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Prevalence of Bacillus spp. in different food products collected in Argentina
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LWT 39 (2006) 105–110 www.elsevier.com/locate/lwt
Prevalence of Bacillus spp. in different food products collected in Argentina Miriam O. Iurlina,1, Amelia I. Saiz1, Sandra R. Fuselli2, Rosalia Fritz1 Departamento de Quı´mica, Bromatologı´a, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Funes 3350, C. P. 7600, Mar del Plata, Provincia de Buenos Aires, Argentina Received 8 November 2004; received in revised form 13 January 2005; accepted 21 January 2005
Abstract The prevalence of Bacillus spp. in 279 samples of different food products collected in Argentina was studied. Bacillus spp. was confirmed in 28 out of 70 honey samples, 29 out of 29 flour samples, 15 out of 50 cheese samples, and 30 out of 30 spice samples, while Bacillus spp. was not found in fresh anchovy. Among the 70 honeys studied, Bacillus cereus, Bacillus pumilus, Bacillus laterosporus and Paenibacillus larvae subspp. larvae showed an incidence of 23%, 4%, 8% and 38%, respectively. More diversity of Bacillus species was found in rye flours than in white flours, Bacillus subtilis being the predominant species isolated from rye flour. B. cereus had an incidence of 50% in Port Salut Argentino cheeses. Meanwhile, B. pumilus was identified in both Port Salut and Quartirolo cheeses with an incidence of 50% and 25%, respectively. All the spices analysed showed Bacillus mycoides as the sole aerobic spore-forming bacilli isolated. The association of the presence of B. cereus, B. subtilis and Bacillus licheniformis with both the potential spoilage of foods and foodborne outbreaks is well known. In this study, Bacillus spp. had an incidence of 38% among all the samples analysed, therefore the monitoring of those species should be routinely done in microbiological food analyses. r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Bacillus spp.; Honeys; Flours; Cheeses; Fresh anchovy; Spices
1. Introduction The genus Bacillus is comprised of a diverse array of Gram-positive, aerobic and facultative anaerobic endospore-forming rods. Bacillus spp. are found in a wide range of habitats, and include species possessing environmental, industrial, and clinical significance (Claus & Berkeley, 1986; Drobniewski, 1993). Many species are common soil inhabitants and may frequently contaminate foods, including dairy products, meats, infant-foods, rice dishes, vegetables, spices and cereals (Chopra, Singh, & Kalra, 1980; Jacquette & Beuchat, Corresponding author. Tel.: +54 223 4756167; fax: +54 223 4753150. E-mail address: [email protected] (M.O. Iurlina). 1 Affiliated to UNMDP (Universidad Nacional de Mar del Plata), Argentina. 2 Affiliated to CIC (Comisio´n de Investigaciones Cientı´ ficas de la Provincia de Buenos Aires), Argentina.
1998; Christiansson, Bertilsson, & Svensson, 1999). The spore-forming ability of Bacillus spp. may allow its survival during food processing treatments; and the spores may then germinate if the food is left at room temperature or even at refrigeration temperatures. In the case of honey, the pysicochemical characteristics like pH, moisture content, oxidation–reduction potential, antimicrobial constituents and sugar concentration, make this food suitable for the survival of spores-forming microorganisms. Bacillus species have been detected in honey (Snowdon & Cleaver, 1996) and feces of bee larvae (Gillian & Prest, 1987; Taormina, Niemira, & Beuchat, 2001). However, Bacillus cereus may reflect a generally higher tolerance among all Bacillus spp. to antimicrobials that may be present in honey (Roth, Kwan, & Sporns, 1986). In wheat and wholemeal flours the main species identified has been Bacillus subtilis. Among 14 species isolated from retail bread and wheat grains, B. subtilis
0023-6438/$30.00 r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2005.01.006
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has been the only species associated with ropiness. Samples of raw materials, particularly bran, seeds and oat products, have shown low levels (100–102 CFU/g) of Bacillus spores, surviving a heat treatment (100 1C, 10 min) corresponding to a baking process (Rosenkvist & Hansen, 1995). Several B. cereus strains have become psychrotrophic over the years, making possible growth at temperatures as low as 4–6 1C (Granum, Brynestad, & Kramer, 1993). Previous work with soft cheeses usually refrigerated below 8 1C, showed a 4% incidence for both B. cereus and Clostridium perfringens (Tham, Hadju, & Danielsson-Tham, 1990). However, in hard cheeses the incidence was 50% in relation to B. cereus (Helmy, El-Bakey, & Mohamed, 1984). Scarce information exists regarding the presence of Bacillus spp. on fish products. Fish muscles are usually sterile but the skin, gills and alimentary tract contain a variable number of microorganisms (Shewan, 1970). In haddock fillets only B. subtilis was isolated (Laycock & Regier, 1971). The level of microorganisms on fish mainly depends on the environment where the fish was caught. Indeed, Bacillus spp. is not normally present in the marine environment (Huss, Dalgoard, & Gram, 1997). Spices are widely used in prepared foods all over the world because of their flavor properties. The most common bacterial isolates in black pepper, oregano and other spices were Bacillus, Clostridium and Micrococcus (Fernandez, Krivoruchco, & Mitschele, 1984; Drobniewski, 1993; Emam, Farag, & Aziz, 1995). The species of Bacillus identified had been B. cereus, B. subtilis, B. polymyxa and B. coagulans (Antai, 1988). Bacillus spp. are among the main spoilage organisms in food due to their versatile metabolism and heatresistant spores. Also, B. cereus, B. subtilis and Bacillus licheniformis have been associated with food poisoning. The aim of this study was to determine the prevalence of Bacillus genus in different raw materials, manufactured products such as honey, cheeses, flours, anchovies; and additives such as spices. This work may have significant interest in understanding the distribution of patterns of Bacillus species in various food products.
