Bacteria from the Bacillus cereus group as contaminants in requeijão curd cheeses and especialidade láctea tipo requeijão

Bactérias do grupo Bacillus cereus como contaminantes em queijos fundidos requeijão e especialidade láctea tipo requeijão

Jamile de Oliveira Hachiya Gabriel Augusto Marques Rossi Higor Oliveira Silva Rafael Akira Sato Ana Maria Centola Vidal Luiz Augusto do Amaral About the authors

ABSTRACT:

This study aimed to evaluate the occurrence of Bacillus cereus group in requeijões and especialidades lácteas tipo requeijão (regular and light) and to verify if there is differences in relation to this occurrence among different categories of these products. A set of 14 (35%) lots was contaminated with this bacterial group from the 40 lots with low counts (maximum 3.1 × 10 CFU/g), and no significant difference regarding counts or presence/absence were observed among the categories of the products. It can be concluded that contamination by B. cereus group in these products is unable to consist in risk to consumers, regarding adequate refrigeration during selling. This study was the first one to report this bacteria group for these dairy products and highlights the needs of further investigations to evaluate the impact of its spoilage during shelf life.

KEYWORDS:
spores, dairy; milk; microbiology; food safety

RESUMO:

Este estudo objetivou avaliar a ocorrência de bactérias do grupo de Bacillus cereus em requeijões e especialidades lácteas (normais e light) e verificar se há diferença em relação a essa ocorrência entre as diferentes categorias desses produtos. Entre os 40 lotes avaliados, 14 (35%) estavam contaminados por bactérias desse grupo, com baixas contagens (máxima de 3,1 × 10 UFC/g) e sem diferenças significativas no que tange às contagens ou à presença/ausência entre as diversas categorias. Conclui-se que as bactérias do grupo de B. cereus em requeijões e especialidades lácteas são incapazes de consistir em risco aos consumidores desses produtos, desde que as condições de refrigeração sejam adequadas durante armazenamento e venda. O presente trabalho é o primeiro a relatar a presença de tal grupo de micro-organismos nesses tipos de produtos, porém, são necessários novos estudos para avaliar o impacto de sua ação deteriorante sobre o período em que passa na prateleira.

PALAVRAS-CHAVE:
alimentos, laticínios; leite, microbiologia; segurança alimentar

The Bacillus cereus group is composed of eight species, including B. cereus sensu stricto (s. s.), Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus anthracis, Bacillus weihenstephanensis, Bacillus cytotoxicus (GUINEBRETIÉRE et al., 2013GUINEBRETIÉRE, M.H.; AUGER, S.; GALLERON, N.; CONTZEN, M.; SARRAU B.; BUYSER, M.L.; LAMBERET, G.; FAGERLUND, A.; GRANUM, P.E.; LARECLUS, D.; VOS, P.; NGUYEN-THE, C.; SOROKIN, A. Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the Bacillus cereus Group occasionally associated with food poisoning. International Journal of Systematic and Evolutionary Microbiology, n.63, p.31-40, 2013.) and Bacillus toyonensis (JIMÉNEZ et al., 2013JIMÉNEZ, G.; URDIAIN, M.; CIFUENTES, A.; LÓPEZ-LÓPEZ, A.; BLANCH, A.R.; TAMAMES, J.; KAMPFER, P.; KOLSTO, A.B.; RAMÓN, D.; MARTÍNEZ, J.F.; CODOÑER, F.M.; ROSSELLÓ-MÓRA, R. Description of Bacillus toyonensis sp. nov., a novel species of the Bacillus cereus group, and pairwise genome comparisons of the species of the group by means of ANI calculations. Systematic and Applied Microbiology, v.36, n.6, p.383-391, 2013. Available from: <Available from: http://dx.doi.org/10.1016/j.syapm.2013.04.008 >. Accessed on: Dec. 05 2016.
http://dx.doi.org/10.1016/j.syapm.2013.0...
). The bacteria belonging to this group are rod-shaped, gram-positive, facultatively anaerobe, spore-producers (allowing their survival to heat-treatment) and widespread on environment (FDA, 2012FDA, Food and Drug Administration. The Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins, Ed. 2, 2012. Available from: <Available from: http://www.fda.gov/downloads/Food/FoodborneIllnessContaminants/UCM297627.pdf >. Accessed on: Dec. 05 2016.
http://www.fda.gov/downloads/Food/Foodbo...
).

