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Effects of high pressure processing (HPP) under 300, 400, 500 and 600 MPa for 5 min on microbiological growth and histamine content of spotted mackerel meats stored at 4 oC were evaluated. It was also found that the L* (lightness), ΔE (color difference), and texture (hardness, cohesiveness and chewiness) of fish meat increased significantly with an increase in pressure, but a* (redness) value decreased. With an increase of pressure, the loads of aerobic plate count (APC), psychrotrophic bacteria count (PBC), H2S-producing bacteria count and coliform in mackerel meat significantly decreased. In addition, HPP significantly delayed the growth of APC and PBC during refrigerated storage for 15 days. Pressure up to 300 MPa significantly inhibited total volatile basic nitrogen (TVBN) and histamine formation, compared to control sample during storage. The results pointed to that the pressurization at least 300 MPa for 5 min on mackerel meat could extend shelf-life during refrigerated storage.

Squid pen sample was treated by high hydrostatic pressure (HHP) prior to the extraction of chitosan. The physicochemical and antioxidant activities of the chitosan obtained with HHP (HHP-CS) were compared with chitosan of untreated squid pen sample (UT-CS). The chitosan extraction yield was optimized using response surface methodology, and the optimum condition was achieved at pressure of 500 MPa, extraction time of 10 min, and 1% (w/w) acetate concentration. The maximum yield of chitosan sample from the chitin of squid pens treated by HHP reached 81.9%. Among the process variables, the combined effects of pressure and acetate concentration significantly enhanced the extraction of chitosan from squid pens. The HHP-CS was found to be significantly effective in enhancing the fat binding capacity, water binding capacity, and water solubility index. SEM image analysis suggested that the HHP-CS had a rough surface with high porosity, while UT-CS exhibited a smooth surface. In vitro antioxidant assay suggested that HHP-CS had significantly higher DPPH radical scavenging activity, greater reducing power, and a stronger ferrous ion chelating effect than did UT-CS. Therefore, HHP can be an excellent alternative method for improving the physicochemical properties and antioxidant activities of chitosan from squid pens.

The effect of polyethylene packaging (PEP) in air cushion and vacuum packaging (VP) on histamine related to the quality of Japanese Spanish mackerel (JS mackerel) was studied with samples stored at -20, 4, 15, and 25°C. The aerobic plate count (APC), total volatile basic nitrogen (TVBN), and histamine concentrations of the PEP and VP samples stored at 25°C increased as the storage time continued. The PEP and VP samples stored at temperatures below 15°C showed lower levels of APC, TVBN, and histamine, with VP samples having considerably lower levels of APC, TVBN, and histamine than PEP samples. For the frozen JS mackerel stored at -20°C for 2 months and then thawed and stored at 25°C, the VP treatment delayed the increases of TVBN and histamine longer than did the PEP treatment. Thus, the storage of VP JS mackerel at temperatures below 4°C could prevent quality deterioration and extend shelf life.

An incident of foodborne poisoning causing illness in seven victims due to ingestion of fried Japanese Spanish mackerel (JS mackerel; Scomberomorus niphonius) meat occurred in September 2014 in Hualien County, eastern Taiwan. Of the two suspected fish meats, one raw sample contained 3,318 ppm of histamine and one fried sample contained 1,906 ppm of histamine, levels which are greater than the potential hazard action level (500 ppm) in most illness cases. Given the allergy-like symptoms of the victims and the high histamine content in the suspected fish samples, this foodborne poisoning was strongly suspected to be caused by histamine intoxication. In addition, five histamine-producing bacterial strains isolated from suspected raw fish samples, capable of producing 152 to 1,020 ppm of histamine in Trypticase soy broth supplemented with 1.0% l-histidine, were identified as Hafnia alvei (one strain), Enterobacter aerogenes (two strains), Raoultella ornithinolytica (one strain), and Morganella morganii (one strain) by 16S rDNA sequencing with PCR amplification. Moreover, 12 raw fish samples and 39 fried fish samples from retail stores were collected and tested to determine the occurrence of histamine. Two of 12 commercial raw fish samples (16.7%) had histamine levels greater than the U.S. Food and Drug Administration guideline for decomposition of 50 ppm for scombroid fish or product or a combination of both. To our knowledge, this is the first report in Taiwan to demonstrate that the JS mackerel meat products could cause histamine intoxication.

