- Email: [email protected]
Survival of amine-forming bacteria during the ice storage of fish and shrimp
Food Microbiology, 2002, 19, 617^625 Available online at http://www.idealibrary.com on
doi:10.1006/yfmic.481
ORIGINAL ARTICLE
Survival of amine-forming bacteria during the ice storage of ¢sh and shrimp R. Lakshmanan1, R. Jeya Shakila*, G. Jeyasekaran Survival of amine-forming bacteria during the ice storage of ¢sh and shrimp was investigated up to 14 days of storage. On iced storage the total bacterial load was reduced to one log from an initial load of 105 cfu g 1 in fresh ¢sh/shrimp due to cold shock. The total incidence of biogenic amine-forming bacteria was found to be 74?63% in ¢sh and the same was recorded as 56?05% in shrimp.The amine-forming bacteria recorded were cadaverine- and putrescine-forming bacteria in ¢sh/shrimp, and no histamine former was detected. Gram-negative, non-fermentative rods, viz. Alcaligenes. Flavobacterium, Acinetobacter. Shewanella and Pseudomonas, were the predominant amine-forming bacteria during the ice storage of ¢sh and shrimp, in addition to the only Gram-positive genus Micrococcus. The genera Aeromonas and Photobacterium also survived ice storage to a certain extent and may also be responsible for the formation of amines in ¢sh and shrimp. # 2002 Published by Elsevier Science Ltd.
Introduction Amines are formed during spoilage of ¢sh as a result of bacterial decarboxylation of free amino acids. Di¡erent bacteria capable of decarboxylating amino acids have been isolated from ¢sh muscle (Taylor 1986, Yoshinaga and Frank 1982, Moori et al. 1988, Okuzumi et al. 1990). They include mesophilic as well as psychrophilic bacteria and most of them possess more than one decarboxylase enzyme (Taylor and Summer 1986). Among the amines, histamine has been more frequently implicated in food poisoning, and the diamines, primarily
*Corresponding author. Fax: +91- 461-340401/ 340574; E-mail: [email protected]; ¢[email protected] 1 Present address: Department of Bioscience and Biotechnology, Royal College Building, Strathclyde University, 204 George Street, Glasgow G 1 1XW, Scotland, UK. E-mail: [email protected] 0740 -0020/02/060617 +09 $35.00/0
putrescine and cadaverine, are known to enhance histamine poisoning (Bjeldanes et al. 1978). Putrescine is the decarboxylation product of ornithine, and cadaverine arises from decarboxylation of lysine. Putrescine and cadaverine have been regarded as the freshness index in ¢sh, as they were the only amines detected before the initial decomposition (Dawood et al. 1988, Fernanadez-Salguero et al. 1987, Yamanaka 1989). In shrimp, putrescine has been suggested as an index of decomposition (Mietz and Karmas 1978, Shakila et al. 1995). Storage of ¢sh and shell¢sh in ice has been the routine practice of preserving ¢sh on board ship and at shore. Many studies have been carried out to examine the e¡ect of temperature on amine formation in ¢sh (Behling and Taylor 1982, Lopez-Sabater et al. 1996, Koutsoumanis et al. 1999). However, reports state that there are di¡erences in the formation of amines in r 2002 Published by Elsevier Science Ltd.
Received: 4 October 2001 Department of Fish Processing Technology, Fisheries College & Research Institute,Tamil Nadu Veterinary and Animal Sciences University, Tuticorin 628 008, Tamilnadu, India
618 R. Lakshmanan et al.
¢sh during ice storage and such di¡erences are mainly due to the type and level of micro£ora present in ¢sh (Middlebrooks et al. 1988; Loperz-Sabater et al. 1996). This work was therefore undertaken with an objective of investigating the survival of the amine-forming bacteria during the storage of ¢sh and shrimp on ice (01C).
Materials and Methods Raw material Fresh emperor ¢sh (Lethrinus miniatus) and shrimp (Penaues semisulcatus) were procured from the local ¢sh market and immediately brought to the laboratory. The average length and weight of ¢sh were 18?22 cm and 108 g, respectively, and that of shrimp were 10 cm and 7 g, respectively. They were washed in potable water to remove blood, slime and any other extraneous material and stored under iced condition in insulated boxes having outlets to remove the melt water. Flake ice was added daily to compensate for melting of ice. Fish and shrimp samples were withdrawn for analysis on day 0, and after 3, 6, 9, 12 and 14 days of storage in ice. Samples were analysed in triplicate for the total bacterial load and their means with standard deviation were calculated.
Enumeration of total bacterial load Fish/shrimp samples (25 g) were homogenized with 225 ml of sterile physiological saline (0?85% NaCl), and serial decimal dilutions of each homogenate were carried out with the same diluent. The diluted suspension was plated onto Trypticase soy agar (TSA) (Hi-Media, Mumbai, India) following spread plate technique. The plates were incubated at 20 and 371C to isolate both the psychrotrophic and mesophilic bacteria, respectively. After an appropriate incubation period, the number of colonies developed on the plates were counted for the total mesophilic and psychrotrophic bacteria and expressed as cfu g 1.
Test for decarboxylase activity Representative isolates were picked up from the TSA plates incubated at 201C, puri¢ed and checked for their decarboxylase activity. Three types of media were prepared using modi¢ed Moeller’s decarboxylase broth base (Hi-Media, Mumbai, India) supplemented with di¡erent amino acids, viz. L -histidine hydrochloride, L -ornithine hydrochloride and L -lysine hydrochloride at 0?5% level (w/v). Medium without added amino acid served as the control. The isolates, which showed positive reaction for the decarboxylase test, were considered as amine-forming bacteria (Okuzumi et al. 1990).
Identi¢cation of positive isolates The amine-forming bacterial (AFB) isolates were identi¢ed by following a series of standard biochemical tests (Lechavelier et al. 1980, Buchanan and Gibbons 1982). The biochemical tests performed include Gram’s reaction, cell morphology, catalase, oxidase, oxidative-fermentative, motility, indole, methyl red, Voges Proskaeur, citrate utilization, urease, sensitivity to O/129 pteridine and ONPG, and fermentation tests for sugars such as glucose, sucrose, lactose and mannitol.