These bacteria are genomically similar besides their phonotypical differences, such as the ability to cause foodborne illness and anthrax, or to be used on biological pest control. The genetic differentiation on these species is possible. However, it still presents some difficulties due the high genetic similarity among these species and plasmids transfer (KOVAC et al., 2016KOVAC, J.; MILLER, R.A.; CARROLL, L.M.; KENT, D.J.; JIAN J.; BENO, S.M.; WIEDMANN, M. Production of hemolysin BL by Bacillus cereus group isolates of dairy origin is associated with whole-genome phylogenetic clade. BMC Genomics, v.17, n.581, 2016. Available from: Doi: 10.1186/s12864-016-2883-z. Accessed on: Dec. 05 2016.
https://doi.org/10.1186/s12864-016-2883-...
).

The species B. cereus s. s. is highlighted in this group due its importance on food production chain considering its ability to cause emetic and diarrheal syndrome (FDA, 2012FDA, Food and Drug Administration. The Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins, Ed. 2, 2012. Available from: <Available from: http://www.fda.gov/downloads/Food/FoodborneIllnessContaminants/UCM297627.pdf >. Accessed on: Dec. 05 2016.
http://www.fda.gov/downloads/Food/Foodbo...
) and foodborne outbreaks (ZHOU et al., 2014ZHOU, G.; BESTER, K.; LIAO, B.; YANG, Z.; JIANG, R.; HENDRIKSEN, N.B. Characterization of three Bacillus cereus strains involved in a major outbreak of food poisoning after consumption of fermented black beans (douchi) in Yunan, China. Foodborne Pathogens & Diseases, v.11, n.10, p.769-774, 2014.). Yet, other species are also considered able to cause foodborne illness (GUINEBRETIÉRE et al., 2013GUINEBRETIÉRE, M.H.; AUGER, S.; GALLERON, N.; CONTZEN, M.; SARRAU B.; BUYSER, M.L.; LAMBERET, G.; FAGERLUND, A.; GRANUM, P.E.; LARECLUS, D.; VOS, P.; NGUYEN-THE, C.; SOROKIN, A. Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the Bacillus cereus Group occasionally associated with food poisoning. International Journal of Systematic and Evolutionary Microbiology, n.63, p.31-40, 2013.). The dairy products are not often involved in these outbreaks (BENNET et al., 2013BENNET, S.D.; WALSH, K.A.; GOULD, L.H. Foodborne disease outbreaks caused by Bacillus cereus, Clostridium perfringens, and Staphylococcus aureus - United States, 1998-2008. Clinical Infectious Diseases, v.57, n.3, p. 425-433, 2013.), but this group is important to dairy industries due its ability to form biofilms and result in food spoilage (KUMARI; SARKAR, 2016KUMARI, S.; SARKAR, P. Bacillus cereus hazard and control in industrial dairy processing environment. Food Control, v.69, p.20-29, 2016.).

