astage check to be used if microbiologicalcontamination occurs in the final product.Methods, culture media and assessmentcriteria are described for both this situationand for routine tests.

Chapter six contains important instructionson the sanitation of systems used in routine winemaking, and chapter sevenprovides information on training in hygienic procedures.

Dr. Elke Just and Hildegard Regnery

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Trivial microbes that enter through water,air or soil do not spoil the product as theycannot proliferate under the conditionsthat prevail in wine. However, if they arepresent in large numbers, they may con-tribute to the degradation of SO2 used as apreservative. Contamination with yeast canbe either more or less critical depending on the type and strain of the yeast encoun-tered. While strongly fermenting yeasts will encourage wine to ferment quickly, (frequently resulting in bottle explosion),weakly fermenting yeasts can cause cloudiness.

In either case, positive microbiologicalfindings will usually result in undesirablechanges to the taste, of the wine.

Here is a brief overview of the wine-spoilage microbes most commonly found:

Bacteria

Trivial bacteria that are not spoilageorganisms (total CFU* count) These microbes do not affect the taste orsmell of the wine. Nevertheless, they can contribute to thedegradation of free SO2, which may affectthe stability of the wine if they are presentin large numbers. These microbes include:– Water bacteria that are transmitted via

water used for washing and rinsing– Hygiene-related bacteria that provide an

insight into the hygienic environment– Airborne bacteria that spread as the

result of their ability to survive.

Product-spoilage bacteriaProduct-spoilage bacteria originate fromthe leaves of the grapevine and the grapesthemselves. Lack of hygiene may result inthem being carried into the finished prod-uct. The subsequent effect on the wine is manifested in various wine diseases, a topicwhich is not discussed in detail here. Inprinciple, it can be said that lactic acid bacteria cause cloudiness and have a nega-tive effect on the taste of the wine if theybegin to grow in the filled bottles. The tasteof lactic or acetic acid is typical, but otherdisruptive taste notes may occur. They cancause acid degradation and give the wine a

slimy, oily consistency. A fact is that aceticacid bacteria cause the wine to taste off.Spore-forming bacteria are sometimesfound in wine. However, these microorgan-isms do not grow at the pH that is normallycharacteristic for wine and thus they oftenoccur only as secondary spoilage organismsafter wine has already been subjected toextensive acid degradation.

Lactic acid bacteria: homofermentative andheterofermentative rod-shaped lactic acid bacteria: Lactobacillus brevis Lactobacillus buchneri Lactobacillus caseiLactobacillus fermentum Lactobacillus fructivoransLactobacillus lindneri Lactobacillus plantarum

Lactic acid cocci: Oenococcus oeni Pediococcus inopinatus Pediococcus damnosusPediococcus pentosaceus

Acetic-acid bacteria: Acetobacter aceti Acetobacter pasteurianusGluconobacter oxydans

2. Which Microbes are Found in the Winery?

The particular microorgansisms present in a product or production area will dependon the relevant ambient conditions in theproduct or production area and on the specific requirements that various micro-organisms have in order to grow.

Certain strains of fermentation yeast are mandatory for the fermentationprocess itself. Likewise, lactic acid bacteria are also oftenused systematically for this purpose. Withinthis context, both groups should not beconsidered as spoilage organisms. However,these microorganisms must be removed ordeactivated at a later stage, once the fin-ished wine has been bottled, so that theydo not alter the wine through renewedgrowth. All microorganisms can thereforebe considered as undesirable in filled winebottles and are regarded as spoilage organ-isms.

Grape must is characterized by a low pH, a good range of nutrients such as sugar,protein and trace elements, and aerobicconditions, allowing the growth of aceticacid bacteria, as well as yeasts, molds, and(in particular) those lactobacilli that devel-op in the presence of oxygen. In contrast,anaerobic conditions and the presence ofalcohol are typical for bottled wine. Bothfactors limit mold growth, meaning thatthis type of microorganism is considered aspoilage organism for grapes and must, butnot for wine. The same applies for aceticacid bacteria whose growth is strictly aero-bic. However, the anaerobic conditions inwine promote the growth of lactic acidbacteria, making these significant spoilagebacteria in bottled wine. Yeasts can endureboth alcohol and anaerobic conditions andfor this reason they are generally consideredas the major spoilage organisms in bottledwine.

Lactobacillus brevis

Leuconostoc mesenteroides

Acetobacter aceti

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YeastsIn principle, yeasts are undesirable in thefinished product, even if they are not ofthe strongly fermenting type that represent a direct risk of secondary fermentation.

Yeasts can survive in spore form in soil, butthey usually originate from the surface ofthe grapes, on which up to 100,000 yeastsper grape can be found. They find naturalaccess to the inside of the grape throughthe stomata. Yeasts are also able to pene-trate if the grapes are damaged by insectbites. Weakly fermenting yeasts are usuallypredominant and only at a later stage arethey outnumbered by more strongly fermenting species. They can impart an off-odor or off-taste to the wine and candecompose the alcohol. Those yeasts thatneed oxygen (aerobic yeast) are a problemin grape must, but not in wine itself as thegrowth conditions are unfavourable.

Yeasts required for fermentation:Saccharomyces cerevisiae, various subspecies and strains

Wild yeasts: Strongly fermenting yeasts: Saccharomyces ludwigii Schizosaccharomyces pombeZygosaccharomyces bailiiZygosaccharomyces florentinus

Fermenting yeasts: Kloeckera apiculataPichia anomala Saccharomyces kluyveri Torulaspora delbrueckii Zygosaccharomyces rouxii Zygosaccharomyces microellipsoides

Weakly fermenting yeasts: Brettanomyces anomalus Brettanomyces bruxellensisCandidaDekkeraHansenula Pichia anomala

Oxygen-requiring yeasts:Candida Cryptococcus albidus Debaromyces hansenii Pichia membranefaciensRhodotorula glutinis

MoldsMolds are normally not a problem in wine,but if the grapes are bruised beforehand,molds can cause an off-taste.

AspergillusAureobasidium pullansBothrytis cinerea Mucor Penicillium

Microbes that grow in grape must are different from those that propagate in fermented wine, and thus there is a shift in the microbial flora from the grape to the bottled wine.

At first, wild yeasts predominate. Thesecannot cause strong fermentation, but theydo reduce the oxygen content in grapemust as they grow, thus paving the way forfermenting yeasts to take over.

Acetic acid bacteria only grow in the presence of oxygen and usually decrease innumber as oxygen levels fall. Even if theydo not die off entirely, they are no longerable to grow and multiply. The same alsoapplies to molds. However, both groups cancause a strong off-taste in grape must thatcannot be removed from the wine later.

Users frequently ask how many bacteriacan be tolerated in filled bottles withoutcausing problems. Unfortunately, you neverget a conclusive reply. We cannot answerthis question, either. It depends too muchon the individual characteristics of theproduct and production plant that general-izations would be of little help. A fewyeasts per bottle will not harm dry, well-fermented wine varieties. This is differentfor wines with a high residual sugar con-tent: even a few yeast cells can lead to sec-ondary fermentation. In addition to resid-ual sugar content, free SO2 content alsoplays a role. And, of course, it depends onwhat type of yeast is present. Is it a fer-menting yeast or only an aerobic yeast? The latter does not pose an acute threat tothe product, but the former does. The sameapplies to bacteria content. Many wineriesonly check for yeast content and ignore thepresence of bacteria. However, this can berisky, as many wine diseases are caused bybacteria. In addition to simply detectingthe presence of bacteria, a general identifi-cation is needed to check the potential risktheir presence actually poses to the product.

Schizosaccharomycespombe

Brettanomyces naardenensis

Zygosaccharomycesrouxii

Pichia membranefaciens

Saccharomyces cerevisiae

Rhodotorula glutinis

Aspergillus

Penicillium

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3. Testing Methods

In a winery, the ultimate goal of microbio-logical monitoring is to ensure the safetyof the end product, in other words, of thebottled wine itself and of its various stagesduring processing, i.e. from grape mustthrough cellarage up to the final filtration.This process involves working with liquidsamples for which the membrane filtermethod is primarily used.

In addition, the production systems alsohave to be monitored as the majority of allmicrobial contamination originates in thefilling or corking machines. Here we are nolonger dealing with liquid samples, butwith the monitoring of surface contamina-tion, for which a different set of tests isrequired. Determining each surface’scolony count is a very important part ofhygiene in wineries and indispensable atnumerous stations.

The risk of airborne microbial contamina-tion is higher or lower depending on thewinery's spatial layout. Airborne microbialcontamination can, however, never beruled out, which is why it is becoming moreand more common to test the air formicroorganisms.

Compressed air poses a relatively low risk.However, a test method is available, shouldtests become necessary.

3.1. Liquid Sample Testing3.1.1. The Membrane Filter MethodThe purpose of the membrane filter method is to isolate a small number ofmicroorganisms from as large a sample aspossible and to demonstrate their presenceas colonies by subsequent incubation on aculture medium. This is the only way atrace infection can be detected at an earlystage. For this purpose, a filter unit consist-ing of a filter holder, filter funnel and suc-tion flask (1) as well as vacuum are used. Inthe filter unit, a membrane filter is placedon the filter holder (2), the sample ispoured into the filter funnel (3) and trans-ferred into the suction flask by applying avacuum. The microorganisms are retainedon the filter surface and concentrated fromthe filtered volume. The filter unit is rinsed

with a few milliliters of sterile water toremove any residues from the sample. Then,the membrane filter is placed on a suitableculture medium (4) where it is incubatedfor the prescribed time. Once this processhas been completed, the number ofcolonies is counted. This count is equivalentto the concentrated number of microor-ganisms present in the sample. The filterunit must always be disinfected betweentwo samples, which is routinely done byflaming the filter support (5), the bottomof the filter funnel (6) and the inside of thefilter funnel (7), or by burning off alcohol

in the filter funnel. The membrane filtersare positioned and removed using forceps(8), which must also be sterilized by flam-ing (9) after each use.