2. Materials and methods
For Paenibacillus larvae the investigation was made according to the method proposed by Hornitzky and Clark (1991). For all samples, 125 ml of honey was mixed with 250 ml phosphate-buffered saline 0.01 mol/l (pH 7.2). The suspension obtained was centrifuged at 3000g for 45 min, and 3 ml of supernatant was re-mixed with the precipitate. The homogenate was heated at 80 1C for 15 min in a water bath in order to eliminate any microbial vegetative forms. A 100 ml-aliquot of suspension was spread in triplicate on Muller–Hintonyeast extract–polymyxin–glucose–sodium pyruvate (MYPGP) agar (Dingman & Stahly, 1983), consisting of: 10 g/l Muller–Hinton broth, 15 g/l yeast extract, 3 g/l K2HPO4, 1 g/l sodium pyruvate, 20 g/l agar, 2 g/l glucose, and supplemented with 6 mg/ml nalidixic acid and 10 mg/ml pipemidic acid. Cultures were incubated under microaerophilic conditions (8–10% CO2) at 3572 1C for 7 days, being initially evaluated at 3 days. In addition, 100 ml was spread on J-agar (JA) (Nordstro¨m & Fries, 1995) without antibiotics. In order to investigate other Bacillus species, cultures on JA were also incubated at 3572 1C for 46 h under aerobic conditions. Presumptive colonies of P. larvae on MYPGP and the isolates on JA were initially identified by catalase reaction, Gram staining and analysis of spores (Claus & Berkeley, 1986). Isolates were maintained on MYPGP and JA agar slants and further characterized and confirmed. 2.1.2. Flours Fourteen samples of rye flours and 15 samples of wheat flour, 000 type, were provided by local mills. The isolation of Bacillus spp. was carried out on dextrose– casein–peptone agar (Williams, 1936). In particular, B. cereus was isolated from mannitol–egg yolk–polymyxin (MYP) agar (Rhodehamel & Harmon, 1998, Chap. 14). For each sample, 25 g was homogenized aseptically in 225 ml of sterile Butterfield‘s phosphate-buffered dilution water (Butterfield, 1960). The dilution was heated at 80 1C for 10 min in order to eliminate microbial vegetative forms and 100 ml was spread on MYP agar and plates were incubated at 30–32 1C for 24–48 h. In addition, 1 ml of homogenate was cultured by the pour plate method on dextrose–casein–peptone agar at 30–32 1C for 48–72 h (Williams, 1936). Colonies from both media were selected and confirmed by Gram staining, shape and position of spores (Claus & Berkeley, 1986).
2.1. Microbiological analyses 2.1.1. Honey Seventy samples of polyfloral honey were analysed. Samples were divided into 3 groups: (i) honey harvested from apiaries (37 samples), (ii) honey purchased at retail market (23 samples), and (iii) bulk honey for industrial use (10 samples). The apiaries were localized in the southeast region of Buenos Aires province.
2.1.3. Cheeses The Port Salut and Quartirolo Argentino cheeses used in this study have the physicochemical characteristics of soft cheeses. The difference between them is the paste cooking process for Port Salut cheeses. A total of 50 samples of trademark cheeses packed in aseptic polyethylene bags were obtained from commercial establishments. Thirty out of 50 concerned Port Salut cheeses,
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while 20 out of 50 concerned Quartirolo cheeses. Samples were kept at 4 1C until being analysed within 24 h after purchasing. Twenty-five grams of sample was homogenized in 225 ml of sterile Butterfield’s phosphate-buffered dilution water in a Stomacher 400 Circulator Homogenizer. For Bacillus genus isolation procedures were performed as described for flours. Plate cultures were incubated at 55 1C for 48 h, at 30 1C for 72 h and at 6 1C for 14 d (Cosentino, Mulargia, Pisano, Tuveri, & Palmas, 1997).
according to the scheme suggested by Claus and Berkeley (1986). The identity of the species isolated was further confirmed by using the API 50 CHB galleries (bioMeriuex, France). Furthermore, isolates of Bacillus spp. obtained from cheeses were also examined for enzymatic activities. Hydrolysis of starch, liquefaction of gelatin, proteolytic and lipolytic activities were tested following the procedure suggested by Claus and Berkeley (1986).
2.1.4. Fresh anchovy Anchovy (Engraulis anchoı´ta) in pre-spawning season was analysed on 5 consecutive catches. This pelagic species was caught between the mouth of De la Plata River and 35–361 South Latitude and was kept in ice during the trip. Anchovy was used as raw material for fish preserves and other fish products. A total of 100 samples was caught and stored at refrigerated temperature until being analysed. Twenty-five grams of sample in saline solution (0.85 g/l) with 0.1 g/l peptone (ICMSF, 1983) was blended in a Stomacher 400 Circulator Homogenizer up to a volume of 250 ml. When necessary, tripotassium phosphate solution 8 g/l for neutralizing the pH was used (Mossel & Moreno Garcı´ a, 1985). Isolation procedures used were as those described for flours.
