Acessibilidade / Reportar erro

Microbiological quality of honey from stingless bee, jandaíra (Melipona subnitida), from the semiarid region of Brazil

Qualidade microbiológica do mel da abelha sem ferrão jandaíra (Melipona subnitida) da região semi-árida do Brasil

ABSTRACT:

The aim of this study was to evaluate the presence of microorganisms in honey produced by the stingless bee (SB) jandaíra (Melipona subnitida) from the semiarid region of Brazil. Thirty-five samples were analyzed and all of them were positive for mesophilic bacteria, coliforms at 45°C, fungi, and yeast. Staphylococcus spp. were identified in 85.7%, while Bacillus spp. were detected in 34.3% of honey samples. DNAs of Clostridium perfringens and C. botulinum were detected in 40% and 2.8% of the samples, respectively. Salmonella spp. and C. difficile were not detected. The present research revealed a great diversity of microorganisms in honey produced by jandaíra.

Key words:
Clostridium botulinum; Clostridium perfringens; stingless bee

RESUMO:

O objetivo deste trabalho foi avaliar a presença de microrganismos no mel produzido pela abelha sem ferrão jandaíra (Melipona subnitida). Trinta e cinco amostras foram avaliadas e todas foram positivas para bactérias mesofílicas, coliformes a 45 ºC, fungos e leveduras. Staphylococcus spp. foi identificado em 85,7% enquanto Bacillus foi detectado em 34,3% das amostras de mel. Clostridium perfringens e C. botulinum foram detectados em 40% e 2,8% das amostras respectivamente. Salmonella spp. e C. difficile não foram detectados. O presente trabalho revelou uma grande diversidade de microrganismos no mel produzido por jandaíra.

Palavras-chave:
Clostridium botulinum; Clostridium perfringens; abelhas sem ferrão

Jandaíra (Melipona subnitida) is a stingless bee (SB) very common in northeastern Brazil that has been increasingly used for honey production (MAIA et al., 2015MAIA, U.M. et al. Meliponicultura no Rio Grande do Norte. Revista Brasileira de Medicina Veterinária, v.37, n.4, p.327-333, 2015. Available from: http://www.rbmv.org/index.php/BJVM/article/download/425/316>. Accessed: Dez. 24, 2017.
http://www.rbmv.org/index.php/BJVM/artic...
). Recently, the Polo de Mel Jandaíra (Honey Pole Jandaíra) was implemented by the Ministério da Integração Nacional (Ministry of National Integration) to organize and encourage the production chain of honey from jandaíra (BRASIL, 2014BRASIL. Portaria n.162, de 24 de abril de 2014. Estabelece as Rotas de Integração Nacional como estratégia de Inclusão Produtiva e Desenvolvimento Regional do Ministério de Integração Nacional. Diário Oficial da União , Poder Executivo, Brasília DF. Abr. 28, 2014. Seção 1, p. 30.). The growing demand for this honey in Brazil has encouraged studies on it and it has been already known that honey from M. subnitida has some remarkable physicochemical and palynological characteristics (ALMEIDA-MURADIAN et al., 2013ALMEIDA-MURADIAN, L.B. et al. Comparative study of the physicochemical and palynological characteristics of honey from Melipona subnitida and Apis mellifera. International Journal of Food Science and Technology, v.48, p.1698-1706, 2013. Available from: https://onlinelibrary.wiley.com/doi/full/10.1111/ijfs.12140>. Accessed: Jan. 30, 2018. doi: 10.1111/ijfs.12140.
https://onlinelibrary.wiley.com/doi/full...
). Despite this, reports about the microbiological quality of honey from SB are rare and there is no specific legislation that deals with it, which hinders the characterization and determination of food safety for this product (MONTE et al., 2013MONTE, A.M. et al. Quality of honey from stingless bees native of Piaui, Brazil. Revista Brasileira de Medicina Veterinária , v.35, n.1, p.48-54, 2013. Available from: http://www.rbmv.com.br/pdf_artigos/02-07-2013_11-21RBMV%20009.pdf>. Accessed: Dez. 20, 2017.
http://www.rbmv.com.br/pdf_artigos/02-07...
). In this study, we evaluated the microbiological quality of the honey produced by SB jandaíra in the semiarid region of Brazil.