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For reasons of convenience, individuallysterile-packaged membrane filters are used.They do not need to be sterilized, and areimmediately ready to use. Disposable filterunits made of plastic are pre-sterilized anddo not have to be disinfected as is the casewith stainless steel filter holders.

The membrane filter should be made ofcellulose nitrate because this material hasthe best growth properties for microorgan-isms. The pore size of the filter is selecteddepending on the objective of testing. Todetect bacteria, a pore size of 0.45 µm iscommonly used. To detect the much largeryeasts and molds, a membrane filter with apore size of 0.65 µm or 0.8 µm is selected.In some cases, 1.2 µm is sufficient. Thesecoarser membranes have a faster flow rate,which saves time during filtration. The riskof microorganisms squeezing through themembrane due to coarser pore size andescaping detection is absolutely non-exis-tent for yeasts and molds.

In cases where the sample volume is toosmall for filtration, as is the case for rinserliquid, a few drops of the sample can bedropped directly onto the agar plate. How-ever, it is not possible to remove inhibitors.Should this be necessary, which certainlycan be the case after disinfection, first fill afew milliliters of sterile water into the filterfunnel and then add a few drops of sample.This way even very small volumes can betested with the membrane filter method,whereby inhibitors, such as disinfectants, are rinsed out.

3.1.2. Bottle Rinsing MethodThe purpose of this method is to submit thecleaned bottles to microbiological testing.This is achieved by filling the cleaned bot-tles with approximately 50 ml of sterilewater and by shaking them vigorously toflush the bacteria from the walls of thebottle so that they can be detected by sub-sequent membrane filtration of the rinsewater.

3.1.3. The Mayer-Vetsch MethodThis method is very easy to carry out anddoes not require any equipment whatsoev-er, howerver it only detects very highcolony counts of some hundred per bottle.

Test tubes containing 20 ml of a specialagar are used, the agar should not be solidand still allow stirring. If need be, the agarcan be prepared by liquefying a solid agarmedium and diluting it with sterile water.With a pipette, 2 ml of sample are stirredcarefully into the medium avoiding airbubbles. During the subsequent incubation, thetransferred microorganisms will grow intocolonies that become visible in the agar.Because the sample volume is limited toapproximately 2 ml, it is not possible todetect trace infections with just a few bac-teria per bottle. Actually, a higher degreeof contamination has to be present beforebacteria can be detected.

3.1.4. Liquid EnrichmentThis method is also easy to use, but becauseof its small sample volume it is not suitablefor the detection of trace infections. Tenmilliliters of the test sample are pipettedinto sterile test-tubes, each of which con-tains 10 ml of grape must. The sampleshould best be taken from the bottom ofthe bottle in order to capture any sedimen-tary bacteria. Both yeasts and bacteriagrow in the grape must and can be identi-fied through cloudiness. In contrast to thepreviously described methods, this methodenables only a qualitative, but not a quan-titative analysis to be made (for example:10 ml without findings).

If wine mixed drinks that contain pulp orother cloudy substances have to be tested,the membrane filter method often cannotbe used. The reason being that it takes nomore than a few milliliters of sample forthe particles to block the membrane filter.In this case, 100 to 200 ml of the productmust be mixed with nutrient broth (20 to200 ml depending on the solution’s con-centration). After incubation, an inoculat-ing loop is used to streak a drop of theenriched liquid on the solid culture medi-um (or on the moist nutrient pad on whicha dry sterile filter has been laid). Two daysare usually long enough to determinewhether the enrichment resulted in micro-bial growth, which will be manifested ascolonies on the culture medium, orwhether the sample was sterile.

3.2. Surface Testing3.2.1. The Contact Plate Method

This method is used to test the bacterialcontamination of relatively smooth surfaces.

Contact can be made directly with an agarplate (RODAC plate). This method uses aspecial agar plate that is filled to the upperrim that makes contact when pressed on asurface. The bacteria stick to the agar and can be counted as colonies afterincubation.

A RODAC plate's lifespan is usually quiteshort because the moisture in the agarevaporates and the surface sinks, makingcontact between the surface and the agarimpossible. This is why membrane filterscan also be used in such cases to collectbacteria, not through filtration, but by thecontact between the membrane and thesurface to be tested. Open a corner of themembrane packaging. Use flamed, sterileforceps to remove the protective paper disk without removing the membrane.Place the membrane and the carrier paperface-up (gridded side) on to the surface to be tested. Rub the back of the carrier paperto charge the membrane with static elec-tricity. The electrical charge ensures thatthe microorganisms on the tested surfacestick to the membrane. Then, remove themembrane and the carrier paper from thesurface. Do this carefully so that the backof the membrane filter remains sterile.Remove the membrane, and, depending on

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the objective, incubate it on the appropri-ate culture medium. When testing moistsurfaces, the back of the carrier paper doesnot have to be rubbed because themicroorganisms and the moisture aredrawn from the pad and adhere to theupper side of the membrane filter. Attrac-tion by electrostatic charge is neither possible nor required for moist surfaces.

3.2.2. The Swab Test MethodSwab tests are suitable for the semi-quan-titative analysis of the colony count inareas that are difficult to reach and cannotbe tested with the contact plate method.Moist cotton swabs are used to test drysurfaces. Dry swabs are used to test moist surfaces.

The cotton swabs must be sterile and can be moistened with sterile physiologicalsaline solution.

In order to obtain approximated quantita-tive evidence, the streaked area should atleast be roughly determined. To collect themicroorganisms, the surface to be tested iswiped with the cotton swab. The swab canthen be directly streaked onto the agarplate, although there is a risk of losing alarge part of the microorganisms becausethey stick to the cotton. A quantitativelybetter result can be achieved by rinsing thecotton swabs in sterile water and testingthe water using the membrane filter

method. For qualitative analysis, which isoften sufficient, the swab can be simplyincubated in a broth. This provides the bestrecovery, but does not allow quantitativeresults to be obtained.

3.3. Testing Methods for AirborneMicroorganisms3.3.1. The Sedimentation MethodFor this method, open petri dishes with cul-ture medium are placed in exposed areas ofthe production facilities (filling area) for adefined time and are then incubated. The disadvantage of this method is the lowvolume of air that can be sampled depend-ing on the movement of the air. It is rec-ommended to keep the Petri dishes openfor approximately 20 to 30 minutesdepending on the expected colony count.This method does not provide a quantita-tive result per defined volume of air, butallows qualitative, comparative evidence tobe obtained for different locations or times of day.

This method can also be carried out by ver-tically attaching petri dishes with culturemedium in the production facilities (mainlyto the filling block, the filter, the labelingmachine etc.) and leaving them there forapproximately 2 hours before removingthem and incubating them for 2 to 3 days.

3.3.2. The Gelatin Membrane FilterMethodThis method is used to quantitativelyestablish the colony count per volume unit(m3) of air. A Rotameter device is used tosample in the air (vacuum cleaner princi-ple). The air flows through a sterile gelatinmembrane filter, which is placed on a filtersupport and attached onto the unit. Thesampling time and the air throughput canbe adjusted. With the direct gelatin mem-brane filter method, the gelatin membranefilter is placed directly onto standard agarand left to incubate for 3 days (ideal time).

The collected microorganisms can be distributed among several culture media bydissolving the gelatin filter in warm sterilewater, filtering the solution in portionsthrough several membrane filters, andincubating these membranes on differentmedia.

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3.4. Which test methods can be usedwhere?Liquid enrichment, swab test, rinsing, contact, membrane filter method, etc.

1. Membrane filtration of the sample andincubation of the filter on nutrient padsets or on agar plates | all sites on alltypes of filter systems, inlet and outlet,filled bottles, rinse water, splash water,etc.

2. Contact plate method and swab testhygiene tests throughout the wineryincluding all vessels, vats, conveyor belts,equipment and machines used to processgrapes, as well as pipes, hoses, tanks orbarrels, filling stations, and corkingmachines.

3. Rinsing, filtration of the solutionempty bottles, connectors, corks or caps.

4. Liquid enrichment in grape juiceconnectors, seals

5. Pour residue out of the bottledirectly onto the agar culture medium or onto the moistened nutrient pad set,with filter | for bottle rinser

6. Filter holder for inline filtration (e.g. Cat. No. 16254) compressed air

7. Airborne microorganism sampler MD8AirPort and gelatin filters that areplaced on the agar | to monitor the airthroughout the winery, particularly inthe critical areas in the filling area.

8. Rapid test equipment only suitablewhen the bacterial count is high, forexample, bioluminescence can be usedto monitor hygiene. Rapid tests are lesssuitable to detect trace infections.Hygiene monitoring to verify the cleanliness of conveyor belts, andemployees' hands and clothes.

3.5. Suitable Culture Media for Wineries 3.5.1. Total Colony CountThe spectrum of microorganisms found inwine and its preliminary stage, grape must,differs considerably from the range ofspecies found in water. This is explained bythe different environments (pH, availablenutrients, inhibiting factors, etc.). The total colony count in wine or grape juiceactually refers to these products' specificproblem microorganisms.

Wineries generally determine the total colony count during their hygiene testing,whereby the focus lies on the total colonycount of aerobic mesophilic bacteria. Areasof application are, for example, the waterused for cleaning or monitoring thehygiene of the equipment, containers,empty bottles or the operators’ clothes.

For this application, universal culture medium with a neutral pH and sufficientpeptone nutrients, but without inhibitingadditives, is used. This includes Standardmedium (with or without TTC), TGE, YeastExtract (particularly suitable for water) or R2A (for ultra-pure water).

