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Bacterial contaminants in carbonated soft drinks sold in Bangladesh markets
International Journal of Food Microbiology 130 (2009) 156–158
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o
Short communication
Bacterial contaminants in carbonated soft drinks sold in Bangladesh markets Muhammad Ali Akond a,b,⁎, Saidul Alam a, S.M.R. Hasan a, Sanzida Mubassara a, Sarder Nasir Uddin c, Momena Shirin d a
Department of Botany, Jahangirnagar University, Dhaka-1342, Bangladesh Lab of Environmental Bioscience, Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh d Institute of Public Health, Mohakhali, Dhaka-1212, Bangladesh b c
a r t i c l e
i n f o
Article history: Received 16 September 2008 Received in revised form 16 January 2009 Accepted 19 January 2009 Keywords: Bangladesh Carbonated soft drinks Public health risks Pathogenic bacteria Coliforms
a b s t r a c t A total of 225 carbonated soft drink (CSD) samples from nine brands, from various locations in five metropolitan cities of Bangladesh were examined to determine their bacteriological quality. Most samples were not in compliance with microbiological standards set by organizations like the World Health Organization (WHO). Pseudomonas aeruginosa was the predominant species with an incidence of 95%. Streptococcus spp. and Bacillus stearothermophilus were the next most prevalent with numbers ranging from 6 to 122 and 9 to 105 cfu/100 ml, respectively. Fifty four percent of the samples yielded Salmonella spp. at numbers ranging from 2 to 90 cfu/100 ml. Total coliform (TC) and faecal coliform (FC) counts were found in 68–100% and 76–100% of samples of individual brands, at numbers ranging from 5 to 213 and 3 to 276 cfu/ 100 ml, respectively. According to WHO standards 60–88% of samples from six brands and 32% and 40% of samples from two other brands belonged to the intermediate risk group with FC counts of 100–1000 cfu/ 100 ml. Heterotrophic plate counts, however, were under the permissible limit in all 225 samples. These findings suggest that carbonated soft drinks commercially available in Bangladesh pose substantial risks to public health. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Carbonated soft drinks (CSDs) were introduced in Bangladesh over 35 years ago, and have since become popular in both cities and rural areas. Since 1996 there have been no standards for, or definition of, soft drinks in the British and EU regulations, except in special cases (Carbonated Drinks, 2004). Protection of all types of drinks from hazardous microbial contaminants is a global issue. Various gastrointestinal illnesses are the most common consequences of consuming contaminated drinks (Frobisher et al., 1974). Good manufacturing practices (GMPs) are not always followed in the food and beverage industries, particularly in developing countries, and this can result in wide variations in the microbial quality of products, such as bottled water and other drinks (Akond et al., 2006; Hara et al., 2005; Venieri et al., 2006). Carbonated soft drinks and bottled waters sold in Bangladesh are claimed to be of excellent microbiological quality in their sales promotion advertisements in both print and electronic media (Akond et al., 2006). This is in contradiction, however, to newspaper reports of extensive algal growth in carbonated soft drink bottles (BELA, 1997; Jaijai Din, 2008; Janakantha, 2004), indicating that the microbiological quality of soft
⁎ Corresponding author. Lab of Environmental Bioscience, Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan. Tel.: +81 80 3056 7275. E-mail address: [email protected] (M.A. Akond). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.01.014
drinks in Bangladesh may actually be poor. Therefore, the present study was undertaken to determine the bacterial quality of some non-alcoholic, carbonated soft drinks available at retail outlets in Bangladesh. 2. Materials and methods 2.1. Sampling Intact containers (plastic and/or glass bottles or aluminum cans) from nine brands of CSD were purchased from various retail outlets in five cities (Dhaka, Khulna, Rajshahi, Chittagong and Barishal) in Bangladesh between August 2006 and March 2008. Contamination with dust during collection and transportation was avoided by placing containers in a sterilized foam box. The containers were washed with sterile distilled water then sterilized by spraying and rubbing with 98% alcohol before opening. Nine brands of carbonated soft drinks were designated arbitrarily CSD-1 to CSD-9. Five containers of each brand from every collection site were examined. 2.2. Bacteriological analysis A heterotrophic plate count (HPC), total coliform count (TCC), faecal coliform count (FCC), and faecal Streptococcus, Bacillus stearothermophilus, Pseudomonas aeruginosa, Salmonella and Shigella
M.A. Akond et al. / International Journal of Food Microbiology 130 (2009) 156–158
157
Table 1 Prevalences (%) of total coliform (TC), faecal coliform (FC) and other bacteria in groups of 25 samples of carbonated soft drinks from Bangladesh Brand Code
Number of positive samples TC
FC
