Fish Sci (2011) 77:161–167 DOI 10.1007/s12562-011-0323-1 ORIGINAL ARTICLE Fisheries Swimming angle and target strength of larval Japanese anchovy (Engraulis japonicus) Yusuke Ito • Hiroki Yasuma • Reiji Masuda Kenji Minami • Ryuichi Matsukura • Saho Morioka • Kazushi Miyashita • Received: 30 June 2010 / Accepted: 16 December 2010 / Published online: 18 February 2011 Ó The Japanese Society of Fisheries Science 2011 Abstract The swimming angle of larval Japanese anchovy (Engraulis japonicus) was measured in a tank, and target strength (TS) was calculated using a theoretical scattering model The mean swimming angle was 12.8° (SD ±22.1) Increased speeds of flow led to increased mean swimming angles The mean swimming angle at flow of cm s-1 was higher than at other speeds TS values were estimated using a distorted-wave Born approximation model for two cases Average values were 1–3 cm s-1 (11.5° ± 22.1) and cm s-1 (16.6° ± 21.7) for cases and 2, respectively For case 1, TS ranged from -92.0 to -74.7 dB with a mean of -79.4 dB at 120 kHz For case 2, TS ranged from -92.2 to -75.2 dB with a mean of -79.9 dB The mean TS in case was lower than that in case 1, with the maximum difference being 1.0 dB at 120 kHz (standard Y Ito (&) Laboratory of Marine Ecosystem Change Analysis, Graduate School of Environmental Science, Hokkaido University, 3-1-1 Minato-cho, Hakodate 041-8611, Japan e-mail: i-you@ees.hokudai.ac.jp H Yasuma Á K Miyashita Laboratory of Marine Ecosystem Change Analysis, Field Science Center for Northern Biosphere, Hokkaido University, 3-1-1 Minato-cho, Hakodate, Hokkaido 041-8611, Japan R Masuda Á K Minami Fisheries Research Station, Kyoto University, Nagahama, Maizuru, Kyoto 625-0086, Japan R Matsukura National Research Institute of Fisheries Engineering, FRA, 7620-7 Hasak, Kamisu, Ibaraki 314-0408, Japan S Morioka Fisheries Research Institute, Tokushima Agriculture, Forestry and Fisheries Technology Support Center, 1-3 Hiwasa, Minami, Kaifu, Tokushima 779-2304, Japan length 22.0 mm) However, there were no significant differences between the regression lines of cases and Thus, changes in flow speed altered the swimming angle of larval Japanese anchovy, but had little influence on TS Keywords Larval Japanese anchovy Á Swimming angle Á Target strength Introduction The Japanese anchovy (Engraulis japonicus) is one of the most important coastal fisheries species in Japan The larvae are an especially important resource in near-shore fisheries According to annual fishery statistics, commercial tow-net harvests were valued at 30 billion yen in 2007 [1] Therefore, data on the distribution and abundance of larvae are important for near-shore fisheries Accurate quantitative data on larvae are particularly important, not only for appropriately managing larvae fisheries but also for predicting the recruitment and management of adults Various studies have used commercial catch data to estimate the abundance and distribution of larval anchovies, often linked to environmental parameters such as current, salinity, and precipitation [2–4] However, such analyses must use data collected over a long period (months or years), and it is thus difficult to obtain quantitative estimates within a single fishery season Conversely, acoustic observations using quantitative echo sounders can provide quantitative data in the short term and have been used for stock assessments of many species [5, 6] In acoustic surveys, a quantitative echo sounder provides reflections from a fish school at various echo intensities This acoustic reflection is converted to quantitative data (e.g., number of individuals, biomass) using the target 123 162 strength (TS) Recently, the TSs of various species have been reported for use in estimating abundance in the field [7, 8] A split-beam echo sounder can be used to measure TS if a target is not too small in comparison with wave length In larval anchovy, however, field measurement of TS is difficult Acoustic reflections from larval anchovies are weak because of the small size of the fish and they form schools during the day influence the way the target detects them Therefore, the TS of larval Japanese anchovy should be estimated using a theoretical sound-scattering model Many theoretical models have been developed for calculating the TS of fish [9] Most of these models use an approximate geometric configuration to represent the swim-bladder (in swim-bladdered fish) or body (in swimbladderless fish) of the target species, which can be obtained from the relationship between TS and swimming angle (pitch or yaw) The Japanese anchovy is a physostomous fish The swim-bladders of their larvae are filled with gas during the night, which helps to reduce energy consumption However, during the day, they discharge gas from their swim-bladders and form schools [10] Miyashita [11] reported that acoustic surveys of larval anchovy should be conducted on high-density schools during the day Moreover, larval anchovy TSs of 50 and 200 kHz were estimated using the DWBA-based deformed cylinder model (DWBA model) [12, 13] that was developed for swim-bladderless species This study suggested that the dorsal average TS is very sensitive to changes in tilt angle Therefore, the TS should be determined using the tilt angle distribution after observation of swimming behavior In this study, we observed the swimming angles of larval Japanese anchovy using a video camera and estimated the TS as a function of tilt angle (TSavg) for use in acoustic surveys We used the swimming angle observation results to calculate the TSavg at 38 and 120 kHz (which are the main frequencies used by coastal research vessels in Japan) using the DWBA model We also considered the availability of TSavg as a scale factor in acoustic surveys, using the relationship between TSavg and the body lengths of larval anchovies Materials and methods Observation of swimming behavior In January and December 2008, live larval anchovies were provided from fixed shore nets in Wakasa Bay During both experimental periods, the larvae were transferred as soon as possible to a black fiberglass cylindrical tank (500 L, 116 cm diameter, 77 cm deep) at the Fisheries Research Station of Kyoto University One day later, we chose live larval anchovies and transported them to an experimental 123 Fish Sci (2011) 77:161–167 116cm 13cm Side Side strut strut 10cm 45cm 77cm 70cm Video camera 97.5cm Fig Schematic diagram of the experimental tank used for the penned Japanese anchovy larvae tank that was maintained at ambient temperature (10°C) in an environment that blocked natural light A 12-h photoperiod was maintained without a dawn or dusk transition in light intensity During the day, incident light at the surface measured approximately 350 lx; no light was provided during the night We conducted the swimming experiment during the day, to simulate the environmental conditions in the bay, and to adjust the flow in the experimental tank, seawater was added as needed The speed of flow was measured before and after video recording using an Electromagnetic Flow Velocity sensor (AEM1-D; ALEC Electronics, Tokyo) within the camera field angle Video recording began 10 after the speed of flow was stable and lasted for h at each speed Video recordings of the swimming behavior of 40 individuals were collected using two underwater cameras (T-WATER-2200c; HERO, Tokyo) (TL 39.0 ± 3.4 mm) in January In December, two digital HD video camera recorders (HDR-SR12; SONY, Tokyo) were used to give high quality image results for 12 individuals (total length 39.2 ± 1.8 mm) Recording systems were placed at a depth of 45 cm (Fig 1), and two additional underwater cameras were used to check the relationships between individuals and the camera lenses In this environment, we observed the swimming behavior of larval anchovies The obtained video recordings were converted to photographs at 1-min intervals for each experimental period Swimming angles were calculated from these photographs using image editing software (SCM Measure; Moritex, Tokyo) (Fig 2) There were two requirements for measurements: the centerline of the individual being measured could not be bending, and the individual had to be perpendicular to the camera The second parameter was confirmed using the dorsal camera The swimming angle was computed Fish Sci (2011) 77:161–167 163 rbs ¼ jfbs j2 , rpos is the position along the axis of the deformed cylinder, and k is the acoustic wave number given by k = 2p/k, where k is the acoustic wavelength The subscript sw refers to the surrounding seawater, the subscript animal refers to larval anchovy, Jl is a Bessel function of the first kind of order l, and ac is the crosssection radius of the cylinder and incident wave We digitized and obtained 200 sets of rpos and ac from the dorsal images of specimens We tried to measure the density contrast (g) and sound-speed contrast (h) of the larval anchovy using the density-bottle [16] and time-of-flight [17] methods These values were applied to the DWBA model as parameters (g = 1.068, h = 1.037) (Ito et al., unpublished) TS (dB) is defined as TS = 10 log10(rbs), and the average TS was defined as the mean TS, ranging from -90° to 90° at 1° steps (tilt angle: head-down, headup position) The tilt-averaged TS (TSavg) was calculated using the probability density function (PDF) of fish tilt angle f(bs) in Eqs and [15]: Dorsal image Target Lateral image 0q Non- target +ǰ -ǰ Target rAvg ¼ Zp=2 rðhÞf ðhÞdh ð2Þ Àp=2 Fig Typical example of a photograph obtained from the observation experiment The upper panel shows a dorsal image and the lower shows a lateral image; h indicates the swimming angle according to the above parameters The angle was that between the centerline of the fish, an imaginary line running from the root of the tail to the tip, and the true horizontal We defined positive angles as those for which the fish was head-up and negative angles as those for which the fish was head-down Theoretical model A total of 200 larval anchovies ranging from 18.0 to 35.7 mm SL (SL = 1.22 TL - 2.41, R2 = 0.99) were used for the TS calculation The sound scattering from specimens was estimated using the DWBA model A modification of the Matlab codes described in McGehee et al [14] ver 6.1 (MathWorks, Natick, MA, USA) was used to estimate the TS of the deformed cylinder, given as   Z ksw ac þ h2 fbs ¼ À 4kanimal gh2 ~ rpos Âe2ikanimal Ár~pos  Jl ð2kanimal ac cosbtilt Þ ~pos  dr cosbtilt ð1Þ where f(bs) is the complex backscattering amplitude, the relation to backscattering cross-section rbs is given by TSAvg ¼ 10 logrAvg ð3Þ where h represents swimming angle; f(h) was assumed to be a truncated normal distribution function The truncations were made at "h À 3Sh and "h þ 3Sh , where " h and Sh denote the mean and standard deviation of the tilt angle, respectively Since the number of samples was different in each photograph, strictly speaking, we should extract the same number of samples from each photograph However, we were not able to so because the experiment was limited Therefore, mean swimming angle and standard deviation were calculated using the limited samples in this study Results Theoretical TS Typical examples of the relationship between variations in TS and body tilt angle at 38 and 120 kHz