Thirty-five beekeeping farms from 12 cities of the semiarid region of Brazil were visited, with all located in a radius of 177km from the geographical loation 4°56’35.9”S 37°18’31.5”W. In each farm, a 500-mL sample was collected directly from the honeycomb with a sterile syringe, packaged in transparent vials of sterile plastics, and kept protected from light with an average temperature of 25°C until microbiological analysis. Detection of mesophilic bacteria, coliforms at 45°C, Salmonella spp., Staphylococcus spp., fungi, and yeast were performed according to the Brazilian legislation (BRASIL, 2003BRASIL. Instrução Normativa n. 62, de 26 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, Poder Executivo, Brasília, DF. Set. 18, 2003. Seção 1, p.14. ). For detection of Clostridium perfringens, C. difficile, and C. botulinum DNA, each sample was inoculated into Cooked Meat Medium (CMM, Difco, USA) broth and Reinforced Clostridial Medium (RCM, Difco, USA) Broth. After incubation at 37°C in an anaerobic atmosphere for 48 hours, a 100-µL aliquot of the broth culture was subjected to thermal DNA extraction and DNA amplification via PCR to identify C. difficile (SILVA et al., 2011SILVA, R.O.S. et al. Detection of enterotoxin A and cytotoxin B, and isolation of Clostridium difficile in piglets in Minas Gerais, Brazil. Ciência Rural , v.41, n.8, p.1130-35, 2011. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-84782011000800021>. Accessed: Dez. 17, 2017. doi: 10.1590/S0103-84782011005000100.
http://www.scielo.br/scielo.php?script=s...
), C. perfringens (VIEIRA et al., 2008VIEIRA, A.A.S. et al. Genotipagem de Clostridium perfringens isolados de leitões diarreicos. Arquivos do Instituto Biológico, v.75, n.4, p.513-516. 2008. Available from: https://www.researchgate.net/publication/258021724_Genotipagem_de_Clostridium_perfringens_isolados_de_leitoes_diarreicos>. Accessed: Dez. 10, 2017.
https://www.researchgate.net/publication...
), and C. botulinum (PRÉVOT et al., 2007PRÉVOT, V. et al. Optimization of polymerase chain reaction for detection of Clostridium botulinum type C and D in bovine samples. Zoonosis Public Health, v.54, n.8, p.320-327, 2007. Available from: http://onlinelibrary.wiley.com/doi/10.1111/j.1863-2378.2007.01070.x/full>. Accessed: Dez. 18, 2017.
http://onlinelibrary.wiley.com/doi/10.11...
). In addition, 10µL of the broth was inoculated on Muller-Hinton agar (Difco, USA), which was supplemented with 5% of sheep blood, and incubated at 37°C in an anaerobic atmosphere for 48 hours. Thus, colonies not confirmed as clostridia in previous PCR analyses were subjected to further PCR assessment of 16S rRNA using universal primers (FOX et al., 1995FOX, J.G. et al. Helicobacter bilis sp. nov.: a novel helicobacter species isolated from bile, livers, and intestines of aged, inbred mice. Journal of Clinical Microbiology, v.33, n.2, p.445-454, 1995. Available from: http://jcm.asm.org/content/33/2/445.full.pdf>. Accessed: Dez. 01, 2017.
http://jcm.asm.org/content/33/2/445.full...
). The amplicons were sequenced and the species were confirmed using BLAST with a similarity of 98% (MIAN et al., 2009MIAN, G.F. et al. Aspects of the natural history and virulence of S. agalactiae infection in Nile tilapia. Veterinary Microbiology, v.136, n.1-2, p.180-183, 2009. Available from: https://www.sciencedirect.com/science/article/pii/S0378113508004926?via%3Dihub>. Accessed: Dez. 20, 2017.
https://www.sciencedirect.com/science/ar...
).

Results of the present research are summarized in Table 1. Brazilian legislation categorizes the honey from SB as a product made by SB or native bees, but no official requirement related to the microbiological quality of the honey from SB is available (BRASIL, 2004BRASIL. Resolução CONAMA n. 346, de 16 de agosto de 2004. Disciplina a utilização de abelhas silvestres nativas, bem como a implantação de meliponários. Diário Oficial da União, Poder Executivo, Brasília, DF. Ago. 14, 2004. Seção 1, p. 70.; BRASIL, 2017BRASIL. Decreto n.9.013, de 29 de março de 2017. Regulamentação da Lei n. 1.283, de 18 de dezembro de 1950 e a Lei n. 7.889, de 23 de novembro de 1989, que dispõem sobre a inspeção industrial e sanitária de produtos de origem animal. Diário Oficial da União , Poder Executivo, Brasília, DF. Mar. 30, 2017. Seção 1, p.3.). Beyond that, the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA) requires the presentation of scientific literature to ensure the quality and innocuous of the honey from SB (CAMARGO et al., 2017CAMARGO, R.C.R et al. Stingless bee honey: technical regulation proposal. Brazilian Journal of Food Technology, v.20, e2016157, 2017. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1981-67232017000100900&lng=pt&tlng=pt>. Accessed: Dez. 26, 2017. doi: 10.1590/1981-6723.15716.
http://www.scielo.br/scielo.php?script=s...
). Considering this, the results of this research will be compared to the microbiological data published in literature for the honey from SB.

