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Microbiological quality of grey-mullet roe
Anaerobe 17 (2011) 273e275
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Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Pathogenesis and Toxins
Microbiological quality of grey-mullet roe C. Voidarou a, A. Alexopoulos b, S. Plessas b, H. Noussias b, E. Stavropoulou c, K. Fotou a, A. Tzora a, I. Skoufos a, E. Bezirtzoglou b, d, K. Demertzi-Akrida e, * a
Technological Educational Institute of Epirus, Arta GR47100, Greece Democritus University of Thrace, Faculty of Agricultural Development, Orestiada GR68200, Greece c Democritus University of Thrace, Medical School, Alexandroupolis GR68100, Greece d Democritus University of Thrace, Faculty of Agricultural Development, Laboratory of Food Processing, Orestiada GR68200, Greece e University of Ioannina, Department of Chemistry, Laboratory of Food Chemistry and Technology, Ioannina GR45110, Greece b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 December 2010 Received in revised form 13 March 2011 Accepted 14 March 2011 Available online 8 April 2011
The Greek grey-mullet roe is produced from the fully developed gonads of the female mullet (Mugil cephalus) couth in lagoons during their reproductive migration. The traditional processing method of the roe includes, air drying, salting, shape formation and covering with multiple layers of natural beeswax for preservation and distribution. Fish Roe brands have been a staple in local diet and is increasingly becoming popular in the international market. As a ready-to-eat food it’s microbial quality should be of concern for the protection of consumers health. In this study, 48 samples of fish roe, just before waxing, were collected from various local processors for microbiological examination by using selective media and incubated under aerobic and anaerobic conditions. The identification of the bacteria was carried out according to the Bergey’s manual. Microscopic examination of Gram stained cells, catalase, oxidase and biochemical tests were performed when necessary to further identify. V. parahaemolyticus, Vibrio spp., Salmonella spp., and Aeromonas hydrophila were detected in one sample (2%). Shigella spp., and Flavobacterium spp. in two samples (4%), Clotriduim perfringens (vegetative forms), E. coli, and spores of Bacillus spp., were detected in three samples (6%), Staphylococcus aureus in four samples (8%). Various Micrococcus spp., and spores of C. perfringens in 16% and 35% of the samples respectively. From the Listeria genus, only the species Listeria innocua, Listeria welshimeri, Listeria seeligeri Listeria ivanovii and Listeria grayi were recovered from 2 to 10% of the samples. Microbiological analyses revealed the presence of a small number of pathogens in grey-mullet roe samples which are in accordance with the findings of similar studies. Traditional processing of the fish roe, seems inadequate to ensure the food safety and even waxing isn’t expected to fully protect them against facultative anaerobes with salt tolerance. Therefore, additional measures should be taken during processing and marketing of fish roe to minimize potential health risks for the consumers. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Mullet roe Fish Quality
1. Introduction Current knowledge in terms of the microbiology of grey-mullet roe seems to be limited only in several countries [1,2]. In Greece, grey-mullet roe is obtained from the pond fish Mugil cephalus, which migrate in lagoons during its reproduction. However, it appears that the likelihood of getting good quality product is becoming less and less, due to improvements in fishing techniques and methodologies [2]. Grey-mullet roe was considered as a delicatessen food since Faraon’s and Byzantine
* Corresponding author. E-mail addresses: [email protected], [email protected] (K. Demertzi-Akrida). 1075-9964/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2011.03.008
years. It contains proteins, u3 lipids, selenium, iron and calcium and it is full of vitamin A, complex vitamin B, vitamin C and vitamin E [3]. Nevertheless, as other fish products, it may become a vehicle and spoiled by many bacterial pathogens. These microorganisms originate from the fish flora and are usually transmitted to the roes during processing in case of limited hygiene conditions [4]. Additionally, its microbial status is strictly depend on the microbiological quality of water [5]. Our study focuses on the evaluation of the microbiological quality of grey-mullet roe caught from a Mediterranean country (Greece) in an attempt to obtain a safe high quality product and explain conditions of bacterial spoilage.