obtained by the DWBA model are shown in Fig The variation in TS versus tilt angle showed peaks at about 0° (dorsal) at both frequencies, and the maximum TS at 120 kHz was higher than those at 38 kHz in all specimens Moreover, these peaks were relatively sharp, especially for the higher frequency, suggesting that slight changes in fish tilt angles have a major effect on TS The maximum TSs at 120 kHz were -85.6 and -68.1 dB in the smallest and the largest 123 164 Fish Sci (2011) 77:161–167 (a) Target strength (dB) -60 SL: 18.0mm -80 -100 -120 㧦 38 kHz 㧦 120 kHz -140 -90 -60 -30 30 60 90 Tilt angle (degree) Target strength (dB) (b) -60 Fig Swimming angles of larval Japanese anchovies Middle squares indicate the mean; whiskers show the standard deviation Numbers in parentheses indicate the maximum and minimum values SL: 35.7mm -80 the swimming angles in cases and There were no differences in either the ranges of values between cases and or their standard deviations (22.1 and 21.7, respectively) In contrast, the mean swimming angles differed by more than 5° (case 1: 11.5°, case 2: 16.5°) -100 㧦38 kHz -120 -90 㧦120 kHz -60 -30 30 60 90 Tilt-averaged TS Tilt angle (degree) Fig Typical variations in the target strength (TS) of minimum (a) and maximum larval length (b) as a function of fish pitch angle, estimated by the distorted-wave Born approximation (DWBA) model at 38 (dotted lines) and 120 (bold lines) kHz Positive angles are headup and negative angles are head-down individuals, respectively In individuals with larger standard length, the tilt angle had a considerable impact on TS Relationship between swimming angle and flow speed The flow speeds in the experimental tank were 1, 2, 3, and cm s-1, respectively We extracted 2,637 angles (2,102 in January, 535 in December) from photographs Figure shows box plots of the swimming angles at each flow speed In this study, the mean and standard deviation of the swimming angle of larval anchovies tended to rise as the flow speed increased However, the standard deviation was not related to the flow speed Larval anchovies were affected by flow variations (P \ 0.001, ANOVA), and the mean swimming angle at cm s-1 was higher than those at the other flow speeds (P \ 0.01, Tukey-Kramer test) Therefore, we separated the calculations of TS into two cases One case combined the data from flow speeds of 1–3 cm s-1 (case 1), and the other used data from flow speeds of cm s-1 (case 2) Figure shows histograms of 123 The values of TS were calculated with respect to fish tiltangle distribution (PDF), which was used for two values (case and case 2) at two frequencies (38 and 120 kHz) TS values are plotted in Fig as functions of fish SL on a logarithmic scale Ranges of TSavg and the equations of the regression lines (the TS-length equation) are shown in Fig and in Table In general, the results of TS can be expressed in terms of the body length L using the following equation: TS ¼ m log10 L þ b ð4Þ where m and L are constants for a given species This equation has been a generally accepted description of the way in which mean TS depends on fish length [18] The slope m and intercept b can be estimated by linearly regressing the TS on log L In this study, the regression line was fitted to the estimated data using cases and The results were TSavg ¼ 60:9 log10 L À 107:4 (R2 = 0.93) for case 1, and TSavg ¼ 60:1 log10 L À 107:5 (R2 = 0.92) for case at 120 kHz The mean TSavg was -79.4 for case and -79.9 dB for case The difference in each case was 0.98 dB (SL 22.0 mm) at the maximum Analysis of covariance (ANCOVA) was used to test for differences in the regression lines The slopes and intercepts of the relationships did not differ among cases (p \ 0.05) TSavg at 38 kHz was weak because L was too small for a wavelength Fish Sci (2011) 77:161–167 165 Fig Frequency distributions of the swimming angles of larval Japanese anchovy under flow rates of 1–3 cm s-1 (a) and cm s-1 (b) (a) (b) 1-3cm/sec 30 30 n = 682 Frequency (%) Frequency (%) n = 1955 Mean: 11.5 20 S.D.: 22.1 10 -90 -60 -30 30 Tilt angle (degree) -80 Target strength (dB) : Case : Case -90 -100 38 kHz -110 0.2 0.4 0.3 0.5 0.6 Log SL (cm) Target strength (dB) -70 : Case : Case -80 -90 120 kHz -100 0.2 0.3 0.4 0.5 5cm/sec 0.6 Log SL (cm) Fig Relationship between TS and log of standard length (in mm) for each case TS is 38 kHz for the upper panel and 120 kHz for the lower panel The circles and crosses indicate calculated TSs for cases (flow speed 1–3 cm s-1) and (flow speed cm s-1), respectively Equations for the regression lines in each panel are given in Table Discussion In this study, we used individuals with total lengths of 28.3–45.4 mm for the observation experiment The swimming angles of these individuals tended to be head-up for each speed of flow However, the relationship between 60 90 20 Mean: 16.5 S.D.: 22.0 10 -90 -60 -30 30 60 90 Tilt angle (degree) swimming angle and body length was not clear We conjecture that the swimming angle of the larvae differs with body length Hunter [19] showed that the swimming speed of northern anchovy (Engraulis mordax) larvae obviously changed with increases in body length Additionally, Batty [20] noted that the speed of larval herring increased with length Thus, the swimming abilities and swimming angles of larvae would also change with increases in body length In the juvenile stage, the swimming angle was almost 0°, and there was no influence of flow speed on the mean swimming angle (1–5 cm s-1 in four steps, one-way ANOVA, P [ 0.05) (Ito et al., unpublished) Thus, the differences in swimming angle between larvae and juveniles are likely caused by body growth Blaxter et al [21], for example, reported that metamorphosis entails the development of fins, resulting in improved swimming ability Therefore, as the body grows, the swimming angle will be close to 0° However, the mean swimming angle of individuals in this study would not be changed by body growth because larval anchovies complete metamorphosis at about 40 mm in length Individuals measuring 20 mm or less in length may swim at various swimming angles Therefore, an experiment examining the swimming angles of smaller fish is necessary to improve the precision of the TS However, our results suggest that the influence of the TS of small individuals is low because the TS of the largest individual was significantly different compared with that of the smallest individual due to changes in fish tilt angles We suggest that swimming angle gives larval anchovy the ability to swim against the flow and is determined by body length In this study, body lengths ranged from 28.3 to 45.4 mm and from 36.4 to 41.3 mm in January and December, respectively The mean swimming angles were 13.6° and 9.6° for each period (t test P \ 0.05) Thus, the swimming angle obviously changed with changes in the body length However, with the exception of the data gathered at cm s-1, these values were not significantly different between periods In individuals with small body lengths, the swimming angle relates to the flow In this study, the flow at cm s-1 affected the mean swimming 123 166 Fish Sci (2011) 77:161–167 Table Equations for the linear regression in Fig and mean target strength at each frequency n Case (1–3 cm/s) y represents TS (dB), and x represents the log of standard length in centimeters Case (5 cm/s) 200 200 angle, and the TSavg was calculated using the PDF for two cases (1–3, cm s-1) However, the regression lines of the calculated TSavg for the two cases did not differ significantly (Table 1) The Japanese anchovy is widely distributed in the northwest Pacific under various environmental conditions, and swimming angles under these conditions have not yet been clarified Therefore, in the future, swimming behavior must be observed under varying speeds of flow Additionally, it is necessary to continuously measure the flow speed during the experiment period In this study, the TS of larval anchovy clearly depended on changes in the swimming angle, which was in turn determined by body length In a previous study, the swimming angle of pelagic fish was set at the TS of larvae because the swimming angle of the larvae was not known As a result, the TSavg will become a source of error when abundance is estimated Therefore, reliable swimming data must be used to calculate TSavg for use in acoustic surveys Additionally, this study shows that when the swimming angle has a large standard deviation it has a little influence on TSavg, if a higher standard deviation of swimming angle (Fig 6) There are also some additional parameters that affect TS Mikami et al [22] measured the density (g) and soundspeed contrast (h) of E pacifica, which has no swimbladder, and thus no air bubbles in the body, between spring and autumn Their results showed that the maximum TS values for E pacifica, calculated from a theoretical scattering model, changed by about dB from spring to autumn Furthermore, Matsukura et al [23] suggested that g and h are affected by seasonal changes in lipid profiles In this study, the larval anchovy g and h values were fixed at 1.068 and 1.037, respectively However, these values must be adjusted to suit different conditions (i.e., season, area), and we must further examine such parameters in the future As mentioned above, these factors are important when attempting to estimate the TS of larval Japanese anchovy In particular, the influence of the swimming angle was high This is the first paper to provide swimming angle data The TSavg values given in Fig are recommended for use in acoustic surveys of fishing grounds Additionally, schools of the larvae of this species can be distinguished from those of other species using the volume back-scattering strength difference method 123 Frequency (kHz) Mean TSavg (dB) y=px?q p q r2 SE 38 -94.2 67.3 -125.3 0.93 1.5 120 -79.4 60.1 -107.4 0.92 1.4 38 -94.5 66.2 -125.1 0.92 1.5 120 -79.9 60.1 -107.5 0.92 1.4 Acknowledgments We thank the captains and crew of Kanagasaki Maru No for their cooperation in collecting specimens We also thank Yukio Ueta, Keisuke Mori Fisheries Research Institute, Tokushima Agriculture and the Forestry and Fisheries Technology Support Center for their support in collecting specimens This study was supported in part by the Fisheries Agency of Japan under the project ‘‘Research and Development Projects for Application in Promoting New Policy of Agriculture Forestry and Fisheries.’’ We thank this institution for their support References Annual Statistics on Fishery and Aquaculture Production 2006 (2008) Statistics Department, Ministry of Agriculture, Forestry and Fisheries, Tokyo Saiura K, Takeda Y (2001) Spring fishing ground formation of anchovy, Engraulis japonica, larvae in 1999 and 2000 in the Kii Channel (in Japanese) Fish Biol Oceanogr Kuroshio 2:109–118 Morioka S (2006) Trial of spring shirasu (Engraulis japonicus larvae) fishery forecast in Kii Channel by using multiple regression analysis (in Japanese) Fish Biol Oceanogr Kuroshio 7:21–27 Mitani I (1988) The biological studies on the larvae of Japanese anchovy, Engraulis japonica Hottuyn, in Sagami Bay (in Japanese) PhD thesis, University of Hokkaido, Hokkaido Honda S (2004) Abundance estimation of the young cohorts of the Japanese Pacific population of walleye pollock (Theragra chalcogramma) by acoustic surveys (In Japanese) PhD thesis, University of Hokkaido, Hokkaido Yasuma H (2004) Studies on the acoustical biomass estimation of myctophid fishes (in Japanese) PhD thesis, University of Tokyo, Tokyo Zhao X, Wang Y, Dai F (2008) Depth-dependent target strength of anchovy 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larvae Fourth European marine biological symposium Cambridge University Press, Cambridge 22 Mikami H, Mukai T, Iida K (2000) Measurements of density and sound speed contrasts for estimating krill target strength using theoretical scattering models (in Japanese with English abstract) Nippon Suisan Gakkaishi 66(4):682–689 23 Matsukura R, Yasuma H, Murase H, Yonezak S, Funamoto T, Honda S, Miyashita K (2009) Measurement of density contrast and sound-speed contrast for target strength estimation of Neocalanus copepods (Neocalanus cristatus and Neocalanus plumchrus) in the North Pacific Ocean Fish Sci 75:1377–1387 123 Fish Sci (2011) 77:169–181 DOI 10.1007/s12562-011-0331-1 ORIGINAL ARTICLE Fisheries Codend selectivity for jack mackerel and whitefin jack and unequal split parameter estimates observed in trouser trawl experiments Mohamed Salah Mahjoub • Seiichi Takeda Toshifumi Hayashi • Daisuke Shiode • Takafumi Arimoto • Tadashi Tokai • Received: 15 September 2010 / Accepted: 27 January 2011 / Published online: 22 February 2011 Ó The Japanese Society of Fisheries Science 2011 Abstract Codend selectivity for the jack mackerel Trachurus japonicus and the whitefin jack Kaiwarinus equula were evaluated based on data from trouser trawl experiments carried out in the East China Sea, using a test codend of 60 mm diamond mesh and a control codend made of minnow net with a square mesh of mm bar length Between-haul variations in parameters and the mean selection curves were tested with the catch data in the SELECT approach, and then the model of between-haul variation in the split parameter with the mean selection curve was chosen as the best fit using Akaike’s information criterion model selection The 50% retention lengths and the selection ranges were 11.4 and 3.36 cm for jack mackerel and 8.83 and 0.93 cm for whitefin jack, respectively The selection curve for whitefin jack was sharp, whereas that for jack mackerel was relatively wide As the estimated split parameters indicated, about 80% of the whitefin jack entered the control codend, but 85 and 90% of the jack mackerel entered the control codend in the second and third hauls, respectively The inequality in the split parameter is discussed from the viewpoint of the animal’s swimming behavior and water movement based on underwater video observations Keywords AIC model selection Á Codend selectivity Á Fish girth Á Jack mackerel Á SELECT Á Swimming ability Á Trouser trawl Á Underwater video observation Á Whitefin jack M S Mahjoub Á S Takeda Á T Hayashi Á D Shiode Á T Arimoto Á T Tokai (&) Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan e-mail: tokai@kaiyodai.ac.jp Introduction Fishing gear selectivity plays an important role in the exploitation of fish stocks and fisheries management A trouser trawl that has two codends (one test codend and another small-mesh control codend) attached to the aft end has often been utilized to estimate codend selectivity [1–3] The trouser trawl method has some advantages in experimental fishing operations; for example, compared with the covered codend method and the twin trawl method, trawl handling more closely resembles standard commercial fishing [3] It is also designed to ensure that similar numbers and size ranges of all species pass down into both codends, in contrast with the other paired gear tests (the alternate haul method and the parallel haul method) [3] However, it was also pointed out that larger fish are caught more in the test codend than in the control one of a trouser trawl [1, 3] Millar and Walsh [4] demonstrated that significantly more fish entered the test codend with a larger mesh than the control codend in a trouser trawl fishing experiment, and estimated the split parameter with codend selectivity parameters by the SELECT method However, only a few studies on the cause of this have been performed The jack mackerel Trachurus japonicus is a commercially valuable species in Japan [5] For example, in 2007, the annual landing and production of jack mackerel in Japan were 170,389 tons and 36,721 million yen, respectively [6] Jack mackerel is caught by trawl as well as purse seine in Japan, and it is one of the important target species of the large trawl in the East China Sea The whitefin jack Kaiwarinus equula is also caught by the large trawl there, and both species belong to the Carangidae family However, the jack mackerel has a spindle-shaped body that is not as compressed as that of the whitefin jack According to 123 170 the definition of fish swimming specialization from Videler and He [7], jack mackerel are more hydrodynamic and can be considered faster swimmers than whitefin jack On the other hand, fish body shape, in particular fish girth, is one of the factors that affect codend selectivity [8–10] This study carried out a trouser trawl with two codends: a test codend with a nominal mesh size of 60 mm and a control codend made of fine minnow netting Based on the data obtained, it was possible to estimate not only logistic curve parameters for expressing the mesh selectivity of the codend with the 60 mm mesh, but also the split parameter indicating the proportions of each species of fish that enter the test codend The cause of an unequal split between the test and control codends in the trouser trawl is also discussed in terms of the animal’s swimming behavior, based on underwater video observations made in front of the mouths of the two codends Materials and methods Fishing experiments A series of trouser trawl fishing experiments were conducted onboard the research and training vessel Umitaka Maru (1886 gross tonnage), Tokyo University of Marine Science and Technology, in the East China Sea in October 2009 A pair of codends consisting of a test codend of nominal 60 mm mesh size and a control codend containing a minnow-net bag were used (Fig 1) The test codend of 60 mm nominal mesh size with a diamond mesh had a mesh opening of 54 mm, which is the current legal minimum mesh size for the large trawl in the East China Sea, according to an Ordinance of the Ministry of Agriculture, Forestry and Fisheries of Japan The control codend was made of minnow net with a square mesh of mm bar length to avoid clogging, and was covered by a strengthening bag with a diamond mesh net with a 28 mm mesh opening in order to stop the minnow net from bursting The trawl net used had a total net length of 39 m and a net mouth with a 28 m headrope and a 37.2 m groundrope Two underwater video system units (DCR-SR100 video camera, Sony Corp.; blimp-shaped underwater housing, Goto Aquatics Corp [11]) were installed facing each other on the upper panel ahead of the codends to observe the behavior of animals in front of the codend mouths (Fig 1) The video camera shot downward at about 20° Two flashlights (Excursion LS24, Tektite Industries Inc.) were attached on both sides of video system #1 The auto-iris video lens of system #2 was illuminated with excessively bright light from the two flashlights, so the video feed from system #2 was out of focus and too bright to be able to observe the animal’s behavior Accordingly, video from system #1 was utilized in this study 123 Fish Sci (2011) 77:169–181 Three hauls were performed at fishing grounds at a depth of about 120 m on 11 October 2009 The water temperatures at the grounds were 17.4°C near the bottom and 26.4°C at m depth according to a conductivity– temperature–depth (CTD) device The warp length was 400 m After the warp was let out, the net was towed for 30 at a vessel speed of approximately 3.5 knots, before it was hauled at a winch speed of 0.44 m/s at a vessel speed of \2 knots Catch numbers (the number of individuals caught) in the test and control codends were recorded for each species, apart from the three-spot swimming crab Ovalipes punctatus, which is usually dominant in the catch in the fishing grounds at around 120 m depth The whole catch of the swimming crab was measured in trays when subsampling, and one tray was randomly chosen for carapace width measurements Lengths of all fish in the codends were measured to the nearest millimeter Fork lengths were measured for jack mackerel, and total lengths for whitefin jack and other fishes Fish length data were grouped into 0.5 cm intervals for further selectivity analysis Fish girth was also measured to the nearest millimeter to examine the relationship between the mesh size and fish girth at a certain retention probability Data analysis Differences in catch number between the test and control codends were statistically tested for significance by the chisquared test for several of the main species in each haul Length distributions of jack mackerel and whitefin jack were analyzed with the SELECT method [4, 12, 13] in order to estimate the selectivity of the test codend, because there were sufficient data to allow this estimate For the dominant species, except for jack mackerel and whitefin jack, there were insufficient data to permit selectivity estimation, so differences in length distributions for the test and control codends were statistically tested with the Kolmogorov–Smirnov test The SELECT approach [4, 12, 13] was applied to estimate mesh selectivity parameters from the data obtained using the paired gear test, including the trouser trawl test For a fish length of lj in the ith haul, the proportion /ij of the catch in the 60 mm mesh test codend NLij compared to the total catch (the catch in the test codend NLij and the catch in the control codend NSij) is defined as follows: /ij ¼ NLij : NLij þ NSij ð1Þ The probability of a fish of length l being retained in the test codend is usually expressed with the logistic function r(l), which has the parameters a and b [3]: Fish Sci (2011) 77:271–277 273 whereas for freezing at -196°C, samples were placed under liquid nitrogen for min, then kept at -80°C until analysis The reactions were terminated by addition of trichloroacetic acid solution to final concentration of 5% Table Comparison of TMAO and components associated with TMAO degradation in frozen minced meat and surimi Croaker Lizard Pollock TMAO (mM) Analysis of the relationship between pH and DMA formation in minced meat Mince 37 ± 32 ± 64 ± Surimi 6±1 3±2 7±2 Fe (lM) The pH of surimi is adjusted using polyphosphate or sodium bicarbonate to stabilize fish proteins during freezing or to improve gel strength We examined the effect of pH on DMA formation in frozen minced croaker, lizardfish, and pollock Minced croaker, lizardfish, and pollock meat were adjusted to pH 5.5–8.5 using 0.1 M HCl or 0.1 M NaOH Samples were blast-frozen at -40°C for 20 h and stored at -10°C for weeks The amount of DMA generated during frozen storage was defined as the difference between the DMA concentration immediately after freezing and the DMA concentration after the storage period Results Comparison of DMA formation in minced meat and surimi Figure shows the amount of DMA