Table 1
Microorganisms identified in 35 samples of stingless honey from Melipona subnitida (jandaíra bees) collected from the semiarid region of Brazil. MB - Mesophilic bacteria; CFU - Colony-forming units; Col. - Coliforms; MPN - Most probable number; FY - fungi and yeast; CPA - Clostridium perfringens type A; CBC - Clostridium botulinum type C; BC - Bacillus cereus; BL - Bacillus licheniformis; BC - Bacillus cereus.

All samples were positive for mesophilic bacteria. Most of them (74.3%) showed counts lower than 25 colony-forming units per gram (CFU/g), 11.4% showed between 25 and 2500CFU/g, and the remaining 14.3% showed more than 2500CFU/g. The presence of mesophilic bacteria is related to the deterioration of food. However, some characteristics of honey, such as pH acidity, low water activity, low protein content, and high sugar, can diminish or stop bacterial activity, contributing to a longer shelf life of the product (EUROPEAN COMMISSION, 2002EUROPEAN COMMISSION. Opinion of the scientific committee on veterinary measures relating to public health on honey and microbiological hazards. Health & Consumer Protection Directorate-General, 19 Jun. 2002. Available from: https://ec.europa.eu/food/sites/food/files/safety/docs/sci-com_scv_out53_en.pdf>. Accessed: Dez. 04, 2017.
https://ec.europa.eu/food/sites/food/fil...
; MONTE et al., 2013MONTE, A.M. et al. Quality of honey from stingless bees native of Piaui, Brazil. Revista Brasileira de Medicina Veterinária , v.35, n.1, p.48-54, 2013. Available from: http://www.rbmv.com.br/pdf_artigos/02-07-2013_11-21RBMV%20009.pdf>. Accessed: Dez. 20, 2017.
http://www.rbmv.com.br/pdf_artigos/02-07...
). However, some samples showed a high count of mesophilic bacteria, which some authors attributed to factors associated with the location of the beekeeping farms that can result in contamination. These sources of contamination include soil, water, air, pollen, and nectar (BÁRBARA et al., 2015BÁRBARA, M.S. et al. Microbiological assessment, nutritional characterization and phenolic compounds of bee pollen from Mellipona mandacaia Smith, 1983. Molecules, v.9, n.7, p.12525-12544, 2015. Available from: http://www.mdpi.com/1420-3049/20/7/12525>. Accessed: Dez. 30, 2017. doi: 10.3390/molecules200712525.
http://www.mdpi.com/1420-3049/20/7/12525...
; OLIVEIRA et al., 2017OLIVEIRA, R.G. et al. Screening for quality indicators and phenolic compounds of biotechnological interest in honey samples from six species of stingless bees (Hymenoptera: Apidae). Food Science and Technology, v.37, n.4, p.552-557, 2017. Available from: http://www.scielo.br/scielo.php?pid=S0101-20612017005005106&script=sci_abstract>. Accessed: Dez. 18, 2017. doi: 10.1590/1678-457x.25716.
http://www.scielo.br/scielo.php?pid=S010...
).