Intensive efforts have been undertaken in the fields of prevention, diagnosis, and therapy of lung cancer. Fucoidans exhibit a wide range of biological activities, which are dependent on the degree of sulfation, sulfation pattern, glycosidic branches, and molecular weight of fucoidan. The determination of oversulfation of fucoidan and its effect on anti-lung cancer activity and related signaling cascades is challenging. In this investigation, we used a previously developed fucoidan (SCA), which served as a native fucoidan, to generate two oversulfated fucoidan derivatives (SCA-S1 and SCA-S2). SCA, SCA-S1, and SCA-S2 showed differences in compositions and had the characteristic structural features of fucoidan by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) analyses. The anticancer properties of SCA, SCA-S1, and SCA-S2 against human lung carcinoma A-549 cells were analyzed in terms of cytotoxicity, cell cycle, Bcl-2 expression, mitochondrial membrane potential (MMP), expression of caspase-3, cytochrome c release, Annexin V/propidium iodide (PI) staining, DNA fragmentation, and the underlying signaling cascades. Our findings indicate that the oversulfation of fucoidan promotes apoptosis of lung cancer cells and the mechanism may involve the Akt/mTOR/S6 pathway. Further in vivo research is needed to establish the precise mechanism whereby oversulfated fucoidan mitigates the progression of lung cancer.

Attempts were made to elucidate the responsible toxins in the gastropod Polinices didyma which recently caused food poisoning incidents in western Taiwan. The remained (cooked) and captured (live) specimens of both gastropods were assayed for toxicity (as tetrodotoxin = TTX). Average toxicity of cooked and live specimens was 118 ± 105 and 47 ± 28 MU/specimen, respectively. In addition, another species Natica lineata collected from the same area was also found to be toxic. The toxin of each gastropod was partially purified from the methanolic extract of the gastropod by ultrafiltration and Bio-Gel P-2 column chromatography. HPLC and GC-MS analyses demonstrated that the toxin consisted of TTX. It was concluded that the causative agent of the above food poisoning was TTX.

It is important to develop rapid methods to detect the presence of histamine producers. In this study, the polymerase chain reaction (PCR) assay yielded a 367-bp DNA amplification fragment from histidine decarboxylase (hdc) of gram-positive bacteria using JV16HC/JV17HC primer, and 534-bp and 709-bp DNA amplification fragments from histidine decarboxylase of gram-negative bacteria using 106/107and hde-f/hde-r primers, respectively. Seven gram-positive histamine-producing strains and 15 gram-negative histamine-producing strains isolated from Taiwanese foods successfully produced the PCR amplification products. The minimum levels of detection of Enterobacter aerogenes or Raoultella ornithinolytica after the PCR amplification using hde-f/hde-r and 106/107 primerswere 10^5 and 10^6 CFU/mL in TSB broth, and 10^6 and 10^7 CFU/g in marlin homogenares, respectively. The hdc amplification using hdc-I/hdc-r primer was detected 2 h earlier than the HPLC detection of histamine in TSBH broth or marlin homogenates inoculated E. aerogenes or R. ornithinolyt icu. However, the detection of hdc amplification using the 106/107 primer and that of histamine by HPLC occurred at the same sampling time. Therefore, the PCR method could be easily used to detect potential histamine-producing bacteria in foods.