3.5.2. Yeasts and MoldsThe above-mentioned culture media usedto determine total colony count are notsuitable for the detection of yeasts andmolds, even if occasionally colonies of isolated yeasts or molds can be formed.Culture media for yeasts and molds needconsiderably more nutrients, especially carbohydrates. A low pH is also beneficial.Wort and Malt Extract are suitable; insome regions, Wallerstein is also common-ly used.

For the detection of Brettanomyces, one ofthese media to which 20 to 50 mg/l ofcycloheximide (Actidione) has been addedcan be used. This additive efficiently suppresses nearly every type of yeast withthe exception of Brettanomyces and somestrains of Kloeckera. Lysine is used todetect wild yeasts, i.e. non-Saccharomycesyeasts. Agar-based culture media have anatural pH limitation. At pH values lowerthan 5, the agar tends to hydrolyze and nolonger solidifies. This is why it is better touse carrier materials other than agar asbasis of the culture medium, such as cellu-lose nutrient pads, which are either alreadyimpregnated with nutrients or are wettedwith nutrient broth. The pH value can thus be adapted to the pH of the product,which, on the one hand, increases theselectivity. This means only the micro-organisms that tolerate the product’s pHwill be able to grow on the media. On theother hand, a low pH prevents the interfer-ing concurrent growth of bacteria of theBacillus genus, which are very similar to theyeast colonies, and therefore often necessi-tate re-examination under the microscope.This additional task is not required whenusing acidic media because the bacillusspecies cannot form colonies in an acidicenvironment. The only exception to thisrule is the acid-tolerant Alicyclobacillus,which, however, does not normally occur in wine.

Standard TTC

Yeast extract

R2A

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3.5.3. Wine-spoilage BacteriaBecause wine has a low pH, wine-spoilagebacteria are acid tolerant. For this reason,the culture media used for their detectionmust also have a low pH.

The most common medium used to detectacid-tolerant bacteria is Orange Serum, a medium on which, primarily the acid-tolerant bacteria of the lactic acid bacteriagroup grow. To detect this group, the cul-ture medium should be incubated underanaerobic to microaerophilic conditionsbecause many of the lactic acid bacteriaare inhibited by oxygen. Acetic acid bacte-ria can also be grown on Orange Serum,but should be aerobically incubated. It isalso recommended to add 5 to 8% ofethanol to promote their growth and sup-press molds. Yeasts and molds can alsogrow on orange serum, but are suppressedif incubated under anaerobic conditions.

The Jus de Tomate (Tomato Juice) culturemedium was specifically developed todetect Oenococcus oeni, it must be anero-bically incubated for the bacterium togrow well.

For liquid enrichment, sterile grape juicecan be used as medium, in which spoilagemicroorganisms such as yeasts, bacteriaand fungi show growth rates equal to thosein the finished product.

The semi-liquid agar medium used in theMayer-Vetsch test is tomato juice agar.

3.6. The Rapid Test MethodRapid tests, for performing microbialanalyses in minutes or a few hours, areavailable on the market. Such tests are, of course, particularly attractive, because they eliminate the quarantine period needed for incubation as in conventionalmicrobiology.

However, before choosing a rapid testmethod, take a close look at how it is conducted and which particular bioburdenis suitable to give a reliable result.

3.6.1. BiosensorsThe rapid test methods, insofar as they arenot pure microscopy tests, are frequentlyconducted with biosensors. A biosensor is adevice that transforms a microorganism'smetabolic reaction into a signal that usual-ly can be measured physically. Because theyare so small, single microbes only produceminute quantities of metabolites. This iswhy a larger number of microbes is neededto achieve a measurable result. Pre-incuba-tion for one or more days depending on thetype of microorganisms is recommended. If a rapid test method is chosen, please bearin mind that although the test time itself isshort as promised, pre-incubation takes upthe saved time, meaning that a quarantineperiod must be observed. The rapid test ishence no longer a true quick test. If pre-enrichment is to be avoided, the detectionsensitivity is lowered so much that a positive signal is received only for a fewhundred or thousand bacteria per milliliter,which is entirely unsuitable for detectingtrace infections in filled bottles. Thesemethods, e.g. the bioluminescence method,which measures the amount of ATP, arerestricted to special applications, such asmonitoring the fermentation process, or the hygiene inspection of equipment orstaff before they start work.

Wallerstein

Lysine

Orange Serum

Jus de Tomate (Tomato Juice)

Wort

Malt Extract

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3.6.2. Microscopy|Epifluorescence

The CFU count can be determined bymicroscopic examination without incuba-tion time or pre-enrichment. Only relative-ly high CFU counts can be immediatelydetermined in a counting chamber, makingthis method unsuitable for monitoring thefilled end product. However, the detectionlimit can be lowered as much as desiredwhen the microorganisms are concentratedon a membrane filter, stained with a dye(usually methylene blue) and directlycounted on the filter. A vital stain, such asacridine orange, which also indicates thecells' viability, can also be used. Microscop-ic examination is definitely recommendedwhen tests need to be conducted quickly.For routine tests, microscopy of an entiremembrane filter is far too time-consuming.Yeast cells are easy to detect under themicroscope. However, if bacteria need to becounted, evaluation must be very accuratebecause bacteria are 5 to 10 times smallerthan yeast cells and are consequently moredifficult to detect.

3.6.3. Immunochemical TechniquesEnzyme-linked immunosorbent assay(ELISA) is a is a well established techniquein the medical sector for detecting patho-genic microorganisms. Monoclonal anti-bodies can be used to correspondinglydetermine specific product-contaminatingmicrobes in the product. ELISA tests usehighly specific antigen-antibody interac-tion and subsequent coupling to secondaryantibody-enzyme-dye complexes. Positiveresults are indicated by color development,allowing easy visual or colorimetric detec-tion of the target microorganism.

3.6.4. PCR and Similar TechniquesPCR is a method that does not involvepropagating the microorganisms, butrather replicating their genetic material,DNA. This process is much faster thangrowing the microorganisms themselves. A short strand of specific DNA is added, i.e., a targeted search is carried out for a spe-cific microorganism. PCR enables specifictarget microorganisms, such as productcontaminants, to be detected in a sample.Gene probes are complementary to typicalsections of the DNA of a specific type ofmicroorganism, its genetic fingerprint, soto speak. A staining agent can be attachedto a gene probe, enabling the targetedmicroorganism to be detected under a fluorescence microscope.

3.7. Additional Identification MethodsIn many cases, routine lab tests end withthe findings detected on the culture medi-um. If necessary, microscopic examinationis carried out to confirm that the colonydetected really is a yeast colony, or to seewhether a bacteria colony shows the typi-cal morphology of, for example, lactic acidbacteria or lactic acid cocci.

3.7.1. YeastsYeasts can be detected under the micro-scope by their size. In fresh cultures stillgrowing logarithmically, budding is anabsolutely reliable indication (unless one is dealing with a representative of thegenus Schizosaccharomyces).

The exact type of the yeast can be deter-mined by conducting biochemical teststhat mainly test the capability of ferment-ing specific sugars. A variety of easy-to-usetest kits are available in lab supply stores.The best-known test kits are API 20 AUX,BBL Mycotube, and RapiD Yeast Plus.Unfortunately, all of these systems havebeen primarily designed for the medicalsector, which is why very few of the typicalbeverage-spoilage yeasts are represented in databases available for evaluation. Theonly system that provides a good overviewof beverage-specific yeasts is that made byBiolog. The evaluation of its results, how-ever, requires an expensive instrument tomeasure turbidity, the cost of which is usually not within a winery’s budget.

A winery, therefore, frequently limits itselfto determining the yeast’s fermentability in order to assess risks to its products. Thisis done by inoculating a yeast colony intosterile grape must and monitoring the sam-ples for growth and gas formation.

Yeast cells under the epifluorescence microscope

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3.7.2. BacteriaThe main tests used to obtain a rough classification of wine-spoilage bacteriainclude:

1. Microscopic examination of the cellmorphology of cultured colonies – rods or cocci, presence or absence of endospores

2. Gram behavior by means of a potassiumhydroxide test

3. Oxidase test 4. Catalase test5. Aerobic or anaerobic growth6. Formation of acetic acid from ethanol

Colonies that grow on a bacteria culturemust be carefully evaluated to identify thepresence of a product spoilage organism or of a commonplace bacterium. This, ofcourse, depends on the medium on whichthe bacteria colony is found. If a universalmedium such as Standard, Plate Count orsimilar is used, wine-spoilage bacteria willprobably not grow because such mediacannot fulfill their nutrient requirements.However, if the colony has been inoculatedfrom a culture medium such as OrangeSerum, especially after microaerophilicincubation, it is very likely that wine-spoilage bacteria are present, and furthertesting is required. There are a number of basic and methodically simple testsincluding microscopic examination, particularly the Gram test and catalase test.

First, the colonies are examined under amicroscope to identify whether they areyeasts, bacteria or molds. Bacteria are 5 to10 times smaller than yeasts, do not bud,and are generally not filamentous likemolds. Identification of a “bacterium”under the microscope also reveals whetherit is a rod or a coccus. A Gram test is thencarried out. The Gram stain targets thestructure of the bacterial cell wall. Gram-positive bacteria have a cell wall with athicker peptidoglycane layer that the Gramstain colors blue. Gram-negative bacteria,on the other hand, have a thinner peptido-glycane layer colored red by the Gramstain. This classic Gram test with stainingagents is time-consuming. The much easierpotassium hydroxide test can be carried

Simplified chart for the rough distinction of wine-spoilage bacteria

Gram staining

G+ G-

Microscopy Catalase test

Rods withoutendospores

Cocci Catalasepositive

Oxidase test

Catalase test Catalase test Ox -

Cat - Cat - Aerobic

Acetic acidfrom ethanol

Lactobacilli LeuconostocOenococcusPediococcus

Acetic acidbacteria

According to Back, modified

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Two to three drops of catalase reagent areplaced on a colony. If the reaction is posi-tive, the mixture foams considerably. Themixture does not develop foam if the out-come is negative. Beverage-contaminatingbacteria are frequently anaerobic, becausethey grow in closed bottles, whether asrods or cocci. Lactic acid bacteria are catalase negative and Gram-positive. Incontrast, acetic acid bacteria are catalasepositive, strictly aerobic, Gram-negativerods or cocci. These few tests provide a rough indication whether a colony maybelong to the group of of wine-spoilagebacteria.