Results and Discussion Fig. 1 represents the total mesophilic and psychrotrophic bacterial load in ¢sh along with the total amine-forming bacteria during their storage on ice. The total mesophilic bacteria in fresh ¢sh was 105 cfu g 1, which on ice storage decreased by one log during the initial period (3^6 days) and later on steadily increased to 106 cfu g 1 at the end of storage. The initial reduction in the total mesophilic bacteria was mainly due to the e¡ect of cold shock (Ingram 1951). The total psychrotrophic bacteria, which was one log lower than the mesophilic bacteria, initially increased steadily on storage and reached a level of 107 cfu g 1 on the 14th day. Of the total psychrotrophic bacteria, the bacteria capable of producing amines were initially very low (101 cfu g 1 ) in ¢sh, but increased progressively upon storage to a ¢nal
Survival of amine-forming bacteria during ice storage 619
Figure 1. Changes in total mesophilic, psychrotrophic and amine-forming bacteria in ¢sh during ice storage.
level of 106 cfu g 1.There was a two-log increase in the total amine-forming bacterial load on the 9th and 14th day of storage in ¢sh. Fig. 2 shows the changes in the total mesophilic, psychrotrophic and amine-forming bacteria of shrimp held on ice.The total mesophilic bacterial load in fresh shrimp was also similar to that observed in ¢sh. Upon initial storage in ice, there was a log reduction in the counts and later it increased to a maximum of 106 cfu g 1 on the 12th day. Upon storage, the psychrotrophic bacteria proliferated slowly and dominated the mesophilic bacterial load, as the low temperature favoured their growth.The amine-
forming bacterial population in fresh shrimp was slightly higher (102 cfu g 1 ) than in ¢sh. On iced storage, their population decreased initially and then steadily increased up to 106 cfu g 1 at the end of storage.Therefore, both the ¢sh and shrimp muscle supports the survival of amine-forming bacteria even after prolonged storage in ice, indicating that the bacteria proliferating at chill environment have the ability to decarboxylate amino acids to form amines. The incidence of di¡erent amine-forming bacteria during the ice storage of ¢sh and shrimp is presented in Fig. 3. The amine-forming
620 R. Lakshmanan et al.
Figure 2. Changes in total mesophilic, psychrotrophic and amine-forming bacteria in shrimp during ice storage.
bacteria recorded in ¢sh and shrimp were putrescine- and cadaverine-forming bacteria and no histamine-forming bacteria were detected. Arnold et al. (1980) and Lopez-Sabater et al. (1996) also could not detect histamine-forming bacteria in tuna until 9^12 days of storage at 01C, which is similar to our observation.The absence of histamine-forming bacteria in ¢sh and shrimp was probably due to the e¡ect of low temperature and the presence of a low level of histidine in their muscle. However, the presence of putrescine- and cadaverine-forming bacteria in ¢sh and shrimp implies that they could grow at 01C and contribute to amine formation. In
fresh ¢sh, the incidence of amine-forming bacteria was relatively low (32%) and remained more or less the same during the early periods of storage (i.e 3^6 days), and later increased to about 96% on the 14th day. It was interesting to note that, during the initial storage in ice (0^6 days), the putrescine-forming bacteria dominated and, at the later stages, the cadaverineforming bacteria were dominant. In fresh shrimp, the incidence of amine-forming bacteria was similar to that observed in fresh ¢sh with about 30% (Fig. 4). But on storage in ice, there was a rapid reduction in their incidence to a level of 14% which then slowly increased and
Survival of amine-forming bacteria during ice storage 621
Figure 3. Incidence of amine-forming bacteria during ice storage of ¢sh. reached a level of 60% on the 12th day. Middlebrooks et al. (1988) have also reported that 65%of the bacterial isolates obtained from tuna held at 11C showed positive decarboxylase activity for lysine and arginine. But, in the present study, the incidence of amine-forming bacteria in the ice-stored ¢sh was comparatively higher (96%). It is also noted by Middlebrooks et al. (1988) that in ¢sh decomposed at 01C, the levels of cadaverine and putrescine began to rise sharply at about 11 days of incubation, while histamine appeared only after 16 days. This could be the reason why the presence of amineforming bacteria was high between 12^14 days of storage in ¢sh and 9^12 days of storage in shrimp. The amine-forming bacteria isolated from ¢sh were identi¢ed and are presented in Table 1. Fresh ¢sh predominantly harboured the genera Bacillus,Vibrio and Flavobacterium. The species of Bacillus isolated from ¢sh had the ability to produce putrescine, but the genera Vibrio and Flavobacterium had the ability
Figure 4. Incidence of amine-forming bacteria during ice storage of shrimp. to produce both cadaverine and putrescine. Among them, only Flavobacterium survived ice storage and was recorded till the end of storage. Bacteria belonging to the family Enterobacteriaceae were detected in few numbers and they were identi¢ed to be Enterobacter aerogenes and Proteus mirabilis, which demonstrated the ability to produce both cadaverine and putrescine. Aeromonas and Photobacterium were recorded on the 12th day of ice storage, which implies that they could survive and become responsible for the formation of cadaverine. Non-fermentative Gram-negative bacteria Shewanella, Acinetobacter, Alcaligenes, Pseudomonas and Moraxella were recorded in relatively large proportions at later stages of ice storage. Almost all the genera under this group had the ability to produce both cadaverine and putrescine, except Shewanella, which was a putrescine former, and Alcaligenes paradoxus,