274
C. Voidarou et al. / Anaerobe 17 (2011) 273e275
2. Materials and methods 2.1. Sampling 48 samples of fish roe (average weight 210e250 gr), just before waxing, were collected from various local processors located at the region of Amvrakikos lagoon (Greece). All samples were harvested during the period of August and September. The samples were introduced aseptically in sterile bags (47 cm 13 cm) and transported into insulated thermo boxes with ice to maintain a temperature around 6 C. Samples were transported to the laboratory and examined within 4 h of collection. 2.2. Microbiological examination For Listeria spp: 25 g of sample were homogenized in 225 ml of UVM I (University of Vermont Listeria enrichment broth [6]. Aliquots of 1 ml of primary enrichments were transferred to 20 ml of UVM II (UVM I with 0.025 g l 1of acriflavine hydrochloride in 10 ml of sterile distilled water, pH 7,2) and incubated again at 37 C for 24e48 h [7]. Aliquots of 0.1 ml of secondary enrichments were plated in duplicate in PALCAM Listeria selective agar (Merck) supplemented with PALCAM Listeria selective supplement (Merck). Suspected colonies were confirmed by Gram staining, motility test (hanging drop), catalase and b-haemolysis tests and sugar fermentation tests for rhamnose, xylose and mannitol [8,9]. Serotypes were determined using Bacto-Listeria-O polyvalent antiserum and Bacto-Listeria-O antisera types 1 and 4 (Difco). For Salmonella spp.: 25 g of sample and 225 ml buffered peptone water were homogenized using a Stomacher. The analysis depends on the principles of preculturing for 18e24 h in Buffered PeptoneWater at a temperature of 35e37 C, the selective culturing for 24 h in Selenith Cystine Broth at a temperature of 35 C. The broths were streaked onto SalmonellaeShigella (SS) agar (Merck) and brilliantgreen phenol-red lactose sucrose (BG) agar (Merck) incubated at 35 C for 24 h. Suspected transparent colonies with or without black center on SS agar and red colonies on BG agar were subjected to biochemical analyses and serotyped using commercial antisera (Difco) [9e11]. The APHA method was used for isolation of Staphylococcus aureus including enrichment of a 1-g sample in 10 ml cooked meat medium (Difco) plus 9% NaCl (w/v), streaking a loopful of the 24 h enrichment culture on BairdeParker agar (Merck) containing egg yolk and potassium tellurite (Merck) and subsequent confirmatory coagulase test of lipase-positive jet-black colonies [8]. To confirm the presence of Clotriduim perfringens, the L.S. (Lactose-Sulfite) broth was used [12,13]. Before use, the medium was boiled for 20 min to reduce the oxygen content and 0.5 ml of a 1.2% solution of anhydrous sodium metabisulfite (Na2S2O2) and 0.2 ml of a 1% solution of ferric ammonium citrate, were added to each tube. The above solutions were prepared and sterilized by filtration (0.45 mm) just prior to use. Incubation was performed aerobically in a water bath at 46 C for 24 h. Additionally, an aliquot of each sample was heated for 20 min at 80 C for detection of germinated spore forms and for each L.S. broth was seeded. Pseudomonas spp.,were enumerated on cetrimide fusidin cephaloridine agar (CFC, Oxoid code CM 559, supplemented with supplement SR 103, Oxoid, Basingstoke, UK) and incubated at 20 C for 2 days [14]. Aeromonas strains were isolated on ampicillin dextrin agar (ADA) [15,16]. Samples were either cultured directly on ADA or cultured after enrichment (1:lO) in tryptic soy broth (Difco) containing 30 mg/ml ampicillin for 24 h at 30 C on ADA. Two or three
typical yellow colonies were selected from ADA medium and cultured on blood agar medium. Only oxidase-positive, glucosefermentative colonies were chosen for further study. For V. parahaemolyticus, the APHA method was used. In brief, 25 g of sample was homogenized in 225 ml alkaline peptone water (APW), using a stomacher and incubated at 35 C for 6e8 h. A loopful from APW was then streaked onto thiosulfate citrate bile salt sucrose agar (TCBS, Merck) and the plate was incubated at 35 C for 24 h. Confirmation tests were then made on blue-green colonies for acid production from cellobiose, sucrose, maltose, mannitol, trehalose and lactose, Gram staining, motility test, VogueseProskauer, oxidase and nitrite reduction tests, growth in 6 and 8% NaCl and growth at 43 C [8]. Mould and yeast: 10 g of sample and 100 ml peptone water were homogenized and further 10 fold dilutions’ were made. The analysis depends on the principles of incubation for 5 days in Dichloran Rose Bengal Chloramphenicol Agar at a temperature of 25 C, moulds and yeasts colonies were counted separately [17]. To enumerate other bacteria, 25-g sample was homogenized with 225 ml of 0.1% (w/v) peptone water (Merck) and 10-fold serial dilutions were prepared. For coliform, Lactobacillus, facultative and strict anaerobes the violet red bile agar (VRBA, Merck), MRS agar (Oxoid -with the pH adjusted to 5.50), Columbia blood agar, Egg Yolk agar and MYP agar (Mannitol Egg Yolk Polymyxin Agar) were used, respectively (NaCl content of all the media was adjusted to 0.5%). Incubation of the plates was performed accordingly to the medium aerobically or anaerobically for 24 h at 37 C. To confirm the suspected coliforms, ten of the purple-red colonies, 0.5 mm in diameter or larger, surrounded by a zone of precipitated bile acids on VRBA were transferred to tubes of brilliant-green lactose bile broth (Merck) at 35 C for 24e48 h. The confirmed coliform colonies were also assessed for detection of E. coli, using eosin- methylene-blue lactose sucrose agar (Merck), and then subjected to biochemical tests [8]. The isolates were further characterized by the sugar fermentation pattern, which was determined by using API microsystem, as specified by the manufacturer (API-BioMerieux, Marcy-l’Etoile, France). Moreover, the VITEK 2 (BioMerieux, Marcy-l’Etoile, France), an automated system for identification, was applied. It detects bacterial growth and metabolic reactions in microwells of plastic test cards by measuring fluorescence. Freshly subcultured inoculums were prepared by adjusting to a McFarland standard of 0.5 in a 0.45% sodium chloride solution. The ID-GPC and ID- GNC card of the Vitek 2 system (BioMerieux, Marcy-l’Etoile, France) served for identification.