Salmonella
Streptococcus
Pseudomonas
Bacillus
CSD-1 CSD-2 CSD-3 CSD-4 CSD-5 CSD-6 CSD-7 CSD-8 CSD-9 Total
17 (68) 23 (92) 25 (100) 23 (92) 23 (92) 22 (88) 25 (100) 22 (88) 25 (100) 205 (91.11)
19 (76) 22 (88) 25 (100) 23 (92) 22 (88) 20 (80) 25 (100) 22 (88) 24 (96) 202 (89.78)
12 (48) 13 (52) 18 (72) 9 (36) 12 (48) 16 (64) 15 (60) 13 (52) 14 (56) 122 (54.22)
24 (96) 25 (100) 25 (100) 25 (100) 17 (68) 25 (100) 25 (100) 25 (100) 21 (84) 212 (94.22)
22 (88) 25 (100) 24 (96) 25 (100) 24 (96) 25 (100) 25 (100) 25 (100) 19 (76) 214 (95.11)
14 (56) 18 (72) 3 (12) 24 (96) 19 (76) 14 (56) 12 (48) 18 (72) 11 (44) 133 (59.11)
counts were obtained for each sample. Heterotrophic bacteria were enumerated by spread plating 0.1 ml and pour plating a 1.0 ml volume of each sample undiluted and in ten-fold dilutions to 10− 3. Other bacteria were enumerated by filtering 10 and 100 ml volumes of each undiluted sample through 0.22 µm membrane filters (Millipore, Bedford, MA, USA), and incubating the filters on selective agars. All bacteria were enumerated on triplicate plates or filters. The culture media used for HPC, TCC, FCC, and faecal Streptococcus, B. stearothermophilus, P. aeruginosa, Salmonella and Shigella counts were, respectively, Nutrient agar (Difco, Becton-Dickinson, Sparks, MD, USA), MacConkey agar (Difco), Membrane Faecal Coliform (MFC) agar (Hi-Media, Mumbai, India), Kenner Faecal Streptococcus (KF-Streptococcus) agar (Hi-Media), 2,3,5-triphenyl tetrazolium chloride (TTC) agar (Sartorius, Goettingen, Germany), Cetrimid agar (Oxoid, Basingstoke, Hampshire, UK), Bismuth Sulfite agar (Oxoid) and Salmonella-Shigella (SS) agar (Oxoid). MFC, SS and TTC agar plates were incubated at 44 °C, 42 °C and 55 °C, respectively, for 24 to 72 h. All other plates were incubated at 37 °C for 24 to 72 h. Characteristic colonies grown on selective and differential agars were confirmed by the morphological and biochemical tests of Buchanan and Gibbons (1974). The tests included gram staining, spore staining, and tests for catalase, coagulase, oxidase, IMViC reactions, starch hydrolysis, sugar fermentation, and nitrate reduction. Salmonella spp., Escherichia coli, P. aeruginosa, and Streptococcus spp. were further confirmed by latex agglutination tests using polyvalent antisera (DENKA SEIKEN Co. Ltd, Tokyo, Japan). Only strong agglutination occurring within 1 min was considered to be a positive reaction. 2.3. Statistical analysis Statistical analyses of the data were carried out using SPSS 11.5 and Microsoft Excel 2003 to calculate means, medians, standard devia-
tions, least significant differences (LSD) and correlations, and to check distribution normality of data sets using Anderson–Darling test. 3. Results and discussion All samples were positive for heterotrophic bacteria (Table 1). The HPCs ranged from 3 to 242 cfu/ml (Table 2), all of which were under the maximum permissible limit of 500 cfu/ml set by the United States Environmental Protection Agency (US EPA, 2003). The low HPC might be due to the incubation temperature of 37 °C, as it has been claimed that the numbers of bacteria recovered from drinks decreases with increasing incubation temperature, from 25 to 35 °C (Armas and Sutherland, 1999; Reasoner, 2004; Rosenberg, 2003; Venieri et al., 2006). However, a 13-fold increase in aerobic bacterial counts of Nigerian carbonated soft drinks was obtained when culture media were incubated at 34 °C rather than 28 °C (Efiuvweuwere and Chynyere, 2001). Several pathogenic and/or opportunistically pathogenic bacteria like Salmonella, P. aeruginosa, and Streptococcus, and B. stearothermophilus were present in CSDs at numbers ranging from 2 to 90, 4 to 162, 6 to 122, and 9 to 105 cfu/100 ml, respectively (Table 2). Salmonella enteritidis, S. typhimurium, E. coli, Pseudomonas spp. and Streptococcus spp. were confirmed by polyvalent antisera agglutination tests. The ranges for TC and FC counts were 5 to 213 and 3 to 276 cfu/ 100 ml, respectively. Ninety percent of samples were non-compliant with the guidelines of the World Health Organization (WHO, 1997); brands CSD-3 and CSD-7 were also 100% non-compliant with guideline values for faecal coliform (Table 3). Sixty five percent of the samples were in the intermediate risk group, while 4% of samples were in the high risk group. Such high counts were found by Kohnen et al. (2005) for both coliform and Pseudomonas spp. in 39 and 12% of samples of home-prepared carbonated water, respectively, as compared to only 12 and 4%, respectively, of samples of tap water used for
Table 2 Medians (ranges) of numbers of bacteria recovered from groups of 25 samples of nine carbonated soft drinks from Bangladesh Brand code CSD-1 CSD-2 CSD-3 CSD-4 CSD-5 CSD-6 CSD-7 CSD-8 CSD-9 a b c d
Median and range for bacterial counts (cfu/100 ml) HPCa 0.8 (0.15–3.25) 12.2 (2.9–23) 3.2 (1.6–10)d 7.2 (2.2–24)d 4 (2.3–8) 1.4 (0.4–3.4)d 7.5 (4–10) 8 (1–24.2)d 7 (1–17)
TCb d
d
9.0 (0–46) 27 (0–213)d 27 (9–48) 28 (0–53) 35 (0–71) 16 (0–51)d 32 (8–146)d 56 (0–110) 32 (9–90)d
Heterotrophic plate count, cfu × 103/100 ml. Total coliform. Faecal coliform. Data set not normally distributed.