This group is reported as contaminant in several dairy products (KUMARI; SARKAR, 2016KUMARI, S.; SARKAR, P. Bacillus cereus hazard and control in industrial dairy processing environment. Food Control, v.69, p.20-29, 2016.), but there are no studies published on literature reporting its presence on requeijão curd cheeses and especialidade láctea tipo requeijão, which are highly consumed in Brazil (ZACARCHENCO et al., 2012ZACARCHENCO, P.B.; VAN DENDER, A.G.F.; SPADOTI, L.M. “Requeijão” culinário: Aspectos históricos, de mercado e tecnológicos. Revista das Indústrias de Laticínios - São Paulo, v.18, p.70-74, 2012.). Requeijão curd cheese is produced using acid coagulation of milk followed by a mass fusion (80ºC during 5 seconds), whey removal and washing, and the addition of fat milk is allowed (BRAZIL, 1997BRAZIL. Ministério da Agricultura, Pecuária e Abastecimento. Portaria nº 359, de 4 de setembro de 1997. Diário Oficial da União, Brasil, Brasília, DF, 8 set. 1997 Seção 1, p.19690-19691.). Furthermore, a very similar dairy product (especialidade láctea tipo requeijão) is often consumed in Brazil, differing from the first product cited by the addition of cornstarch.

Thus, this study aimed to counting bacteria belonging to B. cereus group in requeijão curd cheeses and especialidade láctea tipo requeijão (regular and light) marketed in the state of São Paulo, Brazil, and verify if there is difference in relation to this occurrence among these categories. For this purpose, 200 samples of requeijão and especialidade láctea tipo requeijão were bought on supermarkets located in Jaboticabal, São Paulo, in 2015.

The samples were processed in groups of five unities each (one lot), as it follows:

  1. 50 samples of requeijão normal”, from five commercial brands and ten production lots;

  2. 50 samples of requeijão light”, from five brands and ten lots;

  3. 50 samples of especialidade láctea, from five brands and ten lots;

  4. 50 samples of especialidade láctea light, from three brands and ten production lots (Table 1).

Table 1:
Average counts of bacteria from B. cereus group on requeijão, requeijão light, especialidade láctea tipo requeijão and especialidade láctea tipo requeijão light marketed in Jaboticabal municipality, state of São Paulo, Brazil, in 2015.

The asepsis of package was performed using ethanol 10%, and posteriorly 25 g of the samples were transferred to flasks containing sterile peptone water 0.1% to obtain the initial dilution (10-1). Following, 1 mL of this initial dilution was transferred to a tube containing 9 mL of sterile peptone water 0.1%, obtaining the dilution 10-2, and this step was repeated to obtain the dilution 10-3 (BRAZIL, 2003BRAZIL. Instrução Normativa nº 62, de 28 de Agosto de 2003. Oficializa os Métodos Analíticos Oficiais para Análises Microbiológicas para Controle de Produtos de Origem Animal e Água. Diário Oficial da União, Brasília, DF, 18 de set. 2003. Seção 1, p. 14.).

These dilutions were inoculated in duplicate on plates containing Agar-MYP and incubated on 30ºC during 18-40 hours. The colonies considered as suggestive of B. cereus group were those pink to red and lecithinase-positive (MOSSEL et al., 1967MOSSEL, D.A.A.; KOOPMAN, M.J.; JONGERIUS, E. Enumeration of Bacillus cereus in foods. Applied Microbiology, v.15, n.3, p.650-653, 1967.). Gram stain was performed using three to five suggestive colonies.

In order to verify if there were statistically significant differences on the counts between the different types of evaluated products, the radicalization of the counts values was carried out. When the requirements for the analysis were not a setback, the analysis was performed through non-parametric tests (Kruskal-Wallis). Data analysis was also performed through presence or absence in the products, to verify if there was a statistically significant difference. For this purpose, the products were divided into those with high and low fat (regular and light types), and with or without starch (requeijão and especialidade láctea tipo requeijão). Then, chi-square analysis was performed. These analyzes were carried out adopting the level of significance of 5% (p<0.05).

Among the 40 lots of dairy products, 14 (35%) showed contamination by bacteria from B. cereus group. REZENDE-LAGO et al. (2012REZENDE-LAGO, N.C.M.; ROSSI JUNIOR, O.D.; VIDAL-MARTINS, A.M.C.; AMARAL, L.A. Occurrence of Bacillus cereus in whole milk and enterotoxigenic potential of the isolated strains. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v.59, n.6, p.1563-1569, 2007.) evaluated powdered, raw, pasteurized and ultra-high temperature processing (UHT) milks samples and isolated B. cereus in 73.3, 50.0, 96.7 and 13.3%, respectively, demonstrating the importance of bacteria from this group in dairy products. The result of bacterial counts belonging to the B. cereus group is shown in Table 1.