622 R. Lakshmanan et al.
Table 1. Microbial £oraa isolated from ¢sh during ice storage Days of storage 0
3
6
9
12
14
Total isolates tested for decarboxylase activity
75
48
123
136
90
360
No. of positive isolates
29
43
38
128
67
346
I. Enterobacteriaceae 1. E. aerogenes 2. P. mirabilis
F F
1 F
F F
F 2
F F
F F
II.Vibrionaceae 1.Vibrio 2. Aeromonas 3. Photobacterium
8 F F
F F F
F F F
F F F
F 4 7
F F F
III. Gram-negative, non-fermentative rods 1. Flavobacterium 2. Shewanella 3. Acinetobacter 4. Alcaligenes 5. Alcaligenes paradoxus 6. Pseudomonas 7. Moraxella
3 F F F F F F
6 F F F F F
11 1 7 F F F F
2 3 5 F 23 18 F
18 F 11 7 F 3 1
67 F F 134 F 6 F
IV. Gram-positive bacteria 1. Bacillus 2. Micrococcus luteus 3. M. varians 4. M. roseus 5. Corynebacterium 6. Staphylococcus epidermidis
18 F F F F F
F 5 F F F 1
F F F F 12 5
F 23 26 24 2 F
F 2 14 F F F
F 67 67 F F F
a
Numbers.
which was a cadaverine former. Gram-positive bacteria Bacillus, which was recorded in large numbers in fresh ¢sh, failed to grow during ice storage. On the other hand, Micrococcus sp. survived at low temperature and started proliferating after 9 days. Three species of Micrococcus were isolated and each of them possessed different decarboxylase enzymes. Staphylococcus epidermidis and Corynebacterium were also recorded in high numbers during the initial period and did not survive beyond 9 days of storage in ice. Corynebacterium is a cadaverine-forming bacterium, while S. epidermidis produces both cadaverine and putrescine. The amine-forming bacteria identi¢ed during the ice storage of shrimp are presented in Table 2. Fresh shrimp harboured Gram-positive amine-forming bacteria Bacillus, Corynebacterium and Staphylococcus in relatively high numbers, and they totally diminished on
further storage in ice. The species of Bacillus detected in shrimp had the ability to produce both cadaverine and putrescine, and the genus Corynebacterium was a cadaverine former. The amine-forming bacterial £ora, which survived at 01C throughout the storage period, was the genus Alcaligenes. The bacteria belonging to the family Enterobacteriaceae were recorded in very few numbers with those observed in ¢sh. On the other hand, Aeromonas sp., Vibrio sp. and non-fermentative Gram-negative rods survived ice storage and became the major £ora between 9 and 12 days of storage. Among the Aeromonas, the species A. veronii had the ability to form only putrescine. It was found that the non-fermentative rods Alcaligenes, Shewanella, Acinetobacter and Pseudomonas were the dominant genera responsible for amine formation in shrimp at the ¢nal stages. The only Gram-positive bacteria that could
Survival of amine-forming bacteria during ice storage 623
Table 2. Microbial £oraa isolated from shrimp during ice storage Days of storage 0
3
6
9
12
Total isolates tested for decarboxylase activity
92
69
51
78
115
No. of positive isolates
42
10
23
49
103
I. Enterobacteriaceae 1. E. aerogenes
F
F
F
4
F
II.Vibrionaceae 1.Vibrio 2. Aeromonas 3. Aeromonas veroniiI
F F F
F F F
F 8 F
5 9 8
3 F 5
III. Gram-positive, non-fermentative rods 1. Flavobacterium 2. Shewanella 3. Alcaligenes 4. Acinetobacter 5. Pseudomonas
F F F F F
F F 10 F F
3 3 1 F F
8 8 2 5 F
F F 28 22 42
F F F F
5 3 F F
F F F F
F 3 F F
IV. Gram-positive bacteria 1. Bacillus 2. Micrococcus luteus 3. M. lylae 4. Corynebacterium 5. Staphylococcus epidermidis a
14 F 14 14
Numbers.
survive ice storage belonged to the genus Micrococcus and they were recorded only from the 6th day of storage. Micrococcus detected in shrimp had the ability to form both cadaverine and putrescine. It was clear from this study that during the ice storage of ¢sh and shrimp, amine-forming bacteria could survive and proliferate slowly upon storage, and contribute to amine formation. Fish has been observed to harbour more numbers of amine-forming bacteria than shrimp, this is because of the presence of higher levels of free lysine and ornithine in the ¢sh muscle than in shrimp. Fresh ¢sh and shrimp harboured greater numbers of Gram-positive amine-forming bacteria, fewer numbers of Vibrio and non-fermentative Gram-negative rods. Among these bacteria,Vibrio and Bacillus failed to grow in ice, but the non-fermentative Gram-negative rods survived ice storage.Taylor (1986) has reported that the amines are generally formed in ¢sh by the bacteria belonging to the family Enterobacteriaceae, Lactobacillus and Clostridium. But, this was true only in the
case of ¢sh held at ambient temperature (20^ 371C). In the ¢sh stored in ice, the bacteria belonging to the family Enterobacteriaceae were recorded in low numbers and, therefore, they were not the major amine-forming bacteria. Earlier Moori et al. (1986) also pointed out that, at low temperature, the bacteria belonging to the family Enterobacteriaceae are not very active in forming the amines, which was in agreement with our ¢ndings. However, bacteria belonging to the family Vibrionaceae, Vibrio, Aeromonas and Photobacterium, survived ice storage, proliferated slowly and became the dominant amine-forming bacteria at later stages (9^12 days) in ice-stored ¢sh or shrimp. Photobacterium phosphoreum has been recognized as a principal species responsible for histamine formation in ¢sh at low temperature (Moori et al. 1986). A species of Photobacterium, which has the ability to produce cadaverine, was also detected in horse mackerel held at 51C by Okuzumi et al. (1990). Photobacterium phosphoreum was detected after 12 days of storage in ice, was identi¢ed to be relatively