FCc
Salmonella
Streptococcus
Pseudomonas
Bacillus
8 (0–22)d 20 (0–37) 20 (6–276)d 20 (0–50) 15 (0–45) 9 (0–49)d 21 (5–46) 34 (0–92) 45 (0–160)d
0 (0–58)d 13 (0–87) d 12 (0–52) d 0 (0–17)d 0 (0–45)d 8 (0–27)d 8 (0–69)d 2 (0–20)d 12 (0–90)d
14 (0–33) 88 (12–122)d 27 (9–60)d 19 (6–51) 8 (0–29)d 13 (6–40)d 33 (8–83) 61 (12–83) 11 (0–19)
22 (0–58) 81 (28–128) 20 (0–43) 33 (12–66) 27 (0–61) 29 (9–59) 38 (9–107) 132 (54–162)d 12 (0–36)
9 (0–23)d 22 (0–62) 0 (0–12)d 51 (0–105) 12 (0–45) 9 (0–25)d 0 (0–33)d 21 (0–58) 0 (0–22)d
158
M.A. Akond et al. / International Journal of Food Microbiology 130 (2009) 156–158
Table 3 Numbers of samples (%), in groups of 25, positive for World Health Organization risk groups based on faecal coliform (FC) counts Brand code
Risk category Nonea
Lowb
Intermediatec
Highd
CSD-1 CSD-2 CSD-3 CSD-4 CSD-5 CSD-6 CSD-7 CSD-8 CSD-9 Total
6 (24) 3 (12) 0 (0.0) 2 (8.0) 3 (12) 5 (20) 0 (0.0) 3 (12.0) 1 (4.0) 23 (10.22)
11 2 4 1 5 10 6 3 4 46
8 (32) 20 (8) 16 (64) 22 (88) 17 (68) 10 (40) 19 (76) 19 (76) 15 (60) 146 (64.89)
0 0 5 (20) 0 0 0 0 0 5 (20) 10 (4.44)
a b c d
(44) (8.0) (16) (4.0) (20) (40) (24) (12) (16) (20.44)
FC = 0. FC = 1–10 cfu/100 ml. FC = 10–100 cfu/100 ml. FC = 100–1000 cfu/100 ml.
be causes for the high counts. Extensive algal growth and the presence of sediments in bottles of carbonated soft drinks have been reported in Bangladeshi newspapers (BELA, 1997; Jaijai Din, 2008; Janakantha, 2004), which also indicates the poor quality of carbonated soft drinks. The presence of such materials and the poor microbiological quality reported in the current study are the result of failure to maintain the acceptable quality and to adequately maintain the regulations of Bangladesh Government in the manufacture of carbonated soft drinks. Immediate action to improve the microbiological quality of carbonated beverages in Bangladesh is evidently required. Acknowledgement Authors are thankful to the Lab of Microbiology, Department of Botany, Jahangirnagar University, and Bacteriology Laboratory, Institute of Public Health, Dhaka, Bangladesh. References
their preparation. The high rate of contamination in the carbonated waters was likely due to biofilms in the water bottles. According to WHO (1997) and the International Commission on Microbiological Specifications for Foods (1986), bottled water should be produced without contamination by coliforms and most Pseudomonas. LSD analysis showed significant differences between the mean counts of HPC, TC and FC in most samples (p = 0.01). There was no correlation between the numbers of any two groups of organisms (r = 0.47), except for weak correlations between TC and FC for brands CSD-8 (r = 0.66) and CSD-5 (r = 0.58) and between B. stearothermophilus and P. aeruginosa for brand CSD-5 (r = 0.59). The fractions of the 225 samples positive for other bacteria ranged from 54% for Salmonella spp. to 95% for P. aeruginosa (Table 1). In a Canadian study, Warburton et al. (1998) reported b1 cfu/ml of Pseudomonas spp. in carbonated bottled mineral water in all samples tested. Though Pseudomonas is frequently noticed in bottled water and drinking water (Rosenberg, 2003), it is not normally a health hazard, however, it can cause infections to immunocompromised individuals. Reports of Salmonella spp. in bottled water or carbonated beverages are uncommon. The high counts of Salmonella spp. in carbonated soft drinks in Bangladesh are obviously a risk for public health. The presence of Salmonella spp. is an indication of enteric contamination and may have been introduced to these soft drinks during bottling and/or manufacturing, due to lack of personal hygiene. Of the total samples in the current study, 59% were positive for B. stearothermophilus. This high rate of Bacillus spp. was also found in Cola samples from Nigeria, with incidence of up to 81%. Given the environmental occurrence, as well as the osmotic, thermal and salt stress tolerance of the bacteria, Bacillus spp. could be introduced in these drinks from either or both the environment and/or from raw materials (Efiuvweuwere and Chynyere, 2001; Oranusi et al., 1994). The presence of B. stearothermophilus, which is used as an indicator in sterilization validation tests, indicates improper treatment of water used in the production of these drinks. This is in negligence of the regulations of the Bangladesh Government, which states that the water to be used in the production of carbonated soft drinks must be drawn from an underground depth of at least 300 ft, it must not contain heterotrophic bacteria at more than 50 cfu/ml, and must be negative for all TC and FC counts (BSTI, 2001). The presence of high numbers of TC, FC and various harmful bacteria, especially Salmonella spp., in carbonated soft drinks in Bangladesh poses a serious public health risk. Poor manufacturing processes, and ingredient and/or water source contamination may all