3. Results Our findings are summarized on Table 1. LAB was the dominant microflora (81.25%) followed by coliforms (64.5%) found in 31 samples. E. coli was found in 3 samples (6%). Among Listeria species, Listeria innocua (2%), Listeria welshimeri (4%), Listeria seeligeri (4%), Listeria ivanovii (6%) and Listeria. grayi (10%) were also found. C. perfringens spore forms were isolated in 17 cases (35%). However, isolation of C. perfringens vegetative forms was low (6%). Bacillus spp. (6%) was found only in spore forms. Micrococcus spp. (16.5%) and Staphylococcus aureus (8%) were also present. Other microbial species were also recovered, as V. parahaemolyticus (2%), Vibrio spp. (2%), Salmonella arizonae (2%), Aeromonas hydrophila (2%), Shigella spp. (4%), and Flavobacterium spp (4%). Yeasts (12.5%) were also isolated but mould and Pseudomonas spp. were never found.
C. Voidarou et al. / Anaerobe 17 (2011) 273e275 Table 1 Bacteria strains isolated from grey-mullet roe samples. a/a
Species
(n)
(%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
V. parahaemolyticus V. cholerae Salmonella spp. (S. arizonae) Aeromonas hydrophyla Shigella spp. Flavobacterium spp. C. perfringens (vegetative forms) C. perfringens (spore forms) Bacillus spp. (spore forms) Coliforms E. coli Staphylococcus aureus Micrococcus spp. LAB L. innocua L. welshimeri L. seeligeri L. ivanovii L. grayi Yeasts Moulds Pseudomonas spp.
1 1 1 1 2 2 3 17 3 31 3 4 8 39 1 2 2 3 5 6 nd nd
2 2 2 2 4 4 6 35 6 64.5 6 8 16.5 81.2 2 4 4 6 10 12.5 nd nd
nd: not detected.
4. Discussion Grey-mullet roe is subject to types of spoilage similar to those of fish or other sea-foods. The kind and rate of spoilage is influenced by a number of factors [4,18,19]. Usually, roe originate from fatty fishes spoils rapidly because of the oxidation of the unsaturated fats (oxidative rancidity) [20]. Moreover, it must be mentioned that damage of fish or fish roes by net, hooks, struggling and excessive handling accelerates spoilage. In general, the greater the load of the bacteria on the fish, the more rapid the spoilage occurs and affect the fish roe quality. Grey-mullet roe are salted after their extraction from the fish for several hours, followed by air drying. Natural bee wax is used to cover them; a procedure which permits long-time conservation of the product. Salt-tolerance of Lactobacillus spp. could explain their frequency in roe as many Lactobacillus species were found to be salt and bile tolerant [21]. Moreover, Listeria spp. were also found to be salt tolerant [3,22]. On the present study, different Listeria spp. were present, such as L. innocua (2%), L. welshimeri (4%), L. seeligeri (4%), L. ivanovii (6%) and L. grayi (10%). These species are often found in wetlands and are important animal pathogens. Their presence is probably a result from the heavy livestock and agricultural activities around the Amvrakikos lagoon as well as from the multiple fish farms in that region. Additionally, the cold watery environment favors the growth of Listeria [22]. L. monocytogenes which is a major foodborne pathogen was never recovered. Vibrio spp. (2%) and V.parahaemolyticus (2%) were occasionally found. These microorganisms are usually indigenous to the marine environment and affected by the salinity of waters [19], as well as from the salting procedures of the product. Salinity supports the growth of Micrococcus spp. (16.5%) as well as S. aureus (8%). This last bacterium must be of human or animal origin, as the S. aureus does not appear as a part of the natural microflora of newly caught marine and cultivated fish [18]. Besides that, extensive handling and unproper hygienic conditions are also incriminated [19]. C. perfringens vegetative forms (6%) were found only in 3 cases, compared with C. perfringens spore forms (35%). Salinity together with missing conditions of anaerobiosis seems to affect its presence in vegetative forms. Coliforms (64.5%) and E.coli (6%) were also
275