624 R. Lakshmanan et al.
resistant to cold storage and grew slowly at 41C (Fujii et al. 1994). In the present study also, the amine-forming Photobacterium sp. was detected in the emperor ¢sh on the 12th day of storage in ice. On the other hand, non-fermentative Gramnegative rods, viz. Alcaligenes, Flavobacterium, Acinetobacter, Shewanella and Pseudomonas detectable during initial stages of ice storage, became the predominant amine-forming bacteria in ¢sh and shrimp at later stages. In the tuna ¢sh decomposed at 01C, Pseudomonas sp. were reported to be the most frequently encountered bacteria, besides Aeromonas, Proteus, Citrobacter, Vibrio, Acinetobacter and Morganella morganii (Middlebrooks et al. 1988). It has also been reported that, in horse mackerel held at 51C, Moraxella was dominant up to 2 days and later Pseudomonas sp. showed dominance at the decomposition stage (Okuzumi et al. 1990), which was similar to our observations. Most of the Gram-positive bacteria which were present in ¢sh/shrimp failed to grow in ice, and the only dominant Gram-positive genus which survived ice storage was Micrococcus. It can therefore be concluded that during the ice storage of emperor ¢sh and £ower shrimp, cadaverine- and putrescine-forming bacteria could survive and proliferate rapidly between 9 and 12 days, and contribute to the formation of amines. The amine-forming bacteria recorded in ¢sh and shrimp were not invariably di¡erent. The dominant amine-forming bacteria belonged mainly to the group of non-fermentative Gram-negative rods, Photobacterium, Aeromonas and Micrococcus. The bacteria belonging to the family Enterobacteriaceae were least responsible for amine formation. Hence, control measures to minimize/retard amine formation during the ice storage of ¢sh/shrimp need to be formulated based on the incidence and survival of amine-forming bacteria.
Acknowledgements This study was funded by the Indian council of Agricultural Research (ICAR), New Delhi, Government of India.
References Arnold, S. H., Price, R. J. and Brown,W. D. (1980) Histamine formation by bacteria isolated from skipjack tuna (Katsuwonus pelamis) Nippon Suisan Gakkaishi. 46, 991^995. Behling, A. R. and Taylor, S. L. (1982) Bacterial histamine production as a function of temperature and time of incubation. J Food Sci. 47, 1311^1314, 1317. Buchanan, R. E. and Gibbons N. E. (Eds) (1982) Bergey’s Manual of Determinative Bacteriology. 8th edn. Baltimore, USA,Williams & Wilkins. Bjeldanes, L. F., Shutz, D. E. and Morris, M. M. (1978) Etiology of scombroid poisoningFcadaverine potentiation of histamine toxicity in guinea pigs. Food Cosmet. Toixicol. 16, 157^162. Dawood, A. A., Karkalas, J., Roy, R. N. and Williams, C. A. (1988) The occurrence of non-volatile amines in chilled-stored rainbow trout (Salmo irideus). Food Chem. 27, 33^45. Fernandez-Salguero, J. and Mackie, I. M. (1987) Comparative rates of spoilage of ¢llets and whole ¢sh during storage of haddock (Melanogrammes aegie¢nus) and herring (Clupea herengus) as determined by the formation of non-volatile amines. Int. J. Food Sci. Technol. 22, 385^390. Fujii, T., Kurihara, K. and Okuzumi, M. (1994) Viability and histidine decarboxylase activity of halophilic histamine-forming bacteria during frozen storage. J. Food Prot. 57, 611^613. Ingram, M. (1951) The e¡ect of cold on microorganisms in relation to food. Proc. Soc. Appl. Bacteriol. 14, 243^249. Koutsoumanis, K., Lampropoulou, K. and GeorgeJohn, E. N. (1999) Biogenic amines and sensory changes associated with the microbial £ora of Mediterranean gilt-head sea bream (Sparus aurata) stored aerobically at 0, 8 and 151C. J. Food Prot. 62, 398^402. Lechavelier, M. W., Seider, R. J. and Evans, E. M. (1980) Enumeration and characterization of standard plate count bacteria in chlorinated and raw water supplies. Appl. Environ. Microbial. 40, 922^926. Lopez-Sabater, E. I., Rodriguez-Jerez, J. J., Roig-Sagueus, A. X. and Mora-Ventura, M. A. T. (1996) Bacteriological quality of tuna ¢sh (Thunnus thynnus) destined for canning: e¡ect of handling on the presence of histidine decarboxylase bacteria and histamine level. J. Food Prot. 57, 318^323. Middlebrooks, B. L.,Toom, P. M., Douglas,W. L., Harrison, R. E. and McDowell, S. (1988) E¡ects of storage time and temperature on the micro£ora and amine development in Spanish mackerel (Scomberomorous maculatus). J. Food Sci. 53, 1024^1029. Mietz, J. L. and Karmas, E. (1978) Polyamines and histamine content in rock¢sh, salmon, lobster and shrimp as an indicator of decomposition. J. Assoc. O¡. Anal. Chem. 61, 139^145.
Survival of amine-forming bacteria during ice storage 625
Moori, H., Cann, L. Y. and Taylor, L. Y. (1988) Histamine formation by luminous bacteria in mackerel stored at low temperatures. Nippon Suisan Gakkaishi 54, 299^305. Okuzumi, M., Fukumoto, I. and Fujii, T. (1990) Changes in bacterial £ora and polyamine contents during storage of horse mackerel meat. Nippon Suisan Gakkaishi 56, 1307^1312. Shakila, R. J.,Vasundhara, T. S. and Rao, D.V. (1995) Rapid quality assessment of shrimps during storage by monitoring amines. J. Food Sci. Technol. (Mysore) 32, 310^314. Taylor, S. L. 1986. Histamine food poisoning: toxicology and clinical aspects. CRC Crit. Rev.Toxicol. 17, 91^117.
