ArticlePDF Available

Determination of thermotolerant coliforms present in coconut water produced and bottled in the Northeast of Brazil

Authors:

Abstract and Figures

Coconut water is considered to be a natural isotonic drink and its marketing is gradually increasing. The objective of the present study was to evaluate the microbiological quality of the coconut water produced and bottled in the Northeast of Brazil. Products form ten industries from different states in the Northeast of Brazil were analyzed. The most probable number (MPN) method was used to quantify the coliforms. Samples showing positive for coliforms were seeded on ChromAgar Orient plates and the bacteria identified from isolated colonies using the automated system Vitek 2 (BioMérieux), according to the manufacturer's instructions for the preparation of the inoculum, incubation, reading and interpretation. The samples showed thermotolerant coliform counts between 6.0×102 and 2.6×104 MPN/100 mL. The presence of Klebsiella pneumoniae, Morganella morganii and Providencia alcalifaciens was observed. The implementation of preventive methods and monitoring of the water quality by the industries is required.
Content may be subject to copyright.
Case Study
Campinas, v. 21, e2016166, 2018
http://dx.doi.org/10.1590/1981-6723.16616
ISSN 1981-6723 on-line version
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Determination of thermotolerant coliforms present in coconut water
produced and bottled in the Northeast of Brazil
Determinação da presença de coliformes termotolerantes em águas de coco
produzidas e envasadas no Nordeste brasileiro
Abstract
Coconut water is considered to be a natural isotonic drink and its marketing is gradually increasing. The objective of
the present study was to evaluate the microbiological quality of the coconut water produced and bottled in the Northeast of
Brazil. Products form ten industries from different states in the Northeast of Brazil were analyzed. The most probable number
(MPN) method was used to quantify the coliforms. Samples showing positive for coliforms were seeded on ChromAgar
Orient plates and the bacteria identified from isolated colonies using the automated system Vitek 2 (BioMérieux), according
to the manufacturer’s instructions for the preparation of the inoculum, incubation, reading and interpretation. The samples
showed thermotolerant coliform counts between 6.0×102 and 2.6×104 MPN/100 mL. The presence of Klebsiella pneumoniae,
Morganella morganii and Providencia alcalifaciens was observed. The implementation of preventive methods and monitoring
of the water quality by the industries is required.
Keywords: Natural isotonic drink; MPN; Microbiological quality.
Resumo
A água de coco é considerada uma bebida natural isotônica e sua comercialização tem aumentado gradualmente.
O objetivo do presente estudo foi avaliar a qualidade microbiológica da água de coco produzida e engarrafada na Região
Nordeste do Brasil. Produtos de dez indústrias de diferentes estados foram analisados. Para a quantificação de coliformes, foi
aplicada metodologia de Número Mais Provável (NMP). As amostras positivas para coliformes foram semeadas em placas de
ChromAgar Orient e, a partir de colônias isoladas, foi realizada a identificação bacteriana, utilizando-se o sistema automatizado
Vitek 2 (BioMérieux), de acordo com as instruções do fabricante para a preparação do inóculo, a incubação, a leitura e a
interpretação. As amostras apresentaram contagem de coliformes termotolerantes entre 6,0×102 e 2,6×104 NMP/100 mL.
Foi observada a presença de Klebsiella pneumoniae, Morganella morganii e Providencia alcalifaciens. A implementação de
métodos de prevenção e monitoramento da qualidade da água pelas indústrias é necessária.
Palavras-chave: Bebida natural isotônica; NMP; Qualidade microbiológica.