generated in minced meat and surimi prepared from croaker, lizardfish, and pollock during frozen storage Little or no DMA was generated in minced croaker meat or croaker surimi Substantial DMA was generated in lizardfish and pollock prepared as minced meat, but very little DMA was generated when they were processed into surimi The components associated with TMAO degradation in both minced meat and surimi were compared (Table 1) The amount of TMAO in minced pollock was approximately twofold DMA (mM) Croaker Lizardfish Pollock Mince 82 ± 63 ± 90 ± Surimi 59 ± 33 ± 52 ± Taurine (lg/100 g) Mince 106 ± 87 ± 107 ± 4±1 4±2 Surimi 6±2 Values represent mean ± standard deviation (SD) of triplicate measurements higher than that in minced croaker or lizardfish Minced croaker and pollock contained almost the same amount of iron and taurine, which are known to stimulate DMA formation [23]; minced lizardfish had lower iron and taurine concentrations than croaker and pollock Cysteine has also been reported to increase DMA formation in the presence of Fe2? [23], but no free cysteine was detected in any of the samples examined (data not shown) After processing the meat into surimi, the TMAO content fell to between 11% and 17% of the original content The iron content fell to between 52% and 72% of the original content, and the taurine content fell to between 4% and 6% of the original content in the corresponding species Effect of TMAO, FeSO4, and ascorbic acid on DMA formation in minced meat The addition of TMAO did not stimulate DMA formation in any of the samples (Fig 2) DMA formation was not observed even when all three substances were added together to minced croaker However, in lizardfish and pollock, FeSO4 and ascorbic acid induced DMA formation Most of the TMAO in minced lizardfish degraded into DMA when FeSO4 or ascorbic acid was added In contrast, in minced pollock treated with FeSO4 and/or ascorbic acid, a maximum of only 10% of the total TMAO content was degraded Effect of TMAO, FeSO4, and ascorbic acid on DMA formation in surimi minced surimi Fig Comparison of DMA concentration in minced and surimiprocessed croaker, lizardfish, and pollock following storage at -10°C for weeks DMA concentration was determined by the copperdithiocarbamate method Results are average ± standard deviation (SD) of triplicate measurements Figure shows that, although very little DMA was generated when TMAO, FeSO4 or ascorbic acid was added individually to croaker surimi, when the compounds were added in combination, 0.8 mM DMA was produced; more DMA was generated in the surimi than in the minced croaker in the presence of all three substances DMA generation was not observed when TMAO, FeSO4 or 123 274 Fish Sci (2011) 77:271–277 1.0 initial Croaker -10°C 2wks 0.6 0.4 0.2 0.8 initial Croaker -10°C 2wks 0.6 0.4 0.2 TMAO+Fe+As Fe+As As Control TMAO+Fe+As Fe+As As Fe TMAO control Fe 0.0 0.0 TMAO 0.8 DMA (mM) DMA (mM) 1.0 100 TMAO+Fe+As Fe+As As As TMAO+Fe+As Fe+As As Fe TMAO 20 Fe 20 40 Fe 40 Lizardfish 60 TMAO 60 80 TMAO Lizardfish Control DMA (mM) 80 control DMA (mM) 100 50 20 10 TMAO+Fe+As Fe+As As Fe Fig Effects of exogenously added TMAO, FeSO4 (Fe), and ascorbic acid (As) on DMA concentration in frozen minced meat Minced meat samples were mixed with water, 50 mM TMAO, mM FeSO4, and/or mM ascorbic acid, homogenized, blast-frozen at -40°C for 20 h, and stored at -10°C for weeks Results are average ± standard deviation (SD) of triplicate measurements ascorbic acid was individually added to lizardfish surimi, and 3.3 mM DMA was formed when all three substances were added in combination DMA production in lizardfish surimi was significantly lower than in the minced meat in the presence of all three substances DMA in pollock surimi was produced only when the three substances were added in combination, and the amount of DMA generated was more than twice that in minced pollock under the same conditions Relationship between sucrose and DMA formation in vitro The results are shown in Fig For the solutions frozen at -25°C, DMA formation was suppressed by the addition of 123 30 20 10 TMAO Pollock TMAO+Fe+As 30 40 Fe+As Pollock Control DMA (mM) 40 control DMA (mM) 50 Fig Effect of exogenously added TMAO, FeSO4, and ascorbic acid on the DMA concentration in frozen surimi Surimi samples were treated and analyzed as in Fig Results are average ± standard deviation (SD) of triplicate measurements more than 2.5% sucrose, and DMA formation was reduced by 90% when 10% sucrose was added to the reaction mixture High sucrose concentrations were required to minimize DMA formation when solutions were frozen at -10°C compared with -25°C, but sucrose was not required when solutions were rapidly frozen under liquid nitrogen Relationship between pH and DMA formation Figure shows the relationship between the pH of the minced meat and DMA formation Minimal DMA formation was observed in minced croaker meat at pH 6–8 DMA formation in minced lizardfish increased with decreasing pH; DMA production was the highest at pH 5.7 and lowest at pH 8.6 In minced pollock, the greatest DMA generation was observed at pH 7.5, and DMA production decreased as Fish Sci (2011) 77:271–277 275 DMA (mM) 0.20 -10°C -25°C 0.15 -195°C 0.10 0.05 0.00 10 Sucrose (%) Fig Effects of sucrose concentration and freezing temperature on DMA formation in vitro Sucrose was added at the indicated concentrations to a solution containing 10 mM Tris–acetate (pH 7.0), 50 mM TMAO, 0.1 mM FeSO4, mM EDTA (disodium salt), and mM cysteine Reaction mixtures were prepared on ice and frozen as indicated Results are average ± standard deviation (SD) of triplicate measurements DMA (mM) 40 Croaker Lizardfish Pollock 30 20 10 5.5 6.5 7.5 8.5 pH Fig Relationship between pH and DMA formation in frozen minced meat Minced croaker, lizardfish, and pollock samples were adjusted to pH 5.5–8.5, blast-frozen at -40°C, and stored at -10°C for weeks Results are average ± standard deviation (SD) of triplicate measurements the pH increased or decreased from pH 7.5 Similarly, other research has shown that DMA formation in pollock is maximal at around pH 7.0 [24] Discussion DMA formation in surimi is known to be suppressed during frozen storage [1–6] However, the underlying mechanisms have not yet been revealed We examined the mechanisms of DMA suppression using three fish species After surimi processing, TMAO and taurine were markedly reduced, and iron loss was less substantial than TMAO or taurine loss It is possible that iron is bound to various substances in the muscle, whereas TMAO and taurine exist in free form To investigate whether elimination of substances such as TMAO, iron, and taurine during processing into surimi results in the observed low DMA formation, these components were exogenously added to minced meat and surimi prepared from three fish species The addition of TMAO did not stimulate DMA formation in any of the minced meat Surprisingly, minced croaker did not generate DMA even when TMAO, FeSO4, and ascorbic acid were added Because it has been shown that DMA is formed at subzero temperatures when TMAO, Fe2?, and ascorbic acid are mixed in aqueous solution [22], our data suggest that croaker muscle may contain intense deactivators of FeSO4, ascorbic acid or TMAO Further studies are needed in this area to clarify the mechanism which prevents TMAO from degrading into DMA in croaker during frozen storage Most of the TMAO in minced lizardfish degraded into DMA when FeSO4 or ascorbic acid was added, suggesting that in lizardfish DMA formation depends on the free Fe2? content in the muscle Thus if lizardfish contained enough free iron, all of the TMAO would promptly degrade into DMA and FA In contrast, only 20% of the TMAO in minced pollock degraded when all three substances were added, suggesting that factors other than Fe2? contribute to TMAO decomposition in pollock muscle In the presence of TMAO, FeSO4, and ascorbic acid, croaker surimi produced small amounts of DMA, whereas minced croaker produced almost no DMA Sucrose was added to the surimi samples, and it is known that sugars inhibit DMA formation [4] Therefore, this suggests that croaker may contain suppressors of DMA formation that are removed or changed their functions during the surimi processing DMA production in lizardfish surimi was significantly lower than that in the minced meat in the presence of the three substances added either individually or in combination Therefore, it is thought that intensive DMA production promoters, which are not like irons or reductants, were removed or had their functions changed by the surimi processing DMA in pollock surimi was not produced when TMAO, FeSO4 or ascorbic acid was added individually, but DMA was formed when the three substances were added together; the amount of DMA generated was more than twice that in minced pollock under the same conditions Therefore, although pollock muscle may contain intrinsic DMA formation suppressors, these suppressors may be washed out or have their functions changed by the surimi process We demonstrated that sucrose effectively inhibited DMA formation in vitro Sucrose may prevent freezinginduced concentration of solutes that enhance TMAO degradation Our data also suggest that the pH dependence of DMA formation is species specific In lizardfish, DMA formation increased with decreasing pH This was unexpected because TMAO degradation catalyzed by Fe2? is theoretically suppressed at lower pH due to the oxidation of 123 276 Fe2? Although the mechanisms that underlie the relationship between pH and DMA formation in each fish species remain unclear, our data suggest that factors involved in DMA formation differ between species This study suggests that DMA formation increases in the presence of TMAO, Fe2?, and a reductant in most cases However, unidentified intrinsic components in fish muscle also have a considerable effect on DMA formation Landolt and Hultin [1] reported that, when red hake fillets were soaked in distilled water, not only TMAO and watersoluble proteins were removed, but other substances such as the flavin-reduced nicotinamide adenine dinucleotide system, iron, and some reducing agents were also removed It is suggested that, despite the importance of the behavior of key substances such as TMAO, Fe2?, and reductants during surimi processing, other components such as peptides and proteins, which may interact with Fe2?, also need to be researched Promoters and inhibitors of DMA formation have been reported in tests using methods which were either in vitro or involved adding substances [6, 17, 23, 25–28] However, DMA formation in fish meat should be investigated for each species, because the DMA formation responses to FeSO4, reductants, and pH were very different in each species Fish Sci (2011) 77:271–277 10 11 12 13 14 15 16 17 18 References 19 Landolt LA, Hultin HO (1982) Inhibition of dimethylamine formation in frozen red hake muscle after removal of trimethylamine oxide and soluble proteins J Food Biochem 6:111–125 Adu GA, Babbitt JK, Crawford DL (1983) Effect of washing on the nutritional and quality characteristics of dried minced rockfish flesh J Food Sci 48:1053–1060 Yasui A, Lim PY (1987) Changes in chemical and physical properties of lizardfish meat during ice and frozen