Regarding coliforms at 45°C, all samples showed results lower than 3.0 most probable number per gram (MPN/g), confirming results of previous studies with different SB honeys, including that from jandaíra bees (SOUZA et al., 2009SOUZA, B.A. et al. Avaliação microbiológica de amostras de mel de trigoníneos (Apidae: Trigonini) do estado da Bahia. Ciência e Tecnologia de Alimentos, v.29, n.4, p.798-802, 2009. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0101-20612009000400015>. Accessed: Dez. 22, 2017. doi: 10.1590/S0101-20612009000400015.
http://www.scielo.br/scielo.php?script=s...
; MONTE et al., 2013MONTE, A.M. et al. Quality of honey from stingless bees native of Piaui, Brazil. Revista Brasileira de Medicina Veterinária , v.35, n.1, p.48-54, 2013. Available from: http://www.rbmv.com.br/pdf_artigos/02-07-2013_11-21RBMV%20009.pdf>. Accessed: Dez. 20, 2017.
http://www.rbmv.com.br/pdf_artigos/02-07...
). According to SILVA et al. (2008SILVA, M.B.L. et al. Qualidade microbiológica de méis produzidos por pequenos apicultores e de méis de entrepostos registrados no serviço de inspeção federal no estado de Minas Gerais. Alimentos e Nutrição, v.19, n.4, p.417-420, 2008.), values under 3.0MPN/g indicated high quality of the honey. However, in a proposal of regulation for the honey from SB, CAMARGO et al. (2017CAMARGO, R.C.R et al. Stingless bee honey: technical regulation proposal. Brazilian Journal of Food Technology, v.20, e2016157, 2017. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1981-67232017000100900&lng=pt&tlng=pt>. Accessed: Dez. 26, 2017. doi: 10.1590/1981-6723.15716.
http://www.scielo.br/scielo.php?script=s...
) suggested a three-class plan for coliforms at 45°C, with acceptable samples between 0 and 10MPN/g and intermediate samples until 100MPN/g. These values are based on the habit of SB to use a fine mix of propolis and clay to seal the honeycombs (NOGUEIRA-NETO, 1997NOGUEIRA-NETO, P. Vida e criação de abelhas indígenas sem ferrão. São Paulo: Editora Nogueirapis, 1997. 445p.). Some species even land on feces from vertebrates, which can increase the number of coliforms (SANTOS & ANTONINI, 2008SANTOS, G.M.; ANTONINI, Y. The traditional knowledge on stingless bees (Apidae: Meliponina) used by the Enawene-Nawe tribe in western Brazil. Journal of Ethnobiology and Ethnomedicine, v.4, p.19, 2008. Available from: https://ethnobiomed.biomedcentral.com/track/pdf/10.1186/1746-4269-4-19>. Accessed: Dez. 20, 2017. doi: 10.1186/1746-4269-4-19.
https://ethnobiomed.biomedcentral.com/tr...
).

All samples tested were negative for Salmonella spp., in agreement with other studies evaluating the honey from SB (MONTE et al., 2013MONTE, A.M. et al. Quality of honey from stingless bees native of Piaui, Brazil. Revista Brasileira de Medicina Veterinária , v.35, n.1, p.48-54, 2013. Available from: http://www.rbmv.com.br/pdf_artigos/02-07-2013_11-21RBMV%20009.pdf>. Accessed: Dez. 20, 2017.
http://www.rbmv.com.br/pdf_artigos/02-07...
; PUCCIARELLI et al., 2014PUCCIARELLI, A.B. et al. Microbiological and physicochemical analysis of yateí (Tetragonisca angustula) honey for assessing quality standards and commercialization. Revista Argentina de Microbiología, v.46, n.4, p.325-332, 2014. Available from: https://www.sciencedirect.com/science/article/pii/S0325754114700914>. Accessed: Dez. 18, 2017. doi: 10.1016/S0325-7541(14)70091-4.
https://www.sciencedirect.com/science/ar...
). Some studies have shown the antibacterial effect of honey, and the honey from SB in particular, against a wide range of bacteria, including Salmonella (PIMENTEL et al., 2013PIMENTEL, R.B.Q. et al. Antimicrobial activity and rutin identification of honey produced by the stingless bee Melipona compressipes manaosensis and commercial honey. BMC Complementary and Alternative Medicine, v.13, p.151, 2013. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3702439/pdf/1472-6882-13-151.pdf>. Accessed: Dez. 23, 2017. doi: 10.1186/1472-6882-13-151.
https://www.ncbi.nlm.nih.gov/pmc/article...
; NISHIO et al., 2016NISHIO, E.K. et al. Antibacterial synergic effect of honey from two stingless bees: Scaptotrigona bipunctata Lepeletier, 1836, and S. postica Latreille, 1807. Scientific Reports, v.6, p.21641, 2016. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751499/pdf/srep21641.pdf>. Accessed: Dez. 26, 2017. doi: 10.1038/srep21641.
https://www.ncbi.nlm.nih.gov/pmc/article...
). As Salmonella is a relevant zoonotic pathogen, CAMARGO et al. (2017CAMARGO, R.C.R et al. Stingless bee honey: technical regulation proposal. Brazilian Journal of Food Technology, v.20, e2016157, 2017. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1981-67232017000100900&lng=pt&tlng=pt>. Accessed: Dez. 26, 2017. doi: 10.1590/1981-6723.15716.
http://www.scielo.br/scielo.php?script=s...
) suggested it should be absent in 25g of the honey from SB.