found in our samples probably as a result of the constant contamination of the lagoon from animal and human activities [23]. Yeasts (12.5%) were also found in our samples. Yeasts are incriminated to cause organoleptic spoilage [24] affecting the quality of the product [2]. However, from the microbiological point of view, taking into account the high levels of LAB bacteria (81.25%) which dominated the grey-mullet roe microflora, we can assume that this beneficial flora supported by the acidic conditions of the product (pH 4e5), competes with the putrefactive bacteria. During the traditional handling of fish roe’s, bacteria coming from the fish surfaces, contaminated water or because of inappropriate hygienic practices, could be responsible of roe contamination and subsequent spoilage of the product. Continual hygienic control at all the production stages of the roe chain from the fish caught to the final product should be implemented in order to protect the consumers. It is also recommended the use of advanced packaging to improve the self-life and safety of the product. References [1] Liao I-C. Experiments on induced breeding of the grey mullet in Taiwan from 1963 to 1973. Aquaculture 1975;6:31e58. [2] Altug G, Bayrak Y. Microbiological analysis of caviar from Russia and Iran. Food Microbiol 2003;20:83e6. [3] Bledsoe GE, Bledsoe CD, Rasco B. Caviars and fish roe products. Crit Rev Food Sci Nutr 2003;43:317e56. [4] Gram L, Huss HH. Microbiological spoilage of fish and fish products. Int J Food Microbiol 1996;33:121e37. [5] Bezirtzoglou E, Dimitriou D, Panagiou A, Kagalou I, Demoliates Y. Distribution of C. perfringens in different aquatic environmental in Greece. Microbiol Res 1994;149:129e34. [6] Ryser ET, Arimi SM, Bunduki MM, Donnelly CW. Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media. Appl Environ Microbiol 1996; 62:1781e7. [7] Jorgensen L, Huss H. Prevalence and growth of Listeria monocytogenes in naturally contaminated seafood. Int J Food Microbiol 1998;42:127e31. [8] American Public Health Association (APHA). Compendium of methods for the microbiological examination. 3rd ed.; 1992 [Washington DC]. [9] Harrigan WF. Laboratory methods in food microbiology. San Diego: Academic Press; 1998. [10] ICMSF. Microorganisms in foods, vol. 1. Toronto: University of Toronto Press; 1978. [11] Food and Drug Administration (FDA). Bacterial analytical manual. 8th ed. Washington, DC: AOAC International; 1998. Revision A. [12] Bezirtzoglou E, Dimitriou D, Panagiou A. Occurrence of C. perfringens in river water by using a new procedure. Anaerobe 1996;2:169e73. [13] Savvaidis I, Kegos Th, Papagiannis C, Voidarou C, Tsiotsias A, Maipa V, et al. Bacterial indicators and metal irons in high mountain lake waters. Microb Ecol Health Dis 2001;13:147e52. [14] Mead GC, Adams BW. A selective medium for the rapid isolation of Pseudomonas associated with poultry meat spoilage. Br Poult Sci 1997;18:661e70. [15] Havelaar AD, During M, Versteegh JFM. Ampicillin-dextrin agar medium for the enumeration of Aerromonas species in water by membrane filtration. J Appl Bacteriol 1987;62:279e87. [16] Hanninen ML, Siitonen A. Distribution of Aeromonas phenospecies and genospecies among strains isolated from water, foods or from human clinical samples. Epidemiol Infect 1995;15:39e50. [17] Pit JI, Hocking AD. Methods for isolation enumeration and identification. In: Fungi and food spoilage. London: Blackie Academic and Professional; 1997. [18] Herrero MMH, Sagues RXR, Gerez JJR, Ventura MTM. Halotolerant and halophilic histamine forming bacteria isolated during the ripening of salted anchovies. J Food Prot 1997;62:509e14. [19] Davies AR, Cpell C, Jehanno D, Nychas GJE, Kirby RM. Incidence of foodborne pathogens on European fish. Food Control 2001;12:67e71. [20] Gao MT, Hirata M, Toorisaka E, Hano T. Acid-hydrolysis of fish wastes for lactic acid fermentation. Bioresour Technol 2006;97:2414e20. [21] Charteris WP, Kelly PM, Morelli L, Collins JK. Effect of conjugated bile salts on antibiotic susceptibility of bile salt-tolerant lactobacillus and Bifidobacterium isolates. J Food Prot 2000;63:1369e75. [22] Food and Drug Administration (FDA). Quantitative assessment of relative risk to public health from foodborne Listeria monocytogenes among selected categories of ready-to-eat foods. Cent Food Saf Appl Nutr 2002 [USA]. [23] Voidarou C, Tzora A, Skoufos I, Vassos D, Galogiannis G, Alexopoulos A, et al. Experimental effect of ozone upon some indicator bacteria for preservation of an ecologically protected watery system. Water Air Soil Pollut 2007; 181:161e71. [24] Forsythe SJ, Hayes PR. Food hygiene. Microbiology and HACCP. Gaithersburg: Aspen Publication; 1998.