storage Nippon Shokuhin Kogyo Gakkaishi 34:54–60 MacDonald GA, Willson ND, Lanier TC (1990) Stabilised mince: an alternative to the traditional surimi process In: Chilling and freezing of new fish products International Institute of Refrigeration, Paris, pp 69–76 Yoon KS, Lee CM, Hufnagel LA (1991) Effect of washing on the texture and microstructure of frozen fish meat J Food Sci 56:294–298 Sotelo CG, Pineiro C, Perez-Martin RI (1995) Denaturation of fish proteins during frozen storage: role of formaldehyde Z Lebensm Unters Forsch 200:14–23 (in English with German abstract) Castell CH, Neal WE, Dale J (1973) Comparison of changes in trimethylamine, dimethylamine, and extractable protein in iced and frozen gadoid fillets J Fish Res Board Can 30:1246–1248 Reppond KD, Collins J, Markey D (1985) Walleye pollock (Theragra chalcogramma): changes in quality when held in ice, slush-ice, refrigerated seawater, and CO2-modified refrigerated seawater then stored as blocks of fillets at -18°C J Food Sci 50:985–996 Rehbein H, Orlick B (1990) Comparison of the contribution of formaldehyde and lipid oxidation products to protein denaturation 123 20 21 22 23 24 25 26 and texture deterioration during frozen storage of minced ice-fish fillet (Champsocephalus gunnari and Pseudochaenichthys georgianus) Int J Refrig 13:336–341 Sotelo C, Mackie IM (1993) The effect of formaldehyde on the aggregation behaviour of bovine serum albumin during storage in the frozen and unfrozen states in the presence and absence of cryoprotectants and other low molecular weight hydrophilic compounds Food Chem 47:263–270 Rodger G, Hastings R (1984) Role of trimethylamine oxide in the freeze denaturation of fish muscle—is it simply a precursor of formaldehyde? J Food Sci 49:1640–1641 Lee CM (1985) A pilot plant study of surimi making properties of red hake (Urophycis chuss) In: Martin RE, Collette RL (eds) Proceedings of the international symposium on engineered seafood including surimi National Fisheries Institute, Washington, DC, pp 225–243 Tokunaga T (1974) The effect of decomposed products of trimethylamine oxide on quality of frozen Alaska pollock fillet Bull Jpn Soc Sci Fish 40:167–174 (in Japanese with English abstract) Ang JF, Hultin HO (1989) Denaturation of cod myosin during freezing after modification with formaldehyde J Food Sci 54:814–818 Careche M, Li-chan ECY (1997) Structural changes in cod myosin after modification with formaldehyde or frozen storage J Food Sci 62:717–723 Murata S, Yamasaki N, Tanaka F (1992) Theoretical analysis of chemical reaction in frozen state Nippon Shokuhin Kogyo Gakkaishi 39:972–975 (in Japanese with English abstract) Spinelli J, Koury B (1979) Nonenzymic formation of dimethylamine in dried fishery 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Japanese with English abstract) Spinelli J, Koury BJ (1981) Some new observations on the pathway of formation of dimethylamine in fish muscle and liver J Agric Food Chem 29:327–331 Kimura M, Takeuchi N, Nozawa H, Mizuguchi T, Kimura I, Seki N (2006) The mechanism and oxygen gas inhibition of the degradation of trimethylamine-N-oxide in walleye pollack muscle during storage Nippon Suisan Gakkaishi 72:911–917 (in Japanese with English abstract) Tokunaga T (1980) Biochemical and food scientific study on trimethylamine oxide and its related substances in marine fishes Bull Tokai Reg Fish Res Lab 101:1–129 (in Japanese with English abstract) Rehbein H (1988) Relevance of trimethylamine oxide demethylase activity and haemoglobin content to formaldehyde production and texture deterioration in frozen stored minced fish muscle J Sci Food Agric 43:261–276 Fish Sci (2011) 77:271–277 27 Dingle JR, Hines JA (1975) Protein instability in minced flesh from fillets and frames of several commercial Atlantic fishes during storage at -5°C J Fish Res Board Can 32:775–783 277 28 Reece P (1983) The role of oxygen in the production of formaldehyde in frozen minced cod muscle J Sci Food Agric 34:1108–1112 123 Fish Sci (2011) 77:279–288 DOI 10.1007/s12562-011-0328-9 ORIGINAL ARTICLE Food Science and Technology Isolation, characterization, and utilization of c-aminobutyric acid (GABA)-producing lactic acid bacteria from Myanmar fishery products fermented with boiled rice Su Myo Thwe • Takeshi Kobayashi • Tianyao Luan • Takaaki Shirai • Munenaka Onodera Naoko Hamada-Sato • Chiaki Imada • Received: October 2010 / Accepted: January 2011 / Published online: 18 February 2011 Ó The Japanese Society of Fisheries Science 2011 Abstract c-Aminobutyric acid (GABA)-producing lactic acid bacteria (LAB) were isolated from four types of Myanmar traditional fermented fishery products with boiled rice All of them belonged to the genus Lactobacillus, and comparison of the effects of these representatives on GABA accumulation in fermented fishery products with boiled rice revealed that Lactobacillus farciminis D323 is the most effective strain as a starter culture These results may contribute to the development of traditional fermented fishery products with functional properties In addition, this study is the first to show in detail the distribution of GABA-producing LAB in Southeast Asian fermented fishery products Keywords c-Aminobutyric acid Á Fermentation Á Lactobacillus Á Fermented fishery products Á Myanmar S M Thwe Á T Kobayashi (&) Á T Luan Á N Hamada-Sato Á C Imada Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan e-mail: takeshik@kaiyodai.ac.jp S M Thwe Department of Fisheries, Ministry of Livestock and Fisheries, Yangon, Myanmar T Shirai Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan M Onodera Iwate Industrial Research Institute, 3-35-2 Iioka-Shinden, Morioka, Iwate 020-0852, Japan Introduction c-Aminobutyric acid (GABA) is a four-carbon, nonprotein amino acid that is widely distributed in nature [1] GABA has been proved to be an inhibitory neurotransmitter in mammalian brains [2] In addition, GABA has multiple physiological functions such as tranquilizing, diuretic, and hypotensive activities [3, 4] Furthermore, it has been reported that administration of GABA-enriched food is effective for regulation of depression, sleeplessness, and autonomic disorders [5] It also has a hypotensive effect [6, 7], and can be used for improvement of chronic alcoholrelated symptoms [8], stimulation of immune system cells [9], and prevention of diabetic conditions [10] Therefore, the benefits of GABA on human health have recently attracted increased attention in the food industry, and several GABA-enriched functional foods are currently manufactured, for instance, tea leaves treated unaerobically [11], rice germs soaked in water [12], red mold rice [13], tempeh-like fermented soybeans [14], and dairy products such as yogurt [15], fermented milk products [7], and cheese [16, 17] Various microorganisms, including bacteria such as lactic acid bacteria (LAB), fungi, and yeast, that produce GABA have been reported There have been numerous investigations into GABA-producing LAB from various viewpoints, such as microbiological taxonomy, GABA production from nutrient medium or various fermentation ingredients, and the biochemical and genetic characterization of glutamate decarboxylase (GAD) (EC 4.1.1.15), which catalyzes the primary biosynthesis of GABA, namely a-decarboxylation of L-glutamic acid Ueno et al [18] purified and characterized GAD of Lactobacillus brevis IFO 12005, and a more detailed genetic study on this GAD has recently been performed [19] In addition, this 123 280 strain was also demonstrated as a GABA producer from alcohol distilled lees [20] An earlier report described the GABA production and enzymatic properties of GAD of the Lb paracasei isolated from a Japanese fermented crucian carp, funa-zushi [21, 22] Moreover, some researchers reported GABA-producing Lb buchneri [23], Lb brevis [24, 25], and Lb plantarum [26] in kimchi, a Korean fermented vegetable product Lb brevis was also isolated from fresh milk and plaa-som, a Thai fermented fish product [27, 28] As mentioned earlier, Lactobacillus species are the main GABA-producing LAB; however, other LAB species have also been described as GABA producers Lactococcus lactis has been reported as a producer of carbon dioxide and GABA during cheese ripening [16, 29] The detailed characteristics of GAD of Lc lactis have been elucidated [30] Lactic acid bacteria are some of the most commonly used microorganisms in food fermentation, and have been used not only in natural fermentation, but also are often used as a starter culture to accelerate and direct the fermentation of various types of food fermentations [31] Previously, various studies on the development of fermented food containing high levels of GABA using a starter culture of GABA-producing LAB were reported as follows: Lactobacillus sp L13 was demonstrated as a preferred starter culture for the accumulation of GABA in the Japanese pickled turnip [32] Lb brevis was also used for the fermentation of GABA-enriched blackberry juice [33] In addition, a selection of GABA-producing LAB to be used as a starter culture for cheese making [17], and milk fermentation and cheese manufacturing using Lc lactis and Streptococcus salivarius subsp thermophilus Y2 [15, 16, 34] have been reported These investigations focused on pickled vegetables, fruit products, and dairy products, and we are currently unaware of any reports on the application of GABA-producing LAB in fermented fishery products In Myanmar, there are various types of traditional fermented fishery products, such as fermented fish or shrimp with boiled rice, fish sauce, and fish paste, which are known to be a necessary part of the dietary habits of the Myanmar people as seasonings or side dishes Among them, fermented fishery products with boiled rice are mainly prepared using four types of fishery products, with different names depending on the type of fishery product used They are called ngachin, pazun-chin, ngagyin-chin, and ngaphae-chin when tinfoil barb Puntius schwanenfeldi, speckled prawn Metapenaeus monoceros, rohu Labeo rohita, and featherback fish Notopterus notopterus are used as ingredients for these fermented products, respectively Although Myanmar’s traditional fermented fishery products are very popular and consumed widely in the country, there are only a few early studies, including 123 Fish Sci (2011) 77:279–288 microbiological studies, on fish paste and fish sauce [35, 36] On the other hand, there are no detailed reports on LAB or GABA-producing LAB in these fermented fishery products To contribute to the development of traditional fermented fishery products with functional properties, we focused on screening GABA-producing LAB that are suitable for the fermentation of traditionally fermented fishery products For this purpose, we used four types of Myanmar fermented fishery products with boiled rice with less focus on the microbiological viewpoint We also describe the distribution of GABA-producing LAB in these products and the productivity of each isolate Moreover, GABA-producing LAB were used as starter cultures in the manufacturing of their original fermented fishery products to confirm the accumulation of GABA Materials and methods Fermented fishery products for isolation of GABA-producing LAB A total of 11 samples containing