Fish Sci (2010) 76:177–181 DOI 10.1007/s12562-009-0211-0 ORIGINAL ARTICLE Biology Identification of facultative anaerobic bacteria isolated from the intestine of the minke whale Balaenoptera acutorostrata by 16S rRNA sequencing analysis Go Ogawa • Masami Ishida • Hidehiro Kato Yoshihiro Fujise • Naoto Urano • Received: 23 July 2009 / Accepted: 11 November 2009 / Published online: 22 January 2010 Ó The Japanese Society of Fisheries Science 2010 Keywords Balaenoptera acutorostrata Á Edwardsiella ictaluri Á Enterobacteriaceae Á Intestinal flora Á Minke whale classification of bacteria However, commercial whaling was prohibited after a decision by the International Whaling Commission in 1982 [3], before Woese et al [4] reported their rRNA classification method Therefore, the sampling of whale excrement is a valuable way of studying the microbiology of whales Bacterial infection is one of the most frequent causes of death of marine animals in Japan [5] Additionally, whale meat contamination and food poisoning have been reported [6–8] Studying the intestinal flora of the whale is also important for understanding sickness in whales and potential pathogens that could be passed on to humans We obtained excrement from wild common minke whales Balaenoptera acutorostrata during research whaling carried out in 2008 In this report, we take the first step in the study of the whale intestinal flora, by counting colony-forming units in the whale excrement and identifying facultatively anaerobic bacteria Introduction Materials and methods There have been few reports about the isolation and identification of bacteria from whale intestine Only the Brucella strains have been reportedly isolated from whale skins [1, 2] In recent years, 16S rRNA sequence analysis has become the standard method for identification or Minke whale intestines that contain excrement were obtained from the Japanese Whale Research Program under special permit in Japan’s Whale Research Program in the western North Pacific (JARPN II); the details are shown in Table The samples were transported within a day, on ice, to our laboratory in a sterilized plastic bag The excrement from the intestine was suspended in saline and spread onto each type of agar plate Media, containing 2% agar, were prepared for each of the following: nutrient broth (Eiken Chemical, Tochigi, Japan), GAM broth (Nissui Pharmaceutical, Tokyo, Japan), MRS (Merck, Darmstadt, Germany) and desoxycholate (Wako Pure Chemical industries, Osaka, Japan) Agar plates were Abstract There are few reports in the literature about the isolation of bacteria from whale intestine In this report, we counted colony-forming units in the feces obtained from three female common minke whales (Balaenoptera acutorostrata) The number of colony-forming units ranged from (2.2 ± 0.4) 105 to (8.9 ± 2.0) 108 per gram (wet weight) of excrement 16S rRNA gene sequences of 141 isolates were determined These strains were identified as Enterococcus faecalis, Enterococcus sp., Enterobacter cloacae, Enterobacter sp., Escherichia coli, Edwardsiella ictaluri or Clostridium sp The data suggested that the facultative anaerobic population of the intestinal bacterial flora of the minke whale was similar to that of ground mammals G Ogawa Á M Ishida Á H Kato Á N Urano (&) Tokyo University of Marine Science and Technology, Konan, Minato-ku, Tokyo 108-8477, Japan e-mail: urano@kaiyodai.ac.jp Y Fujise The Institute of Cetacean Research, Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan 123 178 Fish Sci (2010) 76:177–181 Table Individual data for each of the minke whales studied Whale no.a Capture date Location Sex Body length (m) Time after deathb (min) 08NPCS-M020 25 April 2008 38°000 N, 141°090 E Female 7.71 262 08NPCS-M038 May 2008 38°070 N, 141°230 E Female 5.27 169 08NPCS-M050 16 May 2008 38°050 N, 141°260 E Female 5.97 206 a Defined by the Institute of Cetacean Research b Time after death to take out part of the intestine Table Viable bacterial count (cfu/g of wet weight) cultured from the whale feces Whale no.a Large intestine region b Nutrient broth MRS Desoxycholate 08NPCS-M020 Upper (3.3 ± 0.4) 10 (1.3 ± 0.7) 10 Not tested 08NPCS-M038 Upper (2.1 ± 0.5) 107 (2.0 ± 0.5) 107 (8.9 ± 2.0) 108 (1.0 ± 0.5) 106 (2.7 ± 1.1) 108 (5.6 ± 2.4) 107 (4.5 ± 0.3) 108 (4.8 ± 1.3) 106 (3.2 ± 0.4) 10 (3.2 ± 0.3) 10 (2.9 ± 0.5) 106 (1.2 ± 0.3) 10 (2.7 ± 1.1) 10 (3.8 ± 0.6) 105 (2.3 ± 1.0) 10 (1.7 ± 0.4) 10 (2.2 ± 0.4) 105 (3.9 ± 0.1) 10 (1.9 ± 0.4) 10 (7.4 ± 1.0) 105 Middlec Lower 08NPCS-M050 d Upper Middle Lower (1.9 ± 0.2) 10 GAM (5.7 ± 0.6) 10 (1.0 ± 0.1) 10 (5.0 ± 1.8) 10 (1.8 ± 0.4) 10 Each value is mean ± SEM (n = 3) a Defined by the Institute of Cetacean Research b Under 2–3 m from small intestine c Middle of large intestine d Upper about 2–3 m from anus incubated at 35°C for a week The GAM broth plates were incubated anaerobically with Aneropack (Mitsubishi Gas Chemical, Tokyo, Japan) After incubation, bacterial colonies were counted and single colonies were picked at random The cells isolated were stored at 10°C and identified The 16S rRNA sequence analysis was carried out according to our previous study [9] The BLASTN program [10] was then used to determine homology with other organisms The 16S rRNA sequence data obtained in this study (141 sequences) were registered in the DNA Data Bank of Japan (DDBJ) (accession no AB496751– AB496891) Results and discussion The viable bacterial counts of each sample are listed in Table Bacterial counts ranged from 1.3 105 to 8.9 108 cfu/g These results corresponded to previous reports looking at the number of facultative anaerobic bacteria in human and fish intestines [11–13] Therefore, we propose that the number of bacteria in the whale intestine is similar to that of others animals However, a higher viable bacterial count was reported in human feces under 123 completely anaerobic conditions [14] A higher bacterial count may be confirmed if the feces are handled under anaerobic conditions Additionally, the viable bacterial count of feces was similar to those previously reported in whale stomach [15, 16] We predict that about 105–108 (cells/g or ml) viable bacteria live in the digestive tract of whales The viable bacterial count in whale 08NPCS-M020 was lower than that found in the other whales This may just be an individual difference among the whales High numbers of bacteria on the GAM agar suggested that this agar (the composition of nutrients and/or the anaerobic incubation conditions) is more suitable for the isolation of bacteria from whale feces compared with other media Identification of bacterial strains isolated from whale 08NPCS-M020 are shown in Table Twenty-two strains were isolated and identified as Enterococcus faecalis or Enterococcus sp from the nutrient agar and GAM agar These media are used for the growth of a wide range of species, indicating that only a few species of facultatively anaerobic bacteria may inhabit the whale intestine E faecalis is often isolated from the feces of animals and is known to be potentially pathogenic [17] Therefore, whale excrement may be a potential human pathogen The 71 strains isolated and identified from whale 08NPCS-M038 are shown in Table These included Fish Sci (2010) 76:177–181 179 Table BLAST search results based on 16S rRNA gene sequence of bacterial strains isolated from the whale (08NPCS-M020) large intestine Isolated regiona Number of strains (agar plates:numberb) Closest sequence (DDBJ accession no.) Similarity (%) Upper (G:2) Enterococcus sp (FJ463817) 100 20 (G:3 D:8 N:9) Enterococcus faecalis (Y18293) 100 DDBJ accession numbers of these strains are AB496751–AB496891 (22 sequences) G GAM, D desoxycholate, N nutrient broth, M MRS a Same in Table b Detail number of strains isolated from agar plates Table BLAST search results based on 16S rRNA gene sequence of bacterial strains isolated from the whale (08NPCS-M038) large intestine Isolated regiona Upper Middle Lower Number of strains (agar plates:numberb) Closest sequence (DDBJ accession no.) Similarity (%) (G:9) Enterobacter sp (EU162036) 100 (G:3 D:3 N:1) Enterobacter cloacae (Y17665) 100 (G:2 M:3) Enterococcus faecalis (Y18293) 99.7–100 (G:2 N:2) Escherichia coli (CP000970) 99.7–100 (M:2) Enterococcus faecalis (EU547775) 100 (G:1) Enterococcus faecalis (EF653454) 100 (D:1) 10 (G:5 D:4 N:1) Escherichia coli (EU330541) Enterobacter cloacae (Y17665) 99.3 100 (G:1 M:5) Enterococcus faecalis (Y18293) 99.7–100 (G:1 N:2) Escherichia coli (CP000970) 99.7–100 (G:1) Enterococcus faecalis (EF653454) 100 (D:1) Edwardsiella ictaluri (EU285524) 100 (G:3 M:5) Enterococcus faecalis (Y18293) 99.7–100 (G:1 D:2 N:3) Enterobacter cloacae (Y17665) 100 (G:3 D:1 N:2) Escherichia coli (CP000970) 99.7–100 (D:2) Edwardsiella ictaluri (EU285524) 100 DDBJ accession numbers of these strains are AB496773–AB496843 (71 sequences) G GAM, D desoxycholate, N nutrient broth, M MRS a b Same in Table Detail number of strains isolated from agar plates Enterobacter cloacae, Enterobacter sp., Escherichia coli, E faecalis and Edwardsiella ictaluri E coli and Enterobacter sp are known to inhabit the animal intestine [17] However, there have been few reports on the occurrence of E ictaluri in mammal excrement E ictaluri has generally been isolated from fish or freshwater environments [18– 20] Isolation of E ictaluri from whale excrement is a novel finding The 48 strains isolated and identified from whale 08NPCS-M050 are shown in Table These included E faecalis, E coli and Clostridium sp Clostridium sp are anaerobic endospore-forming bacteria [17], and we suppose that a clostridial spore was in the specimen and germinated in anaerobic culture Since several species of Clostridium are human pathogens [17], whale feces possibly contain pathogenic Clostridium spores Among the three whale samples, different kinds of bacterial genera were found Only Enterococcus species were isolated from 08NPCS-M020, whereas species from three different genera, Enterobacter, Enterococcus and Escherichia, were isolated from 08NPCS-038 Therefore, we suggest that individual differences occur in the intestinal bacterial flora of whales, at least with respect to facultatively anaerobic bacteria In contrast, there was no difference in the species level isolated from excrement taken from different regions of the large intestine Among the facultative anaerobic bacteria isolated in this study, most species, except E ictaluri, are representative of 123 180 Fish Sci (2010) 76:177–181 Table BLAST search results based on 16S rRNA gene sequence of bacterial strains isolated from the whale (08NPCS-M050) large intestine Isolated regiona Number of strains (agar plates:numberb) Closest sequence (DDBJ accession no.) Similarity (%) Upper (G:3 N:2 M:3) Enterococcus faecalis (Y18293) 99.7–100 (G:1 D:4 N:2) Escherichia coli (Z83205) 100 (G:1) Enterococcus faecalis (EU547775) 100 (G:4 N:1 M:4) Enterococcus faecalis (Y18293) 100 (D:5 N:2) Escherichia coli (Z83205) 100 (G:1) Enterococcus faecalis (EU547775) 100 (G:2 N:2 M:4) (D:4 N:2) Enterococcus faecalis (Y18293) Escherichia coli (Z83205) 100 100 (G:1) Clostridium sp (DQ298112) 100 Middle Lower DDBJ accession numbers of these strains are AB496844–AB496891 (48 sequences) G GAM, D desoxycholate, N nutrient broth, M MRS a Same in Table b Detail number of strains isolated from agar plates the intestinal bacteria of ground mammals Therefore, we supposed that the intestinal flora of whales would be similar to that of many ground mammals However, the microbial diversity in the intestine of the whale was lower than that of other mammals [21] Such low diversity seems to be related to the predatory behavior of minke whales Ley et al [22] point out that the diversity of intestinal flora of predatory animals is lower than that of herbivorous or omnivorous animals Additionally, there was a difference between the bacterial species in the feces in this study and those found in the stomach of whales [16] In this study, Enterobacteriaceae were mainly isolated from the faces The bile acid of whale’s liver [23] may affect the bacterial flora of the stomach by acting as a selective pressure Saltwater fish have intestinal bacterial flora composed of Vibrio/Photobacterium, Micrococcus, Corynebacterium [24] or Enterobacteriaceae, Aeromonas, Pseudomonas [25] or mainly Vibrio [26] Enterobacteriaceae was a common species between whales (E coli and Enterobacter) and salt fishes There was no other common species This result suggests that whales have a particular intestinal bacterial flora in seawater environments [27, 28] E ictaluri isolated from the whale intestine in this study is a characteristic bacterium of fish [19, 20] Sakata reported that fish intestinal flora was relatively simple compared with that of ground mammals [29] These facts suggest that the whale intestinal flora have characteristic properties of not only ground mammals, but also of fish The 16S rRNA clone library method [14, 30] or PCRDGGE method [31] may be useful for detailed analysis Acknowledgments We thank Dr Genta Yasunaga and members of the Japanese Whale Research Program, who operated under special permit in Japan’s Whale Research Program in the western North 123 Pacific (JARPN II), for preparing whale feces samples and gathering data about whales References Clavareau C, Wellemans V, Walravens K, Tryland M, Verger JM, Grayon M, Cloeckaert A, Letesson JJ, Godfroid J (1998) Phenotypic and molecular characterization of a Brucella strain isolated from a minke whale Balaenoptera acutorostrata Microbiology 144:3267–3273 Tryland M, Kleivane L, Alfredsson A, Kjeld M, Arnason A, Stuen S, Godfroid J (1999) Evidence of Brucella infection in marine mammals in the 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individuals using 16S rDNA library and T-RFLP Microbiol Immunol 47:557–570 Yang JL, Cheng AC, Wang MS, Pan KC, Luo QH, Zhu DK, Chen XY, Qi XF (2009) New strategies for electrophoresis analysis of enterobacterial repetitive intergenic consensus PCR in animal intestinal microflora J Microbiol Methods 77:63–66 123 Fish Sci (2010) 76:183–188 DOI 10.1007/s12562-009-0205-y ORIGINAL ARTICLE Biology Predation dynamics of mackerel on larval and juvenile anchovy: is capture success linked to prey condition? Dominique Robert • Akinori Takasuka • Sayaka Nakatsuka Hiroshi Kubota • Yoshioki Oozeki • Hiroshi Nishida • Louis Fortier • Received: June 2009 / Accepted: 16 November 2009 / Published online: 16 January 2010 Ó The Japanese Society of Fisheries Science 2010 Abstract We tested whether the predation dynamics of chub mackerel Scomber japonicus and spotted mackerel S australasicus on young anchovy Engraulis japonicus relates to individual growth characteristics of the prey and could account for the growth-selective survival predicted by recruitment hypotheses Juvenile and adult mackerel were sampled along with their young anchovy prey field in 2004 (juvenile mackerel and larval anchovy) and 2005 (adult mackerel and juvenile anchovy) off the Pacific coast of Honshu, Japan The recent 5-day mean growth rate of larval and juvenile survivors and prey found in the stomach of mackerel was estimated from the otolith microstructure No significant difference was found between the recent growth of larval or juvenile survivors and that of preyed individuals We conclude that despite a relatively small body size, the high activity level and predation skills displayed by mackerel prevent fast-growing larvae and early juveniles from benefitting in terms of the expected survival advantage over slow-growers Hence, growth-selective predation mortality of larval fish would depend on the feeding ecology of the predator rather than predator size Selection for fast growth is more likely to occur under D Robert (&) Á L Fortier De´partement de Biologie, Que´bec-Oce´an, Pavillon Alexandre-Vachon, Universite´ Laval, Quebec, QC G1V 0A6, Canada e-mail: dominique.robert@qo.ulaval.ca A Takasuka Á H Kubota Á Y Oozeki Á H Nishida National Research Institute of Fisheries Science, Fisheries Research Agency, Yokohama, Kanagawa 236-8648, Japan S Nakatsuka Á Y Oozeki Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan predation pressure from invertebrate organisms and small pelagic fish specialized on zooplankton, such as herring and anchovy Keywords Growth rate Á Growth-selective predation Á Larval and juvenile anchovy Á Mackerel Á Otolith microstructure Á Predation mortality Introduction In marine fish, strong year classes are often associated to a high survival rate during the first weeks or months of life [1–3] Early life survival variability would in turn be driven by variable growth performance Fast-growing individuals generally experience lower cumulative vulnerability to predation, explaining their higher recruitment potential relative to slower growing conspecifics [4–6] Fast growers may also gain a survival advantage due to their larger body size (‘‘bigger is better’’ hypothesis [6]) and the shorter duration of the larval stage (‘‘stage duration’’ hypothesis [7]) during which mortality is maximal Moreover, a high growth rate can directly translate into decreased vulnerability to predation (‘‘growth-selective predation’’ hypothesis [5]) In these mechanisms, predation is assumed to be the major and direct source of mortality Such a potential linkage of growth to survival led to the assumption that the detection of strong selection for fast growth during early life of a given cohort is a symptom of high predation pressure [8–10] However, the typical survival advantage of fast growers under planktivorous predation pressure (e.g., small pelagic fish) tends to disappear when young fish face large piscivorous predators [11] As survival probability at a given growth rate is predator-specific, the predator field encountered during early life stages would 123 184 regulate the characteristics of survivors through selection on growth traits It would therefore appear to be important to describe predation dynamics of the main larval fish predators to determine whether their impact on survival can be resolved by the analysis of selection for fast growth [11] Mackerel Scomber spp are often depicted as one of the main predators of young stages of marine fish [12–15] Their occurrence in most temperate coastal areas of the world implies predation pressure on the larvae and juveniles of a large number of commercially important species Despite representing a major potential source of mortality for young fish, our knowledge of the predation dynamics of mackerel remains limited to the results of a few qualitative [16] and laboratory [12, 17–19] studies The objective of this study was to evaluate the role of mackerel in driving growth-selective survival in young fish Accordingly, we provide a field-based test of the ‘‘growth-selective predation’’ hypothesis [5], proposing that growth rate directly impacts vulnerability to predation, independent of body size or stage duration Two types of predator–prey interactions were assessed: (1) juvenile mackerel preying on larval anchovy and (2) adult mackerel preying on juvenile anchovy in the western North Pacific offshore Japan Materials and methods Japanese anchovy Engraulis japonicus larvae and juveniles, as well as their chub mackerel Scomber japonicus and spotted mackerel S australasicus predators, were collected concurrently offshore Honshu (Japan) in the western North Pacific (Fig 1) Larval and juvenile anchovy of standard length (SL) 20–65 mm (n = 47) and predatory juvenile mackerel of fork length (FL) 52–113 mm Fish Sci (2010) 76:183–188 (n = 28) were simultaneously sampled using an otter trawl (mesh size 10 mm) The trawl was towed for 30 at a speed of 3.5 knots in the surface layer (depth \27 m) after sunset on May 20, 2004 Adult mackerel (219–293 mm FL, n = 61) were collected from two consecutive drift net deployments (mesh size 19–157 mm), each followed by a concurrent kite trawl (mesh size mm) or MOHT [20] net (mesh size 1.59 mm) tow to sample potential anchovy prey The two different gear types used to capture young anchovy allowed us to sample the whole body length interval (11–64 mm SL, n = 180) All of these deployments were realized during nighttime hours on 7–8 June 2005 along the trajectory of a drifting buoy to optimize the probability of repeatedly sampling the same potential prey and predator populations All samples were frozen at -20°C immediately following capture until being processed in the laboratory After taking the FL measurement of juvenile and adult mackerel, we dissected and identified the gut content, sorting out larval and juvenile Japanese anchovy Anchovy prey found in the gut of mackerel were digested to various degrees, but sagittal otoliths were retained in the head of most individuals In total, the otoliths of 13 and 163 individuals ingested by juvenile and adult mackerel, respectively, were available for further analyses Young anchovy collected concurrently with predatory mackerel were regarded as the original populations of these ingested individuals Anchovy prey were pooled among the two mackerel species Sagittal otoliths were extracted from all young anchovy (prey and original populations) and mounted on a glass slide with enamel resin under a binocular microscope Maximum otolith radius (OR) and daily growth increment width were measured to the nearest 0.1 lm along a transect from the nucleus to the outermost margin Otolith Fig Sampling area offshore the Honshu island of Japan in the western North Pacific, with stations where juvenile and adult mackerel were collected along with their young