Contents lists available at ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Pathogenesis and Toxins
Microbiological quality of grey-mullet roe C. Voidarou a, A. Alexopoulos b, S. Plessas b, H. Noussias b, E. Stavropoulou c, K. Fotou a, A. Tzora a, I. Skoufos a, E. Bezirtzoglou b, d, K. Demertzi-Akrida e, * a
Technological Educational Institute of Epirus, Arta GR47100, Greece Democritus University of Thrace, Faculty of Agricultural Development, Orestiada GR68200, Greece c Democritus University of Thrace, Medical School, Alexandroupolis GR68100, Greece d Democritus University of Thrace, Faculty of Agricultural Development, Laboratory of Food Processing, Orestiada GR68200, Greece e University of Ioannina, Department of Chemistry, Laboratory of Food Chemistry and Technology, Ioannina GR45110, Greece b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 December 2010 Received in revised form 13 March 2011 Accepted 14 March 2011 Available online 8 April 2011
The Greek grey-mullet roe is produced from the fully developed gonads of the female mullet (Mugil cephalus) couth in lagoons during their reproductive migration. The traditional processing method of the roe includes, air drying, salting, shape formation and covering with multiple layers of natural beeswax for preservation and distribution. Fish Roe brands have been a staple in local diet and is increasingly becoming popular in the international market. As a ready-to-eat food it’s microbial quality should be of concern for the protection of consumers health. In this study, 48 samples of fish roe, just before waxing, were collected from various local processors for microbiological examination by using selective media and incubated under aerobic and anaerobic conditions. The identification of the bacteria was carried out according to the Bergey’s manual. Microscopic examination of Gram stained cells, catalase, oxidase and biochemical tests were performed when necessary to further identify. V. parahaemolyticus, Vibrio spp., Salmonella spp., and Aeromonas hydrophila were detected in one sample (2%). Shigella spp., and Flavobacterium spp. in two samples (4%), Clotriduim perfringens (vegetative forms), E. coli, and spores of Bacillus spp., were detected in three samples (6%), Staphylococcus aureus in four samples (8%). Various Micrococcus spp., and spores of C. perfringens in 16% and 35% of the samples respectively. From the Listeria genus, only the species Listeria innocua, Listeria welshimeri, Listeria seeligeri Listeria ivanovii and Listeria grayi were recovered from 2 to 10% of the samples. Microbiological analyses revealed the presence of a small number of pathogens in grey-mullet roe samples which are in accordance with the findings of similar studies. Traditional processing of the fish roe, seems inadequate to ensure the food safety and even waxing isn’t expected to fully protect them against facultative anaerobes with salt tolerance. Therefore, additional measures should be taken during processing and marketing of fish roe to minimize potential health risks for the consumers. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Mullet roe Fish Quality
1. Introduction Current knowledge in terms of the microbiology of grey-mullet roe seems to be limited only in several countries [1,2]. In Greece, grey-mullet roe is obtained from the pond fish Mugil cephalus, which migrate in lagoons during its reproduction. However, it appears that the likelihood of getting good quality product is becoming less and less, due to improvements in fishing techniques and methodologies [2]. Grey-mullet roe was considered as a delicatessen food since Faraon’s and Byzantine
* Corresponding author. E-mail addresses: [email protected], [email protected] (K. Demertzi-Akrida). 1075-9964/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2011.03.008
years. It contains proteins, u3 lipids, selenium, iron and calcium and it is full of vitamin A, complex vitamin B, vitamin C and vitamin E [3]. Nevertheless, as other fish products, it may become a vehicle and spoiled by many bacterial pathogens. These microorganisms originate from the fish flora and are usually transmitted to the roes during processing in case of limited hygiene conditions [4]. Additionally, its microbial status is strictly depend on the microbiological quality of water [5]. Our study focuses on the evaluation of the microbiological quality of grey-mullet roe caught from a Mediterranean country (Greece) in an attempt to obtain a safe high quality product and explain conditions of bacterial spoilage.
