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MBAA Technical Quarterly, Vol. 29, pp 86-88, 1992

Control of Biofilms in Breweries through Cleaning and Sanitizing


By Melvin H. Czechowski and Mark Banner
ABSTRACT
Brewery bacteria (Lactobacillus (L.) brevis, Enterobacter (E.) agglom erans, and Acetobacter sp.) were found to readily attach to Buna-n, Teflon and stainless steel surfaces and form biofilms, in test systems. Detergents were evaluated in a recirculating test loop (Tubular Flow System) for their ability to remove brewery bacteria from stain less steel surfaces. Chlorinated alkaline (0.4% v/v), and non-chlori nated alkaline (2% v/v) detergents used at 63C for 15 or 20 min removed greater than 94% bacteria from surfaces. Acid detergents (2% v/v) removed about 74-85%, about the same as water. At 27C, CI-alkaline detergents at 10 and 20 min removed 81 to 91% bacteria, while alkaline and acid detergents removed about as many bacteria as water, 63-84%. At 7C, the cleaners removed no more bacteria than water, at all time intervals. Detergent washing was followed by sanitizing. Detergent/sanitizer combinations which resulted in at least 99% bacterial reduction were chlorinated alkaline/mixed halogen, alkaline/peracetic acid, alkaline/organic bromine-chlorine, and am photeric based acid detergent-sanitizer.

SINTESIS
Bacteria cervecera (Lactobacillus (L.) brevis, Enterobacter (E.) ag g/omerans, y Acetobaete,. sp.) fueron halladas que se adhieren facil mente a superficies de Buna-N, Teflon y acero inoxidable y que for man bio-escamas en sistemas de prueba. Detergentes fueron evaluados en una prueba de vuelta recirculante (Tubular Flow System) - sistema de flujo tubular - por su abilidad de remover bacteria cervecera de superficies de acero inoxidable. De tergentes de alkalina clorinada (0.4% v/v) y alkalina no-clorinada (2% v/v) usados a 63C por 15020 minutos removieron mas de 94% de bacteria de las superficies. Detergentes acidos (2% v/v) removieron cerca del 74-85%, casi 10 mismo que el agua. A 27C, detergentes CI-Alkalina durante lOy 20 minutos removieron 81 a 91% de bac teria, mientras que detergentes de alkalina y acido removieron no mas bacteria que el agua, a todos los intervalos de tiempo. Lavado con detergentes fue seguido por sanitizaci6n. Combinaciones de de tergente/sanitizaci6n que resultaron en 99% de reducci6n bacterial por 10 menos, fueron alkalina clorinada/mezcla halogen, alkalina/ acido paracetico, alkalina/bromina-clorina organica y detergente/san itizante con base anfoterica.

INTRODUCTION

Biofilms may be found on the surfaces of food and beverage processing and packaging equipment; e.g. tanks, lines and fill ers ( Baldock, 1984; Lewis and Gilmour, 19 87; Bolton, et. aI., 198 8; Ashworth, 198 8). They form because microorganisms attach to surfaces and grow, resulting in masses of microbes, called biofilms (Costerton, et. aI., 19 87). The consequences of biofilm formation in food plants can be serious: contami nation of product resulting in reduced quality and shelf-life, decreased heat transfer in pasteurizers, unsightly scums, mal odoTs, corrosion of production equipment, and decreased lu bricity of conveyor line lubricants. Because of these problems, it is extremely important that proper measures be taken to 1) prevent the formation of biofilms, and 2) eliminate biofilms should they inadvertently be allowed to develop. Proper cleaning and sanitizing practices can be highly effective toward both of these goals. Biofilms are readily found in brewery pasteurizers and on conveyor systems (Rossmoore and Lichorat, 1987 and Ross moore, 1991). However, they are less evident in the closed processing areas, such as in tanks, pipes and fillers. Never theless, bacterial species (L. brevis, E. agglomerans, and Ace tobacter sp.) isolated from brewery environments have been found to attach to common surfaces in breweries, such as Buna-n, Teflon and stainless steel. Their removal is important in maintaining product quality. Different detergents alone and in combination with sanitizers were used in cleaning. Effec tiveness of the cleaning procedure was measured in terms of bacteria removed from surfaces within the Tubular Flow Sys tem (TFS), which simulates C IP cleaning systems in brew eries.
MATERIALS AND METHODS Growth

grown in a special beer medium. The medium consisted of 20 g tryptic soy broth, 5 g glucose and 2.5 g yeast extract, in 6 60 ml water and 340 ml beer. The p H of the medium was 6.3. Bacteria were grown at ambient temperature (27C) for 24 or 48 h before tests were performed.
Attachment