3. Results and discussion
2.1.5. Spices A total of 30 randomly collected samples of origanum (Origanum vulgare L.), black pepper (Pipper nigrum L.), paprika (Capsicum annuun L.), bay leaves (Laurus nobilis) and milled red pepper (Capsicum spp.) were analysed. Non-irradiated bulk spices were purchased from the retail market. Products were stored at room temperature (20–25 1C) until being examined. Twentyfive grams of sample were homogenized aseptically in 225 ml of sterile Butterfield’s phosphate-buffered dilution water (Butterfield, 1960) and appropriate dilutions were made. The isolation procedures for Bacillus spp. were performed as described for flours. 2.2. Identification and confirmatory tests The characterization of the isolates maintained both in MYPGP and JA was confirmed by the ability to withstand serial transfer in nutritive broth, Voges– Proskauer and indol tests, nitrate reduction, starch hydrolysis and mannitol utilization (Gordon, Haynes, & Pang, 1973; Priest, Goodfellow, & Todd, 1988). Catalase reaction, motility, growth in anaerobic medium, starch hydrolysis, casein hydrolysis and gelatin hydrolysis were also evaluated (Claus & Berkeley, 1986). Among all isolates obtained from flours, cheeses, fresh anchovy and spices, a further identification and confirmatory test of Bacillus spp. were performed
The number of positive samples for Bacillus spp. obtained in the different foods analysed are shown in Table 1. 3.1. Honey Analyses of honey from apiaries, retail market and bulk showed a range from 10% to 43% of positive samples for P. larvae subspp. larvae (Table 2). These results agreed with studies made by other researchers who have found an incidence of P. larvae in honey products to vary between 12.5% and 16.5% (Hornitzky & Clark, 1991). It is interesting to realize that in this study, more than 40% of the P. larvae isolates from honey belonged to honey harvested from apiaries and purchased from retail market. However, an incidence of 43% positive samples for P. larvae subspp. larvae does not reflect the distribution of this microorganism in the apiaries, due to the spore dissemination that may exist in different steps such as recollection, extraction, division into fractions or throughout robber bees (Hornitzky & Clark, 1991). The bulk honey showed the lowest incidence of Bacillus genus (Table 2). The small number of positive samples obtained in this survey may be due to the fact that each bulk container contained honeys from different beekeepers, and the mixture of samples into the bulk may have had a dilution effect. A previous study has shown an incidence of 20% of B. cereus in Argentinean honey (Alippi, 1995). In Table 1 Isolation of Bacillus spp. from different foods Food
Number of samples
Number of positive samples for Bacillus spp.
Honey Port Salut Argentino cheese Quartirolo cheese Rye flours Wheat flours Fresh anchovy Spices
70 30 20 14 15 100 30
27 15 5 14 15 0 30
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108 Table 2 Isolation of Bacillus spp. from honey Source of samples
Positive samples for P. larvae subspp. larvae
Positive samples for B. cereus
Positive samples for B. pumilus
Positive samples for B. laterosporus
Apiaries and retail market (60) Bulk (10) Positive of total samples (70)
26 1 27
15 1 16
3 0 3
6 0 6
accordance with these results, we found an incidence of B. cereus of about 23% among the 70 samples tested. Smith, Mc Caughey and Kemmerer (1969) demonstrated that untreated and undiluted honeys showed antibacterial activity against B. cereus and B. subtilis, while Roth et al. (1986) found that the use of 0.1 M phosphate buffer (pH 7.0) to dilute honey completely reduced the natural inhibitor effect against B. cereus. Honey has not been involved in foodborne outbreaks caused by B. cereus, although no firm evidence exists that would exclude honey as a potential vehicle of infection.
Bacillus spp. was detected in 100% of rye and wheat samples analysed (Table 1). Rye flour showed the greatest diversity of Bacillus species: B. subtilis, B. pumilus, B. licheniformis, B. macerans, B. lentus and B. megaterium. From wheat flour samples only B. pumilus, B. megaterium and B. subtilis were isolated. The relative incidence of each species in the total population of Bacillus spp., isolated from flours, was about 55% for B. subtilis and 45% for B. pumilus. The remaining species showed a prevalence less than 10%. Among all the samples the count values for Bacillus spp. were lower than 103 cfu/g (data not shown). Dough made with a mixture of 50–50% rye and white flours, along with 3% of Saccharomyces cerevisiae and fermented for a period of 3 h, showed a decrease of Bacillus population of about 60% at the end of fermentation, B. subtilis being the only species isolated. The count values obtained for these doughs showed that Bacillus spp. was found in levels lower than 102 cfu/g. In our study, the incidence of B. subtilis in flours was higher than that reported by te Giffel, Beumer, Leijendekkers, and Rombouts (1996) of 22% for B. subtilis in flour samples. B. subtilis and B. licheniformis can cause alterations in the functional properties of dough modifying starch and gluten giving a viscous dough with off-odors (Pascual Anderson & Caldero´n y Pascual, 2000). Both species have also been associated with food-poisoning cases (te Giffel et al., 1996).