Taylor, S. L. and Sumner, S. S. (1986) Determination of histamine, cadaverine and putrescine. In Seafood Quality Determination (Eds D. E. Kramer and J. Liston) pp. 253^245. Proceedings of an International Symposium. New York, Elsevier Science Publishers. Yamanaka, H. 1989. Changes in polyamines and amino acids in scallop adductor muscle during storage. J. Food Sci. 54, 1113^1115. Yoshinaga, D. H. and Frank, H. A. (1982) Histamineproducing bacteria in decomposing skipjack tuna (Katsuwonus pelamis). Appl. Environ. Microbiol. 44, 447^452.
doi:10.1006/yfmic.481
ORIGINAL ARTICLE
Survival of amine-forming bacteria during the ice storage of ¢sh and shrimp R. Lakshmanan1, R. Jeya Shakila*, G. Jeyasekaran Survival of amine-forming bacteria during the ice storage of ¢sh and shrimp was investigated up to 14 days of storage. On iced storage the total bacterial load was reduced to one log from an initial load of 105 cfu g 1 in fresh ¢sh/shrimp due to cold shock. The total incidence of biogenic amine-forming bacteria was found to be 74?63% in ¢sh and the same was recorded as 56?05% in shrimp.The amine-forming bacteria recorded were cadaverine- and putrescine-forming bacteria in ¢sh/shrimp, and no histamine former was detected. Gram-negative, non-fermentative rods, viz. Alcaligenes. Flavobacterium, Acinetobacter. Shewanella and Pseudomonas, were the predominant amine-forming bacteria during the ice storage of ¢sh and shrimp, in addition to the only Gram-positive genus Micrococcus. The genera Aeromonas and Photobacterium also survived ice storage to a certain extent and may also be responsible for the formation of amines in ¢sh and shrimp. # 2002 Published by Elsevier Science Ltd.
Introduction Amines are formed during spoilage of ¢sh as a result of bacterial decarboxylation of free amino acids. Di¡erent bacteria capable of decarboxylating amino acids have been isolated from ¢sh muscle (Taylor 1986, Yoshinaga and Frank 1982, Moori et al. 1988, Okuzumi et al. 1990). They include mesophilic as well as psychrophilic bacteria and most of them possess more than one decarboxylase enzyme (Taylor and Summer 1986). Among the amines, histamine has been more frequently implicated in food poisoning, and the diamines, primarily
*Corresponding author. Fax: +91- 461-340401/ 340574; E-mail: [email protected]; ¢[email protected] 1 Present address: Department of Bioscience and Biotechnology, Royal College Building, Strathclyde University, 204 George Street, Glasgow G 1 1XW, Scotland, UK. E-mail: [email protected] 0740 -0020/02/060617 +09 $35.00/0
putrescine and cadaverine, are known to enhance histamine poisoning (Bjeldanes et al. 1978). Putrescine is the decarboxylation product of ornithine, and cadaverine arises from decarboxylation of lysine. Putrescine and cadaverine have been regarded as the freshness index in ¢sh, as they were the only amines detected before the initial decomposition (Dawood et al. 1988, Fernanadez-Salguero et al. 1987, Yamanaka 1989). In shrimp, putrescine has been suggested as an index of decomposition (Mietz and Karmas 1978, Shakila et al. 1995). Storage of ¢sh and shell¢sh in ice has been the routine practice of preserving ¢sh on board ship and at shore. Many studies have been carried out to examine the e¡ect of temperature on amine formation in ¢sh (Behling and Taylor 1982, Lopez-Sabater et al. 1996, Koutsoumanis et al. 1999). However, reports state that there are di¡erences in the formation of amines in r 2002 Published by Elsevier Science Ltd.
Received: 4 October 2001 Department of Fish Processing Technology, Fisheries College & Research Institute,Tamil Nadu Veterinary and Animal Sciences University, Tuticorin 628 008, Tamilnadu, India
618 R. Lakshmanan et al.
¢sh during ice storage and such di¡erences are mainly due to the type and level of micro£ora present in ¢sh (Middlebrooks et al. 1988; Loperz-Sabater et al. 1996). This work was therefore undertaken with an objective of investigating the survival of the amine-forming bacteria during the storage of ¢sh and shrimp on ice (01C).
Materials and Methods Raw material Fresh emperor ¢sh (Lethrinus miniatus) and shrimp (Penaues semisulcatus) were procured from the local ¢sh market and immediately brought to the laboratory. The average length and weight of ¢sh were 18?22 cm and 108 g, respectively, and that of shrimp were 10 cm and 7 g, respectively. They were washed in potable water to remove blood, slime and any other extraneous material and stored under iced condition in insulated boxes having outlets to remove the melt water. Flake ice was added daily to compensate for melting of ice. Fish and shrimp samples were withdrawn for analysis on day 0, and after 3, 6, 9, 12 and 14 days of storage in ice. Samples were analysed in triplicate for the total bacterial load and their means with standard deviation were calculated.
Enumeration of total bacterial load Fish/shrimp samples (25 g) were homogenized with 225 ml of sterile physiological saline (0?85% NaCl), and serial decimal dilutions of each homogenate were carried out with the same diluent. The diluted suspension was plated onto Trypticase soy agar (TSA) (Hi-Media, Mumbai, India) following spread plate technique. The plates were incubated at 20 and 371C to isolate both the psychrotrophic and mesophilic bacteria, respectively. After an appropriate incubation period, the number of colonies developed on the plates were counted for the total mesophilic and psychrotrophic bacteria and expressed as cfu g 1.
Test for decarboxylase activity Representative isolates were picked up from the TSA plates incubated at 201C, puri¢ed and checked for their decarboxylase activity. Three types of media were prepared using modi¢ed Moeller’s decarboxylase broth base (Hi-Media, Mumbai, India) supplemented with di¡erent amino acids, viz. L -histidine hydrochloride, L -ornithine hydrochloride and L -lysine hydrochloride at 0?5% level (w/v). Medium without added amino acid served as the control. The isolates, which showed positive reaction for the decarboxylase test, were considered as amine-forming bacteria (Okuzumi et al. 1990).
Identi¢cation of positive isolates The amine-forming bacterial (AFB) isolates were identi¢ed by following a series of standard biochemical tests (Lechavelier et al. 1980, Buchanan and Gibbons 1982). The biochemical tests performed include Gram’s reaction, cell morphology, catalase, oxidase, oxidative-fermentative, motility, indole, methyl red, Voges Proskaeur, citrate utilization, urease, sensitivity to O/129 pteridine and ONPG, and fermentation tests for sugars such as glucose, sucrose, lactose and mannitol.