A high performance liquid chromatography (HPLC) was developed for determining tiamulin residue in chicken and pork. Samples were extracted with acetonitrile, purified by liquid partition separation, and extracted with n-hexane at last. The n-hexane extract was concentrated and eluted through a Bond Elut C18 cartridge for HPLC analysis. The HPLC system was performed on a Lichrospher 100 RP-18 column (5 μm, 4.6 mm I.D. × 250 mm) using a mixture of 80% acetonitrile and 1% ammonia carbonate (90:10, v/v) as mobile phase, and detecting wavelength was set at 210 nm with an UV-Vis detector. The calibration curve (R2 = 0.9995) of tiamulin was highly linear at concentrations of 0.5~8.0 ppm, while the detection limit was 0.025 ppm. Recoveries of tiamulin spiked in chicken and pork samples ranged from 84.3~97.0% and 87.9~105.9%, respectively. Each 10 chicken and pork samples sold in retail markets were tested to detect tiamulin, while none of these samples contained tiamulin.

Fucoidans possess multiple biological functions including anti-cancer activity. Moreover, low-molecular-weight fucoidans are reported to possess more bioactivities than native fucoidans. In the present study, a native fucoidan (SC) was extracted from Sargassum crassifolium pretreated by single-screw extrusion, and three degraded fucoidans, namely, SCA (degradation of SC by ascorbic acid), SCH (degradation of SC by hydrogen peroxide), and SCAH (degradation of SC by ascorbic acid + hydrogen peroxide), were produced. The extrusion pretreatment can increase the extraction yield of fucoidan by approximately 4.2-fold as compared to the non-extruded sample. Among SC, SCA, SCH, and SCAH, the chemical compositions varied but structural features were similar. SC, SCA, SCH, and SCAH showed apoptotic effects on human lung carcinoma A-549 cells, as illustrated by loss of mitochondrial membrane potential (MMP), decreased B-cell leukemia-2 (Bcl-2) expression, increased cytochrome c release, increased active caspase-9 and -3, and increased late apoptosis of A-549 cells. In general, SCA was found to exhibit high cytotoxicity to A-549 cells and a strong ability to suppress Bcl-2 expression. SCA also showed high efficacy to induce cytochrome c release, activate caspase-9 and -3, and promote late apoptosis of A-549 cells. Therefore, our data suggest that SCA could have an adjuvant therapeutic potential in the treatment of lung cancer. Additionally, we explored that the Akt/mammalian target of rapamycin (mTOR) signaling pathway is involved in SC-, SCA-, SCH-, and SCAH-induced apoptosis of A-549 cells.

The inactivation and damage of histamine-forming bacteria (HFB), Enterobacter aerogenes and Staphylococcus capitis, in a 0.1 M potassium phosphate buffer (pH 6.8) and marlin meat slurry by high hydrostatic pressure (HHP) treatments were studied using viability measurement and scanning electron microscopy (SEM). HHP treatments showed first order destruction kinetics to E. aerogenes and S. capitis during the pressure holding period. HFB in marlin meat slurry had higher D values and were more resistant to HHP treatments than in phosphate buffer. In phosphate buffer, E. aerogenes had higher D values than S. capitis at &gt, 380 MPa of pressure, whereas the reverse trend was noticed at lower pressures (&lt, 380 MPa). In marlin meat slurry, S. capitis had a higher D value than E. aerogenes at the same treatment pressure, indicating that S. capitis was more resistant to HHP treatment. To our knowledge, this is the first report to demonstrate that HHP can be used to inactivate HFB, E. aerogenes, and S. capitis, by causing disruption to bacterial cell membrane and cell wall as demonstrated by SEM micrographs.