Akond, M.A., Alam, S., Shil, A., Hasan, S.M.R., 2006. Bacteriological quality of bottled water available commercially in Bangladesh. Journal of Environmental Sciences (Dhaka) 4, 47–52. Armas, A.B., Sutherland, J.P., 1999. A survey of the microbiological quality of bottled water sold in the UK and changes occurring during storage. International Journal of Food Microbiology 48, 59–65. BELA, 1997. PIL (List of Public Interest Litigation), No. 11, Dr. Mohiuddin Farooque v. Bangladesh & others, Writ Petition No. 867/97 (Contaminated Drink). Bangladesh Environmental Lawyers Association (BELA), Segunbagicha, Dhaka. http://www. belabangla.org/html/pil.htm. Accessed on December 30, 2008. BSTI, 2001. Standard for Bottled Drinking Water/ Soft Drinks. BDS:1240, 2001. Bangladesh Standard Testing Institute (BSTI), Dhaka. Buchanan, R.E., Gibbons, N.E. (Eds.), 1974. Bergey's Manual of Determinative Bacteriology, 8th ed. Williams and Willkins Co., Baltimore. Carbonated Drinks, 2004. British Soft Drinks Association, 20–22 Stukeley Street, London. http://www.liquidsmeanlife.org.uk/member/KS3&4%20new%20material/ Teachers%20notes/Carbonated%20Drinks.pdf. Accessed on December 15, 2008. Daily Jaijai Din, 2008. Soft Drinks Shorirer Jonno Soft Noy (Soft Drinks are not soft for human body), February 14, 2008. Jaijai Din Publications Ltd., Tejgaon, Dhaka, Bangladesh, p. 16. Daily Janakantha, 2004. Coker Botole Shaola (Algae in bottle of Coke), March 23, 2004. Janakantha Ltd., Janakantha Bhaban, New Eskaton Road, Dhaka, Bangladesh, p. 1. Efiuvweuwere, B.J.O., Chynyere, M., 2001. The microbiology and deterioration of soft drinks subjected to two different marketing. Global Journal of Pure and Applied Sciences 7, 43–48. Frobisher, M., Hinsdill, R.D., Crabtree, K.T., Goodheart, C.R., 1974. Fundamentals of Microbiology, 9th ed. W.B. Saunders Company, Philadelphia. Hara, M., Fukuyama, M., Furuhata, K., 2005. Isolation of thermotolerant acidophilic bacteria (TAB) from fruit juices. Journal of Antibacterial and Antifungal Agents 9, 447–452. International Commission on Microbiological Specifications for Foods, 1986. Microorganisms in foods, 2, Sampling Plants for Natural Mineral Waters, other bottled waters, Process Waters and Ice, 2nd ed. Blackwell Scientific Publications, London. Kohnen, W., Teske-Keiser, S., Meyer, H.-G., Loos, A.H., Pietsch, M., Jansen, B., 2005. Microbiological quality of carbonated drinking water produced with in-home carbonation system. International Journal of Hygiene and Environmental Health 208, 415–423. Oranusi, S.U., Ezeogu, L.I., Okolo, B.N., 1994. Microbial contaminants of commercially bottled non-alcoholic drinks produced in Nigeria. World Journal of Microbiology and Biotechnology 10, 488–490. Reasoner, D.J., 2004. Heterotrophic plate count methodology in the United States. International Journal of Food Microbiology 92, 307–315. Rosenberg, F.A., 2003. The microbiology of bottled water. Clinical Microbiology Newsletter 25, 41–44. US EPA, 2003. List of drinking water contaminants and maximum contaminant levels, USEPA. Office of GWDW, EPA, Washington, DC. Publication No. 816-F-03-016. Venieri, D., Vantarakis, A., Komninou, G., Papapetropoulou, M., 2006. Microbiological evaluation of bottled non-carbonated (“still”) water from domestic brands in Greece. International Journal of Food Microbiology 107, 68–72. Warburton, D., Harrison, B., Crawford, C., Foster, R., Fox, C., Gour, L., Krol, P., 1998. A further review of the microbiological quality of bottled water sold in Canada: 1992– 1997 survey results. International Journal of Food Microbiology 39, 221–226. WHO, 1997. 2nd ed. Guideline for Drinking Water Quality, vol. 3. World Health Organization, Geneva, Switzerland.