samples analysed. This value was greater than the results reported by Tham, Hadju, and Danielsson-Tham (1990) in other kind of soft and semi-soft cheeses. In relation to the presence of B. cereus in dairy products, the relatively high hydrophobicity of the spores, the low spore surface charge and the spore morphology allow a strong adhesion to different surfaces and the survival through the processing line in the dairy industry (Andersson, Ronner, & Granum, 1995.). Therefore, cheeses are very likely to be affected by the presence and the potential growth of these bacteria in dairy products. On the other hand, the other species identified, B. pumilus, was also found in 50% of Port Salut Argentino samples while in Quartirolo cheeses the incidence was 25%. The absence of B. cereus and the low incidence of other Bacillus species in Quartirolo cheeses could be explained considering that these kinds of cheeses do not undergo the paste cooking process unlike Port Salut Argentino cheeses. After cooking, spores could germinate and vegetative cells of Bacillus spp. could grow well in the absence of competitive microflora (Granum et al., 1993; Granum & Lund, 1997). An incidence of 50% of B. cereus is in accordance with a previous study in hard cheeses (Helmy et al., 1984). The maturation process in these cheeses would reduce the competitive flora, allowing spore-forming microorganisms to predominate. In our study, B. cereus did not reach count values associated with enterotoxin production (Granum, 1997). From the isolates of B.cereus, 75% of them were clearly identified as psychotropic microorganisms (able to grow at 6 1C in 14 d). B. pumilus isolates were identified as mesophilic strains (able to grow at 30 1C for 72 h). Among the B. cereus and B. pumilus isolates obtained from cheeses were measured positive proteolytic and lipolytic activities. B. cereus isolates showed the highest enzymatic activities. Only B. cereus isolates were capable of hydrolysing starch. Proteolytic and lypolitic activities for both B. cereus and B. pumilus from cheeses could be associated with a potential spoilage of cheeses (changes of flavour and texture). However, the inspection of samples during the shelf-life storage period, at 4 1C, did not show any visible spoilage (Table 3).
3.3. Cheeses
3.4. Fresh anchovy
It is worth mentioning that only Port Salut Argentino cheeses showed an incidence of B. cereus in 50% of the
Bacillus spp. were not detected in any of the samples investigated (Table 1). Bacillus spp. may be present on
3.2. Flours
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Table 3 Isolation of Bacillus spp. from cheeses Samples of cheeses
Positive samples for B. cereus
Positive samples for B. pumilus
Port Salut Argentino (30) Quartirolo cheeses (20) Positive of total samples (50)
15 0 15
15 5 20
fresh water fish or fish caught in coastal areas (Huss et al., 1997). Surfaces of fish, gills and gut would have a bacterial flora that mainly reflects their environment. In our study, the anchovy samples came from cold water in the open sea, therefore negative results were obtained for these samples. 3.5. Spices The prevalence of Bacillus spp. was 100% in all the spices investigated (Table 1). The average counts of Bacillus spp. expressed as cfu/g were: 2.2 103 in origanum, 1.4 103 in black pepper, 2.7 103 in paprika, 2.8 102 in bay leaves and 2.5 104 in milled red pepper. These counts were lower than those found in other studies for other Bacillus species (te Giffel et al., 1996). Oregano, black pepper, paprika, bay leaves and milled red pepper only contained Bacillus mycoides. The spice with the highest incidence of Bacillus spp. in our study was the milled red pepper. The grinding process for the dried pepper grains or the handling procedures during packaging could increase the contamination of this product. Considerable variation in the occurrence of Bacillus spp. in spices has been reported, between brands as well as among samples from the same brand. Several spices (alligator pepper, red pepper, black pepper, thyme, curry powder and paprika) were reported to have high loads of B. cereus (Mc Kee, 1995). The absence of B. cereus in the spices currently studied could be explained considering its natural variation in occurrence, and the parameters that are known to affect the microbial contamination such as source, manufacturing process, age and type of spice. Bay leaves showed the lowest bacteriological counts which is in agreement with the results of other authors (Antai, 1988; Garcı´ a, Iracheta, Galvan, & Heredia, 2001). 4. Conclusion The results obtained in this study showed a high incidence (38%) of Bacillus spp. in the food samples examined. The distribution of the Bacillus species among the different foods studied is shown in Fig. 1. The presence of Bacillus spp. seemed to be independent of the nature of each food. However, our study showed a high frequency (100%) of Bacillus genus in foods with lower moisture content as flours and spices.
Fig. 1. Incidence of Bacillus spp. expressed as percentage (%) of positive samples for each one Bacillus species isolated on honeys, flours, cheeses and spices.
Among the 279 samples analysed, the predominant species isolated was B. pumilus, with an incidence of 13%. B. cereus was found in about 11% of the total of samples. Nevertheless, in any case, the level of contamination never reached values associated with toxin production. Besides, of the 279 samples, B. mycoides, P. larvae subspp. larvae, B. subtilis and Bacillus laterosporus were present with an incidence of about 10.9%, 9.8%, 5.8% and 2%, respectively. B. megaterium, B. licheniformis, B. macerans and B. lentus showed a prevalence of 3.6%. It is known that B. subtilis can be affected by processing, storage conditions and preparation of foods (te Giffel et al., 1996). In consequence, it is not surprisingly that these microorganisms did not appear in highly processed foods. Taking into account the association of the presence of B. cereus, B. subtilis and B. licheniformis with both the potential spoilage of foods and foodborne outbreaks, monitoring of these species should be routinely done in microbiological food
Acknowledgements We gratefully acknowledge the financial support of the Universidad Nacional de Mar del Plata (Project N1 EXA 170/00).