Coupons of Buna-n, Teflon and stainless steel were sub merged into a beaker containing slowly circulating beer me dium inoculated with the different bacteria for various time intervals (5 min, 1 h and 24 h). In some experiments, attach ment to stainless steel surfaces (grit 300) was studied in the TFS. Beer medium was circulated thToughout the TFS at a rate of 1 m/sec. To study the role of cleaning (removal of surface-attached bacteria), the inoculated beer medium cir culated through the TFS for 43 to 45 h at about 27C.
Cleaning

Lactobacillus brevis, Enterobacter agglomerans, and Aceto bacter sp. (Obtained from a major brewery in Canada) were
Melvin H. Czechowski, Ph.D. Microbiology, Senior Microbiologist, IBL/CTC, Diversey Corporation, 1532 Biddle Avenue, Wyandotte, MI., USA, 48192. Mark Banner, Ph.D. Food Microbiology, Manager IBL/CTC, Div ersey Corporation, 1532 Biddle Ave, Wyandotte, MI., USA, 48192.

Attached microorganisms (L. brevis and E. agglomerans) were removed by detergent washing followed by sanitizing. The cleaning procedure simulated C IP procedures normally used in breweries (Table 1). Detergents were 1) acids at 2 or 3% v/v of product, con taining surfactants and phosphoric or nitric acid, 2) alkalines (24-39% Na20) at 2% v/v, and 3) chlorinated alkalines (10.513.6 Na20, 2-3% CI) at 0.4% vivo The combinations of de tergents and sanitizers were: 1) acid/mixed halogen (CI-I) (25 ppm I), 2) chlorinated alkaline/mix halogen (CI-I) (25 ppm I), 3) alkaline/organic CI-Br (150 ppm CI), 4) alkaline/peracetic acid (200 ppm), and 5) a combined acid detergent-sanitizer product (1500 ppm amphoteric). In some experiments CO2 was continuously added to the TFS during cleaning because cleaning is often conducted under CO2 in certain areas of a brewery. The product flow rates, times and temperatures of application were those normally used in breweries (Table 1).
Effectiveness of Cleaning and Sanitizing Procedures

The effectiveness of detergents in removing bacteria from surfaces was determined by comparing the total cell numbers on cleaned surfaces to non-cleaned surfaces. The number of viable bacteria on detergent cleaned surfaces was compared

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Czechowski & Banner: Control of Biofilms

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to the total number of bacteria (viable and nonviable) on the cleaned surfaces. In addition, the combined effect of deter gent and sanitizer was evaluated by comparing the number of viable bacteria on cleaned and sanitized surfaces to the number of viable bacteria on non-cleaned and non-sanitized surfaces. Total number of bacteria was determined by counting the cells on the surface after acridine orange staining using epi fluorescence microscopy. Viable bacterial numbers on sur faces was determined by swabbing and rinsing the surface, and plating the swab and rinse solution at appropriate dilu tions with Lactobacilli MRS agar.
RESULTS AND DISCUSSION Bacterial Attachment

Lactobacillus brevis, E. agglomerans and Acetobacter sp. in a circulating beer medium attached to Buna-n, Teflon and stainless steel materials (Table 2). Bacterial attachment was rapid. Within 5 minutes of contact, bacteria adhered to sur faces (Table 2). The longer the contact time, and/or the larger the number of cells in the medium, the more bacteria attached to surfaces (Table 2). After 24 h, the number of attached bacteria was greatest on stainless steel surfaces, followed by Buna-n and then Teflon.
Detergent Washing

less bacteria removed. Washing with chlorinated alkaline de tergents at 27C for 10 and 30 minutes removed about 81 and 9 1% bacteria, respectively, from surfaces, with more bac teria removed the longer the time interval. Washing surfaces with chlorinated alkalines at 7C or with alkalines at 7 or 27C removed no more bacteria than washing with water. Washing with water removed between 52 to 84% of bacteria from surfaces, with best removal at 27C and longer duration cleaning time (Table 3). The number of cells removed by water washing reflect the strength of attachment, which depends upon the soil and mi croorganisms. Some microorganisms, such as some Pseudo monas sp., attach strongly to surfaces and water washing re moves only 10 to 30% of the cells. Acid detergent washing (2% concentration) at all temper atures and time durations removed no more bacteria than water. However, when the acid detergent concentration was increased to 3%, more bacteria were removed, probably re flecting the effect of increased detergent surfactant.
Table 3 Bacterial Removal From Stainless Steel Surfaces with Different Types Detergents Percent Bacteria Removed Temp (C) CI Time (min) Detergents CI-Alkalines 7 27 27