anchovy prey field 123 Fish Sci (2010) 76:183–188 185 measurements were conducted using an otolith measurement system (RATOC System Engineering, Bunkyo-ku, Tokyo, Japan) that consists of a transmitting light microscope equipped with a video camera connected to a computer and monitor Following Searcy and Sponaugle [21], we did not reconstruct each individual somatic growth trajectory or growth rate history via back-calculation techniques in order to avoid potentially important sources of error In particular, the relation between otolith and somatic growth changes through ontogeny in young anchovy (see below), which makes it difficult to derive growth history from a single reliable OR–SL equation Instead, OR and increment width were considered to be proxies for somatic size and daily growth rate, respectively A detrended growth index was computed to reduce potential bias due to sudden departures in the growth trajectory and allow the inclusion of all individuals in the analysis, independent of their age [22, 23]: DGij ¼ ðIWij À IWj ÞSDÀ1 j ð1Þ where DGij is the detrended growth of individual I at age j; and IW and SD are the increment width and standard deviation, respectively This index provides a relative measure of growth performance realized by a given individual at a certain age, with mean growth achieved at the same age for all individuals used as a reference point It thus yields an estimate of growth remaining invulnerable to any ontogenetical shifts in growth patterns Here, we defined recent growth performance of a given individual as the average detrended growth achieved during the last full days of life (i.e., excluding the edge of the otolith) Recent 5-day mean detrended growth was compared between the ingested anchovy and their original population by analysis of variance (ANOVA) or analysis of covariance (ANCOVA) with the OR as a covariate Results The relationship between OR and SL of the original larval populations was expressed by strong allometric functions, indicating that otolith growth is a reliable proxy for somatic growth in young anchovy (Fig 2) The slope of the relationship changed at an OR of approximately 280 lm (SL 30 mm), which corresponds to the transition between the larval and juvenile stages [24] Due to this ontogenetic change in the nature of the OR–SL relationship, as well as the difficulty of precisely measuring the SL of ingested larvae, we considered otolith growth directly for further analyses Fig Relationship between sagittal otolith radius (OR) and standard length (SL) in young anchovy Allometric regressions for the larval and juvenile stages are Log(SL) = 0.101 Log(OR) ? Log(6.79) (n = 101, r2 = 0.95, P \ 0.001) and Log(SL) = 0.068 Log(OR) ? Log(11.69) (n = 527, r2 = 0.86, P \ 0.001), respectively Juvenile mackerel preyed preferentially on larval anchovy (Fig 3a), and we observed only one occurrence of predation on a juvenile anchovy (OR [ 280 lm) A positive linear relation was found between recent 5-day mean detrended growth and OR over the larval size interval consumed by juvenile mackerel (Fig 3b) Both slopes and intercept did not differ between larval anchovy prey (n = 12) and original larval population (n = 34) (ANCOVA, P = 0.15), indicating that juvenile mackerel captured their larval prey independent of prey growth Contrary to juvenile mackerel, adult mackerel mainly consumed juvenile anchovy and seldom preyed on the larvae (OR \ 280 lm) available in the environment (Fig 4a) No difference was observed between the recent 5-day mean detrended growth of juvenile anchovy prey (n = 141) and that of the original juvenile population (n = 114) over the predated size interval (Fig 4b; ANOVA, r2 = 0.01, P = 0.06) Discussion The key role of mackerel Scomber spp as a regulator of fish populations through predation on early ontogenetic stages was proposed nearly three decades ago [13] This hypothesis has been strongly supported by the strong negative relationships between small pelagic fish abundance and demersal fish recruitment reported in recent studies [15, 25] However, while clupeid predation dynamics on larval fish has been relatively well documented [26–28], field reports 123 186 Fish Sci (2010) 76:183–188 Fig Relationship between recent 5-day mean otolith growth rate (a) and recent detrended 5-day mean growth rate (b) over the ingested body size range, and otolith radius for larval anchovy survivors and prey found in the gut of juvenile mackerel Fig Relationship between the recent 5-day mean otolith growth rate (a) and recent detrended 5-day (5d) mean growth rate (b) over the ingested body size range, and otolith radius for juvenile anchovy survivors and prey found in the gut of adult mackerel of mackerel predation on ichthyoplankton remain few in number and only qualitative in nature [16] Our study is the first field-based assessment of both late juvenile and adult mackerel predation dynamics on young fish and the influence of potential prey condition (given by recent growth) on capture success The possibility of a sampling bias in our measurements of the prey field encountered by juvenile mackerel can not be ruled out The 10-mm mesh net probably only retained a partial fraction of the larval anchovy encountered It is also likely that sampling efficiency increased with larval size A possible consequence of such gear selection towards large individuals is the simultaneous selection of the fastest growing larvae The sampled prey field, however, more than adequately covered the size range of larvae found in the gut of juvenile mackerel, and no significant difference in growth was found between ingested larvae and original populations The similarity between growth of the prey and that of the sampled prey field provides evidence that, despite the risks of gear selection for fast-growing individuals, mackerel did not select for slow-growing anchovy Another aspect that needs consideration is that the reported absence of growth selection may partly be attributable to sampling location The intensity of selection may vary with spatial changes in larval growth potential (likely superior in highly productive coastal areas) and with the predator field [10] For example, Takahashi et al [29] observed an 123 inshore–offshore negative growth rate gradient in the Kuroshio–Oyashio transition region, which larvae possibly experienced as a result of transport and migration processes In our study, the reported absence of growth selection in two distinct areas nevertheless suggests that mackerel is not an important agent of growth-selective mortality in young anchovy Despite a relatively small body size, mackerel always captured their anchovy prey independent of growth rate Mackerel thus strongly diverged from the general pattern suggested in a previous study [11] in which we classified the potential fish predators of young anchovy in two categories based on size In that study, small pelagic fish (anchovy, round herring Etrumeus teres, jack mackerel Trachurus japonicus, white croaker Pennahia argentatus) were identified as growth-selective predators, while large predatory fish (sea bass Lateolabrax japonicus, greater amberjack Seriola dumerili, skipjack tuna Katsuwonus pelamis) were identified as non-growth-selective predators consuming larvae randomly, independent of growth rate Such a discrepancy can be resolved by shifting the focus from predator size to feeding strategy The presence of mackerel in the non-growth-selective group along with large predatory fish may be attributable to the high activity level displayed by scombrids relative to other families In particular, mackerel adopt a raptorial feeding mode in the presence of potential fish prey and increase their speed as a Fish Sci (2010) 76:381–388 the same area is under the detection limit [32, 33] Thus, the mussels in the experimental aquarium in this study were exposed to higher concentrations of sulfide than they would have been in their original habitat It is reasonable to assume that mussels produce an increased amount of TAUT to cope with the ambient sulfides, thus facilitating the delivery of more hypotaurine to the gill tissue Our result is interesting because B platifrons harbors a methanotrophic symbiont that requires only methane—not sulfides—for chemosynthesis; thus, they not have to supply sulfur to the symbionts Yancey et al [17] recently reported that the accumulation of thiotaurine in hydrothermal vent polychaetes that did not harbor thioautotrophic endosymbionts was correlated with their sulfur exposure On the basis of these results and reports, we propose that, in our experimental system, B platifrons produced an increased amount of TAUT in the gill to protect the host tissues from the sulfide toxicity As neither the sulfide-binding protein nor modified hemoglobin has ever been found in bathymodiolin mussels, thiotaurine is likely to be the main substance enabling these organisms to adapt to the increase of the ambient sulfide It is probable that these mussels may have established this system to regulate the amount of TAUT in order to obtain precursors for thiotaurine synthesis Measurements of thiotaurine and hypotaurine in the gill are in progress The decrease in the abundance of the symbiont (Fig 4) may have had some influence on TAUT expression However, we did not see any evidence of damage in the host; they actively attached to the wall of aquarium by synthesizing the byssal thread As has been suggested previously [34], the host may be dependent on filter feeding Expression of TAUT in the mantle and foot BpTAUT mRNA was also detected in the foot and mantle (Fig 3) This result agrees with earlier results reported on B septemdierum [22] The level of TAUT mRNA in the mantle was variable among the experimental groups, with that in mussels reared in the absence of sulfide for 35 days being the highest—even higher than that found in the gills of mussels reared under the same conditions However, the mRNA level in the mantle of mussels in the other experimental groups was similar or lower than that in the gill Despite this difference, it is unlikely that the level of TAUT mRNA in the mantle correlates with the presence/ absence of sulfide The mantle is exposed to ambient water as much as the gill, and it has been reported that taurine carrier density on the non-gill epithelia of the shallowwater mussel Mytilus edulis is 7.4-fold higher than that on the gills [35] The mantle may also have considerable numbers of TAUT and respond to other environmental 387 factors in addition to sulfide One possible factor is osmotic change because taurine is an important osmolyte [20, 36– 40] However, in the shallow-water mussel, the response of the TAUT expression has been found to occur in all of the tissues examined to date [40] We are therefore