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o
Short communication
Bacterial contaminants in carbonated soft drinks sold in Bangladesh markets Muhammad Ali Akond a,b,⁎, Saidul Alam a, S.M.R. Hasan a, Sanzida Mubassara a, Sarder Nasir Uddin c, Momena Shirin d a
Department of Botany, Jahangirnagar University, Dhaka-1342, Bangladesh Lab of Environmental Bioscience, Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh d Institute of Public Health, Mohakhali, Dhaka-1212, Bangladesh b c
a r t i c l e
i n f o
Article history: Received 16 September 2008 Received in revised form 16 January 2009 Accepted 19 January 2009 Keywords: Bangladesh Carbonated soft drinks Public health risks Pathogenic bacteria Coliforms
a b s t r a c t A total of 225 carbonated soft drink (CSD) samples from nine brands, from various locations in five metropolitan cities of Bangladesh were examined to determine their bacteriological quality. Most samples were not in compliance with microbiological standards set by organizations like the World Health Organization (WHO). Pseudomonas aeruginosa was the predominant species with an incidence of 95%. Streptococcus spp. and Bacillus stearothermophilus were the next most prevalent with numbers ranging from 6 to 122 and 9 to 105 cfu/100 ml, respectively. Fifty four percent of the samples yielded Salmonella spp. at numbers ranging from 2 to 90 cfu/100 ml. Total coliform (TC) and faecal coliform (FC) counts were found in 68–100% and 76–100% of samples of individual brands, at numbers ranging from 5 to 213 and 3 to 276 cfu/ 100 ml, respectively. According to WHO standards 60–88% of samples from six brands and 32% and 40% of samples from two other brands belonged to the intermediate risk group with FC counts of 100–1000 cfu/ 100 ml. Heterotrophic plate counts, however, were under the permissible limit in all 225 samples. These findings suggest that carbonated soft drinks commercially available in Bangladesh pose substantial risks to public health. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Carbonated soft drinks (CSDs) were introduced in Bangladesh over 35 years ago, and have since become popular in both cities and rural areas. Since 1996 there have been no standards for, or definition of, soft drinks in the British and EU regulations, except in special cases (Carbonated Drinks, 2004). Protection of all types of drinks from hazardous microbial contaminants is a global issue. Various gastrointestinal illnesses are the most common consequences of consuming contaminated drinks (Frobisher et al., 1974). Good manufacturing practices (GMPs) are not always followed in the food and beverage industries, particularly in developing countries, and this can result in wide variations in the microbial quality of products, such as bottled water and other drinks (Akond et al., 2006; Hara et al., 2005; Venieri et al., 2006). Carbonated soft drinks and bottled waters sold in Bangladesh are claimed to be of excellent microbiological quality in their sales promotion advertisements in both print and electronic media (Akond et al., 2006). This is in contradiction, however, to newspaper reports of extensive algal growth in carbonated soft drink bottles (BELA, 1997; Jaijai Din, 2008; Janakantha, 2004), indicating that the microbiological quality of soft
⁎ Corresponding author. Lab of Environmental Bioscience, Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan. Tel.: +81 80 3056 7275. E-mail address: [email protected] (M.A. Akond). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.01.014
drinks in Bangladesh may actually be poor. Therefore, the present study was undertaken to determine the bacterial quality of some non-alcoholic, carbonated soft drinks available at retail outlets in Bangladesh. 2. Materials and methods 2.1. Sampling Intact containers (plastic and/or glass bottles or aluminum cans) from nine brands of CSD were purchased from various retail outlets in five cities (Dhaka, Khulna, Rajshahi, Chittagong and Barishal) in Bangladesh between August 2006 and March 2008. Contamination with dust during collection and transportation was avoided by placing containers in a sterilized foam box. The containers were washed with sterile distilled water then sterilized by spraying and rubbing with 98% alcohol before opening. Nine brands of carbonated soft drinks were designated arbitrarily CSD-1 to CSD-9. Five containers of each brand from every collection site were examined. 2.2. Bacteriological analysis A heterotrophic plate count (HPC), total coliform count (TCC), faecal coliform count (FCC), and faecal Streptococcus, Bacillus stearothermophilus, Pseudomonas aeruginosa, Salmonella and Shigella
M.A. Akond et al. / International Journal of Food Microbiology 130 (2009) 156–158
157
Table 1 Prevalences (%) of total coliform (TC), faecal coliform (FC) and other bacteria in groups of 25 samples of carbonated soft drinks from Bangladesh Brand Code
Number of positive samples TC
FC
Salmonella
Streptococcus
Pseudomonas
Bacillus
CSD-1 CSD-2 CSD-3 CSD-4 CSD-5 CSD-6 CSD-7 CSD-8 CSD-9 Total
