Tuija Pirttijärvi Contaminant Aerobic Sporeforming Bacteria in the Manufacturing Processes of Food Packaging Board and Food Department of Applied Chemistry and Microbiology Division of Microbiology University of Helsinki Academic Dissertation in Microbiology To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in auditorium 1041, at Viikki Biocenter, Viikinkaari 5, on July 21st, 2000, at 12 o’clock noon. Helsingin yliopiston verkkojulkaisut Helsinki 2000 ISBN 952-91-2304-3 (pdf) Supervisor: Prof. dr. Mirja Salkinoja-Salonen Academy Professor Department of Applied Chemistry and Microbiology University of Helsinki Helsinki, Finland Reviewers: Prof. dr. Tuula Honkanen-Buzalski Department of Food Microbiology National Veterinary and Food Research Institute Helsinki, Finland Prof. dr. Seppo Salminen Department of Biochemistry and Food Chemistry University of Turku Turku, Finland Opponent: dr. Anders Christiansson Swedish Dairy Association Research and Development Lund, Sweden To Reijo, Anja and Jutta CONTENTS List of original papers.................................................................................................................1 Abbreviations..............................................................................................................................2 Abstract.......................................................................................................................................3 1. Introduction.............................................................................................................................4 2. Review of the literature...........................................................................................................5 2.1. History and taxonomy of aerobic sporeforming bacteria.................................................5 2.2. Occurrence of bacilli in foods and in food processes.......................................................5 2.3. The significance of bacterial contamination in paper and board production....................9 2.4. Degradation of starch by bacteria....................................................................................9 2.5. Control of contaminant bacteria in paper industry.........................................................11 2.6. Nisin and its applications...............................................................................................12 3. Aims of the study..................................................................................................................14 4. Materials and Methods..........................................................................................................14 5. Results and discussion..........................................................................................................14 5.1. Bacilli contaminating paper and board manufacturing process and their properties related to food spoilage and food safety...............................................................................14 5.2. Detection and typing of B. cereus in industrial processes..............................................20 5.3. Identifying the emetic toxin producing strains of B. cereus...........................................22 5.4. Preservation of surface sizing starch against deterioration by bacilli.............................24 6. Conclusions...........................................................................................................................27 7. Tiivistelmä............................................................................................................................28 8. Acknowledgements...............................................................................................................30 9. References.............................................................................................................................32 Original publications List of original papers I Pirttijärvi, T.S.M., Graeffe, T.H. and Salkinoja-Salonen, M.S. 1996. Bacterial contaminants in liquid packaging boards: assessment of potential for food spoilage. Journal of Applied Bacteriology 81, 445-458. II Pirttijärvi, T.S.M., Ahonen, L.M., Maunuksela, L.M. and Salkinoja-Salonen, M.S. 1998. Bacillus cereus in a whey process. International Journal of Food Microbiology 44, 31-41. III Pirttijärvi, T.S.M., Andersson, M.A., Scoging, A.C. and Salkinoja-Salonen, M.S. 1999. Evaluation of methods for recognising strains of the Bacillus cereus group with food poisoning potential among