Ready-to-use test kits are also available to identify bacteria. However, here again,most of them focus on medically importantbacteria and are frequently not suitable to test for wine-spoilage bacteria.

out instead. This test is conducted by plac-ing two to three drops of a 3% potassiumhydroxide solution on a slide and using aninoculating loop to transfer the colony inquestion. If a filament is formed when theinoculating loop is lifted from the mixture,the colony is Gram-negative. If no filamentis formed, the colony is Gram-positive.

A further test is the catalase test, duringwhich the development of oxygen indicatesa catalase-positive result and is a sign of aerobic and facultative aerobic growth.A catalase-negative result indicates anaer-obic growth. The test is easy to conduct.

Evaluation of the plate Inoculation to a monoculture PCR in the agarose gel Genome PCR

Accurate subsequent identification using the Biolog System

MycoTube test kit API 20 Aux test kit Remel test kit

14

4. Equipment for Routine Microbiological Testing

4.1. Stainless Steel Vacuum Filter Holders and DisposablesFor large numbers of tests, a three-branchor six-branch manifold is used with 500 mlfunnels. The manifold is operated using a laboratory vacuum pump; a flask is con-nected between the pump and the mani-fold to collect the filtrate. A water trap(Vacusart filtration unit) protects the pumpfrom condensate.

Vacuum pumps with direct liquid suctionare also available on the market. As a result, a vacuum flask is no longernecessary.

For small numbers of tests, an individualfilter holder can be used. The filter holdersare disinfected between the individualsamples via flaming or flaming with alcohol. Other disinfection methods canalso be used.

If users wish to avoid the disinfection pro-cedure required for stainless steel funnels,they can go for a single-use option in theform of plastic funnels that are suppliedsterile. Actually, the material can even beautoclaved by the user and can be re-usedseveral times, if necessary.

Three-branch manifold

Water trap (Vacusart)

Individual filter holder

Biosart® 250

Single-use funnels on manifold

Biosart® 100 Monitor

Biosart® 100 Monitors on manifold

Microsart® e.jet vaccum pump; is used without a vacuum flask

15

If disinfection is completely out of thequestion, single-use Biosart® 100 Monitorscan be used but must be disposed of aftersingle use. The particular advantage of theBiosart® 100 Monitor is that it can also beused as a sampling vessel. In this case, theplug supplied with the Biosart® Monitor isused to seal off the base and a sample isthen taken on-site. Inside the monitor, thesample is safely protected from secondarycontamination and can be transported tothe laboratory for subsequent filtration.Afterwards, the Monitor is rinsed with sterile water, an ampoule containing theappropriate liquid culture medium ispoured into the Monitor to wet the incor-porated cellulose pad, the plug is re-insert-ed, and the Monitor is then converted intoa petri dish by removing the filter funneland replacing it by the lid.

4.2. Membrane Filter Types According to ApplicationMembrane filters are ideally suited for collecting smallest quantities of microor-ganisms from large sample volumes, concentrating them and enabling them to grow into visible and countable colonieson suitable culture media.

Bacteria, being much smaller than yeasts,have an average size of approx. 0.2–1 µm1–5 µm. To reliably collect the smallestwine-spoilage bacteria, a membrane filterwith a pore size of 0.45 µm is normallyused. By contrast, a filter with a pore sizeof 0.65–0.8 µm is quite sufficient to retainthe larger cells of yeasts and molds.

The membrane filter for colony countingshould always be made of cellulose nitrate,as this material offers the best growthproperties for the microorganisms.

Membranes are available in a variety of colors: green, white and gray. The reason-ing behind this is to ensure the best possi-ble contrast between the colonies and themembrane filters, as this makes countingeasier. The gray filter is used for examiningyeasts and molds that form white coloniesin their vegetative state; the green filter, for detecting bacteria on colorless culturemedia; and the white filter, for detectingbacteria on culture media that enable adifferentiation of the colonies formed onthe basis of a color reaction.

16

4.3 Culture Media According to Application4.3.1. Nutrient Pad SetsNutrient pad sets (NPS) are a simple andreliable alternative to culture media. Theyare sterile, ready-to-use media, alreadyplaced in petri dishes, supplied with thematching membrane filters. Hence, there isno need to purchase the filters separately.The shelf life of these media is up to twoyears from the date of manufacture,endorsed by an absolutely reliable packag-ing technology that is both lightproof andair-tight and includes a desiccant bag.

To use the nutrient pads, all you need to do is moisten them with sterile water. Thesterile water can be added using a sterilepipette. However, it is simpler and moreaccurate to add the water using a Sartoriusdosing syringe equipped with a sterilizing-grade syringe filter. Sterile water can alsobe added from individual ampoules. Anadditional advantage is that supplementscan be added to the sterile water to makethe medium more selective (e.g., Actidionto detect Brettanomyces) or to facilitatebetter growth of certain microbes (e.g.addition of alcohol for promoting thegrowth of acetic acid bacteria).

Suitable types of nutrient pad sets are available for every routine test requiredby the wine industry: Wort, Malt Extract,Wallerstein Nutrient and Lysine for detect-ing yeasts and molds; Orange Serum, Jus de Tomate (Tomato Juice) for identify-ing wine-spoilage bacteria; and StandardTTC for hygiene testing.

4.3.2. Agar Media Agar media are available as ready-pouredplates, but these are inclined to becomeunsterile and often have shelf life prob-lems. For this reason, agar is often sold inbottles or tubes and is poured into petridishes before use.

The types are similar to those of nutrientpads: Wort, Malt Extract, Lysine, OrangeSerum, Jus de Tomate (Tomato Juice) andStandard.

4.3.3. Ampoules with Liquid Culture Media If monitors are used for filtration, the padsinside must be impregnated with liquidculture media , similar to the media typesused with nutrient pad sets and agar culture media. In terms of handling, theliquid media easiest to use are those that are provided in sterile, ready-to-useampoules. One ampoule is used to wet one cellulose pad.

4.4 Specific Detection of Microorganisms with Culture Media 4.4.1. Yeasts and MoldsCulture Media: Wort, Malt Extract and Wallerstein Nutrient are the most important culturemedia. Other media are also used, e.g.,Schaufus-Pottinger (=mGreen Yeast andMold), Lysine, Orange Serum, etc.

Incubation conditions: The general recommendation is 2–5 days at 25–30°C under aerobic conditions.

The time period can be modified should aparticularly fast growing or slow growingspecies need to be detected. The tempera-ture can also be varied, depending on theparticular requirements of the targetmicrobes. For example, if heat-resistantmolds are to be detected, the incubationtemperature is increased. In the same way,the incubation temperature is loweredaccordingly for psychrophilic organisms. Itmay be necessary to extend the incubationtime, as extreme forms tend to take longerto grow.

Evaluation:Yeasts normally form white, smooth andshiny colonies. However, in exceptionalcases, they are also capable of creating pigments, e.g. Rhodotorula takes on a redcolor. Molds initially grow as white, fluffy,cotton-like colonies. However, as soon asthe mold mycelia start forming spores, thecolor changes to black, brown, red oranother color, depending on the spore pigmentation.

Nutrient pad set with membrane filter

Nutrient pads in original packaging

Agar media in bottles and tubes for pouring plates

Ampoule with liquid culture medium

17

As molds sometimes tend to cover theentire plate very rapidly, making quantita-tive evaluation impossible, it is recom-mended to check the culture after 2–3 daysincubation, then continue with incubationuntil the desired colony growth has beenattained. You can limit mold growth byadding 5–8% ethanol to the medium. Thisis particularly easy in the case of nutrientpads, as the alcohol can simply be added to the water used to wet the pads.

Special Remarks:To detect special types of yeasts or molds,you may need to supplement the culturemedium. For example, the growth of almostall yeasts can be suppressed by addingActidione (cycloheximide) up to a maxi-mum of 50 mg/l, so that only the resistantyeasts continue to grow: Brettanomyces(Dekkera) and Kloeckera. The latter caneven cope with the addition of more than50 mg/l of Actidione. The pH can also belowered to suppress undesired growth ofbacteria.

Similarly, the addition of antibiotics willachieve this result. For agar culture plates,these substances (sterile-filtered) are addedafter autoclaving and are mixed into themedium while it is still in a liquid state. Ifnutrient pads are used, antibiotics can beconveniently added to the sterile waterused to wet the pads.

4.4.2. BacteriaCulture Media: The media for wine-spoilage bacteria aregenerally suitable for detecting acid-toler-ant microbes. The most important of theseis Orange Serum. Wallerstein Nutrient isalso used, however, only in the WL Differ-ential version. Jus de Tomate is also partic-ularly suited for detecting wine-spoilagebacteria.

Incubation Conditions:Microaerophilic to anaerobic incubationfor 3-6 days is recommended to detect lactic acid bacteria. Incubation may beextended over a longer period in specialcases. The temperature is usually 28–30°C.If acetic acid bacteria are to be detected,aerobic incubation conditions must bemaintained.

