International Biodeterioration & Biodegradation 51 (2003) 265–269www.elsevier.com/locate/ibiod

Bio lm and disinfection in meat processing plants

B. Jessena ;∗, L. Lammertb

aDanish Meat Research Institute, Maglegaardsvej 2, DK-4000 Roskilde, DenmarkbNovo Nordisk A/S, Hallas Alle, DK-4400 Kalundborg, Denmark

Abstract

Like other food branches the meat industry is met by increasing demands to cleaning and disinfection in order to remove microbialcoatings such as bio lm. A crucial point is, however, to document the presence of bacteria in bio lm on processing equipment. This paperdescribes an indirect way to detect foodborne bio lms on visually clean equipment surfaces.If at all possible, elimination of bio lm-bound bacteria on processing equipment is an arduous task. If the bio lm is established, it is not

removed in daily sanitation unless extra actions are employed. Various methods such as mechanical treatment as well as extra disinfectionhave been investigated in practice. The results show that a reduction in bacterial load could be achieved, but at present neither one singlemethod nor one single chemical completely eliminated the microorganisms.In conclusion, in order to minimise bio lm-bound bacteria on processing equipment, the critical sites should be identi ed and paid full

attention to during sanitation. Further, the right choice and usage of cleaners and disinfectants as well as an adequate sanitation programmemust be thoroughly considered.? 2003 Elsevier Science Ltd. All rights reserved.

1. Introduction

In terms of food safety, the Danish meat industry regardListeria monocytogenes the most troublesome microorgan-ism due to its capability to survive and even grow at chilltemperature in vacuum packed or modi ed atmospherepacked ready-to-eat meat products.It is well documented that L. monocytogenes can be

found in meat processing areas even at very high incidences(van den Elzen and Snijders, 1993; Wendlandt and Bergann,1994). Further, it is impossible that, given the currentlyavailable technology, to eradicate L. monocytogenes fromthe processing environment or to totally eliminate the poten-tial for contamination of nished products (Tompkin et al.,1999). Pasteurisation or cooking usually guarantee the ab-sence of L. monocytogenes, but as the bacterium can persistin the production environment for years (Nesbakken et al.,1996), probably in foodborne bio lms, it may contaminatethe meat products during processing, e.g. during slicing.The Danish meat industry has initiated a number of

projects in order to control this hazard. Among these, aproject speci cally focusing on the possibility to eliminateL. monocytogenes in bio lms on processing equipment has

∗ Corresponding author.E-mail address: bjs@danishmeat.dk (B. Jessen).

been conducted. Data presented here are primarily based onthe results from this project.

2. What is bio�lm?

Bio lm consists of immobilised bacteria embedded in anorganic polymer matrix of bacterial origin. The bacteria se-crete extracellular polymers by which they adhere to sur-faces. True bio lms may take days or even weeks to developand they are not necessarily uniform in time and space.Pathogenic microorganisms are also known to form or to

be entrapped in bio lms (Johansen, 1997; Holah and Gibson,1999). Therefore, the fact that parts of the bio lm may dis-lodge from the surface is of concern in the food processingindustry due to the risk for contamination of the food prod-ucts. The risk becomes even more serious because bacteriain bio lm may express an increased resistance to disinfec-tants (Frank and KoA, 1990; Mosteller and Bishop, 1993).

3. How can bacterial bio�lms be documented in the meatindustry?

On laboratory scale and via microscopical techniques, itis documented that food industry microorganisms can ad-here to materials used in the construction of food processing

0964-8305/03/$ - see front matter ? 2003 Elsevier Science Ltd. All rights reserved.doi:10.1016/S0964-8305(03)00046-5

266 B. Jessen, L. Lammert / International Biodeterioration & Biodegradation 51 (2003) 265–269

Table 1Evaluation of bio lm on processing equipment by method A (aerobiccount)

Area CFU/area Bio lm Result

Swabbing = scraping Swabbing¿ scraping ⇒ No bio lm 81%(n= 36) Swabbing¡ scraping ⇒ Bio lm 19%Swabbing¿ scraping Swabbing6 scraping ⇒ Bio lm 71%(n= 21) Swabbing¿ scraping ⇒ No bio lm 29%

equipment (Mafu et al., 1990; Blackman and Frank, 1996;Beresford et al., 2001). However, when it comes to docu-mentation in food plants, the same technique is not an idealway of detecting bacteria in bio lm on processing equip-ment. Instead, researchers have invented the use of testcoupons to be glued onto the surfaces and also to be placedin various environmental locations (Holah et al., 1989; Hoodand Zottola, 1997). After a certain time, the coupons are re-moved and inspected for bacterial content. It is a drawbackof the method that it may result in further soil deposits andthereby increase the risk for bio lm formation.