industrial and environmental contaminants. Systematic and Applied Microbiology 22, 133-144. IV Pirttijärvi, T.S.M, Wahlström, G., Rainey, F.A., Saris, P.E.J. and Salkinoja-Salonen, M.S. 200x. Inhibition of bacilli in industrial starches by nisin. Submitted to Journal of Industrial Microbiology and Biotechnology. The author’s contribution Paper I: Tuija Pirttijärvi wrote the paper, is the corresponding author and interpreted the results. She was responsible for all experimental work except for the mass fragmentographic confirmation of the fatty acids and the partial 16S rDNA sequencing. Paper II: Tuija Pirttijärvi wrote the paper, is the corresponding author and interpreted the results. Paper III: Tuija Pirttijärvi wrote the paper, is the corresponding author and interpreted the results. She did all experimental work except for the determination of emetic toxin, a part of the enterotoxin tests, transmission electron microscopy and the serotyping. Paper IV: Tuija Pirttijärvi wrote the paper, is the corresponding author and interpreted the results. She did all experimental work except for nisin activity analysis and the partial 16S rDNA sequencing. 1 Abbreviations AOAC Association of Official Analytical Chemists ATCC American Type Culture Collection ATP adenosine triphosphate cfu colony forming unit BCET-RPLA Bacillus cereus enterotoxin reversed passive latex agglutination BHI brain heart infusion BUGM+G Biolog universal growth medium with 1 % glucose CDase cyclodextrinase CGTase cyclodextrin glycosyl transferase D decimal reduction time at 140 °C 140 DBNPA 2,2-dibromo-3-nitrilopropionamide DSM(DSMZ) Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH EDTA ethylenediaminetetraacetic acid EELA National Veterinary and Food Research Institute ELISA-VIA Bacillus diarrhoeal enterotoxin visual immunoassay FDA Food and Drug Administration HACCP hazard analysis critical control point IDF International Dairy Federation ISO International Organization for Standardization IU international unit kb kilo basepairs m threshold below which all results are considered satisfactory (in microbiological safety criteria of foods) M acceptability threshold, above which results are not considered satisfactory (in microbiological safety criteria of foods) Mb mega basepairs MIC minimum inhibitory concentration MUF methyl umbelliferyl NCFB National Collection of Food Bacteria NMKL The Nordic Committee on Food Analysis O.D. optical density PCA plate count agar PDA potato dextrose agar PFGE pulsed field gel electrophoresis PEG polyethylene glycol SMR Swedish Dairy Association TAPPI Technical Association of the Pulp and Paper Industry TE Tris-EDTA TSA tryptone soy agar TSB tryptone soy broth TSBA tryptone soy broth agar (BBL) UPGMA unweighted pair group method with arithmetic mean 2 Abstract The significance of bacilli in industrial processes was studied, with focus on food grade paper and paperboard production and on a dairy process. Food packaging paper and board contained as contaminants mainly sporeforming bacteria belonging to the genera Bacillus, Paenibacillus and Brevibacillus. The contaminants were usually found in quantities from <50 to 250 cfu g-1 homogenized paperboard, which are lower than in many foods. Of those frequently found, especially the Bacillus cereus group, B. licheniformis, B. subtilis, and Brevibacillus brevis are species known to be capable of hydrolysing food components, and some strains of these species produce food poisoning toxins or may grow at refrigerated temperatures. The diversity among the contaminant B. cereus isolates in the whey processing line of a cheese dairy was large. The whey line of the cheese dairy was colonized by specific communities of phenotypically non-conventional types of B. cereus. Great genetic diversity was found among contaminant B. cereus strains from paper industry and from different dairies. We found that the phenotypic properties on which the standard methods (e.g. ISO, FDA, IDF) rely for recognising B. cereus (lecithinase activity, nitrate reduction), did not match with the properties of B. cereus isolates found as contaminants in industrial processes. Industrial isolates, including the toxigenic ones, often missed one or several of the typical B. cereus characters even in cases where 100 % 16S rDNA identity was found with B. cereus or with B. thuringiensis. The whole cell fatty acid compositions of a group of industrial contaminant B. cereus isolates deviated so much from those in the widely used commercial database that the strains