Only one sample (2.8%) showed the presence of Staphylococcus spp. higher than 20CFU/g, while 97.3% of the samples had 0 to 20CFU/g. The low number of staphylococci can be explained by the aseptic collection of honey, since the presence of Staphylococcus spp. in the honey from SB is rare. Equipment and physical honey handling are considered the main sources of contamination by this microorganism (DÜMEN et al., 2013DÜMEN, E. et al. Microbiological and parasitological quality of honey produced in Ístanbul. Turkish Journal of Veterinary and Animal Sciences, v.37, p.602-607, 2013. Available from: http://dergipark.gov.tr/download/article-file/131957>. Accessed: Dez. 04, 2017. doi: 10.3906/vet-1301-46.
http://dergipark.gov.tr/download/article...
; PUCCIARELLI et al., 2014PUCCIARELLI, A.B. et al. Microbiological and physicochemical analysis of yateí (Tetragonisca angustula) honey for assessing quality standards and commercialization. Revista Argentina de Microbiología, v.46, n.4, p.325-332, 2014. Available from: https://www.sciencedirect.com/science/article/pii/S0325754114700914>. Accessed: Dez. 18, 2017. doi: 10.1016/S0325-7541(14)70091-4.
https://www.sciencedirect.com/science/ar...
). In addition, the antimicrobial activities of honey against gram-positive bacteria, specifically Staphylococcus spp., were also evaluated. Some studies attributed this effect to hydrogen peroxide, methyl-glyoxal (MGO), hydroxymethylfurfural (HMF), flavonoids, and bee-defensins (KWAKMAN et al., 2010KWAKMAN, P.H.S. et al. How honey kills bacteria. The FASEB Journal, v.24, p.2577-2582, 2010. Available from: https://www.fasebj.org/doi/pdf/10.1096/fj.09-150789>. Accessed: Jan. 11, 2018. doi: 10.1096/fj.09-150789.
https://www.fasebj.org/doi/pdf/10.1096/f...
; MERCÊS et al., 2013MERCÊS, M.D. et al. Antimicrobial activity of honey from five species of Brazilian stingless bees. Ciência Rural, v.43, n.4, p.672-675, 2013. Available from: http://www.scielo.br/scielo.php?pid=S0103-84782013000400017&script=sci_abstract&tlng=pt>. Accessed: Jan. 24, 2018.
http://www.scielo.br/scielo.php?pid=S010...
; PIMENTEL et al., 2013PIMENTEL, R.B.Q. et al. Antimicrobial activity and rutin identification of honey produced by the stingless bee Melipona compressipes manaosensis and commercial honey. BMC Complementary and Alternative Medicine, v.13, p.151, 2013. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3702439/pdf/1472-6882-13-151.pdf>. Accessed: Dez. 23, 2017. doi: 10.1186/1472-6882-13-151.
https://www.ncbi.nlm.nih.gov/pmc/article...
; NISHIO et al., 2016NISHIO, E.K. et al. Antibacterial synergic effect of honey from two stingless bees: Scaptotrigona bipunctata Lepeletier, 1836, and S. postica Latreille, 1807. Scientific Reports, v.6, p.21641, 2016. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751499/pdf/srep21641.pdf>. Accessed: Dez. 26, 2017. doi: 10.1038/srep21641.
https://www.ncbi.nlm.nih.gov/pmc/article...
).