3.1. Materials and methods

In the experimental work two methods were explored:Method A: The cleaned surfaces were scraped followed

by swabbing.Method B: The cleaned surfaces were swabbed three con-

secutive times.Both methods were carried out by swabbing huge areas

(70–24; 000 cm2) along the processing line for slicing andpackaging of cooked ham. Usually, the complete food con-tact surface was sampled.Bio lm was evaluated by quantitative enumeration of the

aerobic count (Plate Count Agar, 20◦C, 5 days).In method A, the selected sites were swabbed right

after the regular cleaning and disinfection (=sanitation)programme was nished and next, selected parts of thesame sites were vigorously scraped with a household spat-ula in order to tear apart any bio lm present. The scrapedarea was then swabbed. In some locations, the swabbed re-spectively the scraped area were of the same size, in otherlocations the swabbed area was bigger than the scrapedarea. This diLerence was exploited in the assessment of theresults and also used as an attempt to document bio lm onequipment surfaces.If the swabbed and the scraped area are identical, then

higher count on the scraped surface indicate bacteria presentin a bio lm. On the other hand, if the scraped area is smallerthan the swabbed area, then identical or higher counts onthe scraped area indicate bacteria present in a bio lm.

3.2. Results

The results are illustrated in Table 1. Based on results ob-tained via method A, it was concluded that bio lm is likely

Table 2Evaluation of bio lm on processing equipment by method B (aerobiccfu=cm2)

Plant/Line A B C

Sample 1 ¡ 1 ¡ 1 ¡ 1¡ 1 ¡ 1 ¡ 1¡ 1 ¡ 1 ¡ 1

Sample 2 ¡ 1 ¡ 1 1¡ 1 ¡ 1 1¡ 1 ¡ 1 1

Sample 3 ¡ 1 1 420¡ 1 ¡ 1 200¡ 1 16 325

Sample 4 ¡ 1 ¡ 1 1¡ 1 ¡ 1 1¡ 1 ¡ 1 4

Sample 5 415 700 ¡ 11500 700 ¡ 1495 65 1

Sample 6 ¡ 1 3 16¡ 1 ¡ 1 155¡ 1 ¡ 1 82

Sample 7 96 1 ¡ 1104 1 ¡ 196 22 ¡ 1

Sample 8 1 ¡ 1 2291 ¡ 1 6120 ¡ 1 139

Sample 9 ¡ 1 21 ¡ 1¡ 1 5 ¡ 1¡ 1 7 ¡ 1

Sample 10 ¡ 1 1 ¡ 1¡ 1 ¡ 1 ¡ 1¡ 1 ¡ 1 ¡ 1

Sample 11 7 19 2948 ¡ 1 29411 ¡ 1 352

Sample 12 33 ¡ 1 133 ¡ 1 477 ¡ 1 13

Sample 13 ¡ 1 39 30¡ 1 183 39

6 250 37Sample 14 5 325 1

4 22 12 8 1

to be present on the surface of processing equipment. Therewas, however, some uncertainty whether the bio lm wasgenuinely torn or whether the same results would have beenobtained by swabbing the area twice. Therefore, methodB was developed. In this method, the areas were swabbedthree times after each other. Also by this method the sur-face receives a certain mechanical treatment although not asstrongly as in method A. Results from method B is given inTable 2.Apart from in a few locations it is evident that approx-

imately the same number of bacteria was detected threeconsecutive times on the same site. A decrease in bacterialcount was anticipated but except for a few deviations, the

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bacterial counts remained stable throughout the swabbingsteps. First, this explains that scraping the surface not nec-essarily released cells from bio lm, at least not to a higherdegree than by swabbing the surface. Second, the ability todetect the same bacterial count in three swabbings could beseen as an evidence of the presence of bio lm.It is debatable when a consortium of microorganisms on

surfaces can be named ‘bio lm’. What is important in thiscontext is the fact, that even after three swabbings each in-cluding a mechanical treatment, the same number of bacte-ria was detected. This explains, bio lm or not, that bacteriawere attached to the surface and that they were not easilyremoved.In conclusion, method A was not useful for documenta-

tion of potential bio lm, whereas method B indirectly gaveevidence of bio lm as it proved attachment of bacteria tothe equipment surfaces.