The angiotensin I-converting enzyme (ACE) inhibitory activity and hypocholesterolemic effect of Achatina fulica snail foot muscle protein hydrolysates (SFMPH) and its hydrolysates were studied. The SFMPHs were prepared at a temperature of 121°C for 60 min. To obtain the enzymatic hydrolysates, the SFMPHs were further hydrolysed with three proteases (papain, trypsin, or alcalase). Among all the hydrolysates, alcalase hydrolysate showed the highest degree of hydrolysis and was dominated by a small molecular size fraction (189–686 Da). The SFMPH treated by alcalase was effective in disintegrating intact cholesterol micelles. Furthermore, alcalase hydrolysate with a hydrolysis time of 60 min showed a strong ACE inhibitory activity in vitro with an IC50 of 0.024 mg/mL. Therefore, alcalase hydrolysate may be a promising ingredient for the use in functional foods.

In April 2017, an outbreak of histamine fish poisoning causing illness in nine victims associated with consumption of milkfish surimi products (fish ball) occurred in Kaohsiung City, southern Taiwan. Of the two suspected frozen milkfish surimi samples, one sample contained 91.06 mg/100 g of histamine, levels that are greater than the potential hazard action level (50 mg/100 g) in most illness cases. Moreover, 28 frozen milkfish surimi samples from retail stores were collected and tested to determine the occurrence of histamine. One (3.6%) of 28 commercial surimi samples had histamine levels greater than the U.S. Food and Drug Administration (FDA) guideline for decomposition of 5 mg/100 g for scombroid fish and/or products. Thirteen histamine-producing bacterial strains isolated from suspected and commercial surimi samples were identified as prolific histamine formers, able to produce 98.4 to 121.8 mg/100 mL of histamine in Trypticase soy broth supplemented with 1.0% l-histidine. In addition, milkfish surimi was inoculated with Raoultella ornithinolytica at 5.0 log CFU/g and stored at 4, 15, 25, and 37°C to investigate bacterial growth and formation of histamine. The histamine contents quickly increased to more than 50 mg/100 g in samples stored at 37 and 25°C within 12 and 24 h, respectively, as well those stored at 15°C within 96 h. To our knowledge, this is the first report in Taiwan to demonstrate that milkfish surimi products could cause histamine intoxication. HIGHLIGHTS

Bubble tea beverages (n = 105) purchased from vendors in Taiwan were tested to determine their microbiological and chemical quality. Nearly half of the tested samples (48.6%, 51 of 105) had aerobic plate counts (APCs) higher than the Taiwan Food and Drug Administration guideline of 4.0 log CFU/mL, and 55 (52.4%) had coliform counts (most probable number [MPN]) higher than the 10 MPN/mL guideline. Escherichia coli, Salmonella, Staphylococcus aureus, sweeteners, preservatives, maleic acid, and coumarin were not detected in any sample. However, catechins were not detected to 188 mg/mL, and caffeine was 10.1 to 457.6 mg/mL. Bubble tea samples obtained from vendors in southern Taiwan had a mean APC of 2.6 log CFU/mL and a mean coliform count of 61.7 MPN/mL; these values were significantly lower (P < 0.05) than those from samples collected from vendors in northern, eastern, or central Taiwan. Samples obtained from southern Taiwan had the highest mean catechin concentrations of 21.3 mg/mL (P < 0.05). About 60% (63 of 105) of the bubble tea samples were not labeled with the origin of the tea leaves, which is in violation of Taiwanese food labeling regulations. In general, the bubble tea beverages tested had satisfactory microbial and chemical qualities.

Fish gelatin and its hydrolysates exhibit a variety of biological characteristics, which include antihypertensive and antioxidant properties. In this study, fish gelatins were extracted from extrusion-pretreated tilapia scales, and then subjected to analyses to determine the physicochemical properties and antioxidant activity of the extracted gelatins. Our findings indicate that TSG2 (preconditioned with 1.26% citric acid) possessed the greatest extraction yield, as well as higher antioxidant activities compared with the other extracted gelatins. Hence, TSG2 was subjected to further hydrolyzation using different proteases and ultrafiltration conditions, which yielded four gelatin hydrolysates: TSGH1, TSGH2, TSGH3, and TSGH4. The results showed that TSGH4 (Pepsin + Pancreatin and ultrafiltration &lt, 3000 Da) had a higher yield and greater antioxidant activity in comparison with the other gelatin hydrolysates. As such, TSGH4 was subjected to further fractionation using a Superdex peptide column and two-stage reverse-phase column HPLC chromatography, yielding a subfraction TSGH4-6-2-b, which possessed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity compared with the other fractions. Further LC-ESI/MS/MS analysis of TSGH4-6-2-b suggested two novel peptides (GYDEY and EPGKSGEQGAPGEAGAP), which could have potential as naturally-occurring peptides with antioxidant properties. These promising results suggest that these antioxidant peptides could have applications in food products, nutraceuticals, and cosmetics.