Although bacteria from the B. cereus group may be present in a wide variety of dairy products such as UHT milk, pasteurized milk, powdered milk, cheeses and fermented milks (WONG et al., 1998WONG, H.C.; CHANG, M.H.; FAN, J.Y. Incidence and characterization of Bacillus cereus isolates contaminating dairy products. Applied Environmental Microbiology, v.54, n.3, p.699-702, 1988.; REZENDE-LAGO et al., 2007REZENDE-LAGO, N.C.M.; ROSSI JUNIOR, O.D.; VIDAL-MARTINS, A.M.C.; AMARAL, L.A. Occurrence of Bacillus cereus in whole milk and enterotoxigenic potential of the isolated strains. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v.59, n.6, p.1563-1569, 2007.), it can be observed in Table 1 that the counts of bacteria belonging to the B. cereus group in the evaluated dairy products were considered low. However, although low and unable to put consumers at risk, since the counts were above 105 CFU/g - value required to cause foodborne diseases (FDA, 2012FDA, Food and Drug Administration. The Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins, Ed. 2, 2012. Available from: <Available from: http://www.fda.gov/downloads/Food/FoodborneIllnessContaminants/UCM297627.pdf >. Accessed on: Dec. 05 2016.
http://www.fda.gov/downloads/Food/Foodbo...
) -, the action of their spoilage enzymes can reduce the shelf life of these products (KUMARI; SARKAR, 2016KUMARI, S.; SARKAR, P. Bacillus cereus hazard and control in industrial dairy processing environment. Food Control, v.69, p.20-29, 2016.).

The low occurrence of these bacteria can be explained by the fact that the milk used as raw material has a satisfactory microbiological quality, since the contamination of raw milk by spores of B. cereus is dependent on management in dairy farms (VISSERS et al., 2007VISSERS, M.M.M.; TE GIFFEL, M.C.; DRIEHUIS, F.; DE JONG, P.; LANKVELD, J.M.G. Predictive modeling of Bacillus cereus spores in farm tank milk during grazing and housing periods. Journal of Dairy Science, v.90, n.1, p.281-292, 2007.). Also, the temperature used during curd cheeses processing is able to inactivate most of the microorganisms, with the exception of the sporulating ones. In addition to composition, pH and water activity (Aw), the presence of flux salts and nisin may be inhibitory to sporulating microorganisms (JOHNSON, 2001JOHNSON, M.E. Cheese Products. In: MARTH, E.H.; STEELE, J.L. Applied Dairy Microbiology. Nova Iorque: Marcel Dekker. Cap. 11. 2001).

Another factor that contributes to the low microbial load of these products is the bactofugation process, or Ultra-Fresh Bactofugation combines treatments that target the separation and removal of active, sporulating microorganisms and somatic cell with a subsequent heat treatment by direct injection of steam (PRATA et al., 2012PRATA, L.F.; OLIVEIRA, C.F.; PRATA, A.B.; MACHADO, R.C. Qualidade do leite cru antes e após o processo Ultrafresh. Indústria de Laticínios, v.96, p.58-65, 2012.). In addition, the use of preventive systems such as good manufacturing practices (GMP) and Hazard Analysis and Critical Control Points (HACCP), which are mandatory in Brazilian dairy industries, help to mitigate the risks (KUMARI; SARKAR, 2016KUMARI, S.; SARKAR, P. Bacillus cereus hazard and control in industrial dairy processing environment. Food Control, v.69, p.20-29, 2016.).