4. How can bacteria entrapped in a bio�lm be eliminated?

Regular sanitation programmes comprise basically ofremoval of gross debris, rinsing, presoaking in detergent,rinsing, disinfection and nal rinsing. When sanitation wasmonitored, it became obvious that the disinfectant, appliedvia low-pressure spray, sometimes hit the processing equip-ment sporadically and tended to run oL vertical surfacesquickly or to gather in small pits on horizontal surfaces, i.e.the eLect may be less than expected.The periodic sanitation programme most often consists

of 4 days with alkaline sanitation (alkaline detergent pluschlorine disinfectant) and 1 day with acid sanitation, usuallyFriday, using an acid detergent followed by an acid disin-fectant.From examinations on the capability of detergents and

disinfectants to eliminate L. monocytogenes in bio lm onstainless steel coupons, our laboratory results showed thatthe eLect of detergents was negligible, whereas the most eA-cient disinfectants were able to eliminate L. monocytogenesin model foodborne bio lms. The eLect was only obtainedif the disinfectant was used at the manufacturer’s recom-mendation for high strength and long reaction time and afterpre-treatment with detergent.Acid disinfectants composed of hydrogen peroxide and

peracetic acid were signi cantly more eAcient than chlorinecompounds when tested in meat systems. Similar results arereported from some researchers (Carpentier and Cerf, 1993;Bourion and Cerf, 1996; Fatemi and Frank, 1999), whileothers nd the same eAcacy of the two disinfectants (Frankand KoA, 1990) or even the opposite eAcacy (Rossini andGaylarde, 2000).

4.1. Materials and methods

The experimental work was carried out on process-ing lines for sliced cooked ham products. The eLect was

Table 3The eLect of two sanitation methods in a meat processing company(aerobic cfu=cm2)

Bacterial load=cm2 Alkaline sanitationa Acid sanitationb

¡ 1 64% 40%1–¡ 10 25% 31%10–¡ 100 5% 17%100–¡ 1; 000 2% 6%1,000–10,000 4% 6%Samples in total 55 52

Manufacturer of detergents and disinfectants: SFK, Denmark.aKombinon Special (alkaline detergent)+Blegeessens (solution of

hypochlorite).bAlka 32FF (alkaline detergent), Surklar (acid detergent)+Oxivit Plus

(acid disinfectant: hydrogenperoxide plus peracetic acid).

measured by analysing for aerobic count (Plate Count Agar,20◦C, 5 days), Enterobacteriaceae (NMKL no. 144) inaddition to qualitative L. monocytogenes analysis (NMKLno. 136). In one experiment, L. monocytogenes was alsoanalysed quantitatively (Oxford Agar, 30◦C, 24 hours).The eLect of alkaline sanitation versus acid sanitation

was assessed. Further, it was investigated how much thebacterial standard would be improved, if the disinfection wasmeticulously carried out or if a scrubbing step was includedin the sanitation programme.

4.2. Results

4.2.1. Bacterial level after regular sanitationThe results showed that even visually clean surfaces har-

boured bacteria after regular sanitation, the level rangedfrom less than one bacterium per cm2 up to 3:7 × 104 aer-obic cfu=cm2. The appearance of high counts is due to themethod employed. Huge areas were swabbed, irregular cor-ners included and the samples were stomached, each result-ing in higher counts than using contact plates or swabbingsmall areas.In order to evaluate the eLect of the two sanitation meth-

ods, the aerobic counts were classi ed in ve groups andthe proportion of each was calculated, Table 3.It is interesting that the bacterial level obtained by

chlorine disinfection was better than the level obtained byperacetic acid, which is the opposite of the previously men-tioned laboratory results carried out in a model system. Theresult may, however, rePect variation in the daily hygienestandard as the experiment had to be carried out on diLerentworking days.Further, the results showed that on locations with high

aerobic counts, it was possible to detect Enterobacteriaceaeand in such locations it was likely to demonstrate L. mono-cytogenes. Consequently, the presence ofL. monocytogeneswas not caused by extremely clean conditions, as it was morelikely to exist in locations that also harboured other types ofbacteria. Therefore, in a monitoring programme the aerobiccount could be used as an indicator of potential L. mono-

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Table 4The eLect of extra disinfection next to regular sanitation (aerobic cfu=cm2)

Bacterial load=cm2 Alkaline sanitationa+ Acid sanitationa+extra disinfection (chlorine) extra disinfection (peracetic acid)Before After Before After

¡ 1 64% 58% 40% 27%1–¡ 10 25% 29% 31% 35%10–¡ 100 5% 11% 17% 23%100–¡ 1; 000 2% 2% 6% 12%1,000–10,000 4% 0% 6% 2%Samples in total 55 55 52 52

aSee Table 3.