The average count of fungi and yeast reported in the samples of this study was 9.12×103CFU/g (3.96 log10CFU/g). In Brazil, some authors working with the honey from SB considered the maximum threshold to be 100CFU/g (2.00log10CFU/g) based on national requirements for honey of Apis mellifera (BRASIL, 2000BRASIL. Instrução Normativa n. 11, de 20 de outubro de 2000. Aprova o Regulamento Técnico de Identidade e Qualidade do Mel. Diário Oficial da União, Poder Executivo, Brasília, DF. Out. 23, 2000. Seção 1, p.23.; SOUZA et al., 2009SOUZA, B.A. et al. Avaliação microbiológica de amostras de mel de trigoníneos (Apidae: Trigonini) do estado da Bahia. Ciência e Tecnologia de Alimentos, v.29, n.4, p.798-802, 2009. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0101-20612009000400015>. Accessed: Dez. 22, 2017. doi: 10.1590/S0101-20612009000400015.
http://www.scielo.br/scielo.php?script=s...
; MONTE et al., 2013MONTE, A.M. et al. Quality of honey from stingless bees native of Piaui, Brazil. Revista Brasileira de Medicina Veterinária , v.35, n.1, p.48-54, 2013. Available from: http://www.rbmv.com.br/pdf_artigos/02-07-2013_11-21RBMV%20009.pdf>. Accessed: Dez. 20, 2017.
http://www.rbmv.com.br/pdf_artigos/02-07...
). Despite this, CAMARGO et al. (2017CAMARGO, R.C.R et al. Stingless bee honey: technical regulation proposal. Brazilian Journal of Food Technology, v.20, e2016157, 2017. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1981-67232017000100900&lng=pt&tlng=pt>. Accessed: Dez. 26, 2017. doi: 10.1590/1981-6723.15716.
http://www.scielo.br/scielo.php?script=s...
) suggested a limit of 1.0×104CFU/g (4.00log10CFU/g) for the honey from SB, as the elevated humidity of honey associated with the rich fungi microbiota of SB result in a product with higher counts, as previously described (FERRAZ et al., 2008FERRAZ, R.E. et al. Microbiota fúngica de Melipona subnitida Ducke (Hymenoptera: Apidae). Neotropical Entomology, v.37, n.3, p.345-346, 2008. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1519-566X2008000300017>. Accessed: Dez. 01, 2017. doi: 10.1590/S1519-566X2008000300017.
http://www.scielo.br/scielo.php?script=s...
; MONTE et al., 2013MONTE, A.M. et al. Quality of honey from stingless bees native of Piaui, Brazil. Revista Brasileira de Medicina Veterinária , v.35, n.1, p.48-54, 2013. Available from: http://www.rbmv.com.br/pdf_artigos/02-07-2013_11-21RBMV%20009.pdf>. Accessed: Dez. 20, 2017.
http://www.rbmv.com.br/pdf_artigos/02-07...
). To date, there is no description of diseases caused by the fungi and yeast from the honey from SB. Thus, the main problem caused by their presence is the fermentation of honey, which can reduce the shelf life of the product (GRABOWSKY & KLEIN, 2017GRABOWSKY, N.T.; KLEIN, G. Microbiology and foodborne pathogens in honey. Critical Reviews in Food Science and Nutrition, v.57, n.9, p.1852-1862, 2017. Available from: <http://www.tandfonline.com/doi/abs/10.1080/10408398.2015.1029041?journalCode=bfsn20>. doi: 10.1080/10408398.2015.1029041.
http://www.tandfonline.com/doi/abs/10.10...
).

Bacillus species were reported in 34.3% of samples, including B. cereus (17.1%), B. licheniformis (11.4%), and B. subtilis (5.7%). Bacillus are common in SB’s honey once these microorganisms have a symbiotic relationship to the bees and a strong antibacterial activity against several pathogens, contributing to the stability of the product (GILLIAM, 1997GILLIAM M. Identification and roles of non-pathogenic microflora associated with honey bees. Fems Microbiology Letters, v.155, n.1, p.1-10, 1997. Available from: <http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.1997.tb12678.x/epdf>. doi: 10.1111/j.1574-6968.1997.tb12678.x/epdf.
http://onlinelibrary.wiley.com/doi/10.11...
; TORRES et al., 2015TORRES, M.J. et al. Bacillus subtilis subsp. subtilis CBMDC3F with antimicrobial activity against gram-positive foodborne pathogenic bacteria: UV-MALDI-TOF MS analysis of its bioactive compounds. World Journal of Microbiology and Biotechnology, v.31, n.6, p.929-940, 2015. Available from: https://link.springer.com/article/10.1007%2Fs11274-015-1847-9>. Accessed: Dez. 27, 2017. doi: 10.1007/s11274-015-1847-9.
https://link.springer.com/article/10.100...
). Although, toxigenic strains of B. cereus in honey have been reported, there is no evidence of honey as a vehicle for this pathogen to enter humans (LÓPEZ & ALIPPI, 2010LÓPEZ A.C.; ALIPPI A.M. Enterotoxigenic gene profiles of Bacillus cereus and Bacillus megaterium isolates recovered from honey. Revista Argentina Microbiología, v.42, n.3, p.216-25, 2010. Available from: http://www.scielo.org.ar/pdf/ram/v42n3/v42n3a14.pdf>. Accessed: Nov. 20, 2017.
http://www.scielo.org.ar/pdf/ram/v42n3/v...
; SILVA et al., 2017SILVA, M.S. et al. Microorganisms in honey. In: Toledo, V.A.A. Honey Analysis. Rijeka: In Tech, 2017. Cap.11, p.233-258.).