Evaluation:If the medium contains a yeast inhibitor(Actidione), all the colonies that grow willbe bacteria, predominantly lactic acid bac-teria, if incubation is carried out underanaerobic or microaerophilic conditions.Nevertheless, this result should be con-firmed under the microscope. A furtherbiochemical test is necessary for more precise identification (see chart about basicdifferentiation and section 3.7.2.)

Special Remarks:As with the media used to detect yeastsand molds, the media for testing for bacte-ria can be made more selective by usingadditives, or a certain species of microbecan be promoted by adding nutrients orgrowth-promoting substances (see section3.5.3.).

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5. Flow Chart

Flow Chart of Winemaking

Grape processing:Grape delivery|wine press

1. Transport containers2. Collecting vats3. Conveyor belts|spiral conveyors4. Destemmer5. Grape mills6. Wine presses7. Short-term heating systems8. Clarification of mash (flotation,

separators, diatomaceous earth filter)9. Pipes, tubing and connections

10. Pumps11. Vats and tanks (mash tanks, reservoir

and storage tanks)

Fermentation and young wine:

1. Fermentation tanks2. Barrique vats

Wine processing:

Delivery1. Tank truck upon delivery2. Tank truck after cleaning3. Connections, pipes and tubing 4. Pumps

Cellar1. Storage tanks2. Pipes, tubing and connections3. Pumps4. Dispensing stations5. Collection vessels6. Filter: (inlet and outlet)

Depth filtersDiatomaceous earth filtersFilter cartridges

7. Desulfurization systems

Fill area:Production areas1. Bottling area (wet area)2. Dry area3. Handling of empty bottles:

Bottle rinser, bottle sterilizers (ozone, SO2, steam, chlorine dioxide)

4. Bottles: Empty bottles upon delivery Empty bottles after rinser or sterilizer

5. Supply tanks6. Product pump7. Carbonizer8. Heat exchanger9. Product filter:

Upstream of the prefilterUpstream of the final filterDownstream of the final filter

10. Automatic sampler (downstream of thefinal filter or prefilter|final filter)

11. Bottling machine|Filler:Filler headsInfeed and outfeed star wheels (stations) Filter for washdown equipment for glass fragment removal

12. Return (mix zone) and supply tanks13. Trolleys14. Sealing machinery:

Corking machineManual corker or roll-on capperCrown corker

15. Filled bottle First fill roundPer fill batchThroughout entire production

16. Conveyor beltsBottle conveyor beltsAuxiliary material conveyor belts (corks, crown corks, etc.)

17. Auxiliary material storage hopper18. Media filters

Rinse water filterSterile air filter (O2, N2, CO2)Rinser water filter

19. Filter for washdown equipment forglass fragment removal

Storage:1. Conveyor belts for full crates or boxes|

trays|BIB|Tetra Paks2. Goods in storage positions3. Filled stock storage for damaged

bottles|containers.

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Checklist: Grape processing; Microbiological Tests

Grape delivery|wine press Method Routine test Assessment criteria

1. Transport containers

Empty transport containers Contact plate | swab test weeklyFull transport containers Swab test weeklyCleaned transport containers Contact plate weekly < 10 CFU, no yeast

2. Collecting vatsCleaned collecting vats Contact plate weekly < 10 CFU, no yeastFilled collecting vats Swab test weekly

3. Conveyor belts|spiral conveyorsCleaned Contact plate weekly < 10 CFU, no yeastDuring operation Contact plate weekly

4. DestemmerCleaned Contact plate weekly < 10 CFU, no yeastDuring operation Swab test weekly

5. Grape millsCleaned Contact plate weekly < 10 CFU, no yeastDuring operation Swab test weekly

6. Wine pressesCleaned Contact plate weekly < 10 CFU, no yeastDuring operation Swab test weekly

7. Clarification of mash: Flotation,separator, diatomaceous earth filterCleaned Contact plate weekly < 10 CFU, no yeastDuring operation Swab test weekly

8. Pipes, tubing, connectionsCleaned Swab test weekly < 10 CFU, no yeast

9. Vats and tanks (mash tanks, reservoir and storage tanks)Cleaned Contact plate | swab test weekly < 10 CFU, no yeastFilled Membrane filtration weekly

10. Short-term heating systems Contact plate | swab test weekly

Fermentation and young wine

1. Fermentation tanksCleaned Contact plate | swab test before filling Test filled tank for bacteria, if no Filled Sample volume 0.5–1 ml malolactic fermentation is desired

2. Barrique vats Test filled vat for lactic and Cleaned Contact plate | swab test before filling acetic acid bacteria, possiblyFilled Sample volume 0.5–1 ml for Brettanomyces

The values given here depend on the residual sugar content of the wine, the type of wine (white, red or rosé) and the sulfur content. Different values may therefore apply.All three checklists contain only approximate guidelines.

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Checklist: Wine processing; Microbiological Tests

Wine delivery Method Routine test Stage check Assessment criteria

Unloading station

Sampling point

1. Tank truckTank truck upon delivery 0.5–1 ml sample volume weekly Each tank truck|dailyTank truck after cleaning 1.0 ml sample volume weekly Each tank truck|daily < 10 CFU

2. Connections, pipes, tubingBefore cleaning Swab test weekly daily After cleaning Swab test weekly daily < 10 CFU

3. Unloading pumpBefore cleaning Swab test weekly dailyAfter cleaning Swab test weekly daily < 10 CFU

Cellar

Sampling point

1. Storage tanksAfter cleaning Contact plate | swab test after cleaning daily < 10 CFUFull storage tank Membrane filter method when needed daily

2. Connections, pipes, tubingBefore cleaning Swab test weekly daily After cleaning Swab test weekly daily < 10 CFU

3. Feed pumpsBefore cleaning Swab test weekly dailyAfter cleaning Swab test weekly daily < 10 CFU

4. Dispensing stationsBefore cleaning Swab test weekly dailyAfter cleaning Swab test weekly daily < 10 CFU

5. Collection vesselsBefore cleaning Swab test weekly dailyAfter cleaning Swab test weekly daily < 10 CFU

6. Desulfurization systemsBefore cleaning Membrane filter method weekly daily < 10 CFU, no yeastAfter cleaning Membrane filter method weekly daily No yeast / 100ml

7. Water connectionsHot water Membrane filter method weekly daily No yeast / 100mlCold water Membrane filter method weekly daily No yeast / 100ml

8. Water filter Membrane filter method weekly daily No yeast / 100mlAir filter Sedimentation method weekly daily Negative

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Wine delivery Method Routine test Stage check Assessment criteria

Cellar

Sampling point

9. Filter9.1. Filter sheets

During operationFilter inlet Membrane filter method weekly daily, each batchFilter outlet Membrane filter method weekly daily, each batch Depending on

retention rateAfter cleaning or sterilizationFilter inlet Membrane filter method weekly daily, each batch Filter outlet Membrane filter method weekly daily, each batch Depending on

retention rate

9.2. Diatomaceous earth filterDuring operationFilter inlet Membrane filter method weekly daily, each batch Filter outlet Membrane filter method weekly daily, each batch Depending on

retention rateAfter cleaningFilter inlet Membrane filter method weekly daily, each batchFilter outlet Membrane filter method weekly daily, each batch Depending on

retention rate

9.2.1. Cartridge filterDuring operationFilter inlet Membrane filter method weekly daily, each batch Filter outlet Membrane filter method weekly daily, each batch Depending on

retention rateAfter cleaning or sterilizationFilter inlet Membrane filter method weekly daily, each batch Filter outlet Membrane filter method weekly daily, each batch Depending on

retention rate

9.3. Crossflow filterDuring operationFilter inlet Membrane filter method weekly daily, each batchFilter outlet Membrane filter method weekly daily, each batch Depending on

retention rate

After cleaning or sterilizationFilter inlet Membrane filter method weekly daily, each batchFilter outlet Membrane filter method weekly daily, each batch Depending on

retention rate

22

Checklist – Bottling (Filling) Area, Microbiological Quality Control

Production areas Method Routine test Result

Sampling Point

1. Bottling (filling) areaWet and dry area Airborne microbe collection monthly < 500 CFU|100 liters

2. ContainersEmpty bottles upon delivery Drip test | MF method weekly negativeEmpty bottles after rinseror sterilization Drip test | MF method weekly negative

3. Handling of empty bottles:Bottle rinser Drip test | MF method weekly negativeBottle sterilizers Drip test | MF method weekly negative(ozone, steam, SO2, chlorine dioxide)

4. Collection tanksAfter cleaning or sterilization Swab test | contact plate weekly negativeDuring operation Swab test | contact plate weekly negative

5. Product pumpsAfter sterilization and during operation Swab test | contact plate weekly negative

6. CarbonizerAfter cleaning|sterilization Swab test weekly negativeDuring operation Membrane filter method weekly negative

7. Heat exchangerAfter sterilization Membrane filter method weekly negativeDuring operation Membrane filter method weekly negative

8. Product filterAfter sterilizationFilter inlet, prefilter Membrane filter method dailyOutlet, prefilter | inlet, final filter Membrane filter method daily Depending on retention rateFilter outlet, final filter Membrane filter method daily Sterile or no yeast

9. Bottling machine (filler)Filling area after sterilization Airborne microbe collection | Sedimentation method 0 yeast/100 litersBottling area during operation Airborne microbe collection | Sedimentation method 0 yeast/100 litersBottling area after filling break Airborne microbe collection | Sedimentation method 0 yeast/100 liters

10. Filler headsAfter sterilization Swab test min. 1 - 2 per month negativeDuring operation Swab test min. 1 - 2 per month negativeAfter filling break Swab test min. 1 - 2 per month negative

11. Filler return – supply tankAfter sterilization Membrane filter method weekly negative

12. TrolleysAfter sterilization Swab test weekly negativeDuring operation Swab test weekly negative

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Production areas Method Routine test Result

Sampling Point

13. Sealing machine (corker)Corker after sterilization Swab test weekly negativeCorker during operation Swab test weekly negative

14. Filled bottleFirst fill round after sterilization Membrane filter method 1st bottle,

during production no yeast / 100mlDuring operation: Membrane filter method 1 bottle per hour no yeast / 100mlIn-batch operation: Membrane filter method min. 1 bottle /

fill batch no yeast / 100ml

15. Conveyor beltsBottle conveyor belts Swab test | contact plate monthly no yeastAuxiliary material storage hopper Sedimentation method |

cotton swab monthly no yeast

16. Media filterRinse water filter after sterilization| During operation Membrane filter method weekly no yeast / 100mlSterile air filter (O2, N2, CO2) Sedimentation method weekly no yeast / 100mlRinse water filter after sterilization| During operation Drip test weekly no yeast / 100ml

17. Water filter for shard washdown equipment Membrane filter method weekly no yeast / 100ml

18. Finished goods storageConveyor belts for full crates,boxes, BIB, Tetra Pak Contact plate min. bi-annuallyGoods in storage positions Contact plate min. bi-annuallyFilled stock storage for damagedbottles/containers Contact plate min. bi-annually

Stage check in situations where a contamination in the bottled beverage has been detected: All stations from 1 to 18 must be included in the stage checks.