Vandbergue Santos Pereira1*, Johnatan Wellisson da Silva Mendes2, Lorena Alves Oliveira2,
Carlos Eberton Alves Mangueira2, Edlânia Moraes Rodrigues2, Fernando Gomes Figueredo2
1 Universidade Federal do Ceará (UFC), Centro Especializado em Micologia Médica, Fortaleza/CE - Brazil
2 Centro Universitário Doutor Leão Sampaio (UniLeão), Departamento de Microbiologia, Juazeiro do Norte/CE - Brazil
*Corresponding Author
Vandbergue Santos Pereira, Universidade Federal do Ceará (UFC), Centro Especializado em Micologia Médica, Rua Coronel Nunes de Melo, 1380,
Rodolfo Teófilo, CEP: 60430-275, Fortaleza/CE - Brazil, e-mail: vandbergue.vsp@gmail.com
Cite as: Determination of thermotolerant coliforms present in coconut water produced and bottled in the Northeast of Brazil. Braz. J. Food Technol.,
v. 21, e2016166, 2018.
Received: Nov. 16, 2016; Accepted: July 11, 2017
1 Introduction
In Brazil, the coconut crop is used almost exclusively
for in nature human consumption (coconut water and culinary
use), a practice that involves issues related to transportation,
storage and perishability of the product (MARTINS; JESUS
JÚNIOR, 2011). Brazil currently ranks fourth in the ranking of
the largest coconut producers, with an estimated production of
http://bjft.ital.sp.gov.br
2
Braz. J. Food Technol., Campinas, v. 21, e2016166, 2018
Determination of thermotolerant coliforms present in coconut water produced and bottled in the Northeast of Brazil
Pereira, V. S. et al.
2.8 million tons in an area of 287,000 hectares. The Brazilian
Northeast region accounts for about 70% of this production
(JESUS JÚNIOR et al., 2013).
The continued growth of coconut water consumption
has encouraged the modernization and implementation
of new production, distribution and marketing methods,
which seek to ensure not only the continued use of the
product on the market, but also the extension of product
quality, since several studies have elucidated the risks
and propensity to contamination of the fresh fruit market
due to the precariousness of food handling (FORTUNA;
FORTUNA, 2008).
In Brazil, the agency responsible for the regulation of
the coconut water production is the Ministry of Agriculture,
Livestock and Food Supply, which relates the required
microbiological characteristics of coconut water submitted
to industrial processes and destined for human use as a
beverage in norm nº 27 of July 22nd, 2009. It advocates the
absence of Salmonella spp. and the presence of coliforms
should not exceed 102 cells/100 mL (BRASIL, 2009).
Bacteria belonging to the faecal coliform group have
a great impact as indicators of faecal contamination. These
bacteria belong predominantly to the gastrointestinal tract,
with the fermentation of lactose to acid and gas production at
temperatures between 44.5 to 45.5 °C as their main metabolic
characteristic. Escherichia, Enterobacter and Klebsiella
are the major genera in this group (RECHE et al., 2010).
The microbiological analysis of coconut water samples
is crucial to public health, since the hygienic and sanitary
characteristics during the processing of these products are
directly related to consumer health. Thus, the aim of the
present study was to evaluate the microbiological quality
of coconut water produced and bottled in the Northeast
of Brazil.
2 Material and methods
2.1 Sampling
Bottled coconut water samples from ten manufacturers
distributed throughout the Northeast of Brazil, in the states
of Alagoas (AL), Ceará (CE), Paraíba (PB), Pernambuco
(PE) and Rio Grande do Norte (RN), were used in this study.
Two batches were obtained from each manufacturer and
three samples were examined per batch, giving a total of
60 samples, including shelf-stable and fresh refrigerated
product samples, as described in Table 1. All the tests
were carried out in the Microbiology Laboratory and other
support laboratories at the Leão Sampaio School – Health
Campus in Juazeiro do Norte-CE.
2.2 Determination of thermotolerant coliforms
The multiple-tube technique (APHA, 2015; SILVA et al.,
2010b) was used to determine the coliforms. For this analysis,
100 mL of each sample was seeded into tubes containing
lactose broth and incubated at 37 °C for 24 h, as well as
into EC broth with incubation at 44-45 °C for 24 h. The most
probable number (MPN) method was used for quantification
(SILVA et al., 2010b).