Results and Discussion Fig. 1 represents the total mesophilic and psychrotrophic bacterial load in ¢sh along with the total amine-forming bacteria during their storage on ice. The total mesophilic bacteria in fresh ¢sh was 105 cfu g 1, which on ice storage decreased by one log during the initial period (3^6 days) and later on steadily increased to 106 cfu g 1 at the end of storage. The initial reduction in the total mesophilic bacteria was mainly due to the e¡ect of cold shock (Ingram 1951). The total psychrotrophic bacteria, which was one log lower than the mesophilic bacteria, initially increased steadily on storage and reached a level of 107 cfu g 1 on the 14th day. Of the total psychrotrophic bacteria, the bacteria capable of producing amines were initially very low (101 cfu g 1 ) in ¢sh, but increased progressively upon storage to a ¢nal
Survival of amine-forming bacteria during ice storage 619
Figure 1. Changes in total mesophilic, psychrotrophic and amine-forming bacteria in ¢sh during ice storage.
level of 106 cfu g 1.There was a two-log increase in the total amine-forming bacterial load on the 9th and 14th day of storage in ¢sh. Fig. 2 shows the changes in the total mesophilic, psychrotrophic and amine-forming bacteria of shrimp held on ice.The total mesophilic bacterial load in fresh shrimp was also similar to that observed in ¢sh. Upon initial storage in ice, there was a log reduction in the counts and later it increased to a maximum of 106 cfu g 1 on the 12th day. Upon storage, the psychrotrophic bacteria proliferated slowly and dominated the mesophilic bacterial load, as the low temperature favoured their growth.The amine-
forming bacterial population in fresh shrimp was slightly higher (102 cfu g 1 ) than in ¢sh. On iced storage, their population decreased initially and then steadily increased up to 106 cfu g 1 at the end of storage.Therefore, both the ¢sh and shrimp muscle supports the survival of amine-forming bacteria even after prolonged storage in ice, indicating that the bacteria proliferating at chill environment have the ability to decarboxylate amino acids to form amines. The incidence of di¡erent amine-forming bacteria during the ice storage of ¢sh and shrimp is presented in Fig. 3. The amine-forming
620 R. Lakshmanan et al.
Figure 2. Changes in total mesophilic, psychrotrophic and amine-forming bacteria in shrimp during ice storage.
bacteria recorded in ¢sh and shrimp were putrescine- and cadaverine-forming bacteria and no histamine-forming bacteria were detected. Arnold et al. (1980) and Lopez-Sabater et al. (1996) also could not detect histamine-forming bacteria in tuna until 9^12 days of storage at 01C, which is similar to our observation.The absence of histamine-forming bacteria in ¢sh and shrimp was probably due to the e¡ect of low temperature and the presence of a low level of histidine in their muscle. However, the presence of putrescine- and cadaverine-forming bacteria in ¢sh and shrimp implies that they could grow at 01C and contribute to amine formation. In
fresh ¢sh, the incidence of amine-forming bacteria was relatively low (32%) and remained more or less the same during the early periods of storage (i.e 3^6 days), and later increased to about 96% on the 14th day. It was interesting to note that, during the initial storage in ice (0^6 days), the putrescine-forming bacteria dominated and, at the later stages, the cadaverineforming bacteria were dominant. In fresh shrimp, the incidence of amine-forming bacteria was similar to that observed in fresh ¢sh with about 30% (Fig. 4). But on storage in ice, there was a rapid reduction in their incidence to a level of 14% which then slowly increased and
Survival of amine-forming bacteria during ice storage 621
Figure 3. Incidence of amine-forming bacteria during ice storage of ¢sh. reached a level of 60% on the 12th day. Middlebrooks et al. (1988) have also reported that 65%of the bacterial isolates obtained from tuna held at 11C showed positive decarboxylase activity for lysine and arginine. But, in the present study, the incidence of amine-forming bacteria in the ice-stored ¢sh was comparatively higher (96%). It is also noted by Middlebrooks et al. (1988) that in ¢sh decomposed at 01C, the levels of cadaverine and putrescine began to rise sharply at about 11 days of incubation, while histamine appeared only after 16 days. This could be the reason why the presence of amineforming bacteria was high between 12^14 days of storage in ¢sh and 9^12 days of storage in shrimp. The amine-forming bacteria isolated from ¢sh were identi¢ed and are presented in Table 1. Fresh ¢sh predominantly harboured the genera Bacillus,Vibrio and Flavobacterium. The species of Bacillus isolated from ¢sh had the ability to produce putrescine, but the genera Vibrio and Flavobacterium had the ability
Figure 4. Incidence of amine-forming bacteria during ice storage of shrimp. to produce both cadaverine and putrescine. Among them, only Flavobacterium survived ice storage and was recorded till the end of storage. Bacteria belonging to the family Enterobacteriaceae were detected in few numbers and they were identi¢ed to be Enterobacter aerogenes and Proteus mirabilis, which demonstrated the ability to produce both cadaverine and putrescine. Aeromonas and Photobacterium were recorded on the 12th day of ice storage, which implies that they could survive and become responsible for the formation of cadaverine. Non-fermentative Gram-negative bacteria Shewanella, Acinetobacter, Alcaligenes, Pseudomonas and Moraxella were recorded in relatively large proportions at later stages of ice storage. Almost all the genera under this group had the ability to produce both cadaverine and putrescine, except Shewanella, which was a putrescine former, and Alcaligenes paradoxus,
622 R. Lakshmanan et al.
Table 1. Microbial £oraa isolated from ¢sh during ice storage Days of storage 0
3
6
9
12
14
Total isolates tested for decarboxylase activity
75
48
123
136
90
360
No. of positive isolates
29
43
38
128
67
346
I. Enterobacteriaceae 1. E. aerogenes 2. P. mirabilis
F F
1 F
F F
F 2
F F
F F
II.Vibrionaceae 1.Vibrio 2. Aeromonas 3. Photobacterium
8 F F
F F F
F F F
F F F
F 4 7
F F F
III. Gram-negative, non-fermentative rods 1. Flavobacterium 2. Shewanella 3. Acinetobacter 4. Alcaligenes 5. Alcaligenes paradoxus 6. Pseudomonas 7. Moraxella
3 F F F F F F
6 F F F F F
11 1 7 F F F F
2 3 5 F 23 18 F
18 F 11 7 F 3 1
67 F F 134 F 6 F
IV. Gram-positive bacteria 1. Bacillus 2. Micrococcus luteus 3. M. varians 4. M. roseus 5. Corynebacterium 6. Staphylococcus epidermidis
18 F F F F F
F 5 F F F 1
F F F F 12 5
F 23 26 24 2 F
F 2 14 F F F
F 67 67 F F F
a
Numbers.