274
C. Voidarou et al. / Anaerobe 17 (2011) 273e275
2. Materials and methods 2.1. Sampling 48 samples of fish roe (average weight 210e250 gr), just before waxing, were collected from various local processors located at the region of Amvrakikos lagoon (Greece). All samples were harvested during the period of August and September. The samples were introduced aseptically in sterile bags (47 cm 13 cm) and transported into insulated thermo boxes with ice to maintain a temperature around 6 C. Samples were transported to the laboratory and examined within 4 h of collection. 2.2. Microbiological examination For Listeria spp: 25 g of sample were homogenized in 225 ml of UVM I (University of Vermont Listeria enrichment broth [6]. Aliquots of 1 ml of primary enrichments were transferred to 20 ml of UVM II (UVM I with 0.025 g l 1of acriflavine hydrochloride in 10 ml of sterile distilled water, pH 7,2) and incubated again at 37 C for 24e48 h [7]. Aliquots of 0.1 ml of secondary enrichments were plated in duplicate in PALCAM Listeria selective agar (Merck) supplemented with PALCAM Listeria selective supplement (Merck). Suspected colonies were confirmed by Gram staining, motility test (hanging drop), catalase and b-haemolysis tests and sugar fermentation tests for rhamnose, xylose and mannitol [8,9]. Serotypes were determined using Bacto-Listeria-O polyvalent antiserum and Bacto-Listeria-O antisera types 1 and 4 (Difco). For Salmonella spp.: 25 g of sample and 225 ml buffered peptone water were homogenized using a Stomacher. The analysis depends on the principles of preculturing for 18e24 h in Buffered PeptoneWater at a temperature of 35e37 C, the selective culturing for 24 h in Selenith Cystine Broth at a temperature of 35 C. The broths were streaked onto SalmonellaeShigella (SS) agar (Merck) and brilliantgreen phenol-red lactose sucrose (BG) agar (Merck) incubated at 35 C for 24 h. Suspected transparent colonies with or without black center on SS agar and red colonies on BG agar were subjected to biochemical analyses and serotyped using commercial antisera (Difco) [9e11]. The APHA method was used for isolation of Staphylococcus aureus including enrichment of a 1-g sample in 10 ml cooked meat medium (Difco) plus 9% NaCl (w/v), streaking a loopful of the 24 h enrichment culture on BairdeParker agar (Merck) containing egg yolk and potassium tellurite (Merck) and subsequent confirmatory coagulase test of lipase-positive jet-black colonies [8]. To confirm the presence of Clotriduim perfringens, the L.S. (Lactose-Sulfite) broth was used [12,13]. Before use, the medium was boiled for 20 min to reduce the oxygen content and 0.5 ml of a 1.2% solution of anhydrous sodium metabisulfite (Na2S2O2) and 0.2 ml of a 1% solution of ferric ammonium citrate, were added to each tube. The above solutions were prepared and sterilized by filtration (0.45 mm) just prior to use. Incubation was performed aerobically in a water bath at 46 C for 24 h. Additionally, an aliquot of each sample was heated for 20 min at 80 C for detection of germinated spore forms and for each L.S. broth was seeded. Pseudomonas spp.,were enumerated on cetrimide fusidin cephaloridine agar (CFC, Oxoid code CM 559, supplemented with supplement SR 103, Oxoid, Basingstoke, UK) and incubated at 20 C for 2 days [14]. Aeromonas strains were isolated on ampicillin dextrin agar (ADA) [15,16]. Samples were either cultured directly on ADA or cultured after enrichment (1:lO) in tryptic soy broth (Difco) containing 30 mg/ml ampicillin for 24 h at 30 C on ADA. Two or three
typical yellow colonies were selected from ADA medium and cultured on blood agar medium. Only oxidase-positive, glucosefermentative colonies were chosen for further study. For V. parahaemolyticus, the APHA method was used. In brief, 25 g of sample was homogenized in 225 ml alkaline peptone water (APW), using a stomacher and incubated at 35 C for 6e8 h. A loopful from APW was then streaked onto thiosulfate citrate bile salt sucrose agar (TCBS, Merck) and the plate was incubated at 35 C for 24 h. Confirmation tests were then made on blue-green colonies for acid production from cellobiose, sucrose, maltose, mannitol, trehalose and lactose, Gram staining, motility test, VogueseProskauer, oxidase and nitrite reduction tests, growth in 6 and 8% NaCl and growth at 43 C [8]. Mould and yeast: 10 g of sample and 100 ml peptone water were homogenized and further 10 fold dilutions’ were made. The analysis depends on the principles of incubation for 5 days in Dichloran Rose Bengal Chloramphenicol Agar at a temperature of 25 C, moulds and yeasts colonies were counted separately [17]. To enumerate other bacteria, 25-g sample was homogenized with 225 ml of 0.1% (w/v) peptone water (Merck) and 10-fold serial dilutions were prepared. For coliform, Lactobacillus, facultative and strict anaerobes the violet red bile agar (VRBA, Merck), MRS agar (Oxoid -with the pH adjusted to 5.50), Columbia blood agar, Egg Yolk agar and MYP agar (Mannitol Egg Yolk Polymyxin Agar) were used, respectively (NaCl content of all the media was adjusted to 0.5%). Incubation of the plates was performed accordingly to the medium aerobically or anaerobically for 24 h at 37 C. To confirm the suspected coliforms, ten of the purple-red colonies, 0.5 mm in diameter or larger, surrounded by a zone of precipitated bile acids on VRBA were transferred to tubes of brilliant-green lactose bile broth (Merck) at 35 C for 24e48 h. The confirmed coliform colonies were also assessed for detection of E. coli, using eosin- methylene-blue lactose sucrose agar (Merck), and then subjected to biochemical tests [8]. The isolates were further characterized by the sugar fermentation pattern, which was determined by using API microsystem, as specified by the manufacturer (API-BioMerieux, Marcy-l’Etoile, France). Moreover, the VITEK 2 (BioMerieux, Marcy-l’Etoile, France), an automated system for identification, was applied. It detects bacterial growth and metabolic reactions in microwells of plastic test cards by measuring fluorescence. Freshly subcultured inoculums were prepared by adjusting to a McFarland standard of 0.5 in a 0.45% sodium chloride solution. The ID-GPC and ID- GNC card of the Vitek 2 system (BioMerieux, Marcy-l’Etoile, France) served for identification.