17 (68) 23 (92) 25 (100) 23 (92) 23 (92) 22 (88) 25 (100) 22 (88) 25 (100) 205 (91.11)
19 (76) 22 (88) 25 (100) 23 (92) 22 (88) 20 (80) 25 (100) 22 (88) 24 (96) 202 (89.78)
12 (48) 13 (52) 18 (72) 9 (36) 12 (48) 16 (64) 15 (60) 13 (52) 14 (56) 122 (54.22)
24 (96) 25 (100) 25 (100) 25 (100) 17 (68) 25 (100) 25 (100) 25 (100) 21 (84) 212 (94.22)
22 (88) 25 (100) 24 (96) 25 (100) 24 (96) 25 (100) 25 (100) 25 (100) 19 (76) 214 (95.11)
14 (56) 18 (72) 3 (12) 24 (96) 19 (76) 14 (56) 12 (48) 18 (72) 11 (44) 133 (59.11)
counts were obtained for each sample. Heterotrophic bacteria were enumerated by spread plating 0.1 ml and pour plating a 1.0 ml volume of each sample undiluted and in ten-fold dilutions to 10− 3. Other bacteria were enumerated by filtering 10 and 100 ml volumes of each undiluted sample through 0.22 µm membrane filters (Millipore, Bedford, MA, USA), and incubating the filters on selective agars. All bacteria were enumerated on triplicate plates or filters. The culture media used for HPC, TCC, FCC, and faecal Streptococcus, B. stearothermophilus, P. aeruginosa, Salmonella and Shigella counts were, respectively, Nutrient agar (Difco, Becton-Dickinson, Sparks, MD, USA), MacConkey agar (Difco), Membrane Faecal Coliform (MFC) agar (Hi-Media, Mumbai, India), Kenner Faecal Streptococcus (KF-Streptococcus) agar (Hi-Media), 2,3,5-triphenyl tetrazolium chloride (TTC) agar (Sartorius, Goettingen, Germany), Cetrimid agar (Oxoid, Basingstoke, Hampshire, UK), Bismuth Sulfite agar (Oxoid) and Salmonella-Shigella (SS) agar (Oxoid). MFC, SS and TTC agar plates were incubated at 44 °C, 42 °C and 55 °C, respectively, for 24 to 72 h. All other plates were incubated at 37 °C for 24 to 72 h. Characteristic colonies grown on selective and differential agars were confirmed by the morphological and biochemical tests of Buchanan and Gibbons (1974). The tests included gram staining, spore staining, and tests for catalase, coagulase, oxidase, IMViC reactions, starch hydrolysis, sugar fermentation, and nitrate reduction. Salmonella spp., Escherichia coli, P. aeruginosa, and Streptococcus spp. were further confirmed by latex agglutination tests using polyvalent antisera (DENKA SEIKEN Co. Ltd, Tokyo, Japan). Only strong agglutination occurring within 1 min was considered to be a positive reaction. 2.3. Statistical analysis Statistical analyses of the data were carried out using SPSS 11.5 and Microsoft Excel 2003 to calculate means, medians, standard devia-
tions, least significant differences (LSD) and correlations, and to check distribution normality of data sets using Anderson–Darling test. 3. Results and discussion All samples were positive for heterotrophic bacteria (Table 1). The HPCs ranged from 3 to 242 cfu/ml (Table 2), all of which were under the maximum permissible limit of 500 cfu/ml set by the United States Environmental Protection Agency (US EPA, 2003). The low HPC might be due to the incubation temperature of 37 °C, as it has been claimed that the numbers of bacteria recovered from drinks decreases with increasing incubation temperature, from 25 to 35 °C (Armas and Sutherland, 1999; Reasoner, 2004; Rosenberg, 2003; Venieri et al., 2006). However, a 13-fold increase in aerobic bacterial counts of Nigerian carbonated soft drinks was obtained when culture media were incubated at 34 °C rather than 28 °C (Efiuvweuwere and Chynyere, 2001). Several pathogenic and/or opportunistically pathogenic bacteria like Salmonella, P. aeruginosa, and Streptococcus, and B. stearothermophilus were present in CSDs at numbers ranging from 2 to 90, 4 to 162, 6 to 122, and 9 to 105 cfu/100 ml, respectively (Table 2). Salmonella enteritidis, S. typhimurium, E. coli, Pseudomonas spp. and Streptococcus spp. were confirmed by polyvalent antisera agglutination tests. The ranges for TC and FC counts were 5 to 213 and 3 to 276 cfu/ 100 ml, respectively. Ninety percent of samples were non-compliant with the guidelines of the World Health Organization (WHO, 1997); brands CSD-3 and CSD-7 were also 100% non-compliant with guideline values for faecal coliform (Table 3). Sixty five percent of the samples were in the intermediate risk group, while 4% of samples were in the high risk group. Such high counts were found by Kohnen et al. (2005) for both coliform and Pseudomonas spp. in 39 and 12% of samples of home-prepared carbonated water, respectively, as compared to only 12 and 4%, respectively, of samples of tap water used for
Table 2 Medians (ranges) of numbers of bacteria recovered from groups of 25 samples of nine carbonated soft drinks from Bangladesh Brand code CSD-1 CSD-2 CSD-3 CSD-4 CSD-5 CSD-6 CSD-7 CSD-8 CSD-9 a b c d
Median and range for bacterial counts (cfu/100 ml) HPCa 0.8 (0.15–3.25) 12.2 (2.9–23) 3.2 (1.6–10)d 7.2 (2.2–24)d 4 (2.3–8) 1.4 (0.4–3.4)d 7.5 (4–10) 8 (1–24.2)d 7 (1–17)
TCb d
d
9.0 (0–46) 27 (0–213)d 27 (9–48) 28 (0–53) 35 (0–71) 16 (0–51)d 32 (8–146)d 56 (0–110) 32 (9–90)d
Heterotrophic plate count, cfu × 103/100 ml. Total coliform. Faecal coliform. Data set not normally distributed.