unable to conclude whether the variable mRNA level in the mantle is a response to osmotic changes The level of TAUT mRNA in the foot was low under all experimental conditions These results suggest that the expression of the TAUT gene is regulated differentially in each tissue Members of the Solemya and Calyptogena genera accumulate high concentrations of taurine and/or related compounds in their foot [8, 16, 41] These species inhabit the sediment and take up H2S in the sediment through their foot [42] Thus, they may need taurine-related compounds to detoxify of H2S In contrast, Bathymodiolus species are periphytic and dwell above the sediment; as such, they not have to take up sulfide through the foot Consequently, the low level of expression in the foot is reasonable Although TAUT cDNA has not yet been cloned in Solemya and Calyptogena, an analysis of its expression in the foot of these species may be interesting Acknowledgments We thank the crew and staff of R/V Natsushima and ROV Hyper-Dolphin for their help during the cruise NT06-23 and NT08-03 This work was supported by KAKENHI (No 19380110) References Felbeck H, Childress JJ, Somero GN (1981) Calvin–Benson cycle and sulphide oxidation enzymes in animals from sulphide-rich habitats Nature 293:291–293 Corliss JB, Dymond J, Gordon LI, Edmond JM, von Herzen RP, Ballard RD, Green K, Williams D, Bainbridge A, Crane K, van Andel TH (1979) Submarine thermal springs on the Galapagos Rift Science 203:1073–1083 Cavanaugh CM (1983) Symbiotic chemoautotrophic bacteria in marine invertebrates from sulphide-rich habitats Nature 302:58–61 Terwilliger RC, Terwilliger NB, Arp AJ (1983) Thermal vent clam (Calyptogena magnifica) hemoglobin Science 219:981–983 Kraus DW (1995) Heme proteins in sulfide-oxidizing bacteria/ mollusc symbioses Am Zool 35:112–120 Doeller JE, Kraus DW, Colacino JM, Wittenberg JB (1988) Gill 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KR, Kochevar RK, Nelson DC, Fujiwara Y, Goffredi SK, Hashimoto J (2002) Methane-based symbiosis in a mussel, Bathymodiolus platifrons, from cold seeps in Sagami Bay, Japan Invertebr Biol 121:47–54 35 Rice MA, Stephens GC (1988) Influx and transepithelial flux of amino acids in the mussel, Mytilus edulis J Exp Biol 135:275–287 36 Chesney RW, Gusowski N, Dabbagh S (1985) Renal cortex taurine content regulates renal adaptive response to altered dietary intake of sulfur amino acids J Clin Invest 76:2213–2221 37 Rozen R, Scriver CR (1982) Renal transport of taurine adapts to perturbed taurine homeostasis Proc Natl Acad Sci USA 79:2101– 2105 38 Jayanthi LD, Ramamoorthy S, Mahesh VB, Leibach FH, Ganapathy V (1995) Substrate-specific regulation of the taurine transporter in human placental choriocarcinoma cells (JAR) Biochim Biophys Acta 1235:351–360 39 Tappaz ML (2004) Taurine biosynthetic enzymes and taurine transporter: molecular identification and regulations Neurochem Res 29:83–96 40 Hosoi M, Takeuchi K, Sawada H, Toyohara H (2005) Expression and functional analysis of mussel taurine transporter, as a key molecule in cellular osmoconforming J Exp Biol 208:4203–4211 41 Conway NM, McDowell Capuzzo JE (1992) High taurine levels in the Solemya velum symbiosis Comp Biochem Physiol 102B:175–185 42 Goffredi SK, Barry JP (2002) Species-specific variation in sulfide physiology between closely related Vesicomyid clams Mar Ecol Prog Ser 225:227–238 Fish Sci (2010) 76:389–394 DOI 10.1007/s12562-009-0212-z ORIGINAL ARTICLE Food Science and Technology Effect of bleeding on the quality of amberjack Seriola dumerili and red sea bream Pagrus major muscle tissues during iced storage John Bosco Ahimbisibwe • Kousuke Inoue Toshiyuki Shibata • Takahiko Aoki • Received: 31 August 2009 / Accepted: December 2009 / Published online: February 2010 Ó The Japanese Society of Fisheries Science 2010 Abstract The effect of bleeding on white muscle quality in amberjack and red sea bream was evaluated by measuring ATP-related compounds, volatile basic nitrogen (VBN), and the trimethylamine (TMA) content The freshness was evaluated by the K value, and the degree of spoilage was elucidated by VBN and TMA ATP was rapidly converted to ADP and AMP in the muscle and IMP was the main product of ATP degradation during iced storage For both species, the IMP content was higher in the muscles of fish that were bled than in those unbled during iced storage Conversely, the K value and the levels of hypoxanthine (Hx) and VBN were higher in the muscle tissue of unbled fish than in the bled tissue of both species Similarly, the TMA content was higher in the unbled muscle tissues of both species after a week of storage Keywords Bleeding Á Fish muscle Á IMP Á K value Á TMA Á VBN Introduction Freshness is a subjective attribute that is often associated with food quality, safety, and wholesomeness The freshness of fish muscle tissue is a function of flavor, odor, color, and texture In many instances, commercially caught fish are routinely bled to preserve the freshness of the muscle tissue Bleeding fish prior to iced storage has been reported to delay the process of rigor mortis in the horse mackerel Trachurus japonicus [1] Terayama et al [2] evaluated the effect of bleeding on the quality of skipjack Euthynnus pelamis muscle tissue by measuring extractable nitrogen, pH, and muscle color Similarly, Terayama et al [3] evaluated the effect of bleeding on muscle quality in skipjack sashimi using an organoleptic rating system The authors also measured the changes in breaking force of skipjack and amberjack Seriola dumerili muscle tissues during storage on ice Ando et al [4, 5] measured the effect of bleeding on muscle tissue breaking strength in several fish species However, Ando et al [5] also found that bleeding had no effect on the muscle tissue of marine white-flesh fish species over a period of three days of storage The authors suggested that a collagenolytic enzyme in the blood of marine red-flesh fish species caused postmortem softening of the muscle tissue However, these studies did not evaluate the effect of bleeding on the freshness or degree of spoilage of muscle tissue The objective of this study was to determine the effect of bleeding on muscle quality in amberjack (a marine redflesh fish) and red sea bream Pagrus major (a marine white-flesh fish) during 15 days of iced storage The freshness of muscle samples was determined by estimating the K value The degree of spoilage was quantified by measuring the levels of volatile basic nitrogen (VBN) and trimethylamine (TMA) in the muscle tissue Materials and methods J B Ahimbisibwe Á K Inoue Á T Shibata Á T Aoki (&) Laboratory of Quality in Marine Products, Graduate School of Bioresources, Mie University, Kurima Machiya-cho 1577, Tsu, Mie 514-8507, Japan e-mail: aoki@bio.mie-u.ac.jp Fish samples Live cultured amberjack (average weight 3.7 kg) and red sea bream (average weight 1.4 kg) were obtained from Mie 123 390 Gyoren, Owase, Japan The samples of amberjack and red sea bream muscle were collected and analyzed in May 2008 and October 2008, respectively Bleeding and iced storage The fish were randomly divided into two groups, bled or unbled (n = in each group) Following capture, the fish in the bled group were killed by the quick incision of a sharp knife near the pectoral fin to cut the aorta and spinal cord The fish were paralyzed immediately, and were vertically to bleed out through the mouth The unbled fish were killed by a rapid blow to the cranium This resulted in immediate death and avoided any metabolic stress due to struggling The fish were then immediately immersed in iced water White muscle tissue (7–8 g) was collected from a dorsal site after death (0 days of storage), the carcass was placed in a polyethylene bag, and it was stored in melting ice Additional muscle samples were collected after 1, 3, 5, 7, 10 and 15 days of iced storage in the amberjack, and after 1, 3, 5, 8, 10 and 15 days in the red sea bream Evaluation of muscle quality (ATP-related compounds, K value, VBN, and TMA) The methods of Ryder [6] were followed during preparation of the muscle samples and analysis of ATP-related compounds Five grams of fish muscle tissue were homogenized in ml of 10% perchloric acid (PCA) at 0°C (Physcotron homogenizer, Nition Irika, Tokyo, Japan) The homogenate was then centrifuged at 1,5009g for and the supernatant was collected The residue was then mixed with ml of 5% PCA at 0°C, centrifuged, and the supernatant was collected This process was repeated twice The pH of the supernatant was adjusted to pH 6.4 using 10 N KOH followed by N KOH The mixture was centrifuged at 1,5009g for and the supernatant was collected The residue was then mixed with ml of 1% PCA (adjusted to pH 6.4 with 10 N and N KOH), centrifuged at 1,5009g for min, and the resulting supernatant was collected This stage was repeated and the supernatants were combined, and distilled water was added to the supernatant (up to 25 ml) This stock solution was stored at -20°C until analysis The concentration of ATP-related compounds was measured using high-performance liquid chromatography (HPLC) The HPLC system consisted of a Jasco 980-PU pump, a UV-970 detector, and an LCSS-905 data module (Japan Spectroscopic, Tokyo, Japan) The mobile phase consisted of a 12 mM citric acid buffer (pH 2.5) containing 18 mM 2-diethylaminoethanol The column was a YMCpack ODS-A column (250 4.6 mm ID; YMC, Kyoto, Japan) with a flow rate of 0.5 ml/min and a column 123 Fish Sci (2010) 76:389–394 temperature of 40°C The wavelength was set at 250 nm The K value was calculated according to the method of Saito et al [7] Conway’s microdiffusion method [8] was used to measure VBN content, and the picric acid method [9] was employed to measure TMA content The process involved homogenizing g of white muscle tissue with ml distilled water for 30 at 0°C To the homogenate, ml of 20% trichloroacetic acid (TCA) were added for an additional 10 at 0°C The homogenate was filtered using a filter paper (No 2; Advantec Toyo, Tokyo, Japan), and the filtrate was made up to 10 ml with 20% TCA and stored at -20°C until analysis For each analysis, the measurements were repeated five times, and the statistical significance was determined using Student’s t test Results ADP was detected in both fish muscles throughout iced storage However, mean ADP levels did decrease over time in both bled and unbled muscle tissues in both species (Tables 1, 2) The levels of AMP increased throughout iced storage in amberjack muscle (Table 1) but decreased in red sea bream muscle tissue (Table 2) The primary breakdown product of ATP in both amberjack and red sea bream muscles was IMP The average IMP level was highest on days of storage in the amberjack muscle, and gradually decreased during iced storage (Fig 1a) The content