24

6. Cleaning

Cleaning in the Food and Beverage Sector.In order to ensure consistent product quality under food-safe conditions, cleaning has to be performed periodicallythroughout the whole winery.

The objectives of cleaning are:– to maintain proper hygienic conditions in

buildings, systems and equipment– to increase the life-cycle of systems and

equipment– to ensure the product quality– to reduce costs

As most deposits harden over time (aging)and are thus harder to remove, cleaningshould be carried out immediately afteruse. Cleaning the equipment as soon aspossible minimizes the risk of microbiologi-cal contamination. If production is stoppedfor a longer period, the equipment must becleaned before restarting operation.

The cleaning process is divided into the following steps:– Rinsing of system or equipment– One or two chemical cleaning processes– Neutralization– Final rinse

Most deposits are removed at the rinsingstage. The recommended rinse medium is particle-free DI water.

Chemical cleaning requires careful planning.

The chemicals used must be suitable forloosening and removing the deposits without corroding any components. Additionally, it must be ensured that noresidual cleaning agent is left in the systemas this could subsequently damage theproduct. In many cases, ready-formulatedcleaning chemicals specially designed forcertain cleaning applications are commer-cially available. The components of thesecleaning concentrates are essentially special tensides, acids or bases, complexingagents, enzymes and oxidants.

For each chemical cleaning process, the parameters for the four basic elementsof the process, – Time– Concentration of chemicals– Temperature and– Mechanics of cleaning should be set optimally.

Transport mechanisms and chemical reactions need a certain amount of time.Therefore, chemical cleaning should have a duration of at least 30 minutes. Incertain cases, it may favourable to opt forovernight cleaning.

Chemical concentration depends on thetype and intensity of the soiling. Please follow the instructions issued by the manu-facturers. Usually, a concentration of 1% to3% is recommended. Beware of the mis-conception “using a lot helps a lot” - this isa fallacy. If the concentration of chemicalsis too high, this may lead to undesirabledeposits.

The temperature of the cleaning solution is often a decisive factor for successfulcleaning. At higher temperatures, chemicalreactions are quicker, viscosity decreases,binding forces are reduced and the speedof liquid flow increases. This means that atemperature increase will usually acceler-ate the cleaning speed. However, there areunfortunately some exceptions to this rulewhen selecting the temperature. For enzy-matic cleaning, optimum temperatureshave to be observed. In the case of proteincontamination, the cleaning temperatureshould not be too high , as this could dena-ture the proteins.

Regarding the cleaning mechanics, the following must be observed: cleaningchemicals must be transported to the location of the soiling. A high flow rate willloosen the deposits via shearing forces. Example: crossflow membrane systemsshould be run in the recirculating modewith closed permeate outlet, as this willensure that pressure on the membrane is aslow as possible. For static filtration systems(cartridge filtration), the cleaning solutionshould preferably be applied from theupstream side (in the direction of filtration).

After alkaline cleaning, neutralization with 0.5% phosphoric or citric acid is recommended. This shortens the timeneeded for the final rinse with water.

25

The final rinse water must be withoutflavour and free of foaming after a shaketest. The pH value must be neutral.

As cleaning chemicals are often caustic,attention must be paid to appropriate protective equipment during chemicalcleaning (goggles, gloves).

In general, the following can be said of cleaning and cleaning agents:

Thorough cleaning will remove all productresidues and dirt particles that would otherwise serve as nutrients for microor-ganisms. Such soiling residues consist part-ly of organic substances from the wine itself or from the remains of deadmicroorganisms. These dirt particles alsocontain inorganic substances. The choice of cleaning agent is therefore also depend-ent on the residues themselves, as well asthe equipment components to be cleanedand their chemical resistance.

Acidic cleaning agents are especially suitedfor dissolving limescale, tartar and othermineral deposits. Such deposits are abreeding ground for growth of microbialcommunities, so-called biofilms. Biofilms are very hard to dissolve without leavingresidues, so upfront prevention is the better option.

Acetic acid and citric acid, plus their salts,are the basis for most common acid deter-gents. Their pH values are between 1 and 5. Alkaline detergents are mainlyused to remove organic residues such asprotein, starch or sugar. These residues also provide a basis for thebuild-up of biofilms and they must there-fore be thoroughly removed. The pH valueof alkaline detergents lies between 10

and 14; these agents are based on sodiumhydroxide, potassium hydroxide, potassiumphosphate and sodium phosphate etc.

Effective disinfection is generally only carried out after cleaning, never before. It is also not advantageous to use a combi-nation of detergents and disinfectants dueto potential incompatibilities that mayeven produce environmentally hazardouschemicals if agents are mixed. As a matterof routine, disinfection should be reservedfor the most critical areas and not be carried out throughout the entire winery.In the case of infections, this may have to be reconsidered, if these cannot be eliminated by thorough cleaning. The mostwell-known and effective disinfectant isperacetic acid, frequently used as a combi-nation formula consisting of peracetic acid,acetic acid and hydrogen peroxide.

In general, every winery should establish a cleaning schedule that is followed on a routine basis. This concept must coverevery stage, from the arrival of the grapesthrough the entire production cycle up to the filled and stored bottles. The clean-ing instructions should outline and defineall relevant parameters – time intervals,stages, products and methods. This is bestrepresented in a flow chart, similar to theone we have used for the productionprocess in Chapter 5. The success of thecleaning and (where necessary) disinfectionmeasures is to be checked using micro-biological tests. This is not mandatory after every cleaning process, but – asdefined in the hygiene schedule – suchtests should be carried out at regular intervals, depending on the hygienic stability of the entire winery.

26

Checklist: Grape Processing Cleaning

Grape processing Maintenance cleaning Basic cleaning

Grape delivery|wine press

1. Delivery vessels min. daily, hose out|down min. once a week, hose down with detergents and (where necessary) with disinfectants. Rinse out afterwards!

2. Collecting vats min. daily, hose out|down ditto

3. Conveyor belts|spiral conveyors min. daily, hose out|down ditto

4. Destemmer min. daily, hose out|down ditto

5. Grape mills min. daily, hose out|down ditto

6. Wine presses min. daily, hose out|down ditto

7. Clarification of mash: Flotation, min. daily, hose out|down dittoseparator, diatomaceous earth filter

8. Pipes, tubing, connections min. daily, hose out|down ditto

9. Vats and tanks (mash tanks, reservoir and storage tanks) before filling

10. Short-term heating systems min. daily, hose out|down weekly, rinse in pump-out process

Fermentation and young wine

1. Fermentation tanks rinse before fílling|detergent & rinse out

2. Barrique vats rinse before fílling|detergent & rinse out (where necessary) with disinfectant

27

Checklist: Wine Processing Cleaning

Wine delivery Maintenance cleaning Basic cleaning

Unloading station

Sampling point

1. Tank truck upon delivery hose out daily weekly

2. Connections, pipes, tubing hose out daily weekly

3. Unloading pump hose out daily weekly

Cellar

Sampling point

1. Storage tanks hose out daily with water weeklybefore filling

2. Connections, pipes, tubing ditto weekly

3. Feed pumps ditto weekly

4. Dispensing stations ditto weekly

5. Collection vessels ditto weekly

6. Filter6.1. Filter sheets rinse daily or when changing once/twice weekly,

grape type sterilize with steam or hot water

6.2. Diatomaceous earth filter ditto rinse, using detergent if necessary

6.2.1. Cartridge filter ditto once/twice weekly, oder Heißwassersterilize with steam or hot water

6.3. Crossflow filter according to manufacturer's according to manufacturer's operating instructions operating instructions*

7. Desulfurization systems according to manufacturer's according to manufacturer's operating instructions operating instructions

8. Water connections

9. Water filter weekly, sterilizeAir filter weekly, sterilize

10. Water treatment systems according to manufacturer's according to manufacturer's operating instructions operating instructions

* Once/twice a week, chemical cleaning

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Checklist: Bottling (Filling) Area, Cleaning

Production areas Maintenance cleaning Basic cleaning

1. Filling (bottling) area (wet area) Hose down daily Once a week, apply floor cleaner

2. Dry area Hose down daily Once a week, apply floor cleaner

3. Containers Hose down daily Once a week, CIP

4. Handling of empty bottlesBottle rinser Sterilize each morning Once a week, disinfect depending on typeBottle sterilizer Sterilize each morning Once a week, disinfect depending on type

5. Collection tanks Daily thermal sterilization Once a week, CIP

6. Product pumps Daily thermal sterilization Once a week, CIP

7. Carbonizer Daily thermal sterilization Once a week, CIP

8. Heat exchanger Daily thermal sterilization Once a week, CIP

9. Product filter9.1. Prefilter Rinse and sterilize daily Once a week, CIP9.2. Final filter Rinse and sterilize daily Once a week, CIP

10. Bottling machine/Filler Rinse and sterilize daily Once a week, CIP

11. Filler return (mix zone) and supply tanks Daily thermal sterilization Once a week, CIP

12. Trolleys Hose down daily Once a week, disinfect

13. Sealing machinery Clean|disinfect daily Mechanical cleaning

14. Filled bottles

15. Conveyor belts Hose down daily; clean

16. Auxiliary material storage hopper; Clean daily Once a week, disinfectcorks, MCA, etc.

17. Media filter Daily with steam

18. Water filter for washdown equipment Sterilize dailyfor glass fragment removal

19. Finished goods storage Once a month, clean Conveyor belts for full crates or boxes|trays|BIB|Tetra Paks

29

7. Plant Hygiene Training

7.1 Plant Hygiene in Grape Processing

The objective of plant hygiene in grape processing is to create optimum conditionsfor wine making and wine quality.