3. Results Our findings are summarized on Table 1. LAB was the dominant microflora (81.25%) followed by coliforms (64.5%) found in 31 samples. E. coli was found in 3 samples (6%). Among Listeria species, Listeria innocua (2%), Listeria welshimeri (4%), Listeria seeligeri (4%), Listeria ivanovii (6%) and Listeria. grayi (10%) were also found. C. perfringens spore forms were isolated in 17 cases (35%). However, isolation of C. perfringens vegetative forms was low (6%). Bacillus spp. (6%) was found only in spore forms. Micrococcus spp. (16.5%) and Staphylococcus aureus (8%) were also present. Other microbial species were also recovered, as V. parahaemolyticus (2%), Vibrio spp. (2%), Salmonella arizonae (2%), Aeromonas hydrophila (2%), Shigella spp. (4%), and Flavobacterium spp (4%). Yeasts (12.5%) were also isolated but mould and Pseudomonas spp. were never found.
C. Voidarou et al. / Anaerobe 17 (2011) 273e275 Table 1 Bacteria strains isolated from grey-mullet roe samples. a/a
Species
(n)
(%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
V. parahaemolyticus V. cholerae Salmonella spp. (S. arizonae) Aeromonas hydrophyla Shigella spp. Flavobacterium spp. C. perfringens (vegetative forms) C. perfringens (spore forms) Bacillus spp. (spore forms) Coliforms E. coli Staphylococcus aureus Micrococcus spp. LAB L. innocua L. welshimeri L. seeligeri L. ivanovii L. grayi Yeasts Moulds Pseudomonas spp.
1 1 1 1 2 2 3 17 3 31 3 4 8 39 1 2 2 3 5 6 nd nd
2 2 2 2 4 4 6 35 6 64.5 6 8 16.5 81.2 2 4 4 6 10 12.5 nd nd
nd: not detected.
4. Discussion Grey-mullet roe is subject to types of spoilage similar to those of fish or other sea-foods. The kind and rate of spoilage is influenced by a number of factors [4,18,19]. Usually, roe originate from fatty fishes spoils rapidly because of the oxidation of the unsaturated fats (oxidative rancidity) [20]. Moreover, it must be mentioned that damage of fish or fish roes by net, hooks, struggling and excessive handling accelerates spoilage. In general, the greater the load of the bacteria on the fish, the more rapid the spoilage occurs and affect the fish roe quality. Grey-mullet roe are salted after their extraction from the fish for several hours, followed by air drying. Natural bee wax is used to cover them; a procedure which permits long-time conservation of the product. Salt-tolerance of Lactobacillus spp. could explain their frequency in roe as many Lactobacillus species were found to be salt and bile tolerant [21]. Moreover, Listeria spp. were also found to be salt tolerant [3,22]. On the present study, different Listeria spp. were present, such as L. innocua (2%), L. welshimeri (4%), L. seeligeri (4%), L. ivanovii (6%) and L. grayi (10%). These species are often found in wetlands and are important animal pathogens. Their presence is probably a result from the heavy livestock and agricultural activities around the Amvrakikos lagoon as well as from the multiple fish farms in that region. Additionally, the cold watery environment favors the growth of Listeria [22]. L. monocytogenes which is a major foodborne pathogen was never recovered. Vibrio spp. (2%) and V.parahaemolyticus (2%) were occasionally found. These microorganisms are usually indigenous to the marine environment and affected by the salinity of waters [19], as well as from the salting procedures of the product. Salinity supports the growth of Micrococcus spp. (16.5%) as well as S. aureus (8%). This last bacterium must be of human or animal origin, as the S. aureus does not appear as a part of the natural microflora of newly caught marine and cultivated fish [18]. Besides that, extensive handling and unproper hygienic conditions are also incriminated [19]. C. perfringens vegetative forms (6%) were found only in 3 cases, compared with C. perfringens spore forms (35%). Salinity together with missing conditions of anaerobiosis seems to affect its presence in vegetative forms. Coliforms (64.5%) and E.coli (6%) were also