were only poorly recognised or not recognised at all as B. cereus. Better identification was obtained with signature fatty acids 11-methyldodecanoic acid and trans-9-hexadecenoic acid, which were present without exception in all of the more than 200 industrial contaminant isolates representing B. cereus group and in all of the more than 30 culture collection strains. The detection of these fatty acids provides a secure method for identifying B. cereus. When a biological assay for the dangerous emetic toxin of B. cereus became available, it was possible to scrutinize the emetic toxin producing strains of B. cereus for shared biochemical or chemical properties. Negative reaction for starch hydrolysis and negative reaction for BCET- RPLA test distinguished the emetic toxin producing B. cereus strains from the non-emetic B. cereus strains. B. cereus was found to be particularly rich in ribotype patterns: 40 ribotypes were obtained from 93 strains with the restriction enzyme EcoRI. Enterotoxin producers were found in numerous different ribotypes. Genetic diversity of the emetic strains of B. cereus was narrow: the 15 strains all clustered in one ribotype and its closely similar variants. Modified industrial starches are used for surface sizing of paper and board. The microbiological quality of the starches affects the hygiene of food grade paperboard. The starch spoiling bacteria were B. coagulans, B. licheniformis, B. amyloliquefaciens and B. stearothermophilus. These species grew under mill simulating conditions at 50 °C and depolymerized starch by producing cyclodextrins and α-glucosidase. Nisin, an antimicrobial peptide approved as an antimicrobial additive to prevent spoilage of some foods, inhibited bacterial growth in industrial starches used in paperboard manufacture. It inhibited the Bacillus sp., isolated from starches or from paperboard, equally or up to 1000 times more effectively than the conventionally used biocide 2,2-dibromo-3-nitrilopropionamide. Nisin activity remained stable under conditions simulating cooking and storage of surface sizing starches in the industry. The present high microbiological quality of food grade paper and paperboard could be maintained by replacing part of the 3 presently used process slimicides by this alternative preservative, safe for the consumer and the environment. 1. Introduction Papermaking, production and handling of modified starches, and dairy and some other food processes share the sensitivity of the raw materials and process conditions often favourable for microbial growth. Process equipment has large surfaces for microbes to settle and to form biofilms. Heat in the dry end of the paper machine or during the ‘cooking’ of industrial starches inactivate fungi and vegetative bacteria whereas heat resistant bacterial spores not only survive but may become activated. Similar selection for heat resistant contaminants occurs by the pasteurization of liquid foods. Effective cleaning and preservation techniques and good manufacturing practices are required in these processes to maintain high hygienic quality in the end product. Aerobic endospore forming bacteria are widely distributed in nature. The spores are resistant to heat, desiccation, disinfectants, ionizing radiation and UV light. Bacilli are frequent contaminants in biomaterial-based industrial processes. They represent the major hygienic problem in processes where heat or other treatments kill the competing, non-sporeforming microflora. The significance of bacilli in foods such as milk products has been studied intensively, but little information is available on their role in industrial processes, e.g. in the production of food packaging materials. Paper and paperboard is produced in large volumes worldwide: in 1999 the production was 12.6 million tons in Finland and 79.5 million tons in Europe. Biocides are used to reduce accumulation of slimes and the subsequent production downtime and deterioration of the technical and hygienic quality of paper and board. One kg of each (effective substance) ton-1 of paper is commonly dispensed into the process at the different stages of production. Considering the economic importance of paper and paperboard manufacture, surprisingly little published information is available on the microbes contaminating these processes and the products. This study focuses on the production of food packaging paper and board and a dairy process bringing new knowledge on diverse bacterial contaminants in these biomaterial-based industries. Aerobic