C. botulinum type C and C. perfringens type A were detected through PCR in 2.8% and 40.0% of samples, respectively. PUCCIARELLI et al. (2014PUCCIARELLI, A.B. et al. Microbiological and physicochemical analysis of yateí (Tetragonisca angustula) honey for assessing quality standards and commercialization. Revista Argentina de Microbiología, v.46, n.4, p.325-332, 2014. Available from: https://www.sciencedirect.com/science/article/pii/S0325754114700914>. Accessed: Dez. 18, 2017. doi: 10.1016/S0325-7541(14)70091-4.
https://www.sciencedirect.com/science/ar...
) reported that 64% of the samples tested positive for Clostridium spp. in the honey from SB. Studies investigating the presence of C. botulinum in honey from A. melifera bees are very common but, to the best of our knowledge, this is the first study to confirm the presence of C. botulinum and C. perfringens in samples of the honey from SB (NAKANO et al., 1990NAKANO, H. et al. Incidence of Clostridium botulinum in honey of various origins. Japanese Journal of Medical Science and Biology, v.43, p.183-185, 1990. Available from: https://www.jstage.jst.go.jp/article/yoken1952/43/5/43_5_183/_article>. Accessed: Dez. 20, 2017. doi: 10.7883/yoken1952.43.183.
https://www.jstage.jst.go.jp/article/yok...
; NEVAS et al., 2005NEVAS, M. et al. Prevalence and diversity of Clostridium botulinum types A, B, E and F in honey produced in the Nordic countries. International Journal of Food Microbiology, v.105, n.2, p.145-151, 2005. Available from: https://www.sciencedirect.com/science/article/pii/S016816050500320X>. Accessed: Dez. 21, 2017. doi: 10.1016/j.ijfoodmicro.2005.04.007.
https://www.sciencedirect.com/science/ar...
).

The presence of C. perfringens in the honey from SB is relevant once this product is being used for therapeutic purposes to heal wounds as this pathogen is commonly associated with gas gangrene in deep wounds (OLAITAN et al., 2007OLAITAN, P.B. et al. Honey: a reservoir for microorganisms and an inhibitory agent for microbes. African Health Sciences, v.7, n.3, p.159-165, 2007. Available from: https://www.ajol.info/index.php/ahs/article/view/7009/58269>. Accessed: Dez. 23, 2017.
https://www.ajol.info/index.php/ahs/arti...
; MERCÊS et al., 2013MERCÊS, M.D. et al. Antimicrobial activity of honey from five species of Brazilian stingless bees. Ciência Rural, v.43, n.4, p.672-675, 2013. Available from: http://www.scielo.br/scielo.php?pid=S0103-84782013000400017&script=sci_abstract&tlng=pt>. Accessed: Jan. 24, 2018.
http://www.scielo.br/scielo.php?pid=S010...
). In addition, the detection of C. botulinum is of great significance in the present study even though the number of positive samples was lower than those in honey from A. mellifera bees (NAKANO et al., 1990NAKANO, H. et al. Incidence of Clostridium botulinum in honey of various origins. Japanese Journal of Medical Science and Biology, v.43, p.183-185, 1990. Available from: https://www.jstage.jst.go.jp/article/yoken1952/43/5/43_5_183/_article>. Accessed: Dez. 20, 2017. doi: 10.7883/yoken1952.43.183.
https://www.jstage.jst.go.jp/article/yok...
; RAGAZANI et al., 2008RAGAZANI, A.V.F. et al. Esporos de Clostridium botulinum em mel comercializado no estado de São Paulo e em outros estados brasileiros. Ciência Rural , v.38, n.2, p.396-399, 2008. Available from: http://www.scielo.br/pdf/cr/v38n2/a16v38n2.pdf>. Accessed: Dez. 18, 2017. doi: 10.1590/S0103-84782008000200016.
http://www.scielo.br/pdf/cr/v38n2/a16v38...
). C. botulinum is responsible for infant botulism, which is commonly associated with honey consumption (CAGAN et al., 2010CAGAN, E. Infant botulism. The Eurasian Journal of Medicine, v.42, p.92-94, 2010. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4261338/pdf/eajm-42-2-92.pdf>. Accessed: Dez. 26, 2017.
https://www.ncbi.nlm.nih.gov/pmc/article...
; DABRITZ et al., 2014DABRITZ, H. A.; et al. Molecular epidemiology of infant botulism in California and elsewhere, 1976-2010. The Journal of Infectious Diseases, v.210, p.1711-22, 2014. Available from: https://www.ncbi.nlm.nih.gov/pubmed/24924163>. Accessed: Jan. 30, 2018. doi: 10.1093/infdis/jiu331.
https://www.ncbi.nlm.nih.gov/pubmed/2492...
). Interestingly, most cases of botulism are caused by C. botulinum type A and B. However, one case of infant botulism was caused by C. botulinum type C, the same type detected in the present study that had already been described previously (OGUMA et al., 1990OGUMA, K. et al. Infant botulism due Clostridium botulinum type C toxin. The Lancet, v.336, n.8728, 1449-1450, 1990. Available from: https://www.sciencedirect.com/science/article/pii/014067369093157K?_rdoc=1&_fmt=high&_origin=gateway&_docanchor=&md5=b8429449ccfc9c30159a5f9aeaa92ffb>. Accessed: Dez. 26, 2017. doi: 10.1016/0140-6736(90)93157-K.
https://www.sciencedirect.com/science/ar...
; BROWN & DESAI, 2013BROWN, N.; DESAI, S. Infantile botulism: a case report and review. The Journal of Emergence Medicine, v.45, n.6, p.842-845, 2013. Available from: https://www.sciencedirect.com/science/article/pii/S0736467913005131?via%3Dihub>. Accessed: Dez. 30, 2017. doi: 10.1016/j.jemermed.2013.05.017.
https://www.sciencedirect.com/science/ar...
). Thus, the detection of C. botulinum type C suggested that honey produced by jandaíra bees might pose a risk to infants under one year of age and consumers should be clearly warned about this risk on the product label (EUROPEAN COMMISSION, 2002EUROPEAN COMMISSION. Opinion of the scientific committee on veterinary measures relating to public health on honey and microbiological hazards. Health & Consumer Protection Directorate-General, 19 Jun. 2002. Available from: https://ec.europa.eu/food/sites/food/files/safety/docs/sci-com_scv_out53_en.pdf>. Accessed: Dez. 04, 2017.
https://ec.europa.eu/food/sites/food/fil...
; BRASIL, 2017BRASIL. Decreto n.9.013, de 29 de março de 2017. Regulamentação da Lei n. 1.283, de 18 de dezembro de 1950 e a Lei n. 7.889, de 23 de novembro de 1989, que dispõem sobre a inspeção industrial e sanitária de produtos de origem animal. Diário Oficial da União , Poder Executivo, Brasília, DF. Mar. 30, 2017. Seção 1, p.3.).