four types of fermented fishery products with boiled rice were purchased from a market in Yangon City, Myanmar in 2006 and 2008, as shown in Table Tinfoil barb and speckled prawn were used for fermentation, and the products were called ngachin (N) and pazun-chin (PC-1 and PC-2), respectively In addition, rohu and featherback fish were also used for fermentation of ngagyin-chin (NGC-1 to 4) and ngaphaechin (NPC-1 to 4), respectively These purchased samples were transported to our laboratory by air and immediately stored at 4°C Chemical properties of fermented fishery products The pH values were measured using a pH meter (M-11; Horiba, Kyoto, Japan), and the salt concentrations were measured using a NaCl meter (C-121; Horiba, Kyoto, Japan) Before these measurements, one gram of each fermented fishery product was mixed well with ml distilled water and homogenized For the amino acid analysis, g of a fermented product was homogenized with volumes of 6% perchloric acid (PCA) and centrifuged at 18,8009g for 10 at 4°C The obtained supernatant was neutralized by adding N KOH to adjust the pH to 7.0 and filtered through 0.22-lm cellulose acetate membranes The neutralized supernatants were analyzed for GABA content using a JLC-500 automatic amino acid analyzer (JEOL, Tokyo, Japan) Unless otherwise stated, all experiments were performed in triplicate Fish Sci (2011) 77:279–288 281 Table Chemical properties and distribution of isolated GABA-producing LAB in four types of fermented fishery products Fermented products pH NaCl GABA amount of fermented products (mg/100 g) No of isolates No of GABA producers Isolation medium NaCl 5%-GYP Tinfoil barb (Ngachin, N) 4.8 ± 0.1 3.8 ± 1.1 193.4 ± 6.5 Speckled prawn (Pazun-chin, PC-1) 4.3 ± 0.1 4.1 ± 0.8 26.2a 13 NaCl 5%-GYP Speckled prawn (Pazun-chin, PC-2) Rohu (Ngagyin-chin, NGC-1) 3.9 ± 0.1 4.2 ± 0.2 3.9 ± 0.3 6.6 ± 0.4 0.5 ± 0.1 4.0 ± 6.5 12 12 0 NaCl 10%-GYP NaCl 7%-MRS Rohu (Ngagyin-chin, NGC-2) 4.5 ± 0.1 8.6 ± 1.7 1.5 ± 2.1 12 NaCl 7%-MRS Rohu (Ngagyin-chin, NGC-3) 4.2 ± 0.3 7.2 ± 0.4 10.0 ± 13.6 12 NaCl 7%-MRS Rohu (Ngagyin-chin, NGC-4) 4.3 ± 0.2 7.1 ± 0.2 1.9 ± 1.9 12 NaCl 7%-MRS Featherback fish (Ngaphae-chin, NPC-1) 3.8 ± 0.1 6.9 ± 0.5 0.3 ± 0.5 12 NaCl 3%-MRS Featherback fish (Ngaphae-chin, NPC-2) 4.1 ± 0.1 4.1 ± 1.4 0.2 ± 0.4 12 NaCl 3%-MRS Featherback fish (Ngaphae-chin, NPC-3) 3.9 ± 0.2 5.2 ± 0.6 ND 12 NaCl 3%-MRS Featherback fish (Ngaphae-chin, NPC-4) 3.9 ± 0.1 5.1 ± 0.6 ND 12 NaCl 3%-MRS Data expressed as mean ± standard deviation (SD) from three independent experiments ND not detected a n=2 Isolation of GABA-producing bacteria An enrichment culture was carried out to isolate GABAproducing LAB Five grams of each of the fermented products was inoculated into 35 ml de Man, Rogosa, and Sharpe (MRS) broth [37] or glucose yeast extract polypepton (GYP) broth [18] containing 5% monosodium glutamate (MSG) adjusted to initial pH of 7.0 An appropriate amount of NaCl corresponding to each of the fermented products was added to each enrichment culture medium; the detailed conditions are shown in Table After days of incubation at 27°C, an aliquot of the culture broth was transferred to a new liquid medium for further cultivation This procedure was repeated three times in total, and the final culture broth was diluted serially and then spread on 1.2% agar medium corresponding to each enrichment culture broth After incubation at 27°C for week under anaerobic condition using an AnaeroPack system (Mitsubishi Gas Chemical, Tokyo, Japan), individual colonies were isolated and purified These isolates were stored at -80°C in MRS broth containing 20% glycerol (w/v) Confirmation of GABA production of isolates All the isolates were cultured in ml 5% MSG-MRS broth (pH 7.0) at 27°C for days The GABA-producing abilities of the isolates were then confirmed using thin-layer chromatography (TLC) One microliter of supernatant from each culture broth was spotted onto a silica plate (Silicagel 60 F254; Merck, Darmstadt, Germany) TLC was conducted using a solvent mixture [1-butanol:acetic acid:distilled water (3:2:1)], and a spot of GABA was treated on a hot plate for a few minutes after being detected by ninhydrin spray (Wako, Tokyo, Japan) In addition, the GABA-producing abilities determined from the TLC results were reconfirmed using an automatic amino acid analyzer as follows: GABA-producing candidates were cultured once again in ml 5% MSG-MRS broth supplemented with 0.1 mM pyridoxal-50 -phosphate (PLP) or without PLP at 27°C for days Culture supernatants were mixed with volumes of 6% PCA and centrifuged at 18,8009g for 10 at 4°C The supernatant was neutralized by adding N KOH and filtered through 0.22-lm cellulose acetate membranes The neutralized supernatants were analyzed for GABA content using the automatic amino acid analyzer described above All experiments were performed in triplicate Phenotypical identification of GABA-producing LAB Gram stain, cell form, and cell motility were observed by light microscopy (BX 51; Olympus, Tokyo, Japan) Catalase activity was tested with 3% H2O2 The acid production from D-glucose was determined using MRS agar medium (pH 7.0) on the basis of a clear zone indicating CaCO3 dissolution, and MRS broth with a Durham’s tube was used to determine gas production from D-glucose To determine the oxygen requirement for growth, the isolates were incubated anaerobically on MRS agar medium in a GasPack system The growth behavior under different NaCl and temperature conditions was also determined using MRS broth The fermentation of various carbohydrates was determined using API CH strips with API CHL medium (Bio Merieux, Lyon, France) The LAB strains were grown in the MRS broth at 27°C for 24 h The MRS 123 282 Fish Sci (2011) 77:279–288 broth was centrifuged at 5,0009g for 10 at 4°C, and the collected cells were washed twice with distilled water The washed cells were suspended in ml distilled water and inoculated into the API CH strips The strips were incubated at 27°C for 48 h, and then the isolates were identified using apiweb version 1.2.1 (Bio Merieux, Lyon, France) after the acid formation was determined visually of 2:1 by weight Lyophilized cells (0.3 g) of each starter culture (approximately 1010 cfu/g) were inoculated, and then the mixture was fermented in a plastic jar for days at room temperature During this fermentation process, degassing was not performed The GABA content of trial fermented products was measured using the automatic amino acid analyzer described above Genotypical identification of GABA-producing LAB Statistics Deoxyribonucleic acid (DNA) was extracted by the method of Marmur [38] Oligonucleotides 50 -AGT TTG ATC CTG GCT AG-30 (primer 27F) and 50 -GTT ACC TTG TTA CGA CTT C-30 (primer 1492R) were used as primers for amplification of the 16S rRNA gene via polymerase chain reaction (PCR) using a Takara rTaq gene amplification PCR kit (Takara Bio, Shiga, Japan) [39] The PCR products were purified by polyethylene glycol (PEG) precipitation Then, the PCR products for sequencing were prepared using BigDye Terminator Cycle Sequencing Kit version 3.1 (Applied Biosystems, Foster City, CA, USA), and DNA sequencing was carried out using an Applied Biosystems 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA) The sequence results were compiled from overlapping sequence data using the GENETYX computer program (GENETYX, Tokyo, Japan) The resulting sequences were compared with known sequences using the basic local alignment search tool (BLAST, http://www.ncbi.nlm.nih.gov/BLAST/) The statistical significance of GABA contents of trial fermented fishery products was evaluated using Student’s t test, and differences between trial products were compared by one-way analysis of variance (ANOVA) using Statistic version 6.0 (Statsoft, Tulsa, OK, USA) and Tukey’s multiple-range test at P \ 0.05 Preparation of trial fermented products using three representative GABA-producing LAB All experiments were performed in quadruplicate Before preparations of the trial fermented products, all cookware was sterilized Three types of fermented fishery products with boiled rice were prepared experimentally at approximately 300 g, laboratory scale using the method traditionally used in Myanmar Trial fermentation experiments were performed twice In the first trial, tinfoil barb (ngachin, N-T1), speckle prawn (pazun-chin, PC-T1), and rohu (ngagyin-chin, NGC-T1) were fermented with boiled rice using GABA-producing isolates, namely strains D323, F311, and K35 as starter cultures, respectively In the second trial, tinfoil barb (ngachin, N-T2), speckle prawn (pazun-chin, PC-T2), and rohu (ngagyin-chin, NGC-T2) were also fermented with boiled rice using strain D323 The process is described as follows: Fresh fish or shrimp were purchased from the fish market in Yangon City They were washed, eviscerated, cut into small pieces, and then salted at 10:1 weight ratio of fishery products to salt For the preparation of boiled rice, long-grain rice was cooked with a rice cooker After salting for h, boiled rice was added at the ratio of salted fishery products to boiled rice 123 Results Chemical properties of the fermented fishery products Table presents the basic chemical properties of the four types of fermented fishery products used for the screening of GABA-producing LAB The pH values of these fermented fishery products were kept under acidic conditions of 3.8–4.8, and the NaCl concentrations were between 3.8% and 8.6% The lowest pH was found in fermented featherback fish (NPC-1) at 3.8, and the highest pH was 4.8 in fermented tinfoil barb (N) On the other hand, the highest NaCl concentration was 8.6% for fermented rohu (NGC-2), whereas the lowest was 3.8% for fermented tinfoil barb (N) The amount of GABA in fermented tinfoil barb was 193.4 mM, whereas in fermented speckled prawn, fermented rohu, and fermented featherback fish, it ranged from 0.5 to 26.2 mM, 1.5 to 10.0 mM, and ND to 0.3 mM, respectively Screening of GABA-producing LAB The number of GABA-producing LAB isolated from the four types of fermented fishery products with boiled rice are indicated in Table A total of 130 bacteria were obtained from the enrichment culture broth, and among them, 12 isolates showed GABA production from the TLC results (Fig 1); all of them were confirmed as GABA producers using the automatic amino acid analyzer A GABA producer (strain D323) was isolated from the fermented tinfoil barb sample (N), and four producers (F311, F332, F341, and F342) were also isolated from a sample of fermented speckled prawn (PC-1) Moreover, four strains (K35, K39, K42 and K47) and three strains (P41, P43 and P44) were also isolated from the two Fish Sci (2011) 77:279–288 283 GABA MSG 10 11 12 13 14 Fig GABA production of isolated LAB determined by TLC Analyses were performed after incubation for days in MRS medium Lane 1% GABA solution, lane 5% MSG in MRS broth, lane strain K35, lane strain K39, lane strain K42, lane strain K47, lane strain P41, lane strain P43, lane strain P44, lane 10 strain D323, lane 11 strain F311, lane 12 strain F332, lane 13 strain F341, lane 14 strain F342 samples of fermented rohu (NGC-3 and NGC-4) and a sample of fermented featherback fish (NPC-4), respectively However, we could not isolate