Surfaces were rinsed and washed following brewery CIP protocols (Table 1). Rinsing surfaces with water does not ad equately remove or kill bacteria from surfaces. The most im portant step in controlling biofilms is detergent washing. Therefore, detergents used in brewery washing were evalu ated for their ability to remove bacteria from stainless steel surfaces. Chlorinated alkaline and alkaline detergents were more effective than acids at removing bacteria from surfaces. Washing at 63C for 15 min with 0.4% v/v chlorinated al kaline or 2% v/v alkaline detergents removed more than 94% of bacteria from surfaces. Almost all of the remaining bacteria on the surfaces were dead. As washing temperature decreased, fewer bacteria were removed from the surfaces. Duration of cleaning time also affected bacterial removal, the shorter the cleaning time the
Table 1 CIP Procedure for Cleaning Stainless Steel Surfaces

10-30

10-30

30

63 15-20

% Viable Bact Clean Surface (7 or 27C)

Conc (% v/v) A (pH: 11.9) B (pH: 11.5)


Alkalines

0.4 69-73

0.4 87-91 81-90

0.4 95.4 99.5

62 0.1-31

Conc (% v/v) C (pH: 13) D (pH: 12.5)


Acids

2 61-71

2 77-80 75-85

2 94 95-97

16

Conc (% v/v) E (pH: 1.8) F (pH: 1.4) w and w/o CO,


Water

2 56-81

2 73-73 66-80

3 91

2 85 74

5-38 1-5

1. Three to five water burst rinses (30-50 sec). 2. Detergent wash: 15-30 min detergent circulation (1.3 m/s) at 7, 27 and 63C (45,80 and 145F). 3. Three water burst rinses (30-60 sec). 4. Sanitizer treatment: 2, 10 and 20 min at 26C (80F) with 1.3 m/s flow rate using sanitizer.

52-80

63-84

82

90-100

Bacteria were removed from the surface by washing with detergents in the TFS. The number of viable bacteria on clean surfaces was compared to the total number of bacteria on the surface (viable and dead), and is reported as a percentage (%). The number of bacteria on non-cleaned surfaces was between 40,000 and 100,000/cm'.

Table 2 Bacterial Attachment to Buna-N, Teflon and Stainless Steel Surfaces Time of Attachment Bact/MI in Medium Beaker System Attachment to Surfaces (Bact/cm") Buna-N Teflon St. Steel

Bacteria

Lactobacillus Brevis

5 Min 1H 24 H 5 Min 24 H 5 Min 24 H

3.9 X 10 1.9 X 10' 1.6 X 107 1.5 X 10 2.7 X 107 3.5 X 10


Tubular Flow System

1000 6300 30200 100 52800 2700 42200

500 1900 17100 300 3200 2000 53600

900 4500 43100 300 53600 1600 89000

Acetobacter sp. Enterobacter Agglomerans

Lactobacillus and Enterobacter:

5 Min 1H

2.3 X 10

29500 68500

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Czechowski & Banner: Control of Biofilms


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MBAA T.Q., Vol. 29

Table 4 Bacterial Reduction From Surfaces with Different Detergent/Sanitizer Combinations 99% or Greater Bacteria Reduction Detergent (Conc)/Sanitizer (Conc) Temperature (OC) Time (min)

VIABLE BACTERIA REDUCTION

CI-Alk (0.4% v/v)/CI-I (25 ppm I) CI-Alk (0.4% v/v)/CI-I (25 ppm I) Alk (2% v/v)/Org-CI (150 ppm CI) Alk (2% v/v)/Oreg-CI (150 ppm CI Alk (2% v/v)/PAA (200 ppm) Acid Det/San (1.5%) without CO. with CO.

26/26 63/26 26/26 63/26 26/26 26 63 26

15/10,20 15/2 15/2,10,20 15/2 15/2,10,20 15,30 15 15

CL-ALK/CI-I (2epPM)

ALK/ORG-CI (1eOPPM)

ALK/PAA (200PPM)

10 MIN 1IIIIII 20 MIN SANITIZER APPLI CATION TIME DETERGENT (26C)/SANITIZER (26C)
Fig. 1. Cleaning and sanitizing stainless steel surfaces soiled with beer and bacteria.