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LWT 39 (2006) 105–110 www.elsevier.com/locate/lwt
Prevalence of Bacillus spp. in different food products collected in Argentina Miriam O. Iurlina,1, Amelia I. Saiz1, Sandra R. Fuselli2, Rosalia Fritz1 Departamento de Quı´mica, Bromatologı´a, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Funes 3350, C. P. 7600, Mar del Plata, Provincia de Buenos Aires, Argentina Received 8 November 2004; received in revised form 13 January 2005; accepted 21 January 2005
Abstract The prevalence of Bacillus spp. in 279 samples of different food products collected in Argentina was studied. Bacillus spp. was confirmed in 28 out of 70 honey samples, 29 out of 29 flour samples, 15 out of 50 cheese samples, and 30 out of 30 spice samples, while Bacillus spp. was not found in fresh anchovy. Among the 70 honeys studied, Bacillus cereus, Bacillus pumilus, Bacillus laterosporus and Paenibacillus larvae subspp. larvae showed an incidence of 23%, 4%, 8% and 38%, respectively. More diversity of Bacillus species was found in rye flours than in white flours, Bacillus subtilis being the predominant species isolated from rye flour. B. cereus had an incidence of 50% in Port Salut Argentino cheeses. Meanwhile, B. pumilus was identified in both Port Salut and Quartirolo cheeses with an incidence of 50% and 25%, respectively. All the spices analysed showed Bacillus mycoides as the sole aerobic spore-forming bacilli isolated. The association of the presence of B. cereus, B. subtilis and Bacillus licheniformis with both the potential spoilage of foods and foodborne outbreaks is well known. In this study, Bacillus spp. had an incidence of 38% among all the samples analysed, therefore the monitoring of those species should be routinely done in microbiological food analyses. r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Bacillus spp.; Honeys; Flours; Cheeses; Fresh anchovy; Spices
1. Introduction The genus Bacillus is comprised of a diverse array of Gram-positive, aerobic and facultative anaerobic endospore-forming rods. Bacillus spp. are found in a wide range of habitats, and include species possessing environmental, industrial, and clinical significance (Claus & Berkeley, 1986; Drobniewski, 1993). Many species are common soil inhabitants and may frequently contaminate foods, including dairy products, meats, infant-foods, rice dishes, vegetables, spices and cereals (Chopra, Singh, & Kalra, 1980; Jacquette & Beuchat, Corresponding author. Tel.: +54 223 4756167; fax: +54 223 4753150. E-mail address: [email protected] (M.O. Iurlina). 1 Affiliated to UNMDP (Universidad Nacional de Mar del Plata), Argentina. 2 Affiliated to CIC (Comisio´n de Investigaciones Cientı´ ficas de la Provincia de Buenos Aires), Argentina.
1998; Christiansson, Bertilsson, & Svensson, 1999). The spore-forming ability of Bacillus spp. may allow its survival during food processing treatments; and the spores may then germinate if the food is left at room temperature or even at refrigeration temperatures. In the case of honey, the pysicochemical characteristics like pH, moisture content, oxidation–reduction potential, antimicrobial constituents and sugar concentration, make this food suitable for the survival of spores-forming microorganisms. Bacillus species have been detected in honey (Snowdon & Cleaver, 1996) and feces of bee larvae (Gillian & Prest, 1987; Taormina, Niemira, & Beuchat, 2001). However, Bacillus cereus may reflect a generally higher tolerance among all Bacillus spp. to antimicrobials that may be present in honey (Roth, Kwan, & Sporns, 1986). In wheat and wholemeal flours the main species identified has been Bacillus subtilis. Among 14 species isolated from retail bread and wheat grains, B. subtilis
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has been the only species associated with ropiness. Samples of raw materials, particularly bran, seeds and oat products, have shown low levels (100–102 CFU/g) of Bacillus spores, surviving a heat treatment (100 1C, 10 min) corresponding to a baking process (Rosenkvist & Hansen, 1995). Several B. cereus strains have become psychrotrophic over the years, making possible growth at temperatures as low as 4–6 1C (Granum, Brynestad, & Kramer, 1993). Previous work with soft cheeses usually refrigerated below 8 1C, showed a 4% incidence for both B. cereus and Clostridium perfringens (Tham, Hadju, & Danielsson-Tham, 1990). However, in hard cheeses the incidence was 50% in relation to B. cereus (Helmy, El-Bakey, & Mohamed, 1984). Scarce information exists regarding the presence of Bacillus spp. on fish products. Fish muscles are usually sterile but the skin, gills and alimentary tract contain a variable number of microorganisms (Shewan, 1970). In haddock fillets only B. subtilis was isolated (Laycock & Regier, 1971). The level of microorganisms on fish mainly depends on the environment where the fish was caught. Indeed, Bacillus spp. is not normally present in the marine environment (Huss, Dalgoard, & Gram, 1997). Spices are widely used in prepared foods all over the world because of their flavor properties. The most common bacterial isolates in black pepper, oregano and other spices were Bacillus, Clostridium and Micrococcus (Fernandez, Krivoruchco, & Mitschele, 1984; Drobniewski, 1993; Emam, Farag, & Aziz, 1995). The species of Bacillus identified had been B. cereus, B. subtilis, B. polymyxa and B. coagulans (Antai, 1988). Bacillus spp. are among the main spoilage organisms in food due to their versatile metabolism and heatresistant spores. Also, B. cereus, B. subtilis and Bacillus licheniformis have been associated with food poisoning. The aim of this study was to determine the prevalence of Bacillus genus in different raw materials, manufactured products such as honey, cheeses, flours, anchovies; and additives such as spices. This work may have significant interest in understanding the distribution of patterns of Bacillus species in various food products.