any GABA producers from the remaining fermented products 16S rRNA gene sequencing analysis of GABA-producing LAB GABA productivity of isolates The GABA production of the twelve isolates after incubation for days is shown in Fig The amounts of GABA produced in the MRS broth reached 97.5– 798.0 mM, and the highest amount was produced by strain F311 All the isolates were divided into two groups on the basis of their GABA productivity Eleven strains showed high GABA productivity, which ranged from 472.7 to 798.0 mM, whereas strain D323 produced a small amount of GABA (97.5 mM) The effect of addition of PLP to the culture medium on GABA productivity was also examined, because GAD has been described as a PLP-dependent enzyme [40] The GABA productivity of all the isolates seemed to be enhanced by the addition of PLP, and the level of increase was different depending on the type of isolate In particular, the GABA production of strain D323 increased 5.1-fold and reached 493.6 mM in the growth medium containing PLP General phenotypic characterization of GABA-producing LAB The twelve GABA-producing bacteria were Gram-positive, catalase-negative, and homofermentative rod-shaped bacteria They can grow in 0%, 3%, 5%, 7%, and 10% NaCl media In addition, eleven isolates could grow at 15°C and 45°C, whereas only one isolate, strain D323, could not grow above 40°C These results indicate that all the GABA producers should be classified into one genus, Lactobacillus, on the basis of the identification protocols used for lactic acid bacteria [41] Among the 12 isolated GABA producers, three strains were used for processing trial fermentation products on the laboratory scale Therefore, an additional taxonomical study of these representatives was performed with detailed fermentation potential tests using 16S rRNA gene sequencing analysis and API rapid CH fermentation strips First, we performed 16S rRNA gene sequencing analysis to clarify the taxonomic positions of strains F311, K35, and D323 from the phylogenetic viewpoint The 16S rRNA gene of strain F311 showed 99.9% sequence similarity to that of strain K35 with a single base difference in 1,488 positions From comparison with the sequences of the type strains of Lactobacillus species in the public DDBJ database, strains F311 and K35 were shown to be closely related to both Lb plantarum JCM 1149T (D79210) and Lb pentosus JCM 1558T (D79211) The comparison of the 16S rRNA gene sequence revealed no difference between strain F311 and Lb plantarum JCM 1149T (D79210), and only a single difference (99.9% similarity) between strain F311 and Lb pentosus JCM 1558T (D79211) in 1,488 positions In addition, no nucleotide difference was observed between strain K35 and Lb pentosus JCM 1558T (D79211), and only a single nucleotide difference (99.9% similarity) was found between strain K35 and Lb plantarum JCM 1149T (D79210) On the other hand, the 16S rRNA gene sequence of strain D323 showed 100% similarity to Lb farciminis DSM 21850 (AJ417499) in 1,505 positions There are twenty unknown nucleotides in the sequence of Lb farciminis ATCC 29644T (M58817) Therefore, realignments were carried out using the revised sequence excluding these ambiguous nucleotides, and a significantly high similarity between strain D323 and Lb farciminis ATCC 29644T (M58817) was observed with only one nucleotide difference in 1,495 positions 123 284 1200 GABA content (mM) Fig GABA productivity of LAB isolated from Myanmar fermented fishery products; GABA amounts were determined using an amino acid analyzer after incubation for days in MRS medium with 0.1 mM PLP (white) or without PLP (black) Data expressed as mean ± SD from three independent experiments Fish Sci (2011) 77:279–288 1000 800 600 400 200 K35 K39 K42 K47 P41 P43 P44 D323 F311 F332 F341 F342 GABA-producing bacteria Carbohydrate fermentation profiles of GABA-producing LAB Table Carbohydrate fermentation profiles of three representative GABA producers isolated from Myanmar fermented fishery products Substrate The carbohydrate fermentation profiles of three representative strains are shown in Table The results of the computer-aided identification based on the fermentation profiles using apiweb version 1.2.1 indicate that strains F311 and K35 have 99.7% and 98.8% identities to Lb plantarum and Lb pentosus, respectively The profiles of strains F311 and K35 appeared to be similar, but the profile of strain D323 was different from those of strains F311 and K35 All three strains fermented D-glucose, D-fructose, D-mannose, N-acetylglucosamine, esculin, salicin, D-maltose, and D-trehalose In addition to these carbohydrates, both strains F311 and K35 fermented L-arabinose, D-ribose, D-galactose, D-mannitol, arbutin, D-cellobiose, D-lactose, D-melibiose, and gentiobiose Methyl-a-D-mannopyranoside, D-raffinose, D-turanose, and D-arabitol were fermented only by strain F311, and glycerol, sorbitol, methyl-a-D-glucopyranoside, and gluconate were fermented only by strain K35 On the other hand, strain D323 could not be identified as any specific Lactobacillus species owing to its ambiguous identification result, because Lb farciminis was not found on the identification list in the instruction manual of API CH GABA accumulation in trial fermented products inoculated with GABA-producing LAB Three isolates (strains D323, F311, and K35) were used as starter cultures to elucidate the accumulation of GABA while processing trial fermentation products These individuals were selected from each of three fermented products containing higher amounts of GABA (N, PC-1, and NGC-3) In the first experiment, as indicated in Fig 3, three representative strains were inoculated into trial fermented products corresponding to the isolation source of each strain When strain D323 was inoculated into tinfoil barb (N-T1) as a starter culture, the highest GABA amount 123 Isolates D323 F311 K35 Glycerol - - ? L(?)-Arabinose - ? ? D-Ribose D-Galactose - ? ? ? ? D-Glucose ? ? ? D-Fructose ? ? ? D-Mannose ? ? ? L-Rhamnose - - ND D-Mannitol - ? ? D-Sorbitol - - ? Methyl-a-D-mannopyranoside - ? - Methyl-a-D-glucopyranoside - - ? N-Acetylglucosamine ? ? ? Amygdalin - ? ND Arbutin - ? ? Esculin ? ? ? Salicin ? ? ? D-Cellobiose D-Maltose ? ? ? ? ? D-Lactose - ? ? D-Melibiose - ? ? D-Sucrose - - ND D-Trehalose ? ? ? D-Raffinose - ? - Gentiobiose - ? ? D-Turanose - ? - D-Arabitol - ? - Gluconate - - ? Erythritol, D-arabinose, D-xylose, L-xylose, D-adonitol, methyl-b-Dxylopyranoside, L-sorbose, dulcitol, inositol, inulin, D-melezitose, starch, glycogen, xylitol, D-tagatose, D-fucose, L-fucose, L-arabitol, 2-keto-gluconate, and 5-keto-gluconate were not fermented by three isolates ?, positive; -, negative; ND not determined 285 300 c 200 150 b 100 ab ab 50 ab a Tinfoil barb Speckle prawn (Ngachin, N-T1) (Pazun-chin, PC-T1) (Ngagyin-chin, NGC-T1) Rohu Fig Accumulation of GABA in three types of trial fishery products fermented with different LAB as starter cultures N-T1, PC-T1, and NC-T1 were inoculated with strains D323, F331, and K32, respectively Means with different letters within the same column are significantly different (P \ 0.05) GABA amounts in samples inoculated with each strain are indicated by black bars, and GABA amounts in uninoculated samples are indicated by white bars Data expressed as mean ± SD from four independent experiments was detected in the final fermentation product, reaching 169.7 mg/100 g On the other hand, a smaller amount of GABA accumulation was observed in fermented speckled prawn (PC-T1) inoculated with strain F311 and in fermented rohu (NGC-T1) inoculated with strain K35 Comparison of the GABA amounts in each of the reference products that were not inoculated with starter cultures revealed that there were markedly significant differences in the GABA amounts between tinfoil barb (N-T1) fermented with and without strain D323 These results suggested that strain D323 is the most effective as a starter culture in fermented fishery products with boiled rice Therefore, a second trial processing the fermented products was performed to confirm the GABA accumulation effect of strain D323 (Fig 4) When three types of fermented products inoculated with strain D323 [tinfoil barb (N-T2), speckled prawn (PC-T2), and rohu (NGC-T2)] were compared, the highest GABA amount was obtained in fermented tinfoil barb (N-T2), reaching 231.9 mg/100 g, where significant increases were also found in the inoculated and noninoculated products In the case of the other two fermentations inoculated with strain D323, a higher GABA amount was found in fermented speckled prawn (PC-T2), whereas no effect on GABA accumulation was observed in comparison with the reference product Although the GABA amount was low, there was a significant difference between the inoculated and noninoculated products of fermented rohu (NGC-T2) Discussion Traditional fermented fish products are classified into two groups, namely the more widely known fish/salt formulations, such as fish paste and sauce, and fish/salt/ GABA amount (mg/100g) GABA amount (mg/100g) Fish Sci (2011) 77:279–288 c 250 200 b 150 b b 100 ab a 50 Tinfoil barb Speckle prawn ( Ngachin, N-T2) (Pazun-chin, PC-T2) Rohu (Ngagyin-chin, NGC-T2) Fig Accumulation of GABA in three types of trial fishery products fermented with strain D323 as a starter culture Means with different letters within the same column are significantly different (P \ 0.05) GABA amounts in samples inoculated with strain D323 are indicated by black bars, and GABA amounts in uninoculated samples are indicated by white bars Data expressed as mean ± SD from four independent experiments carbohydrate blends [42] Among them, fermented fishery products with boiled rice, which are called by different names locally, are manufactured and consumed widely in East and Southeast Asia Except for the Myanmar fermented products used in this study, they are known to include, for example, pla-ra, plaa-som, pla-chom, and kung-chom in Thailand, burong-isda in the Philippines, phaak in Cambodia, and funa-zushi in Japan All the Myanmar fermented fishery products used in this study had acidic pH ranging from 3.8 to 4.8 owing to the LAB On the other hand, the highest NaCl concentration was 8.6%, whereas the lowest was 3.8% The pH and NaCl concentration of other fishery products fermented with boiled rice in neighboring countries have previously been shown The fermented fish product pla-ra was found to have high NaCl concentration around 11.5–23.7% In contrast, the fermented small fish product pla-chom and the small shrimp product kung-chom had NaCl concentrations similar to those of our products, ranging from 3.2% to 9.4% (w/w) [43] Pla-ra, pla-chom, and kung-chom were also found to be acidic, with pH ranging from 4.3 to 5.6, 4.2 to 6.2, and 3.9 to 4.7, respectively Despite the geographic distance, the properties of these products seemed to be similar to those of the Japanese funa-zushi Kubo et al [44] analyzed four types of Japanese funa-zushi and reported that these products contain 2.1–5.8% (w/w) NaCl with pH from 3.7 to 4.0 In this study, 12 GABA-producing LAB were isolated from four types of Myanmar fermented fishery products with boiled rice in accordance with the results of both TLC and amino acid analysis These producers