of IMP in the bled muscle of amberjack was significantly higher (p B 0.05) than in the unbled muscle at all times during iced storage In the red sea bream, IMP levels were only higher in the bled muscle after days of storage and tended to decrease throughout storage (Fig 1b) The inosine (HxR) content increased between and 15 days of storage in the amberjack muscle (Table 1) The HxR content of the bled muscle was significantly higher than in the unbled muscle (p B 0.05) after days of storage In the red sea bream, the average HxR content also increased between and 10 days of storage in both bled and unbled muscle tissues (Table 2) Hypoxanthine (Hx) was detected after day of storage in both amberjack and red sea bream (Tables 1, 2) The average Hx content increased between and 15 days of storage in the bled and unbled muscles of both species The Hx content of the unbled muscle was significantly higher than that in the bled muscle (p B 0.05) of amberjack (after days of storage) and red sea bream (after days of storage) The K value was significantly higher (p B 0.05) in the unbled muscle than in the bled muscle of amberjack (after days of storage) and red sea bream (between and 10 days of storage) (Fig 2) The K value was also higher in Fish Sci (2010) 76:389–394 391 Table Changes in the contents of ATP-related compounds (lmol/g) in amberjack muscle tissue during iced storage ATP-related compound Storage time (days) 10 15 ATP Bled 0.24 ± 0.02 – – – – – – Unbled 0.22 ± 0.03 – – – – – – ADP Bled 0.16 ± 0.06 0.13 ± 0.03 0.11 ± 0.02 0.10 ± 0.02 0.07 ± 0.01 0.06 ± 0.02 0.05 ± 0.01 Unbled 0.17 ± 0.05 0.12 ± 0.01 0.10 ± 0.01 0.09 ± 0.01 0.07 ± 0.02 0.06 ± 0.02 0.04 ± 0.00 AMP Bled Unbled 0.24 ± 0.00 0.36 ± 0.07 0.44 ± 0.08 0.67 ± 0.07 0.92 ± 0.08 1.13 ± 0.10 1.62 ± 0.10 0.22 ± 0.05 0.34 ± 0.04 0.43 ± 0.04 0.54 ± 0.07 0.68 ± 0.08 1.00 ± 0.05 1.46 ± 0.05 HxR Bled 0.99 ± 0.07 1.27 ± 0.04 1.64 ± 0.13 2.14 ± 0.12 2.62 ± 0.11 3.12 ± 0.14 3.45 ± 0.01 Unbled 0.99 ± 0.04 1.22 ± 0.07 1.62 ± 0.13 2.04 ± 0.09 2.46 ± 0.05 2.81 ± 0.08 3.11 ± 0.01 Hx Bled – 0.90 ± 0.05 1.34 ± 0.06 1.58 ± 0.07 1.85 ± 0.05 1.98 ± 0.40 2.11 ± 0.11 Unbled – 1.01 ± 0.08 1.64 ± 0.13 1.88 ± 0.11 2.04 ± 0.09 2.23 ± 0.13 2.43 ± 0.80 Values were obtained by averaging samples –, not detected Table Changes in the contents of ATP-related compounds (lmol/g) in red sea bream muscle tissue during iced storage ATP-related compounds Storage time (days) 10 15 ATP Bled 3.00 ± 0.10 – – – – – – Unbled 3.20 ± 0.20 – – – – – – ADP Bled 0.46 ± 0.04 0.11 ± 0.03 0.11 ± 0.02 0.11 ± 0.05 0.11 ± 0.05 0.11 ± 0.04 0.11 ± 0.05 Unbled 0.46 ± 0.04 0.11 ± 0.06 0.10 ± 0.06 0.11 ± 0.04 0.11 ± 0.05 0.11 ± 0.09 0.11 ± 0.09 AMP Bled Unbled 0.26 ± 0.05 0.12 ± 0.05 0.12 ± 0.05 0.12 ± 0.05 0.04 ± 0.04 0.04 ± 0.03 0.04 ± 0.02 0.28 ± 0.04 0.12 ± 0.04 0.08 ± 0.04 0.06 ± 0.05 0.06 ± 0.04 0.06 ± 0.04 0.06 ± 0.03 HxR Bled 0.68 ± 0.01 1.18 ± 0.01 1.44 ± 0.01 1.60 ± 0.10 2.32 ± 0.04 2.90 ± 0.07 2.32 ± 0.03 Unbled 0.70 ± 0.01 1.30 ± 0.01 1.58 ± 0.06 1.66 ± 0.07 2.72 ± 0.08 2.96 ± 0.03 2.28 ± 0.02 Hx Bled – 0.04 ± 0.01 0.10 ± 0.03 0.20 ± 0.06 0.48 ± 0.03 0.68 ± 0.01 0.84 ± 0.05 Unbled – 0.04 ± 0.01 0.14 ± 0.01 0.28 ± 0.03 0.62 ± 0.05 0.78 ± 0.06 0.98 ± 0.04 Values were obtained by averaging samples –, not detected the amberjack muscle than in the red sea bream at days of storage The VBN content was significantly higher (p B 0.05) in the unbled muscle than in the bled muscle of both amberjack (after days of storage) and red sea bream (after days of storage) (Fig 3) A rapid increase in TMA content was observed after a week of storage in both fish species (Fig 4) After days of storage, the unbled muscle of the red sea bream had higher levels of TMA than the bled muscle (p B 0.05) In contrast, this difference was not observed until 15 days of storage in the amberjack muscle 123 392 Fish Sci (2010) 76:389–394 Fig IMP content (lmol/g) in bled (unfilled circles) and unbled (filled circles) muscle tissue during iced storage a Amberjack b Red sea bream The bars represent 95% confidence intervals The letter S indicates a value which is higher significantly (p B 0.05) than that of the unbled muscle tissue Fig K value (%) in bled (unfilled circles) and unbled (filled circles) muscle tissue during iced storage a Amberjack b Red sea bream The bars represent 95% confidence intervals The letter S indicates a value which is higher significantly (p B 0.05) than that of the bled muscle tissue Fig VBN content (mg/ 100 g) in bled (unfilled circles) and unbled (filled circles) muscle tissue during iced storage a Amberjack b Red sea bream The bars represent 95% confidence intervals The letter S indicates a value which is higher significantly (p B 0.05) than that of the bled muscle tissue Fig TMA content (mg/ 100 g) in bled (unfilled circles) and unbled (filled circles) muscle tissue during iced storage a Amberjack b Red sea bream The bars represent 95% confidence intervals The letter S indicates a value which is higher significantly (p B 0.05) than that of the bled muscle tissue Discussion The levels of ATP were very low in the both amberjack and red sea bream muscle tissues, and ATP was not detected after day of storage (Tables 1, 2) Other studies have also 123 shown that ATP is rapidly degraded, within 24 h of death at 0°C [10] Based on the results we obtained by measuring the IMP content, the average IMP contents in both fish species were higher than those of other ATP-related compounds Ehira and Uchiyama [11] documented the changes Fish Sci (2010) 76:389–394 in ATP-related compounds during the iced storage of muscle tissue from several fish species In their study, the levels of IMP in black sea bream Acanthopagrus schlegeli (Mylio macrocephalus) muscle (approx 9.5 lmol/g at days of storage) and red sea bream (approx 7.5 lmol/g) were similar to those we measured in the unbled muscle of amberjack and red sea bream (Fig 1) The IMP content affects the change in the K value, and is thought to be responsible for the fresh flavor generally associated with fish [12, 13] The HxR and Hx contents increased between and 10 days of storage in both fish species, and the sum of the HxR and Hx contents seen in our study was similar to those seen in the black sea bream (approx 5.5 lmol/g after 12 days of storage) and red sea bream (approx lmol/g after 10 days of storage) muscle in the study of Ehira and Uchiyama [11] The HxR and Hx contents affected the change in K value, and high levels of Hx are associated with a bitter offtaste and a loss of fish flavor [14] Ehira and Uchiyama [11] also reported the changes in the K values of black sea bream and red sea bream during iced storage, and the values they observed are similar to those seen for the unbled muscles of amberjack and red sea bream in our study In their study of the changes in the value of K after 12 days storage, they found that the k values were unstable in both fish species This instability is assumed to affect the k value in red sea bream muscle after 15 days of storage in our study (Fig 2b) As shown in Fig 3, the average VBN content increased throughout the period of iced storage in both fish species Increases in VBN content can be attributed to the production of ammonia [15, 16] from the deamination of ATP-related compounds and free amino acids [17] Ehira and Uchiyama [11] also measured the VBN contents of the muscles of black sea bream and red sea bream In their study, the levels of VBN in the black sea bream and red sea bream muscles (approx 17 mg/100 g after 10 days of storage in both fish species) were similar to those we measured in the unbled muscles of amberjack and red sea bream (Fig 3) Based on the results we obtained by measuring TMA content, the average TMA level was higher in amberjack muscle than in red sea bream muscle (Fig 4) The TMA content was higher in the unbled muscles than the bled muscles of both species only after a week of storage TMA has a characteristic odor associated with spoiled marine fish [18], and it is produced by the bacterial reduction of trimethylamine oxide (TMAO) during the spoilage process [19, 20] Uchiyama et al [21] studied the relationship between freshness and biochemical changes in fish muscle during iced storage In their study, the changes in VBN and TMAN levels in skipjack muscle were similar to those we documented in the unbled muscles of amberjack and red 393 sea bream Furthermore, the levels of TMA-N in skipjack muscle (approx 0.3 mg/100 g after days of storage) were similar to those we measured in the unbled muscle of amberjack (0.24 mg/100 g) Ando et al [5] evaluated the effect of bleeding on the muscle tissues of a range of fish species using electron microscopy and measurements of breaking strength The authors reported a delay in muscle softening in yellowtail Seriola quinqueradiata, horse mackerel, and striped jack Caranx delicatissimus However, they also reported that bleeding had no effect on this process during days of storage in red sea bream, flatfish Paralichthys olivaceus, and rudderfish Girella punctata, all of which are marine white-flesh fish We evaluated the effect of bleeding over a much greater period of storage time, so it is difficult to compare these two studies Mochizuki et al [1] noted that the rate of rigor mortis in the bled muscle of horse mackerel was lower than that in unbled muscle up to 12 h after death However, they found no significant difference between the K values of the bled and unbled muscle tissues The authors hypothesized that this may have been due to bleeding in the control fish In our study, the unbled fish were killed by a rapid blow to the cranium to avoid any bleeding Taken together, our results suggest that bleeding has a preferable effect on the quality of both amberjack (IMP, HxR, Hx, K value, VBN, and TMA) and red sea bream (IMP, Hx, K value, VBN, and TMA) muscle tissues, and in our study the bleeding method was more effective in the former (a marine red-flesh fish) than in the latter (a marine white-flesh fish) Acknowledgments The authors are indebted to Yoshiyuki Kurata for fish preparation References Mochizuki S, Norita Y, Maeno K (1998) Effects of bleeding on post-mortem changes in the muscle of horse mackerel (in Japanese with English abstract) Nippon Suisan Gakkaishi 64:276– 279 Terayama M, Yamanaka H (2000) Effects of bleeding on the quality of skipjack (in Japanese with English abstract) Nippon Suisan Gakkaishi 66:852–858 Terayama M, Ohshima T, Ushio H, Yamanaka H (2002) Effects of instant killing and bleeding machine on quality of spindleshape fish Fish Sci 68:1651–1652 Ando M (1996) A study on the mechanism of post-mortem tenderization of fish muscle (in Japanese) Nippon Suisan Gakkaishi 62:555–558 Ando M, Nishiyabu A, Tsukamasa Y, Makinodan Y (1999) Postmortem softening of fish muscle