2. Materials and methods
For Paenibacillus larvae the investigation was made according to the method proposed by Hornitzky and Clark (1991). For all samples, 125 ml of honey was mixed with 250 ml phosphate-buffered saline 0.01 mol/l (pH 7.2). The suspension obtained was centrifuged at 3000g for 45 min, and 3 ml of supernatant was re-mixed with the precipitate. The homogenate was heated at 80 1C for 15 min in a water bath in order to eliminate any microbial vegetative forms. A 100 ml-aliquot of suspension was spread in triplicate on Muller–Hintonyeast extract–polymyxin–glucose–sodium pyruvate (MYPGP) agar (Dingman & Stahly, 1983), consisting of: 10 g/l Muller–Hinton broth, 15 g/l yeast extract, 3 g/l K2HPO4, 1 g/l sodium pyruvate, 20 g/l agar, 2 g/l glucose, and supplemented with 6 mg/ml nalidixic acid and 10 mg/ml pipemidic acid. Cultures were incubated under microaerophilic conditions (8–10% CO2) at 3572 1C for 7 days, being initially evaluated at 3 days. In addition, 100 ml was spread on J-agar (JA) (Nordstro¨m & Fries, 1995) without antibiotics. In order to investigate other Bacillus species, cultures on JA were also incubated at 3572 1C for 46 h under aerobic conditions. Presumptive colonies of P. larvae on MYPGP and the isolates on JA were initially identified by catalase reaction, Gram staining and analysis of spores (Claus & Berkeley, 1986). Isolates were maintained on MYPGP and JA agar slants and further characterized and confirmed. 2.1.2. Flours Fourteen samples of rye flours and 15 samples of wheat flour, 000 type, were provided by local mills. The isolation of Bacillus spp. was carried out on dextrose– casein–peptone agar (Williams, 1936). In particular, B. cereus was isolated from mannitol–egg yolk–polymyxin (MYP) agar (Rhodehamel & Harmon, 1998, Chap. 14). For each sample, 25 g was homogenized aseptically in 225 ml of sterile Butterfield‘s phosphate-buffered dilution water (Butterfield, 1960). The dilution was heated at 80 1C for 10 min in order to eliminate microbial vegetative forms and 100 ml was spread on MYP agar and plates were incubated at 30–32 1C for 24–48 h. In addition, 1 ml of homogenate was cultured by the pour plate method on dextrose–casein–peptone agar at 30–32 1C for 48–72 h (Williams, 1936). Colonies from both media were selected and confirmed by Gram staining, shape and position of spores (Claus & Berkeley, 1986).
2.1. Microbiological analyses 2.1.1. Honey Seventy samples of polyfloral honey were analysed. Samples were divided into 3 groups: (i) honey harvested from apiaries (37 samples), (ii) honey purchased at retail market (23 samples), and (iii) bulk honey for industrial use (10 samples). The apiaries were localized in the southeast region of Buenos Aires province.
2.1.3. Cheeses The Port Salut and Quartirolo Argentino cheeses used in this study have the physicochemical characteristics of soft cheeses. The difference between them is the paste cooking process for Port Salut cheeses. A total of 50 samples of trademark cheeses packed in aseptic polyethylene bags were obtained from commercial establishments. Thirty out of 50 concerned Port Salut cheeses,
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while 20 out of 50 concerned Quartirolo cheeses. Samples were kept at 4 1C until being analysed within 24 h after purchasing. Twenty-five grams of sample was homogenized in 225 ml of sterile Butterfield’s phosphate-buffered dilution water in a Stomacher 400 Circulator Homogenizer. For Bacillus genus isolation procedures were performed as described for flours. Plate cultures were incubated at 55 1C for 48 h, at 30 1C for 72 h and at 6 1C for 14 d (Cosentino, Mulargia, Pisano, Tuveri, & Palmas, 1997).
according to the scheme suggested by Claus and Berkeley (1986). The identity of the species isolated was further confirmed by using the API 50 CHB galleries (bioMeriuex, France). Furthermore, isolates of Bacillus spp. obtained from cheeses were also examined for enzymatic activities. Hydrolysis of starch, liquefaction of gelatin, proteolytic and lipolytic activities were tested following the procedure suggested by Claus and Berkeley (1986).
2.1.4. Fresh anchovy Anchovy (Engraulis anchoı´ta) in pre-spawning season was analysed on 5 consecutive catches. This pelagic species was caught between the mouth of De la Plata River and 35–361 South Latitude and was kept in ice during the trip. Anchovy was used as raw material for fish preserves and other fish products. A total of 100 samples was caught and stored at refrigerated temperature until being analysed. Twenty-five grams of sample in saline solution (0.85 g/l) with 0.1 g/l peptone (ICMSF, 1983) was blended in a Stomacher 400 Circulator Homogenizer up to a volume of 250 ml. When necessary, tripotassium phosphate solution 8 g/l for neutralizing the pH was used (Mossel & Moreno Garcı´ a, 1985). Isolation procedures used were as those described for flours.