Healthiness, ripeness of the grapes, weath-er conditions and picking temperatureshave considerable influence on the typeand frequency of the hygiene measures to be implemented.

See the checklist Monitoring of Grape Processing and Cleaning

In autumn, microorganisms rarely spreadfrom the grape processing area into theother production areas or into the fillingarea if these are sufficiently partitionedfrom each other.

7.2 Plant Hygiene in the Production and Cellar Rooms

The objective of plant hygiene in the pro-duction and cellar rooms is the hygienicand clean production, processing and storage of beverages under food-safe conditions.

A distinction is made between the follow-ing areas:– Wine delivery– Tank storage– Work area

centrifuges, filters, desulfurization systems, heat exchangers, pumps, etc.

– Filling cellar

From a microbiological perspective, theseareas have to be assessed individually andlimit values for bacterial load must bedefined plant-specifically. The checklistsMonitoring of Wine Processing and Cleaning serve as the basis for an individualdefinition of the monitoring points.

The various seasons must be taken intoaccount for the monitoring time periodand evaluation of the individual areas. Thecleaning measures and cleaning intervalsand repetitions are based on the respectiveresults of the microbiological testing.

A distinction is made between:– Maintenance cleaning– Basic cleaning

Maintenance cleaning takes place duringongoing production operations, and intensive basic cleaning takes place atdefined intervals.

In principle, the following must beobserved: a cleaning certificate mustaccompany each tanker delivery. Pumps, pipes, receiver containers, connec-tions and hoses must be regularly cleanedwith cold and warm water, sterilized andsubjected at regular intervals to CIP cleaning with caustic solution. Storagetanks must be cleaned and, if necessary,disinfected before filling. Regeneration and sterilization of the filters takes placeaccording to the operating instructions of the manufacturer.

The work area should be kept as clean and dry as possible, cleaning lines must becollected into gully lines. No mildew or mold can be tolerated in the productionrooms. Infected areas must be treatedappropriately or painted with anti-moldpaint.

Auxiliary materials and treatment agentsshould be packaged and stored cleanly and dryly in specially separated rooms orstorage areas. Cleaning agents and toolsmust be stored in a separate room on special collection trays.

Auxiliary media: water, air, C02, nitrogenmust be equipped with corresponding filters, regularly regenerated and sterilized.

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7.3 Plant Hygiene in the Bottling AreaThe objective of plant hygiene in the bottling area is the safe and reliable fillingof beverages under food-safe conditions.

The filled product and the plant processesin the production chain are subjected toregular microbiological monitoring. Theoverall production safety is increasedwhen, in addition to the finished product,the product path is also analyzed, andwhen the effectiveness of the cleaningand/or sterilization of the individual pro-duction units is verified microbiologically.

In the production rooms, the analysisfocuses on the wet area. In the less sensitive dry area, the analysis intervals can be extended.Another important point is personalhygiene in the filling area and the behaviorof the employees during production. Ingeneral, all machines, auxiliary materialsand persons in the bottling hall that comeinto contact with the product must be rated as sensitive.

Of the filled product, at least the first andlast bottle and one bottle from the middleof the batch must be taken from everyindividual filling batch and then analyzed;for larger filling batches, samples must betaken during the entire production at 1-hourly intervals.

The individual hygiene areas in filling arelisted and described below. The type of the sample and the analysis method areindicated in the checklists in table formand divided into routine monitoring andstaged inspections in the event of micro-biological findings. Following the microbiological assessmentof the current situation, an evaluation ofthe effectiveness of the measures takenshould always be performed.

Flow chart: Product path of an automatedcartridge filter system

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Machines and units of the product path:– Collecting tanks– Pumps– Wine heating– Filter system– Bottling machine (filler)– Corker

The entire production line, includingreceiver tanks, pumps, wine heating, filtersystems and fillers are subject to a morningroutine in preparation for filling and thenafter filling, an evening routine. The morn-ing routine includes rinsing the productionline with cold water and warm water, ster-ilization with steam or hot water, coolingwith cold water and the integrity test ofthe filter membranes. Following this, theproduct is added to the filler and the pro-duction takes place. The evening routine after production ends, includes pressurized

emptying of the production line with air,followed by regeneration with cold andwarm water.

For collecting tanks and pipes as well asfillers, sterilization with steam or hotwater, a CIP cleaning with base and subse-quent neutralization are required at regularintervals in addition to cleaning with coldand hot water. The feed pumps and theresidue drains (sink) must also be inspected.

The bottling machine, or filler, is one of themicrobiologically most sensitive units inthe production chain. During sterilizationof the filler, the temperature and steriliza-tion time at the filler discharge and the filling heads must be regularly monitoredand documented. The protective panelingof the filler must always be closed duringproduction, a corresponding inspection of

the ambient air in the filling room shouldtake place regularly. After every shutdown,filling intermission, repair work or otherproduction delay, an alcohol disinfectionwith 70% ethanol should be performed.

The filler operating personnel and fillermaintenance personnel must undergo special hygiene training. In principle, nocleaning work with compressed air, waterhose or especially high-pressure cleanersmay take place during ongoing production.No openwaste containers are permitted inthe filling area. For the sealers, attentionmust be paid to the temperature of thecork seal heating (if present and if process-ing of the seal material permits this). Closure supply paths must be kept dry, free of product and must also be sterilizedwith ethanol.

Example: Collecting tank

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7.3.1 Auxiliary Materials– Containers (bottles, soft packs, etc.)– Closures (cork, plastic cork, screw top

crown cork, etc.)

All auxiliary materials should be stored in dry and undamaged packaging.The filling containers must be clean andfree of particles and microorganisms harm-ful to the product; in order to ensure this,all bottles must pass through the rinser.

7.3.2 Units for Filling Preparation, Conveyance and Provisioning of theAccessory Materials– Rinser– Conveyor belts for containers, seals, finished packaging, boxes, etc.– Conveyor belt covers– Reserve hoppers for closures

For the bottle rinser and sterilizer, depend-ing on the design and type of the bottlesterilization, a morning routine with coldand warm water and subsequent steriliza-tion is generally carried out before filling.The evening routine after productionincludes cold and warm water rinsing. Theconcentration of sterilization media(ozone, peracetic acid, chlorine dioxide,etc.) must be checked regularly. For sterilewater sprays, the water filter must beequipped with membrane filter cartridges,which must be sterilized and tested forintegrity during the morning routine.

The conveyor belt covers should be clean,dry and free from product residue. Afterthe production run, the belt can be cleanedmechanically with an alkaline solution and,if necessary, sterilization with alcohol cantake place. Reserve containers for sealsmust be kept closed, clean and dry.

The floor should be clean and – insofar as possible – dry. In the vicinity of the filler,which is a particularly sensitive zone, thefloor should not be hosed down with wateror cleaned with a steam blaster to avoidgerms spreading via aerosols to the filler.Rinsing lines must lead directly to the gullywhen possible. During production, nocleaning agents should be used in proximi-ty to the filler.

7.3.3 Bottling HallAll doors must be kept closed. In particular,the doors to the outside area but also thoseto the workshop area and social rooms.When possible, the bottling hall should beseparated from the rest of the productionfloor. For sterilization with steam, theexhaust should lead out of the productionhall.

The bottling hall must be free of insectsand other bugs. Insect lamps have proveneffective for this. Auxiliary materials mayonly be stored at the designated storagelocations. The objects required for cleaningmust be stored in the designated storagelocations. The forklift transports are onlypermitted along the designated forkliftpaths.

7.3.4 Behavior during ProductionNo eating, drinking or smoking is permitted in the production area. Handsmust be washed and disinfected beforeentering the sensitive production area!

No jewelry may be worn whilst workingwith open product containers.Hands must be disinfected before workingon machines or auxiliary materials thatcome into direct contact with the product.

Work clothing must be worn, includingsafety shoes and, in the filling area, a head cover as well.

7.3.5 Less Sensitive Areas– Storage– Auxiliary materials receiving– Auxiliary material storage– Finished products storage

These areas should be clean, dry and free of bugs. Cleaning agents must be stored inappropriate protective trays according totheir hazard class.

In all areas, the hygiene status must bemonitored at appropriate intervals andwith corresponding analysis proceduresdepending on the sensitivity of the respective area.

Please see section 3.

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7.4 Procedure in the Event of Microbio-logical Findings in Filled ProductIn ongoing production, a CIP cleaning ofthe CIP-capable units and subsequent ster-ilization of the entire production lineshould take place immediately after detection of an abnormality.