during chilled storage as affected by bleeding J Food Sci 64:423–428 Ryder JM (1985) Determination of adenosine triphosphate and its breakdown products in fish muscle by high-performance liquid chromatography J Agric Food Chem 33:678–680 123 394 Saito T, Arai K, Matsuyoshi M (1959) A new method for estimating the freshness of fish Nippon Suisan Gakkaishi 24:749– 750 Conway EJ (1947) Micro-diffusion analysis and volumetric error Crosby, Lockwood & Son, London Dyer WJ, Dyer FE, Snow JM (1952) Amines in fish muscle V Trimethylamine oxide estimation J Fish Res Bd Can 8:309–313 10 Hamada-Sato N, Usui K, Kobayashi T, Imada C, Watanabe E (2005) Quality assurance of raw fish based on HACCP concept Food Control 16:301–307 11 Ehira S, Uchiyama H (1974) Freshness-lowering rates of cod and sea bream viewed from changes in bacterial count, total volatile base- and trimethylamine-nitrogen, and ATP related compounds Nippon Suisan Gakkaishi 40:479–487 12 Howgate P (2005) Kinetics of degradation of adenosine triphosphate in chill-stored rainbow trout (Oncorhynchus mykiss) Int J Food Sci 40:579–588 13 Bremner HA, Olley J, Statham JA, Vail AMA (1988) Nucleotide catabolism: Influence on the storage life of tropical species of fish from the north west shelf of Australia J Food Sci 53:6–11 14 Fletcher GC, Statham JA (1988) Shelf-life of sterile yellow-eyed mullet (Aldrichetta forsteri) at 4°C J Food Sci 53:1030–1035 123 Fish Sci (2010) 76:389–394 15 Gill TA (1990) Objective analysis of seafood quality Food Rev Int 6:681–714 16 Oehlenschla¨ger J (1992) Evaluation of some well established and some underrated indices for the determination of freshness and/or spoilage of ice stored wet fish In: Huss HH et al (eds) Quality assurance in the fish industry Elsevier, Amsterdam, pp 339–350 17 Ababouch LH, Souibri L, Rhaliby K, Ouahdi O, Battal M, Busta FF (1996) Quality changes in sardines (Sardina pilchardus) stored in ice and at ambient temperature Food Microbiol 13:123– 132 18 Ringø E, Stenberg E, Strøm AR (1984) Amino acid and lactate catabolism in trimethylamine oxide respiration of Alteromonas putrefaciens NCMB 1735 Appl Environ Microbiol 47:1084– 1089 19 Malle P, Eb P, Tailliez R (1986) Determination of the quality of fish by measuring trimethylamine oxide reduction Int J Food Microbiol 3:225–235 20 Gram L, Dalgaard P (2002) Fish spoilage bacteria—problems and solutions Curr Opin Biotechnol 13:262–266 21 Uchiyama H, Suzuki T, Ehira S, Noguchi E (1966) Studies on relation between freshness and biochemical changes of fish muscle during ice storage (in Japanese with English abstract) Nippon Suisan Gakkaishi 32:280–285 Fish Sci (2010) 76:395–401 DOI 10.1007/s12562-010-0217-7 ORIGINAL ARTICLE Food Science and Technology Assessing and enhancing the antimicrobial effect of nisin in soy-seasoned salmon Oncorynchus keta roe using a Pediococcus pentosaceus fermentate and pectin Dominic Kasujja Bagenda • Koji Yamazaki Tetsuya Kobayashi • Yuji Kawai • Received: September 2009 / Accepted: 28 December 2009 / Published online: 19 February 2010 Ó The Japanese Society of Fisheries Science 2010 Abstract The compatibility and potential of nisin, a Pediococcus pentosaceus fermentate, and pectin to inhibit Listeria monocytogenes in soy-seasoned salmon roe at and 12°C was studied The compatibility of nisin and soyseasoning was assessed by adding 6.3 lg/g of nisin to freshly washed or soy-seasoned (60% soy, 15% sweetsake, 5% sugar, and 15% water) roe and monitoring the growth of an inoculum of L monocytogenes (5.6 Log CFU/ g; strains IID 578, IID 581, ATCC 7644) as well as residual nisin concentrations at 12°C The combined antimicrobial potential was determined by monitoring growth of the inoculum (5.2 Log CFU/g) in roe when the soy-seasoning contained nisin (0.05 g/ml), fermentate powder made by freeze-drying a broth culture of P pentosaceus (0.1 g/ml), and 2% pectin at or 12°C Chloride content, total acid content, and the effect of pectin on acceptability of roe were also determined Nisin was more effective in soyseasoned roe than in freshly washed roe Microbial growth was completely inhibited by a combination of soy-seasoning, nisin, the fermentate, and pectin Despite its viscosity, pectin did not significantly affect the chemical properties or acceptability of roe relative to pectin-free roe samples (P \ 0.05) Based on these results, we conclude that nisin’s potential as an antimicrobial agent in soy-seasoned roe can be enhanced using fermentates and pectin D K Bagenda (&) Department of Media Architecture, Future University-Hakodate, Hakodate, Hokkaido 041-8655, Japan e-mail: bkdnic2@yahoo.co.jp K Yamazaki Á T Kobayashi Á Y Kawai Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan Keywords Listeria Á Pectin Á Pediococcus Á Nisin Á Soy-seasoning Á Roe Introduction The Japanese government officially recognized the antimicrobial protein nisin as a food additive on March 2, 2009 (JFCRF website: http://www.ffcr.or.jp/zaidan/MHWinfo nsf/ Accessed 22 July 2009) Nisin is a heat-stable bacteriocin that kills sensitive pathogens by disrupting their cell membranes, leading to the leakage of cellular material and ultimately cell death [1] The efficacy of nisin as an antimicrobial agent in raw seafood (characteristic of Japanese cuisine) is limited by several factors These include the potential for proteolytic activity in raw food to cause the rapid degradation of bacteriocins [2] as well as the rapid decrease in the antimicrobial activity of nisin with time As a result, in the absence of supplementary antimicrobial hurdles, food pathogens gradually develop a tolerance to nisin [3] The fermentates of bacteriocin-producing bacteria have been suggested for use in food safety applications [4] Fermentates are easily prepared by concentrating (usually by freeze-drying) the liquid medium in which a beneficial bacterial strain has been allowed to grow These fermentates may contain cells of the beneficial bacterium (bacteriocin producer) as well as secondary metabolites that may have additional antimicrobial effects [5] Unlike many bacteriocins that have been discovered to date, fermentates are readily accepted and used as food additives In Japan, for example, seasoning mixtures used in salmon roe processing often contain fermentates [6] Salmon roe, a popular item in Japanese cuisine, is extremely heat labile Consequently, the control of 123 396 microbial growth in this food product is limited to refrigeration and to the use food preservatives, such as soy sauce [6, 7] The preservative (or antimicrobial) effect of soy sauce is attributed to its high salt ([10%) and acidic nature (sometimes pH 4.5) [8] However, despite the antimicrobial potential of soy sauce, soy-seasoned salmon roe caused an Escherichia coli O157 outbreak in 1998 [6] The risk of contamination of roe with Listeria monocytogenes is also a primary concern L monocytogenes causes listeriosis, a food-borne disease with a fatality rate of 25–30% [9] Based on epidemiological studies on ready-to-eat raw seafood products in Tokyo, 10.4% of fish roe is contaminated with L monocytogenes [10] L monocytogenes may grow in refrigerated ready-to-eat foods even if they have a high salt concentration [11] Moreover, it has recently been shown that salt enhances biofilm formation in L monocytogenes and may therefore enhance rather than inhibit the growth of this pathogen [12] The risk of post-processing contamination of soy-seasoned roe products with L monocytogenes and the potential or limitations of nisin in such situations have not been sufficiently dealt with [3, 4, 11] The aims of our study were to assess and enhance the post-processing antimicrobial effect of nisin in soy-seasoned roe In an attempt to successfully achieve the latter goal, we used a Pediococcus pentosaceus fermentate (obtained by freeze-drying a broth culture of P pentosaceus) and pectin Materials and methods Growth media and bacterial strains Pediococcus pentosaceus Iz3.13, previously isolated from fermented Japanese marine food, was used as a bacteriocin (pediocin) producer [13] A cocktail of three strains of L monocytogenes (ATCC 7644, IID 578, IID 580) was used as the target food-borne pathogen Inoculum levels were log 5.2–5.6 Log10CFU/g Micrococcus luteus IFO 13867 was used as a nisin-sensitive indicator strain The strains were stored on slants prepared from tryptic soy broth (Oxoid, Basingstoke, Hants, UK) and supplemented with 0.6% yeast extract (Oxoid) (TSBYE) and 1.5% agar (Wako) at 4°C and propagated for 24 h in TSBYE at 30°C prior to use To make the cocktail, we separately centrifuged (2 min, 10,000 rpm) the three broth-cultured L monocytogenes strains (approximately 106 CFU), washed the harvested pellets three times, and then resuspended (mixed) all of the pellets together in phosphate-buffered saline (PBS) The listerial load of the suspension was confirmed by plating on TSBYE agar (1.5%) plates This cocktail suspension was used to inoculate roe samples 123 Fish Sci (2010) 76:395–401 Preparation of nisin and fermentate of P pentosacues Iz3.13 The bacteriocins used in this study were nisin powder (Sigma, St Louis, MO) containing 2.5% nisin and a fermentate containing pediocin To prepare the fermentate, we inoculated sterile de Man, Rogosa, Sharpe (MRS; Oxoid) broth medium with a pre-incubated (18 h, 30°C, MRS) culture of P pentosaceus Iz3.13 The final inoculum-tomedium ratio was 0.01 Fermentation of the medium was monitored (Twin pH meter B-211; Horiba, Japan) until the pH reached 3.8 The medium was then heated to 80°C for 15 to inhibit enzyme activity, transferred into freezetolerant containers, and frozen (-20°C, overnight) The frozen medium was freeze-dried using a freeze-dryer (FD5N; EYELA, Tokyo, Japan) The freeze-dried powder was referred to as the fermentate of P pentosaceus Iz3.13 and kept in an air-tight container at 4°C until use The desired quantities of nisin were dissolved in 0.02 M HCl and heated at 80°C for immediately prior to use Preparation of roe samples Five types of roe samples were used for laboratory analysis, while three types of roe samples were used for organoleptic analysis The samples used for laboratory analysis were freshly washed roe (FWR), nisin-containing freshly washed roe (NFWR), standard soy-seasoned roe (SSSR), nisin soyseasoned roe (NSSR), and pectin–bacteriocin soy-seasoned roe (PBSSR) Those samples used for organoleptic analysis were commercial soy-seasoned roe (CSSR), SSSR, and soyseasoned roe containing pectin (PSSR) To prepare these samples, we first purchased fresh roe locally, washed it thoroughly in warm sterile saline water, and stored it at -80°C This stored roe was referred to as the FWR A portion of the FWR was seasoned in sterile soy-seasoning (60% soy, 15% sweet-sake, 5% sugar and 15% water heated to 110°C