2.3 Isolation and identification of the
microorganisms found
Samples from lactose broth tubes showing growth
were seeded onto CHROMagar Orientation medium
(BD Diagnostics, Franklin Lakes, NJ), re-isolated when
necessary, and then tested for Gram coloration performed
for their classification as gram positive or gram negative
bacteria. The bacteria were identified from isolated colonies
using the automated system Vitek 2 (BioMérieux) according
to the manufacturer’s instructions for the preparation of the
inoculum, incubation, reading and interpretation.
3 Results
Table 1
shows the results for the microbiological
analysis of the thermotolerant coliforms present in the
coconut water obtained from the different manufacturers,
produced and bottled in the Northeast of Brazil. Of the ten
industries analyzed, six did not conform to the microbiological
standards determined by the legislation.
Table 2
shows the results of the biochemical
identification of the contaminating bacteria present in the
coconut water samples as determined by the Vitek 2 system.
The presence of three different bacterial species was
observed: Klebsiella pneumoniae; Providencia alcalifaciens
and Morganella morganii.
4 Discussion
The results observed for the presence of thermotolerant
coliforms showed samples which did not conform to norm
nº 27 of the Ministry of Agriculture, Livestock and Food
Table 1. Microbiological analysis of faecal coliforms present in
coconut water from the Northeast of Brazil.
Samples
State
1
st
Batch
(MPN/100 mL)
2
nd
Batch
(MPN/100 mL)
Classificationc
Industry A
a
CE 2.4×10
4
6×10
2
Improper
Industry B
a
CE 1.4×10
4
1.4×10
4
Improper
Industry C
a
PE 1.7×10
3
3.3×10
3
Improper
Industry D
a
PE 3.2×10
3
2.6×10
4
Improper
Industry E
a
PB 1.7×10
3
3.3×10
3
Improper
Industry F
a
RN 1.4×10
3
1.4×10
3
Improper
Industry G
b
RN <10
2
<10
2
Proper
Industry H
a
AL <10
2
<10
2
Proper
Industry I
b
AL <10
2
<10
2
Proper
Industry J
a
PB <10
2
<10
2
Proper
Results expressed as the arithmetic mean of quantifications carried
out in triplicate;
a
Shelf-stable product;
b
Fresh refrigerated product;
c
Limit permitted by the legislation: 10
2
MPN/100 mL.
http://bjft.ital.sp.gov.br
3
Braz. J. Food Technol., Campinas, v. 21, e2016166, 2018
Determination of thermotolerant coliforms present in coconut water produced and bottled in the Northeast of Brazil
Pereira, V. S. et al.
Supply (BRASIL, 2009) which regulates the microbiological
characteristics of the food and demands the absence
of Salmonella sp. and the presence of thermotolerant
coliforms below the limit of 102 cells/mL for coconut water.
Lima et al. (2015), when analysing industrialized coconut
water produced in the states of Ceará and Paraíba, found
similar results for the presence of species of this group.
The presence of these organisms indicates contact with
faecal contaminants during the filling process, which may
be associated with a deficit in the control of manipulation.
Another highlight of this study was the absence of
bacteria reported as potential contaminants of water and
food in the microbiological analysis, such as Escherichia coli
and Salmonella sp. In a study of the microbiological quality
of coconut water marketed in Currais Novos/RN, Silva et al.
(2010a) obtained similar results regarding the absence
of those microorganisms, which may be explained by
the reduction in temperature during both the production
(24 ºC) and storage processes (5 ºC).
The presence of Klebsiella pneumoniae was observed
amongst the thermotolerant coliforms, which is associated
with gastrointestinal disorders. A study by Santos et al.
(2005) showed the presence of K. pneumoniae in 80% of
the food samples prepared by hand manipulation. These
bacteria were found mainly in samples containing fresh
fruit as a major element of their compositions. This species
is indicated as a cause of meningitis, endocarditis and
pneumonia, amongst other diseases of the respiratory
tract (FORSYTHE et al., 2015).