The results of the B. cereus counts did not meet the requirements to be able to perform the analysis of variance. Then, the Kruskal-Wallis test was used. There were no statistically significant differences between the B. cereus counts (p>0.05) among the different evaluated product types. The results of chi-square analysis for the presence/absence of B. cereus did not reveal any statistically significant difference (p>0.05) between the different products. Thus, it is believed that the presence of starches or vegetable oils (requeijão × especialidade láctea) and fat (regular × light) in the products did not significantly influence the occurrence of B. cereus group.

Psychrotrophic strains of B. cereus present greater germination and multiplication capacity in high fat dairy products due to their hydrophobic spores (LARSEN; JORGENSEN, 1997LARSEN, H.D.; JORGENSEN, K. The occurrence of Bacillus cereus in Danish pasteurized milk. International Journal of Food Microbiology, v.34. p.179-186, 1997.). In addition, lipids confer a protective characteristic on the spores, since fat has low-thermal conductivity (MEER et al., 1991MEER, R.R.; BAKER, J.; BODYFELT F.W.; GRIFFITHS, M.W. Psychrotrophic Bacillus spp. in fluid milk products: a review. Journal of Food Protection, v.54, n.12, p.969-979, 1991.). Consequently, the presence of lipids in food could be an important factor in the microorganism adaptation to these conditions, but no influence was observed in this study.

The counts of the B. cereus group in requeijão and especialidade láctea tipo requeijão were low and unable to jeopardize the consumers of these products if proper conditions of refrigeration during storage and sale are adopted. However, the present study is the first one to report the presence of such group of microorganisms in these dairy products, which requires new studies to evaluate the impact of its spoilage on the shelf life of these products routinely consumed in Brazil.

ACKNOWLEDGEMENTS

To Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) - grant no. 2014/21534-6