3. Results and discussion
2.1.5. Spices A total of 30 randomly collected samples of origanum (Origanum vulgare L.), black pepper (Pipper nigrum L.), paprika (Capsicum annuun L.), bay leaves (Laurus nobilis) and milled red pepper (Capsicum spp.) were analysed. Non-irradiated bulk spices were purchased from the retail market. Products were stored at room temperature (20–25 1C) until being examined. Twentyfive grams of sample were homogenized aseptically in 225 ml of sterile Butterfield’s phosphate-buffered dilution water (Butterfield, 1960) and appropriate dilutions were made. The isolation procedures for Bacillus spp. were performed as described for flours. 2.2. Identification and confirmatory tests The characterization of the isolates maintained both in MYPGP and JA was confirmed by the ability to withstand serial transfer in nutritive broth, Voges– Proskauer and indol tests, nitrate reduction, starch hydrolysis and mannitol utilization (Gordon, Haynes, & Pang, 1973; Priest, Goodfellow, & Todd, 1988). Catalase reaction, motility, growth in anaerobic medium, starch hydrolysis, casein hydrolysis and gelatin hydrolysis were also evaluated (Claus & Berkeley, 1986). Among all isolates obtained from flours, cheeses, fresh anchovy and spices, a further identification and confirmatory test of Bacillus spp. were performed
The number of positive samples for Bacillus spp. obtained in the different foods analysed are shown in Table 1. 3.1. Honey Analyses of honey from apiaries, retail market and bulk showed a range from 10% to 43% of positive samples for P. larvae subspp. larvae (Table 2). These results agreed with studies made by other researchers who have found an incidence of P. larvae in honey products to vary between 12.5% and 16.5% (Hornitzky & Clark, 1991). It is interesting to realize that in this study, more than 40% of the P. larvae isolates from honey belonged to honey harvested from apiaries and purchased from retail market. However, an incidence of 43% positive samples for P. larvae subspp. larvae does not reflect the distribution of this microorganism in the apiaries, due to the spore dissemination that may exist in different steps such as recollection, extraction, division into fractions or throughout robber bees (Hornitzky & Clark, 1991). The bulk honey showed the lowest incidence of Bacillus genus (Table 2). The small number of positive samples obtained in this survey may be due to the fact that each bulk container contained honeys from different beekeepers, and the mixture of samples into the bulk may have had a dilution effect. A previous study has shown an incidence of 20% of B. cereus in Argentinean honey (Alippi, 1995). In Table 1 Isolation of Bacillus spp. from different foods Food
Number of samples
Number of positive samples for Bacillus spp.
Honey Port Salut Argentino cheese Quartirolo cheese Rye flours Wheat flours Fresh anchovy Spices
70 30 20 14 15 100 30
27 15 5 14 15 0 30
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108 Table 2 Isolation of Bacillus spp. from honey Source of samples
Positive samples for P. larvae subspp. larvae
Positive samples for B. cereus
Positive samples for B. pumilus
Positive samples for B. laterosporus
Apiaries and retail market (60) Bulk (10) Positive of total samples (70)
26 1 27
15 1 16
3 0 3
6 0 6
accordance with these results, we found an incidence of B. cereus of about 23% among the 70 samples tested. Smith, Mc Caughey and Kemmerer (1969) demonstrated that untreated and undiluted honeys showed antibacterial activity against B. cereus and B. subtilis, while Roth et al. (1986) found that the use of 0.1 M phosphate buffer (pH 7.0) to dilute honey completely reduced the natural inhibitor effect against B. cereus. Honey has not been involved in foodborne outbreaks caused by B. cereus, although no firm evidence exists that would exclude honey as a potential vehicle of infection.
Bacillus spp. was detected in 100% of rye and wheat samples analysed (Table 1). Rye flour showed the greatest diversity of Bacillus species: B. subtilis, B. pumilus, B. licheniformis, B. macerans, B. lentus and B. megaterium. From wheat flour samples only B. pumilus, B. megaterium and B. subtilis were isolated. The relative incidence of each species in the total population of Bacillus spp., isolated from flours, was about 55% for B. subtilis and 45% for B. pumilus. The remaining species showed a prevalence less than 10%. Among all the samples the count values for Bacillus spp. were lower than 103 cfu/g (data not shown). Dough made with a mixture of 50–50% rye and white flours, along with 3% of Saccharomyces cerevisiae and fermented for a period of 3 h, showed a decrease of Bacillus population of about 60% at the end of fermentation, B. subtilis being the only species isolated. The count values obtained for these doughs showed that Bacillus spp. was found in levels lower than 102 cfu/g. In our study, the incidence of B. subtilis in flours was higher than that reported by te Giffel, Beumer, Leijendekkers, and Rombouts (1996) of 22% for B. subtilis in flour samples. B. subtilis and B. licheniformis can cause alterations in the functional properties of dough modifying starch and gluten giving a viscous dough with off-odors (Pascual Anderson & Caldero´n y Pascual, 2000). Both species have also been associated with food-poisoning cases (te Giffel et al., 1996).