Moreover a significant presence of Morganella morganii
and Providencia alcalifaciens was detected, organisms
occasionally found in food contamination or on inanimate
surfaces where food manipulation occurs. They are clinically
associated with gastroenteritis, P. alcalifaciens being more
associated with intestinal infections in children. This species
has been recovered from urine, stool, throat, perineum,
axilla, blood, and wound specimens (O’HARA et al., 2000).
The contamination of coconut water commercialized
in cups with thermo welded lids may be associated with
poor storage conditions of the product or contamination
of the raw material used in production, since all the
processing of this type of packaging is carried out
using specific industrial equipment which minimizes the
chances of contamination during the production process.
Regarding the long-life packed samples analyzed, all the
microbiological parameters conformed with the legislation.
Long-life packages are aseptic, ideal for the packaging
of food due to their structural composition, allowing for
better food preservation (RAI; SHARMA, 2014).
5 Conclusions
Of the samples obtained from the ten industries
analyzed, half did not conform to the microbiological
standards established by the legislation with respect to
the presence of faecal coliforms amongst other potentially
pathogenic microorganisms, which makes the product
improper for consumption. It is worth noting the absence of
Salmonella spp., Escherichia coli, and Staphylococcus spp.
in all the samples.
The results suggest that contamination may have
occurred due to the use of contaminated raw material and
inadequate storage times and temperatures, important
factors in maintaining the microbiological quality of any
food. It was noticed that long-life packaging had the
greatest potential for conservation of the microbiological
characteristics of coconut water.
It is recommended that the public entities enforce
the use of preventive actions by the industries to stimulate
better practices in production, as well as the adoption of
control methods aimed at excellence in product quality,
with a view to offering products that do not offer any risk
to public health.
Acknowledgements
This work was made possible due to financial and
technical support from University Center Doctor Leão
Sampaio (UniLeão), Ceará, Brazil.
References
AMERICAN PUBLIC HEALTH ASSOCIATION – APHA. Compendium
of methods for the microbiological examination of foods.
Washinton: APHA, 2015.
BRASIL. Ministério da Agricultura, Pecuária e Abastecimento.
Normativa nº 27, de 22 de julho de 2009. Estabelece os
procedimentos mínimos de controle higiênico-sanitário, padrões
de identidade e características mínimas de qualidade gerais
Table 2. Biochemical identification of the bacterial contaminants in coconut water from the Northeast of Brazil by the VITEK2 system.
Samples State 1
st
Batch 2
nd
Batch
Industry A CE Klebsiella pneumoniae Klebsiella pneumoniae
Industry B CE Providencia alcalifaciens Morganella morganii
Industry C PE Klebsiella pneumoniae Klebsiella pneumoniae
Industry D PE Morganella morganii Morganella morganii
Industry E PB Klebsiella pneumoniae Klebsiella pneumoniae
Industry F RN Morganella morganii Morganella morganii
http://bjft.ital.sp.gov.br
4
Braz. J. Food Technol., Campinas, v. 21, e2016166, 2018
Determination of thermotolerant coliforms present in coconut water produced and bottled in the Northeast of Brazil
Pereira, V. S. et al.
para a água de coco. Diário Oficial [da] República Federativa
do Brasil, Brasília, DF, 2009.
FORSYTHE, S. J.; ABBOTT, S. L.; PITOUT, J. Klebsiella,
Enterobacter, Citrobacter, Cronobacter, Serratia, Plesiomonas,
and other Enterobacteriaceae. In: AMERICAN SOCIETY OF
MICROBIOLOGY – ASM. Manual of clinical microbiology.
10th ed. Washington: ASM, 2015. p. 714-737. http://dx.doi.
org/10.1128/9781555817381.ch38.
FORTUNA, D. B. S.; FORTUNA, J. L. Avaliação da qualidade
microbiológica e higiênico sanitária da água de coco comercializada
em carrinhos ambulantes nos logradouros do município de
Teixeira de Freitas (BA). Revista Baiana Saúde Pública, v. 32,
p. 203-217, 2008.