[2] 2 MIN

After detergent washing many bacteria remaining on sur faces were dead, while after cleaning with water many of the residual surface bacteria were viable.
DETERGENT WASHING AND SANITIZING

The control of microorganisms in biofilms is best accom plished by cleaning with a detergent followed by the appli cation of a sanitizer. In fact, applying a sanitizer to an unclean surface has been shown to be ineffective in controlling mi croorganisms, particularly those enmeshed in bionlms (Le Chevalier, et. al. 1987). However, sanitizer application after detergent washing will kill those viable microorganisms left on the surface after cleaning. Selected combinations of de tergent/sanitizer, therefore, were evaluated for their effec tiveness in reducing viable bacteria on surfaces (Table 4). Detergent washing at 63C followed by sanitizer (2 min) reduced viable bacteria on surfaces by 99% or more (Table 4). In fact, almost all remaining bacteria on surfaces after washing at 63C, were dead. When surfaces were washed at low temperature (26C) fol lowed by sanitizer application for 2, 10 and 20 min, almost all the surfaces were bacteriologically clean (99% or greater bacterial removal). The detergent/sanitizer combinations were Cl-alkaline/CI-I ( 10 and 20 min), alkaline/organic Cl (2, 10 or 20 min), alkaline/PAA (2, 10 and 20 min), and the combined acid detergent-sanitizer in the absence of CO2 ( 15 and 30 min), and with CO2 (30 min) (Table 4). Even though sanitizer is normally applied for only a few minutes, longer application times may be required when processing systems exhibit chronic contamination problems due to microbes. The longer sanitizer application times ( 10 and 20 min) increased efficacy of the sanitizers (Figure 1). In general, all the detergent/sanitizer combinations work well. This is mostly due to the detergent, especially the al kaline and chlorinated alkaline types, which remove many of the cells from the surface, or makes those viable cells still on the surface more susceptible to the effects of the chemical sanitizer.
SUMMARY

may form tenacious biofilms. This was demonstrated by laboratory experiments using a model pipeline system and a beaker test method. 2. In order to remove these bionlms, the surfaces must first be cleaned and then sanitized. Water rinsing is not ade quate to remove and kill bacteria on surfaces. 3. Factors afrecting cleaning are: detergent type, temperature of application and time interval of cleaning. Chlorinated alkalines at 27 or 63C and alkaline detergents at 63C are very effective at removing microorganisms from surfaces. Increasing times for cleaning enhances bacteria removal. 4. Detergent/sanitizer combinations, when used as recom mended, reduce viable microorganisms from surfaces 99% or more. Increased sanitizer efficacy is found with longer application times.
REFERENCES
1. ASH WORT H ,

J., Bioftlm Fonnation and the Hygenic Operation of

Drinks Vending Machines, In Biofilms, ed. L. H. G. Morton &


A. H. L. Chamberlain. Biodeterioration Society, Kew, pp. 26-34. 2. BALDOCK, J. D., Microbiological Monitoring of the Food Plant: Methods to Assess Bacterial Contamination on Surfaces, J. Milk Food Techno!., 37:361-368, 1984. 3. BOLTON, K. J., DODD, C. E. R., MEAD, G. C. and WAITES, W. M.,

Chlorine Resistance of Strai,lS of Staphylococcus Aureus Isolated from Poultry Processing Plants, Lett. App!. Microbio!., 6:31-34,
1988. 4. COSTERTON,

J. W., CHENG, K. J., GEESEY, G. G., LADD, T.I. , NICKEL, J. c., DASGUPTA, M., and MARRIE, T. J., Bacterial Bioftlms
Surfaces of Rubber and Stai,lless Steel Milk Transfer Pipeline,

in Nature and Disease, Ann. Rev. Microbio!. 41:435-465, 1987. 5. LEWIS, S. and GILMOUR, A., MicrojloraAssociated with the Intemal

J.

App!. Bact. 62:327-333, 1987. 6. LE CHEVALIER, M. W., CAWTHON, C. D. and LEE, R. G., Factors

Promoting Survival of Bacteria in Chlorinated Water Supplies,


App!. Environ. Microbio!. , 54:649-654, 1988. 7. ROSSMOORE, K. and LICHORAT, J. J. Source and Identification of Contamination of a Brewenj Conveyor Lubricant. Biodeterioration VI, 1988. 8. ROSSMOORE, K., Microbial Ecology of Brewery Pasteurizers. SIM Abstracts, 1986.

1. Common brewery spoilage bacteria can readily and quickly attach to typical brewery equipment surfaces where they

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