FCc
Salmonella
Streptococcus
Pseudomonas
Bacillus
8 (0–22)d 20 (0–37) 20 (6–276)d 20 (0–50) 15 (0–45) 9 (0–49)d 21 (5–46) 34 (0–92) 45 (0–160)d
0 (0–58)d 13 (0–87) d 12 (0–52) d 0 (0–17)d 0 (0–45)d 8 (0–27)d 8 (0–69)d 2 (0–20)d 12 (0–90)d
14 (0–33) 88 (12–122)d 27 (9–60)d 19 (6–51) 8 (0–29)d 13 (6–40)d 33 (8–83) 61 (12–83) 11 (0–19)
22 (0–58) 81 (28–128) 20 (0–43) 33 (12–66) 27 (0–61) 29 (9–59) 38 (9–107) 132 (54–162)d 12 (0–36)
9 (0–23)d 22 (0–62) 0 (0–12)d 51 (0–105) 12 (0–45) 9 (0–25)d 0 (0–33)d 21 (0–58) 0 (0–22)d
158
M.A. Akond et al. / International Journal of Food Microbiology 130 (2009) 156–158
Table 3 Numbers of samples (%), in groups of 25, positive for World Health Organization risk groups based on faecal coliform (FC) counts Brand code
Risk category Nonea
Lowb
Intermediatec
Highd
CSD-1 CSD-2 CSD-3 CSD-4 CSD-5 CSD-6 CSD-7 CSD-8 CSD-9 Total
6 (24) 3 (12) 0 (0.0) 2 (8.0) 3 (12) 5 (20) 0 (0.0) 3 (12.0) 1 (4.0) 23 (10.22)
11 2 4 1 5 10 6 3 4 46
8 (32) 20 (8) 16 (64) 22 (88) 17 (68) 10 (40) 19 (76) 19 (76) 15 (60) 146 (64.89)
0 0 5 (20) 0 0 0 0 0 5 (20) 10 (4.44)
a b c d
(44) (8.0) (16) (4.0) (20) (40) (24) (12) (16) (20.44)
FC = 0. FC = 1–10 cfu/100 ml. FC = 10–100 cfu/100 ml. FC = 100–1000 cfu/100 ml.
be causes for the high counts. Extensive algal growth and the presence of sediments in bottles of carbonated soft drinks have been reported in Bangladeshi newspapers (BELA, 1997; Jaijai Din, 2008; Janakantha, 2004), which also indicates the poor quality of carbonated soft drinks. The presence of such materials and the poor microbiological quality reported in the current study are the result of failure to maintain the acceptable quality and to adequately maintain the regulations of Bangladesh Government in the manufacture of carbonated soft drinks. Immediate action to improve the microbiological quality of carbonated beverages in Bangladesh is evidently required. Acknowledgement Authors are thankful to the Lab of Microbiology, Department of Botany, Jahangirnagar University, and Bacteriology Laboratory, Institute of Public Health, Dhaka, Bangladesh. References
their preparation. The high rate of contamination in the carbonated waters was likely due to biofilms in the water bottles. According to WHO (1997) and the International Commission on Microbiological Specifications for Foods (1986), bottled water should be produced without contamination by coliforms and most Pseudomonas. LSD analysis showed significant differences between the mean counts of HPC, TC and FC in most samples (p = 0.01). There was no correlation between the numbers of any two groups of organisms (r = 0.47), except for weak correlations between TC and FC for brands CSD-8 (r = 0.66) and CSD-5 (r = 0.58) and between B. stearothermophilus and P. aeruginosa for brand CSD-5 (r = 0.59). The fractions of the 225 samples positive for other bacteria ranged from 54% for Salmonella spp. to 95% for P. aeruginosa (Table 1). In a Canadian study, Warburton et al. (1998) reported b1 cfu/ml of Pseudomonas spp. in carbonated bottled mineral water in all samples tested. Though Pseudomonas is frequently noticed in bottled water and drinking water (Rosenberg, 2003), it is not normally a health hazard, however, it can cause infections to immunocompromised individuals. Reports of Salmonella spp. in bottled water or carbonated beverages are uncommon. The high counts of Salmonella spp. in carbonated soft drinks in Bangladesh are obviously a risk for public health. The presence of Salmonella spp. is an indication of enteric contamination and may have been introduced to these soft drinks during bottling and/or manufacturing, due to lack of personal hygiene. Of the total samples in the current study, 59% were positive for B. stearothermophilus. This high rate of Bacillus spp. was also found in Cola samples from Nigeria, with incidence of up to 81%. Given the environmental occurrence, as well as the osmotic, thermal and salt stress tolerance of the bacteria, Bacillus spp. could be introduced in these drinks from either or both the environment and/or from raw materials (Efiuvweuwere and Chynyere, 2001; Oranusi et al., 1994). The presence of B. stearothermophilus, which is used as an indicator in sterilization validation tests, indicates improper treatment of water used in the production of these drinks. This is in negligence of the regulations of the Bangladesh Government, which states that the water to be used in the production of carbonated soft drinks must be drawn from an underground depth of at least 300 ft, it must not contain heterotrophic bacteria at more than 50 cfu/ml, and must be negative for all TC and FC counts (BSTI, 2001). The presence of high numbers of TC, FC and various harmful bacteria, especially Salmonella spp., in carbonated soft drinks in Bangladesh poses a serious public health risk. Poor manufacturing processes, and ingredient and/or water source contamination may all