for min) to make a SSSR sample for the preliminary experiments in which the microbiological and chemical (NaCl and acid content) properties of FWR and SSSR were determined These tests revealed that the total aerobic microbial cell counts in the FWR samples were less than the detection limit (2 Log CFU/g) and that there was no significant increase in inhibitory activity at concentrations of [0.05 g nisin powder and [0.1 g fermentate/ml seasoning Assessing antimicrobial effect and stability of nisin in soy-seasoned roe We used batches of FWR and SSSR in the experiments to assess the antimicrobial effect of nisin in soy-seasoned roe Nisin powder at concentrations equivalent to 6.3 lg/g roe Fish Sci (2010) 76:395–401 (suspended in 0.02 M HCl) was added to batches of FWR or SSSR to make the NFWR and NSSR samples, respectively The NFWR and NSSR samples were air dried to ensure that all of the bacteriocin was taken up Samples were inoculated with the L monocytogenes cocktail (5.6 Log10CFU/g), incubated at 12°C, and monitored regularly for microbial counts and residual nisin concentration The residual nisin concentration in the NFWR and NSSR samples was determined using the method of AlHoly et al [3] with minor modifications The nisin activity of the extracts was determined by the agar-well method as described by Yamazaki et al [14] One milliliter of an overnight culture of M luteus IFO 13867 was used as the nisin-sensitive indicator The diameters of the inhibition zones of roe extracts containing known concentrations of nisin were used to prepare standard curves These standard curves were used to estimate the residual concentration of nisin in the roe samples Enhancing the antimicrobial effect of nisin in soy-seasoned roe using the P pentosaceus fermentate and pectin A PBSSR batch was used to determine the combined effect of soy-seasoning, nisin, the P pentosaceus fermentate, and pectin on bacterial growth The PBSSR batch was made by seasoning FWR in sterile soy-seasoning (composition is described above) containing 0.05 g/ml nisin powder and 0.1 g/ml MRS fermentate of P pentosaceus Iz3.13 stabilized in 2% pectin (Wako, Osaka, Japan) Pectin was dissolved in the soy-seasoning by heating and stirring before the bacteriocins were added The duration of the seasoning process was 60 min, at a seasoning-to-roe ratio of 1:1 for all batches with the exception of when the seasoning contained pectin (ratio was 1:3 in this case) The seasoning solution was subsequently drained off and the roe samples aseptically packed The packed roe was then inoculated with the L monocytogenes cocktail (IID 578, IID 581, ATCC 7644) at an inoculum level of about 5.2 Log10CFU/g and incubated at and 12°C till analysis for chemical and microbiological properties 397 containing the listeria selective supplement SR150E (Oxoid) according to the manufacturer’s instructions Characteristic colonies were then counted after a 36-h incubation The results were expressed as counts of Listeria spp because the presence of undetected Listeria spp in the raw material could not be ruled out Assessing the effect of pectin and P pentosaceus fermentate on the total acid and chloride content of soy-seasoned roe To confirm that the P pentosaceus fermentate and pectin did not have a major effect of the chemical properties of roe, we determined and compared the chloride and total acid content of the FWR, SSSR, PBSSR, and CSSR samples Chloride content was determined by the Mohr’s method [15] The total acid content was obtained by direct titration with NaOH using phenolphthalein indicator and was expressed as the amount of 0.1 M NaOH required to neutralize 100 g of sample Assessing the effect of pectin on organoleptic acceptability of soy-seasoned salmon roe In a separate experiment, the effect of pectin on organoleptic acceptability of soy-seasoned roe was determined by means of a discriminative sensory evaluation by 20 untrained panelists The sensory properties of samples of SSSR, PSSR, and CSSR were assessed in which the panelists awarded scores for appearance, odor, flavor, texture, and overall attributes on a scale of 1–9 (1 = strongly disliked, = strongly liked) Statistical analyses Scores from the analyses of organoleptic attributes were ranked and compared using the Wilcoxon–Mann–Whitney two-sample rank-sum test Results Determination of total aerobic and Listeria spp cell counts in salmon roe A 9-ml aliquot of sterile PBS was added to each sample of roe (1 g) This was followed by homogenization for 30 s, serial dilution, and spread plating Total aerobic microbial counts for each sample were determined by spread plating 0.1 ml of each sample onto plates of TSBYE (Oxoid) supplemented with 1.5% agar (Wako), incubating the plates at 30°C for 24 h, and counting the colonies Listeria spp microbial counts were determined by spread plating 0.1 ml of sample onto plates of PALCAM agar base Antimicrobial effect and stability of nisin in soy-seasoned roe The antimicrobial agent nisin was more effective and stable in NSSR than in NFWR at 12°C (Table 1) In NFWR, Listeria spp counts decreased during the first day, but total aerobic counts increased In comparison, in NSSR, both total aerobic counts and Listeria spp counts dropped during the first day of incubation Although total aerobic counts recovered after the third day in both NFWR and NSSR samples, they remained lower in the NSSR sample 123 398 Fish Sci (2010) 76:395–401 Table Antimicrobial effect and stability of nisin in fresh and soy-seasoned roe incubated at 12°C Parameter Incubation time (days) 12 NFWR Total plate counts 7.4 ± 0.3 9.0 ± 0.2 8.9 ± 0.1 8.8 ± 0.1 9.5 ± 0.8 L monocytogenes counts 3.9 ± 0.4 6.5 ± 0.2 6.9 ± 0.1 6.9 ± 0.4 6.8 ± 0.5 Residual nisin (lg/g) 2.4 ± 0.1 0.7 ± 0.1 0.6 ± 0.3 0.6 ± 0.1 0.4 ± 0.4 NSSR Total plate counts 4.8 ± 0.1 6.3 ± 1.0 7.8 ± 0.2 7.5 ± 0.2 7.6 ± 0.4 L monocytogenes counts Residual nisin (lg/g) 4.2 ± 0.5 5.7 ± 0.1 2.9 ± 1.8 5.6 ± 0.4 4.8 ± 1.0 4.0 ± 0.2 4.2 ± 0.6 3.2 ± 0.7 4.2 ± 0.9 1.9 ± 0.7 Initial nisin concentration was 6.3 lg/g of roe and initial inoculum level was 5.6 Log10 CFU/g of roe Data are given as the mean ± standard deviation (SD) of three measurements NFWR Freshly washed roe samples containing nisin, NSSR soy-seasoned roe samples containing nisin than in the NFWR sample Nisin controlled the growth of Listeria spp (kept it below the inoculum level) in NSSR till the 12th day, but it was not as effective in FWR In terms of stability, and 90% of the nisin had been degraded in NSSR and FWR, respectively, by the third day of incubation Nisin degradation in NSSR was still less than 50% by the ninth day of incubation Enhancing the antimicrobial effect of nisin in soy-seasoned roe using the P pentosaceus fermentate and pectin Antimicrobial activity in soy-seasoned roe was enhanced when nisin, the P pentosaceus fermentate, and pectin were combined (PBSSR) (Fig 1a, b) This enhancement was better at 12°C than at 5°C In PBSSR, total microbial aerobic counts fell to below the detection limit after and days when incubation temperatures were 12 and 5°C, respectively At both and 12°C Listeria spp counts reduced to below detection limits after day of incubation Effect of pectin and P pentosaceus fermentate on the total acid and chloride content of soy-seasoned roe Although the total acid content was the same among the samples, the chloride content of PBSSR was 0.53% higher than that of SSSR (Table 2) Both PBSSR and SSSR had higher chloride and acid contents than CSSR The seasoning mixtures for PBSSR had a higher total acid but lower chloride content than those of SSSR Effect of pectin on organoleptic acceptability of soy-seasoned salmon roe The overall organoleptic properties of soy-seasoned roe prepared in the laboratory according to standard procedures 123 Fig Survival and growth of mesophiles (a) and Listeria spp (b) in pectin–bacteriocin soy-seasoned roe (PBSSR) at 5°C (circles) and 12°C (squares) Arrows Readings below the detection limit (2 Log CFU/g), points means (n = 3), vertical bars standard deviations (SSSR) and a randomly purchased commercial sample (CSSR) were not significantly different (P \ 0.05) from those of roe seasoned with pectin-supplemented soy-seasoning (PSSR) (Table 3) Furthermore there were no Fish Sci (2010) 76:395–401 399 Table Results from the analysis of roe samples for chloride and total acid contents Sample Chloride content (%) FWR Total acida 70 SSSR 2.12 90 PBSSR 2.65 90 CSSR Seasoning mixture for SSSR Seasoning mixture for PBSSR 1.6 70 14.5 240 6.4 320 FWR Freshly washed roe, LSSR laboratory soy-seasoned roe, PBSSR pectin–bacteriocin soy-seasoned roe, CSSR commercial soy-seasoned roe a Quantity (ml) of 0.1 M NaOH required to neutralize 100 g of sample Table Scores for organoleptic attributes of roe samples Organoleptic attributes SSSR PSSR CSSR Appearance 5.3 ± 1.6 a 6.3 ± 1.6 a 7.6 ± 1.7 b Odor 5.4 ± 1.4 b, c 4.9 ± 2.0 b 4.0 ± 2.0 c Flavor 6.0 ± 1.6 d, e 5.6 ± 1.7 d 4.2 ± 2.3 e Texture 5.0 ± 1.4 4.9 ± 1.5 6.0 ± 2.5 Overall 5.9 ± 1.5 5.4 ± 1.1 4.7 ± 2.3 Scores are based on a 1–9 scale, with indicating an unfavorable assessment and indicating a favorable assessment Data are given as the mean ± SD (n = 20) Means in the same row followed by different lower case letters are significantly different (P \ 0.05) significant differences in texture of samples prepared in this study (SSSR and PSSR) and the purchased sample (CSSR) (P \ 0.05) Although flavor scores for the SSSR samples were not significantly different from those for the PSSR samples, they were significantly higher than those for the CSSR sample (P \ 0.05) It must be noted that the CSSR sample had a significantly better appearance than the samples prepared in this study (SSSR and PSSR) (P \ 0.05) Discussion The contamination rate of fish roe with L monocytogenes in Japan is reported to be 10.4% [10], with these high contamination rates often attributed to post-processing contamination [9] In the case of soy-seasoned salmon roe, seasonal and geographic limitations in availability as well as extensive processing requirements have led to extended refrigeration and wide distribution chains This inevitably leads to the additional problem is that proper refrigeration (below 4°C) cannot be guaranteed during processing and distribution Consequently, the control of pathogens like L monocytogenes in salmon roe remains a challenge for the fish roe industry The common practice of the food processing industry faced with the challenge of controlling microbes in fish roe without sacrificing organoleptic attributes is to apply a combination of mild and sub-lethal methods (hurdle technology) rather than using a single strong and lethal method In Japan, for example, seasoning in soy sauce (high salt content) and refrigeration are often