The present study revealed a broader microbiological profile of the honey from SB compared to that of the honey from A. mellifera, which draws attention to the need for a specific legislation for this product and the adoption of measures to reduce the number of contaminants (SILVA et al., 2008SILVA, M.B.L. et al. Qualidade microbiológica de méis produzidos por pequenos apicultores e de méis de entrepostos registrados no serviço de inspeção federal no estado de Minas Gerais. Alimentos e Nutrição, v.19, n.4, p.417-420, 2008.). Good beekeeping practices associated with conservation techniques are important in guaranteeing a product of high quality. However, more studies are necessary to evaluate the effects of such practices on the physical and chemical characteristics of the honey from SB, specifically jandaíra honey (SOUZA et al., 2009SOUZA, B.A. et al. Avaliação microbiológica de amostras de mel de trigoníneos (Apidae: Trigonini) do estado da Bahia. Ciência e Tecnologia de Alimentos, v.29, n.4, p.798-802, 2009. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0101-20612009000400015>. Accessed: Dez. 22, 2017. doi: 10.1590/S0101-20612009000400015.
http://www.scielo.br/scielo.php?script=s...
; MOURA et al., 2014MOURA, J.Z. et al. Qualidade do mel de Apis mellifera L. relacionadas às boas práticas apícolas. Revista Brasileira de Saúde e Produção Animal, v.15, n.3, p.731-739, 2014. Available from: http://www.scielo.br/scielo.php?pid=S1519-99402014000300021&script=sci_abstract&tlng=pt>. Accessed: Dez. 23, 2017. doi: 10.1590/S1519-99402014000300021.
http://www.scielo.br/scielo.php?pid=S151...
).

ACKNOWLEDGMENTS

This research was supported by funds from Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Pró-Reitoria de Pesquisa Universidade Federal de Minas Gerais (PRPq-UFMG).

REFERENCES:

  • 0
    CR-2018-0151.R1

Publication Dates

  • Publication in this collection
    2018

History

  • Received
    26 Feb 2018
  • Accepted
    10 July 2018
  • Reviewed
    31 July 2018
Universidade Federal de Santa Maria Universidade Federal de Santa Maria, Centro de Ciências Rurais , 97105-900 Santa Maria RS Brazil , Tel.: +55 55 3220-8698 , Fax: +55 55 3220-8695 - Santa Maria - RS - Brazil
E-mail: cienciarural@mail.ufsm.br