which was a cadaverine former. Gram-positive bacteria Bacillus, which was recorded in large numbers in fresh ¢sh, failed to grow during ice storage. On the other hand, Micrococcus sp. survived at low temperature and started proliferating after 9 days. Three species of Micrococcus were isolated and each of them possessed different decarboxylase enzymes. Staphylococcus epidermidis and Corynebacterium were also recorded in high numbers during the initial period and did not survive beyond 9 days of storage in ice. Corynebacterium is a cadaverine-forming bacterium, while S. epidermidis produces both cadaverine and putrescine. The amine-forming bacteria identi¢ed during the ice storage of shrimp are presented in Table 2. Fresh shrimp harboured Gram-positive amine-forming bacteria Bacillus, Corynebacterium and Staphylococcus in relatively high numbers, and they totally diminished on
further storage in ice. The species of Bacillus detected in shrimp had the ability to produce both cadaverine and putrescine, and the genus Corynebacterium was a cadaverine former. The amine-forming bacterial £ora, which survived at 01C throughout the storage period, was the genus Alcaligenes. The bacteria belonging to the family Enterobacteriaceae were recorded in very few numbers with those observed in ¢sh. On the other hand, Aeromonas sp., Vibrio sp. and non-fermentative Gram-negative rods survived ice storage and became the major £ora between 9 and 12 days of storage. Among the Aeromonas, the species A. veronii had the ability to form only putrescine. It was found that the non-fermentative rods Alcaligenes, Shewanella, Acinetobacter and Pseudomonas were the dominant genera responsible for amine formation in shrimp at the ¢nal stages. The only Gram-positive bacteria that could
Survival of amine-forming bacteria during ice storage 623
Table 2. Microbial £oraa isolated from shrimp during ice storage Days of storage 0
3
6
9
12
Total isolates tested for decarboxylase activity
92
69
51
78
115
No. of positive isolates
42
10
23
49
103
I. Enterobacteriaceae 1. E. aerogenes
F
F
F
4
F
II.Vibrionaceae 1.Vibrio 2. Aeromonas 3. Aeromonas veroniiI
F F F
F F F
F 8 F
5 9 8
3 F 5
III. Gram-positive, non-fermentative rods 1. Flavobacterium 2. Shewanella 3. Alcaligenes 4. Acinetobacter 5. Pseudomonas
F F F F F
F F 10 F F
3 3 1 F F
8 8 2 5 F
F F 28 22 42
F F F F
5 3 F F
F F F F
F 3 F F
IV. Gram-positive bacteria 1. Bacillus 2. Micrococcus luteus 3. M. lylae 4. Corynebacterium 5. Staphylococcus epidermidis a
14 F 14 14
Numbers.
survive ice storage belonged to the genus Micrococcus and they were recorded only from the 6th day of storage. Micrococcus detected in shrimp had the ability to form both cadaverine and putrescine. It was clear from this study that during the ice storage of ¢sh and shrimp, amine-forming bacteria could survive and proliferate slowly upon storage, and contribute to amine formation. Fish has been observed to harbour more numbers of amine-forming bacteria than shrimp, this is because of the presence of higher levels of free lysine and ornithine in the ¢sh muscle than in shrimp. Fresh ¢sh and shrimp harboured greater numbers of Gram-positive amine-forming bacteria, fewer numbers of Vibrio and non-fermentative Gram-negative rods. Among these bacteria,Vibrio and Bacillus failed to grow in ice, but the non-fermentative Gram-negative rods survived ice storage.Taylor (1986) has reported that the amines are generally formed in ¢sh by the bacteria belonging to the family Enterobacteriaceae, Lactobacillus and Clostridium. But, this was true only in the
case of ¢sh held at ambient temperature (20^ 371C). In the ¢sh stored in ice, the bacteria belonging to the family Enterobacteriaceae were recorded in low numbers and, therefore, they were not the major amine-forming bacteria. Earlier Moori et al. (1986) also pointed out that, at low temperature, the bacteria belonging to the family Enterobacteriaceae are not very active in forming the amines, which was in agreement with our ¢ndings. However, bacteria belonging to the family Vibrionaceae, Vibrio, Aeromonas and Photobacterium, survived ice storage, proliferated slowly and became the dominant amine-forming bacteria at later stages (9^12 days) in ice-stored ¢sh or shrimp. Photobacterium phosphoreum has been recognized as a principal species responsible for histamine formation in ¢sh at low temperature (Moori et al. 1986). A species of Photobacterium, which has the ability to produce cadaverine, was also detected in horse mackerel held at 51C by Okuzumi et al. (1990). Photobacterium phosphoreum was detected after 12 days of storage in ice, was identi¢ed to be relatively