Table 5The eLect of scrubbing next to regular sanitation (aerobic cfu=cm2)

Bacterial load=cm2 After regular sanitation After scrubbing,disinfection, and rinsing

¡ 1 47% 59%1–¡ 10 16% 19%10–¡ 100 22% 22%100–¡ 1; 000 13% 0%1,000–10,000 3% 0%Samples in total 32 32

cytogenes contamination. When L. monocytogenes was de-tected, it was in low numbers, 0.025 cfu=cm2 or less.Another outcome of the investigations was that, based on

contamination frequency and bacterial load, selected areascould be categorised as critical sites respectively observationsites, i.e. sites that were contaminated occasionally. Typi-cally, critical sites were control panels and rollers along theconveyor belt while observation sites were knives and hold-ers for keeping the cooked meat sausage in place.

4.2.2. The e5ect of extra disinfectionIt was believed that a thorough disinfection could improve

the bacteriological level of the equipment. Therefore, after nishing regular sanitation, the eLect of carrying out an extracareful disinfection was investigated. The extra disinfectionwas followed by a rinse prior to sampling. The results aregiven in Table 4.Comparison of the results before and after performing the

extra disinfection step shows that apart from a reduction inthe count of the most contaminated sites, it is evident thatthe general bacteriological standard was not improved.

4.2.3. The e5ect of mechanical treatmentThe results obtained by extra disinfection were discourag-

ing. Therefore, another method that preliminarily had shownvery promising results was transferred for investigation in ameat processing plant. After nishing regular sanitation themethod was to scrub the surfaces with a household spongefollowed by disinfection and rinsing. The eLect was assessedby swabbing the surface and by determination of the aerobiccount, Table 5.

The high counts in certain sites were removed by scrub-bing, and the percentage of sites with¡ 1 aerobic cfu=cm2

was increased. Scrubbing was therefore more eAcient than‘extra disinfection’ although in practice not as eAcient asthe preliminary results, which all showed results down to¡ 0:05 aerobic cfu=cm2.

5. Discussion

It was shown that bacteria established in a bio lm couldnot be eradicated by using one single treatment or one singledetergent or disinfectant. The most eAcient method wasto scrub the surfaces. Other researchers have also reportedbrushing as an eLective way of removing bio lm (Gibsonet al., 1999). Scrubbing or brushing can be an ardous task.Consequently, it is usually not part of the regular sanitationprogramme. Instead, it is recommended as part of rotationsanitation respectively frequency sanitation.In addition to an adequate sanitation programme, the in-

dustry must realise that consistency and attention to detailare necessary in order to avoid bacteria in bio lms or tosolve the problem in case of their appearance. The followingprecautions and actions are recommended:Introduce good manufacturing practice:

• Ensure hygienic pre-slicing procedures.• Introduce high-risk production zone.• Employ frequent hygiene training of personnel.

Make a risk assessment in order to introduce good sanitationpractice:

• De ne critical sites.• De ne observation sites.

Introduce good sanitation practice:

• Expose critical sites to rotation sanitation.• Expose observation sites to frequency sanitation.• Measure the bacteriological eLect of the sanitation regu-larly.

• Choose appropriate detergents and disinfectants as wellas sanitation programme.

B. Jessen, L. Lammert / International Biodeterioration & Biodegradation 51 (2003) 265–269 269

Rotation sanitation means that critical sites are given spe-cial attention in daily turns and if necessary, special treat-ment. Frequency sanitation means that certain parts of theprocessing equipment are thoroughly sanitised at e.g. 7-daysor 14-days intervals. Scrubbing is recommended as specialtreatment.

6. Conclusion

The presence of bio lm on equipment in meat process-ing plants could not be de nitively concluded. It was, how-ever, shown that the same number of bacteria was presentafter three consecutive swabbings each including a mechan-ical treatment and thus indicating a strong attachment tothe equipment surfaces. To avoid the entrance and the dis-persion of bacteria in general and of L. monocytogenes inparticular into the processing areas, it is recommended toimplement good manufacturing practice.Bacteria attached to surfaces could be reduced but not

eradicated by using one single method or one single deter-gent or disinfectant. Laboratory experiments and many re-ports have claimed disinfectants containing peracetic acid tobe the most eAcient to remove bio lm, whereas chlorine inpractice appeared more eAcient in meat processing plants.Extra disinfection on top of the regular sanitation did notimprove the hygienic level, but the introduction of a scrub-bing step reduced the bacterial load and is recommended oncritical sites and observation sites as well.In order to minimise the risk of bio lm it is recommended

to introduce good sanitation practice, i.e. identi ed criticalsites should undergo rotation sanitation and observation sitesfrequency sanitation.

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