bacteria producing heat-tolerant spores (named bacilli in this thesis), are the main contaminant group. The properties of these bacteria relevant to hygiene, food spoilage and food poisoning were studied in this work. The most prevalent pathogenic bacillus in industrial processes is Bacillus cereus, classified to hazard category 2 (Anon., 1995; European Commission, 1993). An industrially applicable method for differentiating the potentially dangerous, emetic toxin producing strains of B. cereus from the non-producing strains, did not exist when this work was initiated. Strains of B. cereus isolated from food poisoning incidents, from the products and process of food packaging paper and board manufacture and from dairy processes were characterized chemically, biochemically and genotypically. Starch constitutes one to two percent of the dry weight of paper products other than newsprint and some brands of grease proof paper. Paper industry is thus a major user of modified starches. Over 90 % of the starches used in Finland are used by paper industry, 250 000 tons in 1999. Preservation of industrial starches against microbial deterioration is difficult. The organisms responsible for spoilage have seldom been identified. This gap of knowledge hampered the design of rational strategies for preserving industrial starches against microbial spoilage. Good novel preservation methods with minimal usage of toxic or sensitising chemicals are needed. 4 Understanding the microbial spoilage and the antimicrobial sensitivities of the spoilage organisms will facilitate the development of preservation strategies friendly towards the environment and the consumer. 2. Review of the literature 2.1. History and taxonomy of aerobic sporeforming bacteria The genus Bacillus was created in 1872 by Ferdinand Cohn to include rod-shaped bacteria that grow in filaments (Claus and Fritze, 1989; Slepecky and Hemphill, 1992). Four years later Cohn and Robert Koch independently detected that two species of the genus Bacillus were able to form resting stages which were not easily killed by boiling (Claus and Fritze, 1989). From 1923, after the publication of the first edition of Bergey’s Manual of Determinative Bacteriology, it was generally accepted that the genus Bacillus should be restricted to the rod- shaped bacteria forming endospores and growing aerobically (Fritze and Claus, 1995). In the latest edition of Bergey’s Manual of Determinative Bacteriology in 1986, genus Bacillus was described as Gram-positive, aerobic or facultatively anaerobic, mostly catalase positive rods forming endospores very resistant to many adverse conditions (Claus and Berkeley, 1986). At that time the genus comprised 34 validly described species (and 25 species incertae sedis) having guanine plus cytosine (GC) content varying from 32 to 69 mol % (Claus and Berkeley, 1986). This indicates that the genus is heterogenous as the DNA base compositions of species belonging to one genus should not differ by more than 10 mol % GC (Bull et al., 1992). The genotypic and phenotypic heterogeneities of the genus Bacillus, evident already for a long time, have supported the subdivision of this genus. Comparisons of rDNA sequences of the type strains of many Bacillus species (Ash et al., 1991) generated the outline of phylogenetic relations within this genus. In the beginning of the year 2000 the genus Bacillus comprised 75 validly described species. Since 1992, seven new genera were separated from the genus Bacillus: Alicyclobacillus (four species), Paenibacillus (27 species), Brevibacillus (ten species), Aneurinibacillus (three species), Virgibacillus (two species), Gracilibacillus (two species), and Salibacillus (one species) (see for the valid nomenclature, for instance DSMZ, 2000). The term ‘bacilli’ in this thesis is used for all these genera; aerobic rod-shaped bacteria capable of forming endospores that are more resistant than the vegetative cells to heat, disinfectants, drying and other unfavourable conditions. These genera mainly belong to the low GC% lineages of the phylogenetic map of eubacteria, together with clostridia and lactobacilli. Classification, isolation and identification of bacilli have in the past 10 years been subject to many reviews, e.g. by Claus and Fritze (1989), Priest (1989, 1993), Varnam and Evans (1991), Slepecky and Hemphill (1992), and Berkeley and Ali (1994). 2.2. Occurrence of bacilli in foods and in food processes Bacilli contaminate many kinds of foods. Some well documented cases are cited in Table 1. 