Action needed– Re-inspection of the filled product – Identification of the microorganisms– Localization of the source of

contamination– Elimination of the source of

contamination– Subsequent inspection

A subsequent re-inspection of the filledproduct serves to determine the scope ofthe damage. Is it a widespread infection,are parts of the filling affected or theentire filling batch or filling day?

Sample Volumes for Subsequent Inspection1. Retention samples2. Finished goods:

– At least 1 box per filled pallet– Start and end of the filling batch– Or 1 bottle every half hour

On identification of the microorganism, see the preceding section.

Inspection Points (marked with red arrows):1 Bottle inspection2 Bottle neck sterilization 3 Filler (bottling machine)4 Corker5 Collecting tank6 Prefilter7 Final filter

Localization of the damage source throughstaged inspections: – see checklist –

In cases of severe contamination, causal research in the form of a sample filling is advisable.

Particular attention should be paid to thefiller and rinser, which in 60% of the casescause the contamination. The 1st filler cir-cuit after filling starts gives informationabout the success of the filler sterilizationand how clean the filling valves are.Inspection of the sterilization temperatureat the filling valve and filler discharge isalso useful.

Sampling from the product line, eitherfrom the filter outlet or the filler inlet rulesout contamination in the product path line.It is also important to draw samples duringthe pressurized draining of both the prod-uct line and the filter into the filler.

Automatic sampling devices that place several ml of product in a sterile analysisbottle at defined intervals are advanta-geous; in routine testing they are regardedas a sterile control for the entire produc-tion time . Rinser water, rinsed bottles,monitoring of the sealing units and con-tainers as well as defective valves, bypassesand return flow lines in the sterile area ofthe filler node can also be sources of infec-tion.

When the source of damage has beenlocalized and eliminated, a further stagecheck inspection must always be per-formed as a final test to validate theresults.

34

8. Risk Assessment and HACCP

HACCP is a preventive system that isintended to ensure the safety of foods and consumers.

HACCP stands for Hazard Analysis CriticalControl Points. It is a systematic preventiveapproach intended to accompany theentire production process and to identifypotential safety hazards, so that keyactions, known as Critical Control Points(CCP's) can be taken to reduce or eliminatethe risk of the hazards actually occuring.The goal is to protect the consumers fromhealth risks.

The HACCP concept is based on 7 principles:

1. Conduct risk analysis

2. Identify control points

3. Establish critical limit values

4. Establish monitoring and inspectionprocesses

5. Establish monitoring actions

6. Establish review procedures

7. Establish regulatory documentation

These principles will now be explained ingreater detail. However, one should alwaysconsider that the concept was originallydeveloped for foodstuffs that could presenta health risk for consumers if improperlyproduced. For one thing, there is the micro-biological risk, in other words, the presenceof pathogens in the product; for anotherthing there is the risk of chemical andphysical dangers. The microbiological risk ispractically nonexistent in the wine industrysince microorganisms that are capable ofsurviving and reproducing in wine do notfall into the category of pathogens. Listeriain cheese, Salmonella in dried milk orClostridium botulinum in ham on the bone– such dangers are luckily not present inwine.

1. Conduct risk analysisThe first step is hazard or risk analysis. This involves identifying all potential haz-ards of the individual manufacturingprocess steps that could negatively impactthe food hygiene of the finished product.

Every hazard is analyzed and assessed with regard to its significance for foodsafety aswell as the probability of its occur-rence. This establishes risk transparency.The result is a list of clearly defined individ-ual risks. This risk analysis should be per-formed by an interdisciplinary team that isappointed specifically for this purpose. Thisteam creates a detailed description of theproduct and a flow chart of its typical production cycle at the plant and includesa detailed list of possible risks. This flowchart must be tested for validity by theteam through an on-site inspection. Risksto be considered are microbiological risks(in wine, as previously mentioned, insignifi-cant), chemical risks (disinfectants, clean-ing agents, pesticides, etc.) and physicalrisks (glass splinters, metal parts, etc.). The production process must be inspected forpotentially problematic environmental conditions, such as bugs, waste disposal,hard-to-clean surfaces, employees withpoor hygiene discipline, etc.

The risk group into which the product fallsmust also be defined; for example “suscep-tible,” “perishable” or “re-heated beforeconsumption”. The consumer group forwhom the product is intended must also beprecisely defined, such as “healthy adults,”“diet food” or other target groups. Thesefew examples make clear that risks withregard to shelf life and consumer group are low in combination with the practicallynonexistent microbiological risk.

2. Identifying Control PointsIn this step, the critical control points, orCCPs, at which the identified hazard can bebrought under control are identified. Thiscan be a site or working area, such as a coldroom or the pasteurizer. It can be a rawmaterial, which may be contaminated withpesticides or other substances. It can be acritical process, such as water softening, ora critical final product that only has limitedshelf life due to its pH value or other prop-erties. The determination of the controlpoints is made based on a so-called deci-sion tree. The result must be a complete listof the CCPs. Once this list is drawn up,every CCP should be re-evaluated to deter-mine whether it can be modified in such a way as to remove it from the risk area,making it no longer a CCP. If this is not possible, the risk should at least beminimized.

3. Establish Critical Limit ValuesAfter the control points have been identi-fied, the set values and tolerance limitsmust be determined. This can be donebased on physical parameters, such as thetemperature (e.g. temperature during fermentation), or chemical parameters,such as the pH value and the level of sulfuric acid content in the wine. Limit values can be defined for such parametersthat must be met in order for the productto achieve the desired stability.

4. Establish Monitoring and InspectionProcessesFor the set values defined under item 3,measurement and testing processes mustnow be defined that allow for continuousmonitoring of every CCP and indicate when it exceeds the established limits.Here, in-process controls should always be preferred over spot sampling.

35

5. Monitoring ActionsIf deviations from the set values are discovered during monitoring, the processmust be stopped immediately, if possible,and corrective action must be taken beforethe deviation jeopardizes product safety. To this end, suitable measures must bedefined under item 5. This makes it possibleto manipulate and effectively master the control point locally. Measures can include,for example, increasing the pressure, cool-ing or heating, pH modification, etc.

6. Establish Review ProceduresTo check the efficiency of a system, additional, system-independent monitoringprocedures must be established, such asaudits, the analysis of random samples bythird parties, testing monitoring equipment,the availability of work flow plans, etc.

7. Establish DocumentationRecords are to be kept of all processes and instructions, written documentation of all findings and occurrences during theplanning, construction, application andmodification of the system. These alsoinclude test procedures, process, sequenceand data collection plans, accumulatedperformance data together with all statisti-cal analyses. A reliable administrative procedure for change notification must be ensured.

In summary, the following can be stated:

A CCP can only be established in a placewhere process deviations can be measured,e.g. process deviations from a critical limitvalue. Further, it can only be established ina place where steps can be taken in orderto correct this process during ongoing production.

It must be considered here that the focusis on the health safety of the manufacturedproduct and not on the quality of taste, the filling quantity, an undesired odor orsuchlike.

In practice, this is handled less strictly inthe wine industry, and quality-relevantpoints are frequently included in theHACCP system, points that do not endangerconsumers’ health if not complied with(e.g. the filling level of the bottle). Everyplant must decide individually on its CCPs.

Below some guidelines for the establish-ment of CCPs

1. After bottle cleaning. It must be ensuredthat no residue of alkali or cleaningagents is left in the bottle.– Test using pH meter or litmus paper.

2. After cleaning of crossflow systems. Also here, the risk that cleaning agentsmay not be correctly rinsed out cannotbe completely excluded.– Test using pH meter or litmus paper.

3. Bottle inspector. Empty bottles thatexhibit broken glass or damaged necksmust be reliably detected and excluded.– Visual check or via optical sensor.

4. Labeling. It must be excluded that, forinstance, a bottle or filling series labeledas wine for diabetics contains a productthat does not meet these requirements.– Visual check or via a scanning device.

Finally, below some experiences and resultswe have observed during our extensivemicrobiological analyses in various wineryoperations.

If excessively high bacterial counts arefound, the cause usually lies in insufficientcleaning of the production systems.

Cleaning of tanks is often inadequate. Thiswill result in high bacterial counts in theproduct, either prior to filling, to the effectthat the filter systems block at an earlypoint, or, if there is no sterile filtration,even in the filled end product itself.

The hygiene of hoses and lines often leaves much to be desired, which can resultin correspondingly high bacterial counts.

Example: Collecting tank

36

It generally proves practical to place petridishes with agar or with moistened nutri-ent pads plus a membrane filter, directly inthe filling room and to leave them exposedthere for several hours – for instance during a filling shift. This offers a directcorrelation to the bacterial load that thefilling heads are exposed to. As can beclearly seen from the adjacent images, thebacterial load can vary greatly.

Cleaning is crucial and should be per-formed according to a cleaning plan thattakes the specific conditions of a plant intoconsideration. Whilst filling is in process ,care must be taken not to further increasethe bacterial loads in filling room. Forinstance, measurements have shown thathosing down the floor and the devicesresults in a multiplication of the airborne bacterial load.

However, most infections stem from thefiller or corker. Regular inspection is just asimportant as cleaning and disinfection.Contaminated filling points will always leadto product alterations as there is no way toavert infection – with the exception of hotfilling. Areas such as transport rails for thecorks are often neglected during cleaningand inspection, although sometimes highbacterial loads can be detected there.

Only under strictest observation of hygienecontrol throughout the complete plant,with sufficient sampling and microbiologicalanalysis of the entire process chain, onlythen can a constant long term quality beassured and errors avoided that can lead toimpaired taste and customer complaints.

Note: The controls and practices providedin this brochure are recommendations andguidance for the wine industry. This guid-ance is not a set of binding requirements.

Sales and Service ContactsFor further contacts, visit www.sartorius-stedim.com

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Phone +61.3.8762.1800Fax +61.3.8762.1828 Sp

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