JESUS JÚNIOR, L. A.; TOMMASI, A. C.; OLIVEIRA JÚNIOR,
A. M.; RUSSO, S. L. Análise da produção de coco no Estado
de Sergipe frente ao crescimento da cultura no Nordeste e no
Brasil. Revista GEINTEC, v. 3, p. 400-408, 2013.
LIMA, S. A. J.; MACHADO, A. V.; CAVALCANTI, M. T.; LISBÔA, C.
G. C. Análise sensorial da água de coco anão verde processada
e comercializada no sertão paraibano e cearense. Revista Verde
de Agroecologia e Desenvolvimento Sustentável, v. 10, n. 2,
p. 165-169, 2015. http://dx.doi.org/10.18378/rvads.v10i1.3466.
MARTINS, C. A.; JESUS JÚNIOR, L. A. Evolução da produção
de coco no Brasil e o comércio internacional: panorama 2010.
Aracaju: Embrapa Tabuleiros Costeiros, 2011.
O’HARA, C. M.; BRENNER, F. W.; MILLER JUNIOR, M. Classification,
identification, and clinical significance of Proteus, Providencia,
and Morganella. Clinical Microbiology Reviews, v. 13, n. 4,
p. 534-546, 2000. PMid:11023955. http://dx.doi.org/10.1128/
CMR.13.4.534-546.2000.
RAI, M. M.; SHARMA, T. R. Some recent developments in food
packaging. Defence Science Journal, v. 16, n. 4A, p. 141-146,
2014.
RECHE, M. H. L. R.; PITTOL, M.; FIUZA, L. M. Bactérias e
bioindicadores de qualidade de águas de ecossistemas orizícolas
da Região Sul do Brasil. Oecologia Australis, v. 14, n. 2, p.
452-463, 2010. http://dx.doi.org/10.4257/oeco.2010.1402.08.
SANTOS, B. H.; COSTA, A. C.; SOUZA, E. L. D. SOUSA, C. P.
D. Klebsiella pneumoniae como agente contaminante de dietas
enterais artesanais. Higiene Alimentar, v. 19, n. 131, p. 58-60,
2005.
SILVA, J. L. A.; DANTAS, F. A. V.; SILVA, F. C. Qualidade
microbiológica de águas de coco comercializadas no município
de Currais Novos/RN. Holos, v. 34, n. 41, p. 34-41, 2010a. http://
dx.doi.org/10.15628/holos.2009.255.
SILVA, N.; JUNQUEIRA, V. C. A.; SILVEIRA, N. F. A.; TANIWAKI,
M. H.; SANTOS, R. F. S.; GOMES, R. A. R. Manual de métodos
de análise microbiológica de alimentos e água. 4. ed. São
Paulo: Varela, 2010b.
ResearchGate has not been able to resolve any citations for this publication.
Article
Full-text available
O Brasil, atualmente, possui uma grande tendência de crescimento do cultivo do coqueiro anão verde, onde o principal objetivo deste se da para a produção da água de coco, sua cultura esta distribuída em todo o território nacional, destacadamente na região Nordeste onde se concentram os maiores plantios do país. A água de coco é um produto natural que vem ganhando espaço no mercado como uma bebida de vasto potencial comercial, pois além de ser um produto natural é praticamente livre de contaminações, possui baixo teor calórico, considerável valor nutricional e apresenta aroma e sabor suaves e agradáveis. Neste contexto se faz necessário a avaliação da qualidade sensorial da água de coco industrializada e comercializada de forma a garantir a qualidade do produto para o consumidor. Com isso este trabalho objetivou avaliar sensorialmente á água de coco anão verde, produzidas comercialmente e envasadas, por indústrias do Sertão Paraibano e do Ceará. As amostras de água de coco proveniente das indústrias, foram analisadas sensorialmente no laboratório de microbiologia do CVT – UFCG - Pombal, utilizando o teste de comparação múltiplas utilizando uma escala hedônica de 9 pontos (9 = gostei extremamente, até 1 desgostei), também foi aplicado o teste de intenção de compra utilizando-se uma escala estruturada de 5 pontos (1 = certamente compraria, até 5 = Certamente compraria), conforme metodologia do Instituto Adolfo Lutz. Os resultados dos testes somente se deram até o 15 dias de armazenamento, onde 70% dos copos de água de coco armazenados, apresentaram-se turvos e com produção de gás, demonstrando apartir deste ponto impróprios para o consumo. Com 30 dias de armazenamento todas as amostras apresentaram insatisfatórios para consumo, demonstrando resultados preocupantes quanto a estabilidade da qualidade do produto durante sua validade comercial. Sugere-se como forma de garantia e manutenção da qualidade da água de coco à implantação e monitoramento de Boas Práticas de Fabricação para as unidades produtoras.