samples analysed. This value was greater than the results reported by Tham, Hadju, and Danielsson-Tham (1990) in other kind of soft and semi-soft cheeses. In relation to the presence of B. cereus in dairy products, the relatively high hydrophobicity of the spores, the low spore surface charge and the spore morphology allow a strong adhesion to different surfaces and the survival through the processing line in the dairy industry (Andersson, Ronner, & Granum, 1995.). Therefore, cheeses are very likely to be affected by the presence and the potential growth of these bacteria in dairy products. On the other hand, the other species identified, B. pumilus, was also found in 50% of Port Salut Argentino samples while in Quartirolo cheeses the incidence was 25%. The absence of B. cereus and the low incidence of other Bacillus species in Quartirolo cheeses could be explained considering that these kinds of cheeses do not undergo the paste cooking process unlike Port Salut Argentino cheeses. After cooking, spores could germinate and vegetative cells of Bacillus spp. could grow well in the absence of competitive microflora (Granum et al., 1993; Granum & Lund, 1997). An incidence of 50% of B. cereus is in accordance with a previous study in hard cheeses (Helmy et al., 1984). The maturation process in these cheeses would reduce the competitive flora, allowing spore-forming microorganisms to predominate. In our study, B. cereus did not reach count values associated with enterotoxin production (Granum, 1997). From the isolates of B.cereus, 75% of them were clearly identified as psychotropic microorganisms (able to grow at 6 1C in 14 d). B. pumilus isolates were identified as mesophilic strains (able to grow at 30 1C for 72 h). Among the B. cereus and B. pumilus isolates obtained from cheeses were measured positive proteolytic and lipolytic activities. B. cereus isolates showed the highest enzymatic activities. Only B. cereus isolates were capable of hydrolysing starch. Proteolytic and lypolitic activities for both B. cereus and B. pumilus from cheeses could be associated with a potential spoilage of cheeses (changes of flavour and texture). However, the inspection of samples during the shelf-life storage period, at 4 1C, did not show any visible spoilage (Table 3).
3.3. Cheeses
3.4. Fresh anchovy
It is worth mentioning that only Port Salut Argentino cheeses showed an incidence of B. cereus in 50% of the
Bacillus spp. were not detected in any of the samples investigated (Table 1). Bacillus spp. may be present on
3.2. Flours
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Table 3 Isolation of Bacillus spp. from cheeses Samples of cheeses
Positive samples for B. cereus
Positive samples for B. pumilus
Port Salut Argentino (30) Quartirolo cheeses (20) Positive of total samples (50)
15 0 15
15 5 20
fresh water fish or fish caught in coastal areas (Huss et al., 1997). Surfaces of fish, gills and gut would have a bacterial flora that mainly reflects their environment. In our study, the anchovy samples came from cold water in the open sea, therefore negative results were obtained for these samples. 3.5. Spices The prevalence of Bacillus spp. was 100% in all the spices investigated (Table 1). The average counts of Bacillus spp. expressed as cfu/g were: 2.2 103 in origanum, 1.4 103 in black pepper, 2.7 103 in paprika, 2.8 102 in bay leaves and 2.5 104 in milled red pepper. These counts were lower than those found in other studies for other Bacillus species (te Giffel et al., 1996). Oregano, black pepper, paprika, bay leaves and milled red pepper only contained Bacillus mycoides. The spice with the highest incidence of Bacillus spp. in our study was the milled red pepper. The grinding process for the dried pepper grains or the handling procedures during packaging could increase the contamination of this product. Considerable variation in the occurrence of Bacillus spp. in spices has been reported, between brands as well as among samples from the same brand. Several spices (alligator pepper, red pepper, black pepper, thyme, curry powder and paprika) were reported to have high loads of B. cereus (Mc Kee, 1995). The absence of B. cereus in the spices currently studied could be explained considering its natural variation in occurrence, and the parameters that are known to affect the microbial contamination such as source, manufacturing process, age and type of spice. Bay leaves showed the lowest bacteriological counts which is in agreement with the results of other authors (Antai, 1988; Garcı´ a, Iracheta, Galvan, & Heredia, 2001). 4. Conclusion The results obtained in this study showed a high incidence (38%) of Bacillus spp. in the food samples examined. The distribution of the Bacillus species among the different foods studied is shown in Fig. 1. The presence of Bacillus spp. seemed to be independent of the nature of each food. However, our study showed a high frequency (100%) of Bacillus genus in foods with lower moisture content as flours and spices.
Fig. 1. Incidence of Bacillus spp. expressed as percentage (%) of positive samples for each one Bacillus species isolated on honeys, flours, cheeses and spices.
Among the 279 samples analysed, the predominant species isolated was B. pumilus, with an incidence of 13%. B. cereus was found in about 11% of the total of samples. Nevertheless, in any case, the level of contamination never reached values associated with toxin production. Besides, of the 279 samples, B. mycoides, P. larvae subspp. larvae, B. subtilis and Bacillus laterosporus were present with an incidence of about 10.9%, 9.8%, 5.8% and 2%, respectively. B. megaterium, B. licheniformis, B. macerans and B. lentus showed a prevalence of 3.6%. It is known that B. subtilis can be affected by processing, storage conditions and preparation of foods (te Giffel et al., 1996). In consequence, it is not surprisingly that these microorganisms did not appear in highly processed foods. Taking into account the association of the presence of B. cereus, B. subtilis and B. licheniformis with both the potential spoilage of foods and foodborne outbreaks, monitoring of these species should be routinely done in microbiological food
Acknowledgements We gratefully acknowledge the financial support of the Universidad Nacional de Mar del Plata (Project N1 EXA 170/00).
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