5 Table 1. Occurrence of bacilli in foods Species Food Reference Alicyclobacillus acidoterrestris apple juice Cerny et al., 1984a Alicyclobacillus acidoterrestris fruit juices Splittstoesser et al., 1994b Alicyclobacillus acidoterrestris acidic beverages Yamazaki et al., 1996 Bacillus cereus rice and oriental foods, milk and dairy products, spices, and other dried products, meat products, salads, pastries, dried potatoes, frozen fish croquettes Kramer and Gilbert, 1989 Bacillus cereus milk products including UHT milk and yoghurt, meat products, cereal products (flour, bread, rice, corn starch), dry foods, infant feeds, spices, bean salad, canned tuna, egg mayonnaise sandwich Varnam and Evans, 1991 Bacillus cereus spices, egg yolk, custard, cream pastry, past. milk, roasted /fried meats and poultry, rice meals, fish pate, pea soup, lasagne, cheeses, vegetable salads van Netten et al., 1990 Bacillus cereus pasteurized milk, yeast, flour, cocoa, bakery products, spices, meat products, pasta, chinese meals te Giffel et al., 1996, 1997 Bacillus cereus pasteurized milk, cheese Cosentino et al., 1997 Bacillus cereus pasteurized milk, fermented milks, ice cream, milk powder Wong et al., 1988 Bacillus cereus pasteurized milk Larsen and Jørgensen, 1997 Bacillus cereus pasteurized milk, milk products Ternström et al., 1993 Bacillus cereus dairy products Väisänen et al., 1991b Bacillus cereus infant foods and dried milk products Becker et al., 1994 Bacillus cereus infant foods (milk based) Rowan et al., 1997 Bacillus cereus bread Rosenkvist and Hansen, 1995 Bacillus cereus noodles, spices, legume products, cooked foods Rusul and Yaacob, 1995 Bacillus cereus soybean and cereal based vegetarian foods Fang et al., 1999 Bacillus circulans pasteurized milk Chromie et al., 1989 Bacillus circulans pasteurized milk, cheese Cosentino et al., 1997 Bacillus coagulans pasteurized milk, UHT milk, cheese Cosentino et al., 1997 Bacillus coagulans acidic juices and other acidic foods Brown, 1995 Bacillus coagulans evaporated milk (flat sour spoilage) Kalogridou-Vassiliadou, 1992 Bacillus coagulans canned foods (flat sour spoilage) Setlow and Johnson, 1997 Bacillus lentus pasteurized milk, cheese Cosentino et al., 1997 6 Bacillus licheniformis pasteurized milk, UHT milk, cheese Cosentino et al., 1997 Bacillus licheniformis evaporated milk (flat sour spoilage) Kalogridou-Vassiliadou, 1992 Bacillus licheniformis infant foods (milk based) Rowan et al., 1997 Bacillus licheniformis infant feed formula, vanilla sauce Salkinoja-Salonen et al., 1999 Bacillus licheniformis bread, meat dishes Varnam and Evans, 1991 Bacillus licheniformis bread Bailey and von Holy, 1993 Bacillus licheniformis bread Rosenkvist and Hansen, 1995 Bacillus megaterium pasteurized milk, cheese Cosentino et al., 1997 Bacillus megaterium bread Bailey and von Holy, 1993 Bacillus mycoides pasteurized milk, cheese Cosentino et al., 1997 Bacillus pumilus pasteurized milk, UHT milk, cheese Cosentino et al., 1997 Bacillus pumilus bread Bailey and von Holy, 1993 Bacillus pumilus bread Rosenkvist and Hansen, 1995 Bacillus sphaericus pasteurized milk, UHT milk, cheese Cosentino et al., 1997 Bacillus sporothermodurans UHT-treated milk Pettersson et al., 1996 Bacillus stearothermophilus pasteurized milk, cheese Cosentino et al., 1997 Bacillus stearothermophilus evaporated milk (flat sour spoilage) Kalogridou-Vassiliadou, 1992 Bacillus stearothermophilus canned foods (flat sour spoilage) Setlow and Johnson, 1997 Bacillus subtilis flour, cocoa, bakery products, spices, meat products, pasta, chinese meals te Giffel et al., 1996 Bacillus subtilis bread, meat dishes Varnam and Evans, 1991 Bacillus subtilis pasteurized milk, cheese Cosentino et al., 1997 Bacillus subtilis evaporated milk (flat sour spoilage) Kalogridou-Vassiliadou, 1992 Bacillus subtilis infant foods (milk based) Rowan et al., 1997 Bacillus subtilis bread Rosenkvist and Hansen, 1995 Bacillus subtilis bread Bailey and von Holy, 1993 Bacillus thuringiensis pasta, pitta bread, milk Damgaard et al., 1996 Brevibacillus brevis UHT milk, cheese Cosentino et al., 1997 Brevibacillus laterosporus pasteurized milk, UHT milk, cheese Cosentino et al., 1997 Paenibacillus macerans pasteurized milk, cheese Cosentino et al., 1997 Paenibacillus polymyxa pasteurized milk and milk products Ternström et al., 1993 Paenibacillus macerans evaporated milk (flat sour spoilage) Kalogridou-Vassiliadou, 1992 aidentified by Deinhard et al. (1987) bidentified by Pontius et al. (1989) 7