Article
Full-text available
A água de coco é uma bebida obtida do fruto verde de coqueiro, sendo um líquido refrescante, isotônico e cada vez mais consumido por suas características organolépticas e nutricionais. As etapas de extração e engarrafamento são bastante susceptíveis ao ataque microbiano resultando em perda da qualidade do produto e conseqüente diminuição do seu valor comercial. O presente trabalho teve como objetivo verificar a qualidade microbiológica da água de coco engarrafada e comercializada na cidade de Currais Novos/RN. Observou-se que, das 22 amostras analisadas, a presença de coliformes totais foi verificada em 82% das amostras (dezoito amostras) sugerindo o contato do coco com o habitat da referida bactéria bem como falhas no processo de higienização do fruto. Com relação à contagem de coliformes fecais, 22,7% apresentava-se fora dos padrões estabelecidos pela Legislação Brasileira, no entanto não foi constatada a presença de Salmonella sp e Staphylococcus coagulase positiva. Bactérias mesófilas e bolores e leveduras foram encontrados em níveis de 6,0 x 10 UFC/mL a 3,0 x 105 UFC/mL e de zero a 1,7 x 105 respectivamente, sugerindo a necessidade de melhorias durante o processo de envase de água de coco verde resfriada.
Article
Full-text available
The presence of heterotrophic bacteria in aquatic environments, such as those in the agricultural systems of irrigated rice fields, is determined by biotic and abiotic processes, with the focus on physical-chemical factors and highly complex interactions with other organisms. In this study, we address aspects related to these bacteria, such as: bacterial diversity in rice growing areas, total coliforms and thermotolerant as environmental indicators. Are also mentioned topics related to natural and artificial wetlands, the viability of enteric bacteria, the environmental impacts, reuse and monitoring of water in rice fields ecosystems.
Article
Full-text available
This review presents the current taxonomy of the genera Proteus, Providencia, and Morganella, along with the current methods for the identification of each species within the three genera, incorporating both conventional biochemical and commercial methods. While all of these organisms are ubiquitous in the environment, individual case reports and nosocomial outbreak reports that demonstrate their ability to cause major infectious disease problems are presented. Lastly, anticipated antimicrobial susceptibility patterns are reviewed. Many of these organisms are easily controlled, but the advent of newer and more powerful antimicrobial agents has led to some problems of which laboratorians need to be aware.
Article
The paper deals with various types of recently developed food packing materials specially suitable for dehydrated food-stuffs. Though mettalic cans continue to enjoy important place in packing industry, laminated flexible pouches of desired machanical strength and durability have also been found to be equally suitable with less weight and cost.
Klebsiella pneumoniae como agente contaminante de dietas enterais artesanais
  • B H Santos
  • A C Costa
  • E L D Souza
  • C P Sousa
SANTOS, B. H.; COSTA, A. C.; SOUZA, E. L. D. SOUSA, C. P. D. Klebsiella pneumoniae como agente contaminante de dietas enterais artesanais. Higiene Alimentar, v. 19, n. 131, p. 58-60, 2005.