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found in our samples probably as a result of the constant contamination of the lagoon from animal and human activities [23]. Yeasts (12.5%) were also found in our samples. Yeasts are incriminated to cause organoleptic spoilage [24] affecting the quality of the product [2]. However, from the microbiological point of view, taking into account the high levels of LAB bacteria (81.25%) which dominated the grey-mullet roe microflora, we can assume that this beneficial flora supported by the acidic conditions of the product (pH 4e5), competes with the putrefactive bacteria. During the traditional handling of fish roe’s, bacteria coming from the fish surfaces, contaminated water or because of inappropriate hygienic practices, could be responsible of roe contamination and subsequent spoilage of the product. Continual hygienic control at all the production stages of the roe chain from the fish caught to the final product should be implemented in order to protect the consumers. It is also recommended the use of advanced packaging to improve the self-life and safety of the product. References [1] Liao I-C. Experiments on induced breeding of the grey mullet in Taiwan from 1963 to 1973. Aquaculture 1975;6:31e58. [2] Altug G, Bayrak Y. Microbiological analysis of caviar from Russia and Iran. Food Microbiol 2003;20:83e6. [3] Bledsoe GE, Bledsoe CD, Rasco B. Caviars and fish roe products. Crit Rev Food Sci Nutr 2003;43:317e56. [4] Gram L, Huss HH. Microbiological spoilage of fish and fish products. Int J Food Microbiol 1996;33:121e37. [5] Bezirtzoglou E, Dimitriou D, Panagiou A, Kagalou I, Demoliates Y. Distribution of C. perfringens in different aquatic environmental in Greece. Microbiol Res 1994;149:129e34. [6] Ryser ET, Arimi SM, Bunduki MM, Donnelly CW. Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media. Appl Environ Microbiol 1996; 62:1781e7. [7] Jorgensen L, Huss H. Prevalence and growth of Listeria monocytogenes in naturally contaminated seafood. Int J Food Microbiol 1998;42:127e31. [8] American Public Health Association (APHA). Compendium of methods for the microbiological examination. 3rd ed.; 1992 [Washington DC]. [9] Harrigan WF. Laboratory methods in food microbiology. San Diego: Academic Press; 1998. [10] ICMSF. Microorganisms in foods, vol. 1. Toronto: University of Toronto Press; 1978. [11] Food and Drug Administration (FDA). Bacterial analytical manual. 8th ed. Washington, DC: AOAC International; 1998. Revision A. [12] Bezirtzoglou E, Dimitriou D, Panagiou A. Occurrence of C. perfringens in river water by using a new procedure. Anaerobe 1996;2:169e73. [13] Savvaidis I, Kegos Th, Papagiannis C, Voidarou C, Tsiotsias A, Maipa V, et al. Bacterial indicators and metal irons in high mountain lake waters. Microb Ecol Health Dis 2001;13:147e52. [14] Mead GC, Adams BW. A selective medium for the rapid isolation of Pseudomonas associated with poultry meat spoilage. Br Poult Sci 1997;18:661e70. [15] Havelaar AD, During M, Versteegh JFM. Ampicillin-dextrin agar medium for the enumeration of Aerromonas species in water by membrane filtration. J Appl Bacteriol 1987;62:279e87. [16] Hanninen ML, Siitonen A. Distribution of Aeromonas phenospecies and genospecies among strains isolated from water, foods or from human clinical samples. Epidemiol Infect 1995;15:39e50. [17] Pit JI, Hocking AD. Methods for isolation enumeration and identification. In: Fungi and food spoilage. London: Blackie Academic and Professional; 1997. [18] Herrero MMH, Sagues RXR, Gerez JJR, Ventura MTM. Halotolerant and halophilic histamine forming bacteria isolated during the ripening of salted anchovies. J Food Prot 1997;62:509e14. [19] Davies AR, Cpell C, Jehanno D, Nychas GJE, Kirby RM. Incidence of foodborne pathogens on European fish. Food Control 2001;12:67e71. [20] Gao MT, Hirata M, Toorisaka E, Hano T. Acid-hydrolysis of fish wastes for lactic acid fermentation. Bioresour Technol 2006;97:2414e20. [21] Charteris WP, Kelly PM, Morelli L, Collins JK. Effect of conjugated bile salts on antibiotic susceptibility of bile salt-tolerant lactobacillus and Bifidobacterium isolates. J Food Prot 2000;63:1369e75. [22] Food and Drug Administration (FDA). Quantitative assessment of relative risk to public health from foodborne Listeria monocytogenes among selected categories of ready-to-eat foods. Cent Food Saf Appl Nutr 2002 [USA]. [23] Voidarou C, Tzora A, Skoufos I, Vassos D, Galogiannis G, Alexopoulos A, et al. Experimental effect of ozone upon some indicator bacteria for preservation of an ecologically protected watery system. Water Air Soil Pollut 2007; 181:161e71. [24] Forsythe SJ, Hayes PR. Food hygiene. Microbiology and HACCP. Gaithersburg: Aspen Publication; 1998.