REFERENCES

  • BENNET, S.D.; WALSH, K.A.; GOULD, L.H. Foodborne disease outbreaks caused by Bacillus cereus, Clostridium perfringens, and Staphylococcus aureus - United States, 1998-2008. Clinical Infectious Diseases, v.57, n.3, p. 425-433, 2013.
  • BRAZIL. Ministério da Agricultura, Pecuária e Abastecimento. Portaria nº 359, de 4 de setembro de 1997. Diário Oficial da União, Brasil, Brasília, DF, 8 set. 1997 Seção 1, p.19690-19691.
  • BRAZIL. Instrução Normativa nº 62, de 28 de Agosto de 2003. Oficializa os Métodos Analíticos Oficiais para Análises Microbiológicas para Controle de Produtos de Origem Animal e Água. Diário Oficial da União, Brasília, DF, 18 de set. 2003. Seção 1, p. 14.
  • FDA, Food and Drug Administration. The Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins, Ed. 2, 2012. Available from: <Available from: http://www.fda.gov/downloads/Food/FoodborneIllnessContaminants/UCM297627.pdf >. Accessed on: Dec. 05 2016.
    » http://www.fda.gov/downloads/Food/FoodborneIllnessContaminants/UCM297627.pdf
  • GUINEBRETIÉRE, M.H.; AUGER, S.; GALLERON, N.; CONTZEN, M.; SARRAU B.; BUYSER, M.L.; LAMBERET, G.; FAGERLUND, A.; GRANUM, P.E.; LARECLUS, D.; VOS, P.; NGUYEN-THE, C.; SOROKIN, A. Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the Bacillus cereus Group occasionally associated with food poisoning. International Journal of Systematic and Evolutionary Microbiology, n.63, p.31-40, 2013.
  • JIMÉNEZ, G.; URDIAIN, M.; CIFUENTES, A.; LÓPEZ-LÓPEZ, A.; BLANCH, A.R.; TAMAMES, J.; KAMPFER, P.; KOLSTO, A.B.; RAMÓN, D.; MARTÍNEZ, J.F.; CODOÑER, F.M.; ROSSELLÓ-MÓRA, R. Description of Bacillus toyonensis sp. nov., a novel species of the Bacillus cereus group, and pairwise genome comparisons of the species of the group by means of ANI calculations. Systematic and Applied Microbiology, v.36, n.6, p.383-391, 2013. Available from: <Available from: http://dx.doi.org/10.1016/j.syapm.2013.04.008 >. Accessed on: Dec. 05 2016.
    » http://dx.doi.org/10.1016/j.syapm.2013.04.008
  • JOHNSON, M.E. Cheese Products. In: MARTH, E.H.; STEELE, J.L. Applied Dairy Microbiology. Nova Iorque: Marcel Dekker. Cap. 11. 2001
  • KOVAC, J.; MILLER, R.A.; CARROLL, L.M.; KENT, D.J.; JIAN J.; BENO, S.M.; WIEDMANN, M. Production of hemolysin BL by Bacillus cereus group isolates of dairy origin is associated with whole-genome phylogenetic clade. BMC Genomics, v.17, n.581, 2016. Available from: Doi: 10.1186/s12864-016-2883-z. Accessed on: Dec. 05 2016.
    » https://doi.org/10.1186/s12864-016-2883-z
  • KUMARI, S.; SARKAR, P. Bacillus cereus hazard and control in industrial dairy processing environment. Food Control, v.69, p.20-29, 2016.
  • LARSEN, H.D.; JORGENSEN, K. The occurrence of Bacillus cereus in Danish pasteurized milk. International Journal of Food Microbiology, v.34. p.179-186, 1997.
  • MEER, R.R.; BAKER, J.; BODYFELT F.W.; GRIFFITHS, M.W. Psychrotrophic Bacillus spp in fluid milk products: a review. Journal of Food Protection, v.54, n.12, p.969-979, 1991.
  • MOSSEL, D.A.A.; KOOPMAN, M.J.; JONGERIUS, E. Enumeration of Bacillus cereus in foods. Applied Microbiology, v.15, n.3, p.650-653, 1967.
  • PRATA, L.F.; OLIVEIRA, C.F.; PRATA, A.B.; MACHADO, R.C. Qualidade do leite cru antes e após o processo Ultrafresh. Indústria de Laticínios, v.96, p.58-65, 2012.
  • REZENDE-LAGO, N.C.M.; ROSSI JUNIOR, O.D.; VIDAL-MARTINS, A.M.C.; AMARAL, L.A. Occurrence of Bacillus cereus in whole milk and enterotoxigenic potential of the isolated strains. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v.59, n.6, p.1563-1569, 2007.
  • VISSERS, M.M.M.; TE GIFFEL, M.C.; DRIEHUIS, F.; DE JONG, P.; LANKVELD, J.M.G. Predictive modeling of Bacillus cereus spores in farm tank milk during grazing and housing periods. Journal of Dairy Science, v.90, n.1, p.281-292, 2007.
  • ZACARCHENCO, P.B.; VAN DENDER, A.G.F.; SPADOTI, L.M. “Requeijão” culinário: Aspectos históricos, de mercado e tecnológicos. Revista das Indústrias de Laticínios - São Paulo, v.18, p.70-74, 2012.
  • ZHOU, G.; BESTER, K.; LIAO, B.; YANG, Z.; JIANG, R.; HENDRIKSEN, N.B. Characterization of three Bacillus cereus strains involved in a major outbreak of food poisoning after consumption of fermented black beans (douchi) in Yunan, China. Foodborne Pathogens & Diseases, v.11, n.10, p.769-774, 2014.
  • WONG, H.C.; CHANG, M.H.; FAN, J.Y. Incidence and characterization of Bacillus cereus isolates contaminating dairy products. Applied Environmental Microbiology, v.54, n.3, p.699-702, 1988.

Publication Dates

  • Publication in this collection
    2018

History

  • Received
    16 Dec 2016
  • Accepted
    21 Mar 2018
Instituto Biológico Av. Conselheiro Rodrigues Alves, 1252 - Vila Mariana - São Paulo - SP, 04014-002 - São Paulo - SP - Brazil
E-mail: arquivos@biologico.sp.gov.br