accounted for approximately 10% of the total number of isolates, and they were isolated from five fermented samples These results indicate that GABA-producing LAB are widely distributed in Myanmar fermented fishery products with 123 286 boiled rice In a previous study, GABA-producing Lb paracasei was isolated from Japanese crucian carp fermented with boiled rice [21] Moreover, Lb brevis was also isolated as a GABA producer from plaa-som, a Thai fermented fish product with boiled rice [28] Although this finding in plaa-som encouraged us to undertake our investigation, their detailed results concerning Lb brevis have not been clarified at present Therefore, our report is not only the first to show that GABA-producing LAB are distributed in Myanmar fermented fishery products, but also the first detailed one concerning GABA-producing LAB in Southeast Asia fermented fishery products with boiled rice Prior to the application of GABA-producing LAB for GABA accumulation in trial fermented fishery products, we carried out several experiments to clarify GABA productivity and the taxonomical characteristics of our isolates Addition of PLP to the culture media was also examined in relation to GABA productivity, because PLP has been described as a necessary coenzyme of GAD [40] Therefore, we expected that addition of PLP to the culture medium would affect the GABA production of our isolates In previous study, Komatsuzaki et al [21] described that even 0.01 mM PLP effectively enhanced the GABA production of Lb paracasei isolated from funa-zushi Therefore, although our isolates were identified as a different species, in our experiment for the enhancement of GABA production, 0.1 mM PLP was supplemented to the culture media, which was expected to be an adequate amount GABA producers in this study were divided into two groups on the basis of their GABA production In the case of strain D323, addition of PLP contributed markedly to the enhancement of GABA productivity In contrast, the effects of PLP addition on GABA production in the other eleven isolates seemed to be smaller than that in the case of strain D323 PLP addition was found to effectively promote GABA production in the culture medium during incubation with Lb paracasei [21] However, PLP has not always been shown to effectively increase GABA production in all LAB described previously as GABA producers Jun et al [27] reported that the activity of the purified GAD of Lb brevis is slightly increased by addition of PLP, and they also mentioned a possible reason, namely that the strong integration of PLP with GAD of this strain results in a holoenzyme, causing minimal enhancement of enzymatic activity Denaturation of PLP in the culture medium during cultivation was also considered as a reason for the minimal enhancement of GABA production of St salivarius subsp thermophilus Y2 [34] From the results of the phenotypic characterization, all the GABA producers isolated in this study belonged to the genus Lactobacillus In addition, 16S rRNA gene sequencing results and carbohydrate fermentation ability 123 Fish Sci (2011) 77:279–288 determined using API CH indicate that three representative strains (F311, K35, and D323) having different GABA productivities can be identified as Lb plantarum, Lb pentosus, and Lb farciminis, respectively These Lactobacillus species, including Lb plantarum, Lb pentosus, and Lb farciminis, were previously isolated from various types of similar fermented products in Thailand [43] In this study, trial fermented fishery products were processed using three representative strains (F311, K35, and D323), which were isolated from different types of fermented fishery products Therefore, in the first experiment, we used them as starter cultures for the trial fermentation corresponding to the origin of each isolate, and the highest GABA accumulation was observed in the fermented tinfoil barb product (N-T1) inoculated with strain D323 In addition, in the second experiment, in which strain D323 was used as the starter culture, the highest amount of GABA accumulated in the fermented tinfoil barb product (N-T2) as well as in the former product (N-T1) These results indicate that strain D323 is a useful starter culture in terms of GABA accumulation, and can be used as a starter culture for manufacturing of GABAenriched tinfoil barb fermented in boiled rice At present, the reason why strain D323 is so useful for GABA accumulation in tinfoil barb fermentation is unknown, because suitable and optimal culture conditions for GABA production in vitro are not currently understood, although the behaviors of starter cultures are known to be affected by various complex factors in the fermentation environment Therefore, detailed investigation of strain D323, in terms of the effect of culture conditions on GABA productivity under various broth culture conditions, and the biochemical and enzymatic characterization of GAD, as well as gene cloning, are underway Moreover, detailed study on other GABA producers, such as strains F311 and K35 in comparison with strain D323, should be performed We are currently unaware of any reports on GABA amount in Myanmar fermented fishery products Therefore, we also investigated GABA amount in several samples of four types of fermented fishery products with boiled rice purchased in Myanmar As shown in Table 1, among them, the highest GABA amount was detected in fermented tinfoil barb, compared with those in fermented speckled prawn, fermented rohu, and fermented featherback fish These results indicate that the isolation source of strain D323, which was shown to be a useful starter culture in this study, corresponds to the fermented fishery product containing the highest GABA amount Although tinfoil barb fermentation itself might provide a suitable environment for GABA accumulation because of some factors, these results suggested that GABA producers belonging to Lb farciminis, such as strain D323, contribute to GABA accumulation during tinfoil barb fermentation owing to the Fish Sci (2011) 77:279–288 wide distribution of Lb farciminis, just like in Thai fermented fishery products described previously [43] In addition to that, GABA producers were isolated in the fermented product containing no GABA (NPC-4) However, in most cases GABA producers were not isolated in the fermented products containing small amounts or no GABA In conclusion, this study is the first to show in detail the distribution of GABA-producing LAB in Southeast Asian fermented fishery products In addition, to our knowledge, this is the first time that GABA-producing LAB has been applied as a starter culture for the fermentation of fishery products The accumulation of GABA in the Myanmar fermented tinfoil barb product in boiled rice (ngachin) using strain D323 as a starter culture appears to be of considerable interest for the manufacturing of traditional fishery products These findings may contribute to enhancing the health benefits and increasing the commercial value of traditional fermented fishery products 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Fonseca [33 ] Campos et al [34 ] Mediterranean horse mackerel Trachurus mediterraneus Selection parameters in total length (cm) l50inTL SRinTL SF DM 64 14.4 3. 3 2 .3 DM 69 14.7 2.9 2.1 DM 79 16.0 3. 7 2.0 SM 63 21.9 8 .3 3.5 DM 55 18.0 3. 8 3. 3 DM 60 19.8 3. 6 3. 3 DM 71 SM 55 21.9 21.7 4.9 5.0 3. 1 3. 9 Campos et al [35 ] Tosunog˘lu et al [36 ] SM 63 27 .3 3.4 4 .3 DM 50 15.6 5.5 3. 15 Aydın and Tosunog˘lu [37 ] DM... 20/PBS, No 9 03 3, Takara Bio, Shiga, Japan) at 37 °C for 15 min, fixed with 4% paraformaldehyde (162-1606 5, Wako, Osaka, Japan) in PBS Then the sections were preincubated at 42°C for 30 min in a prehybridization buffer [50% formamide, 29 SSC (0 .3 M NaCl, 0 .33 M trisodium citrate dehydrate, pH 7.0)] and incubated at the same temperature for 20 h in a hybridization buffer [ISHR 7, NipponGene, Tokyo, Japan... longnose gar Lepisosteus osseus [33 ], Amia calva [34 ], whitefish Coregonus clupeaformis [35 ], and stickleback Gasterosteus aculeatus [36 ], but not in any clupeids However, the development of a blastocoel has been reported in the Japanese eel Anguilla japonica [37 ], rainbow trout Onchorhynchus masou [38 ], and Cynolebias viarius [39 ], but it is not obvious in American shad [ 23 ], and is absent in zebrafish... DM 44 14.7 4.6 3. 29 SM 42 15.9 5.6 3. 75 DM 40 13. 70 2.10 3. 43 SM 40 15.20 3. 00 3. 80 DM 39 9.71 2.75 2.51 SM 40 13. 12 2. 43 3.40 DM 54 12.95 3. 57 2.40 Ordines et al [38 ] Sala et al [39 ] Jack mackerel Trachurus japonicus Mesh shape and mesh opening (mm) Present study DM diamond mesh, SM square mesh, l50inTL total length corresponding to 50% retention, SR selection range in total length, SF selection factor... Logistic parameters a b Split parameters 0.654 (0.469) 2 .35 (0.628) p1 0. 534 (0.0902) 0.848 (0. 033 8) p2 0.147 (0. 033 1) 0.8 13 (0.0291) p3 0.0 93 (0. 030 5) 0.762 (0.0296) Selection parameters l50 S.R 11 .39 (1.05) 8. 83 (0. 236 ) 3. 36 (2.410) 0. 93 (0.250) Values in parentheses are standard errors of the codends passed into either codend without any struggle to swim, while a few animals attempted to swim in a random... Griffin FJ, Smith EH, Vines CA, Cherr GN (2009) Impacts of suspended sediments on fertilization, embryonic development, and early larval life stages of the Pacific herring, Clupea pallasi Biol Bull 216:175–187 16 Uchida K, Imai S, Mito S, Fujita S, Ueno M, Shojima Y, Senta T, Tahuku M, Dotu Y (1958) Studies on the egg, larvae and juvenile of Japanese fishes Series I (in Japanese) Second Laboratory of Fisheries. .. 75:17 38 29 Mito S (1979) Fish egg (in Japanese) Mar Sci Mon 11:126– 130 30 McGowan MF, Berry FH (1984) Clupeiformes: development and relationships In: Moser HG, Richards WJ, Cohen DM, Fahay MP, Kendall AW, Richardson SL (eds) Ontogeny and systematics of fishes Allen, Lawrence, pp 108–126 31 Jones PW, Martin FD, Hardy JD (1978) Development of fishes in the Mid-Atlantic Bight: an atlas of egg, larval, and... Segmentation: 20-somite 16–18 Segmentation: 35 -somite 19 20 21 Pharyngula 25 25 l 26 27 m 16 Organogenesis: hatch 28 Hatching period 1 23 188 pectoral fin, and hatching occurred at 3 0, 59 0, 83 0, 95 1, 95 1, 1 ,3 11 and 2,6 56 degree-hours, respectively Newly hatched larvae All of the newly hatched larvae observed had straightened bodies and were regarded as exhibiting normal development, except for one that had a contorted... stage, 5 h 15 min after fertilization b Blastula, 17 h 55 min c 50% epiboly, 42 h 30 min d 70% epiboly, 48 h 28 min e 90% epiboly, 54 h 20 min f Appearance of optic vesicle, 10 myomeres present, 66 h 22 min g Appearance of lens and otic vesicle, 30 myomeres present, 90 h 23 min h Beginning of body movements and heartbeat, 102 h 32 min i Appearance of melanophores on the eye and pectoral fin rudiments,... Wiley-Blackwell, Ames, pp 25– 43 30 Tokai T, Omoto T, Matsuda K (1994) Mesh selectivity of unmarketable trash fish by a small trawl fishery in the Seto Inland Sea Nippon Suisan Gakkaishi 60 :34 7 35 2 (in Japanese with English abstract) 31 Matsushita Y, Ali R (1997) Investigation of trawl landings for the purpose of reducing the capture of non-target species and sizes of fish Fish Res 29: 133 –1 43 32 Liang Z, Horikawa