combined to preserve salmon roe [6] It has also been shown that salt and nisin, if combined with suitable hurdles (such as acids and mild heating) can control the growth of L monocytogenes in fish roe at 4°C [3, 16] However, the risks of post-processing contamination and improper refrigeration must always be considered With the official acceptance of nisin as a safe food additive by the Japanese government, its relevance to post-processing contamination, especially in soy-seasoned roe, needs to be considered With this aim, we examined the potential of nisin as a bacteriocin in soy-seasoned salmon roe as well as the possible enhancement of this potential with a P pentosaceus fermentate and pectin Nisin was found to be more effective in soy-seasoned roe than in FWR The total microbial aerobic counts and Listeria spp counts were comparatively lower in the SSSR samples than in the FWR samples This difference was expected because soy-seasoning is known to be effective even against Gram-negative bacteria such as E coli due to a combined presence of antimicrobial agents, such as sodium chloride, ethanol, and lactic acid [8] Moreover, salt extracts of salmon roe have been shown to contain listeriostatic substances (similar to lysozyme and phosvitin) which may combine with nisin to increase antimicrobial activity [7] Our initial premise was that combining nisin and the P pentosaceus fermentate (which contains pediocin) would significantly increase the antimicrobial effect at this stage, but this was not the case (results not shown), possibly due to a higher rate of degradation of pediocin in roe rendering the addition of the fermentate ineffective Nisin was degraded more slowly in SSSR than in FWR The degradation of bacteriocins in food matrices through binding and enzymatic processes remains a challenge in industrial applications In the case of meat products, heating reduces the degradation of nisin in the food matrix by reducing the sulphydryl groups [2, 17] It has been postulated that soy-seasoning slows the biochemical processes that binds nisin in roe Although it has been reported that sodium chloride enhances adherence and biofilm formation in L monocytogenes, this phenomenon was not evident in our study [12] It must be noted that nisin retarded but did not inhibit microbial growth in the SSSR samples To enhance the antimicrobial effect, we supplemented soy-seasoning with nisin, the P pentosaceus fermentate, 123 400 and pectin (PBSSR samples) This specific fermentate was selected because it contains pediocin, which has a different mode of action from nisin Pectin was used because of its potential to form gels that can protect bacteriocins from degradation [18, 19] We found that this method effectively inhibited microbial growth without significantly affecting organoleptic or chemical properties Moreover, the enhancement in antimicrobial effect was more significant at 12°C than it was at 5°C The Listeria spp counts dropped faster than the total aerobic counts at both temperatures The result that Listeria spp was more sensitive than total aerobes was expected because the bacteriocins used are listeriostatic, and bacteriocins are generally known to be strain-dependant [20] It is possible that the pathogen experienced more injury and less mortality at the lower temperature, resulting in the absence of counts on selective media (due to selective agents in palcam agar) and recovery on tryptic soy agar It has also been reported that L monocytogenes can modify its cell envelope charge or density to increase its tolerance to nisin [21] How this process is affected by temperature and the presence of other listeriostatic agents is a subject for future studies Improved bacteriostatic activity from the pectin–bacteriocin supplemented soy-seasoning was expected because nisin and pediocins have been shown to combine well in hurdle technology systems [13, 22, 23] In addition, pectin may have reduced bacteriocin degradation that normally results from the interaction of bacteriocins with the food matrix (roe fluids) The reduction of bacteriocin degradation in the presence of pectin is thought to be similar to the protective effect of cell immobilization by gel entrapment Gels have been reported to increase the tolerance of bacterial cells to bile acid and gastric juice, thereby promoting their survival and activity in human intestinal tracts [18, 19] Based on the improved antimicrobial effects of nisin in the presence of pectin, we postulate that the pectin used in this study provided a similar protective effect to the bacteriocins and cells of the bacteriocin-producer (from the fermentate) Unfortunately, pectin interfered with the analyses (as shown by the apparently low chloride content of PBSSR seasoning), and we were therefore unable to directly confirm the protective effect of pectin on nisin In view of its viscosity, pectin is thought to have formed a coating around the individual eggs that would effectively interfere with colonization of the roe by Listeria spp in the post-processing contamination simulations As shown in our results, pectin did not significantly affect the organoleptic attributes of the roe samples, primarily because only a thin layer of the seasoning was sufficient to protect the roe from the Listeria spp inoculum The comparatively lower scores for appearance were thought to be related to the raw material used rather than the presence of pectin in the seasoning Also, any 123 Fish Sci (2010) 76:395–401 off-flavors from the bacteriocins or pectin would be masked by the strong characteristic flavor of the soy sauce in the seasoning Pectin is an important polysaccharide with applications in the food, pharmaceutical, and a number of other industries It forms gels in the presence of solutes at low pH In the food industry, pectin is used in jams, jellies, frozen foods and, more recently, in low-calorie foods as a fat and/or sugar replacer [24] The results of our study show that pectin can be used for the delivery of bacteriocins in food safety applications As the bacteriocins contained non-food grade substances (especially the P pentosaceus fermentate that was produced in MRS broth), they were eliminated from roe batches subjected to sensory evaluation Although the fermentate may have increased the total acidity of the seasoning mixtures, it is thought that its overall effect on organoleptic acceptability would not be significant because bacteriocins were added to the seasoning mixtures (most of which were later discarded) and not directly to the final food product The control of Listeria spp is a challenge to the roe industry in Japan Nisin, because of its listeriostatic potential and its recent official acceptance as a food additive in Japan, has a potential for improving the safety of roe The results from our study show that although nisin is of little use on its own, it can be combined with a P pentosaceus fermentate and pectin to inhibit microbial growth in soy-seasoned salmon roe even in the case of post-processing contamination Acknowledgments This work was supported in part by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Research project for ensuring food safety from farm to table FP-6105) Additional support was provided by JSPS Grant-in-Aid for Scientific ResearchÓ (20580216) References Winkowski K, Ludescher RD, Montville TJ (1996) Physiochemical characterization of the nisin 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In each plot, the number of aquatic organisms ranging in size from 30 lm to 5 mm in the water, as well as those from 63 lm to 5 mm in size in the surface sediments, was surveyed 6, 1 3, 2 0, 2 6, 3 4, and 41 days after the onset of irrigation Three-day-old fish larvae were released on day 10 Undigested organisms in the gut contents of the larvae or fry were identified on days 2 0, 2 6, 3 4, and 4 1, respectively... (19 93) Osmotic and ionic regulation In: Evans DH (ed) The physiology of fishes CRC, Boca Raton, pp 31 5 34 1 2 Smith HW (1 930 ) The absorption and excretion of water and salts by marine teleosts Am J Physiol 93: 480–505 3 Marshall WS, Grosell M (2006) Ion transport, osmoregulation, and acid–base balance In: Evans DH, Claiborne JB (eds) The physiology of fishes CRC, Boca Raton, pp 179– 230 4 Hirano T, Mayer-Gostan... treatments (in spring, in autumn, and none) and sampling days (days 20–2 1, 26–2 7, 3 4, and 41 ), and Ai 9 Bj, Ai 9 Ck and Bj 9 Ck are their interactions Between-subject effects, Ai and Bj, were tested by Ai 9 Bj Within-subject effects, Ck, Ai 9 Ck and Bj 9 Ck, were tested by eijk, with the Huynh–Feldt adjustment The dependent variables, except for pH and the number of taxonomic groups, were converted into common... contain key molecules that promote calcification N Ogawa Á K Ura Á Y Takagi Graduate School of Fisheries Sciences, Hokkaido University, 3- 1-1 Minato-cho, Hakodate, Hokkaido 041-861 1, Japan Present Address: N Ogawa (&) Biomaterial Center, National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki 30 5-004 7, Japan e-mail: OGAWA.Nobuhiro@nims.go.jp Keywords Biomineralization Á Carasssius auratus... mineralization within biomineralization foci J Biol Chem 282:26002–260 13 27 Gorski JP, Kremer EA, Chen Y (1996) Bone acidic glycoprotein75 self-associates to form large macromolecular complexes Connect Tissue Res 35 : 137 –1 43 1 23 Fish Sci (2010) 76:189–198 28 Gorski JP, Kremer EA, Chen Y, Ryan S, Fullenkamp C, Delviscio J, Jensen K, McKee MD (1997) Bone acidic glycoprotein-75 self-associates to form macromolecular... indicate the samples in the plots with and without fish, respectively Circles, triangles, and squares indicate the samples from the plot n, the plot a, and the plot s, respectively Fish larvae were released on day 13 2 13 105 Prorodontida 104 1 03 0 106 Euglenales 105 Turbellaria 104 1 03 0 105 Halteriida Ploimida 104 1 03 0 6 13 20 26 34 41 6 13 20 26 34 41 Days after onset of paddy flooding Podocopida tended... 206 :34 95 35 05 9 Mackenzie FT, Bischoff WB, Bishop FC, Loijens M (19 83) Magnesian calcites: low-temperature occurrence solubility and solid-solution behavior In: Reeder RJ (ed) Carbonates Mineral Soc Am, Michigan, pp 97–144 10 Delpire E, Mount DB (2002) Human and murine phenotypes associated with defects in cation-chloride cotransport Annu Rev Physiol 64:8 03 8 43 11 Walsh PJ, Blackwelder PK, Gill A, Danulat... 199:2 231 – 234 3 14 Wilson RW, Grosell M (20 03) Intestinal bicarbonate secretion in marine teleost fish-source of bicarbonate, pH sensitivity, and consequence for whole animal acid–base and divalent cation homeostasis Biochim Biophys Acta 1618:1 63 1 93 15 Shedadeh ZH, Gordon MS (1969) The role of intestine salinity adaptation of the rainbow trout, Salmo gairdneri Comp Biochem Physiol 30 :39 7–418 16 Humbert W,