Akond, M.A., Alam, S., Shil, A., Hasan, S.M.R., 2006. Bacteriological quality of bottled water available commercially in Bangladesh. Journal of Environmental Sciences (Dhaka) 4, 47–52. Armas, A.B., Sutherland, J.P., 1999. A survey of the microbiological quality of bottled water sold in the UK and changes occurring during storage. International Journal of Food Microbiology 48, 59–65. BELA, 1997. PIL (List of Public Interest Litigation), No. 11, Dr. Mohiuddin Farooque v. Bangladesh & others, Writ Petition No. 867/97 (Contaminated Drink). Bangladesh Environmental Lawyers Association (BELA), Segunbagicha, Dhaka. http://www. belabangla.org/html/pil.htm. Accessed on December 30, 2008. BSTI, 2001. Standard for Bottled Drinking Water/ Soft Drinks. BDS:1240, 2001. Bangladesh Standard Testing Institute (BSTI), Dhaka. Buchanan, R.E., Gibbons, N.E. (Eds.), 1974. Bergey's Manual of Determinative Bacteriology, 8th ed. Williams and Willkins Co., Baltimore. Carbonated Drinks, 2004. British Soft Drinks Association, 20–22 Stukeley Street, London. http://www.liquidsmeanlife.org.uk/member/KS3&4%20new%20material/ Teachers%20notes/Carbonated%20Drinks.pdf. Accessed on December 15, 2008. Daily Jaijai Din, 2008. Soft Drinks Shorirer Jonno Soft Noy (Soft Drinks are not soft for human body), February 14, 2008. Jaijai Din Publications Ltd., Tejgaon, Dhaka, Bangladesh, p. 16. Daily Janakantha, 2004. Coker Botole Shaola (Algae in bottle of Coke), March 23, 2004. Janakantha Ltd., Janakantha Bhaban, New Eskaton Road, Dhaka, Bangladesh, p. 1. Efiuvweuwere, B.J.O., Chynyere, M., 2001. The microbiology and deterioration of soft drinks subjected to two different marketing. Global Journal of Pure and Applied Sciences 7, 43–48. Frobisher, M., Hinsdill, R.D., Crabtree, K.T., Goodheart, C.R., 1974. Fundamentals of Microbiology, 9th ed. W.B. Saunders Company, Philadelphia. Hara, M., Fukuyama, M., Furuhata, K., 2005. Isolation of thermotolerant acidophilic bacteria (TAB) from fruit juices. Journal of Antibacterial and Antifungal Agents 9, 447–452. International Commission on Microbiological Specifications for Foods, 1986. Microorganisms in foods, 2, Sampling Plants for Natural Mineral Waters, other bottled waters, Process Waters and Ice, 2nd ed. Blackwell Scientific Publications, London. Kohnen, W., Teske-Keiser, S., Meyer, H.-G., Loos, A.H., Pietsch, M., Jansen, B., 2005. Microbiological quality of carbonated drinking water produced with in-home carbonation system. International Journal of Hygiene and Environmental Health 208, 415–423. Oranusi, S.U., Ezeogu, L.I., Okolo, B.N., 1994. Microbial contaminants of commercially bottled non-alcoholic drinks produced in Nigeria. World Journal of Microbiology and Biotechnology 10, 488–490. Reasoner, D.J., 2004. Heterotrophic plate count methodology in the United States. International Journal of Food Microbiology 92, 307–315. Rosenberg, F.A., 2003. The microbiology of bottled water. Clinical Microbiology Newsletter 25, 41–44. US EPA, 2003. List of drinking water contaminants and maximum contaminant levels, USEPA. Office of GWDW, EPA, Washington, DC. Publication No. 816-F-03-016. Venieri, D., Vantarakis, A., Komninou, G., Papapetropoulou, M., 2006. Microbiological evaluation of bottled non-carbonated (“still”) water from domestic brands in Greece. International Journal of Food Microbiology 107, 68–72. Warburton, D., Harrison, B., Crawford, C., Foster, R., Fox, C., Gour, L., Krol, P., 1998. A further review of the microbiological quality of bottled water sold in Canada: 1992– 1997 survey results. International Journal of Food Microbiology 39, 221–226. WHO, 1997. 2nd ed. Guideline for Drinking Water Quality, vol. 3. World Health Organization, Geneva, Switzerland.