624 R. Lakshmanan et al.
resistant to cold storage and grew slowly at 41C (Fujii et al. 1994). In the present study also, the amine-forming Photobacterium sp. was detected in the emperor ¢sh on the 12th day of storage in ice. On the other hand, non-fermentative Gramnegative rods, viz. Alcaligenes, Flavobacterium, Acinetobacter, Shewanella and Pseudomonas detectable during initial stages of ice storage, became the predominant amine-forming bacteria in ¢sh and shrimp at later stages. In the tuna ¢sh decomposed at 01C, Pseudomonas sp. were reported to be the most frequently encountered bacteria, besides Aeromonas, Proteus, Citrobacter, Vibrio, Acinetobacter and Morganella morganii (Middlebrooks et al. 1988). It has also been reported that, in horse mackerel held at 51C, Moraxella was dominant up to 2 days and later Pseudomonas sp. showed dominance at the decomposition stage (Okuzumi et al. 1990), which was similar to our observations. Most of the Gram-positive bacteria which were present in ¢sh/shrimp failed to grow in ice, and the only dominant Gram-positive genus which survived ice storage was Micrococcus. It can therefore be concluded that during the ice storage of emperor ¢sh and £ower shrimp, cadaverine- and putrescine-forming bacteria could survive and proliferate rapidly between 9 and 12 days, and contribute to the formation of amines. The amine-forming bacteria recorded in ¢sh and shrimp were not invariably di¡erent. The dominant amine-forming bacteria belonged mainly to the group of non-fermentative Gram-negative rods, Photobacterium, Aeromonas and Micrococcus. The bacteria belonging to the family Enterobacteriaceae were least responsible for amine formation. Hence, control measures to minimize/retard amine formation during the ice storage of ¢sh/shrimp need to be formulated based on the incidence and survival of amine-forming bacteria.
Acknowledgements This study was funded by the Indian council of Agricultural Research (ICAR), New Delhi, Government of India.
References Arnold, S. H., Price, R. J. and Brown,W. D. (1980) Histamine formation by bacteria isolated from skipjack tuna (Katsuwonus pelamis) Nippon Suisan Gakkaishi. 46, 991^995. Behling, A. R. and Taylor, S. L. (1982) Bacterial histamine production as a function of temperature and time of incubation. J Food Sci. 47, 1311^1314, 1317. Buchanan, R. E. and Gibbons N. E. (Eds) (1982) Bergey’s Manual of Determinative Bacteriology. 8th edn. Baltimore, USA,Williams & Wilkins. Bjeldanes, L. F., Shutz, D. E. and Morris, M. M. (1978) Etiology of scombroid poisoningFcadaverine potentiation of histamine toxicity in guinea pigs. Food Cosmet. Toixicol. 16, 157^162. Dawood, A. A., Karkalas, J., Roy, R. N. and Williams, C. A. (1988) The occurrence of non-volatile amines in chilled-stored rainbow trout (Salmo irideus). Food Chem. 27, 33^45. Fernandez-Salguero, J. and Mackie, I. M. (1987) Comparative rates of spoilage of ¢llets and whole ¢sh during storage of haddock (Melanogrammes aegie¢nus) and herring (Clupea herengus) as determined by the formation of non-volatile amines. Int. J. Food Sci. Technol. 22, 385^390. Fujii, T., Kurihara, K. and Okuzumi, M. (1994) Viability and histidine decarboxylase activity of halophilic histamine-forming bacteria during frozen storage. J. Food Prot. 57, 611^613. Ingram, M. (1951) The e¡ect of cold on microorganisms in relation to food. Proc. Soc. Appl. Bacteriol. 14, 243^249. Koutsoumanis, K., Lampropoulou, K. and GeorgeJohn, E. N. (1999) Biogenic amines and sensory changes associated with the microbial £ora of Mediterranean gilt-head sea bream (Sparus aurata) stored aerobically at 0, 8 and 151C. J. Food Prot. 62, 398^402. Lechavelier, M. W., Seider, R. J. and Evans, E. M. (1980) Enumeration and characterization of standard plate count bacteria in chlorinated and raw water supplies. Appl. Environ. Microbial. 40, 922^926. Lopez-Sabater, E. I., Rodriguez-Jerez, J. J., Roig-Sagueus, A. X. and Mora-Ventura, M. A. T. (1996) Bacteriological quality of tuna ¢sh (Thunnus thynnus) destined for canning: e¡ect of handling on the presence of histidine decarboxylase bacteria and histamine level. J. Food Prot. 57, 318^323. Middlebrooks, B. L.,Toom, P. M., Douglas,W. L., Harrison, R. E. and McDowell, S. (1988) E¡ects of storage time and temperature on the micro£ora and amine development in Spanish mackerel (Scomberomorous maculatus). J. Food Sci. 53, 1024^1029. Mietz, J. L. and Karmas, E. (1978) Polyamines and histamine content in rock¢sh, salmon, lobster and shrimp as an indicator of decomposition. J. Assoc. O¡. Anal. Chem. 61, 139^145.
Survival of amine-forming bacteria during ice storage 625
Moori, H., Cann, L. Y. and Taylor, L. Y. (1988) Histamine formation by luminous bacteria in mackerel stored at low temperatures. Nippon Suisan Gakkaishi 54, 299^305. Okuzumi, M., Fukumoto, I. and Fujii, T. (1990) Changes in bacterial £ora and polyamine contents during storage of horse mackerel meat. Nippon Suisan Gakkaishi 56, 1307^1312. Shakila, R. J.,Vasundhara, T. S. and Rao, D.V. (1995) Rapid quality assessment of shrimps during storage by monitoring amines. J. Food Sci. Technol. (Mysore) 32, 310^314. Taylor, S. L. 1986. Histamine food poisoning: toxicology and clinical aspects. CRC Crit. Rev.Toxicol. 17, 91^117.
Taylor, S. L. and Sumner, S. S. (1986) Determination of histamine, cadaverine and putrescine. In Seafood Quality Determination (Eds D. E. Kramer and J. Liston) pp. 253^245. Proceedings of an International Symposium. New York, Elsevier Science Publishers. Yamanaka, H. 1989. Changes in polyamines and amino acids in scallop adductor muscle during storage. J. Food Sci. 54, 1113^1115. Yoshinaga, D. H. and Frank, H. A. (1982) Histamineproducing bacteria in decomposing skipjack tuna (Katsuwonus pelamis). Appl. Environ. Microbiol. 44, 447^452.