The microwave-assisted induction heating (MAIH) system provides comprehensive heating by combining microwave heating (with 1300 W of power and 2450 MHz of frequency) in the top part and induction heating (with 1800 W of power) in the bottom part. In this study, fresh white shrimps were placed in a sealed crystallized polyethylene terephthalate (CPET) container and heated in the MAIH system at two temperatures (130 and 90 °C) from 60 to 120 s. Afterwards, the shrimp samples were rapidly cooled, and the changes in the shrimp quality, including the appearance, cook loss, aerobic plate count (APC), color values, and texture, during the heating process were analyzed. The results demonstrate that longer heating times decrease the APC levels, but increase the cook loss, color values (lightness, redness, and whiteness), and texture (hardness, cohesiveness and chewiness) of the white shrimp samples. In particular, the white shrimp is fully cooked and gains a completely red appearance, along with no APC detected after heating in the MAIH system at 130 °C for at least 80 s or at 90 °C for at least 100 s. In summary, to achieve a good appearance, no APC detected, and low cook loss, the following heating conditions are recommended for cooking white shrimp in the MAIH system: heating at 130 °C for 80 s or at 90 °C for 100 s. This novel MAIH technology allows food to be heated and sterilized after being packed, thereby eliminating the post-pollution issue. To the best of the authors’ knowledge, this is the first study to evaluate the use of MAIH in the application of food processing.

Ten milkfish dumpling products purchased from retail stores in southern Taiwan were collected to determine the occurrence of biogenic amines, histamine-forming bacteria, and adulteration of pork. This study showed the high contents of aerobic plate count (APC), total coliforms (TC) and Escherichia coli in tested milkfish dumpling samples, whereas the average content of various biogenic amines in all tested samples was < 1.6 mg/100 g (< 0.05 to 1.54 mg/100 g). Three histamine-producing bacterial strains (2 isolates of Raoultella ornithinolytica and 1 isolate of Enterobacter aerogenes) isolated from tested samples produced 276.6 ppm to 561.8 ppm of histamine in trypticase soy broth supplemented with 1.0% L-histidine (TSBH). Assay of multiplex polymerase chain reaction (PCR) revealed that the adulteration rates were 50% (5/10) for pork in milkfish dumplings. In addition, milkfish dumpling stuffing was inoculated with R. ornithinolytica at 5.0 log colony forming units (CFU)/g and stored at various temperatures from 4°C to 37°C to investigate bacterial growth and formation of histamine. The histamine contents quickly increased to higher than 50 mg/100 g in samples stored at 37°C and 25°C within 24 hours and 36 hours, respectively, as well as stored at 15°C within 48 hours. Therefore, bacterial growth and histamine formation were controlled by cold storage of the samples at 4°C.

Mahi-mahi meat was inoculated with Raoultella ornithinolytica at 5.0 log CFU/g and stored at −20°C, 4°C, 15°C, 25°C, or 37°C to investigate bacterial growth and formation of total volatile base nitrogen and histamine in mahi-mahi meat. R. ornithinolytica grew rapidly in samples stored at temperature above 15°C. The histamine contents quickly increased to higher than 50 mg/100 g in samples stored at 25°C and 37°C within 12 hours as well as those stored at 15°C within 48 hours. The total volatile base nitrogen contents increased to higher than the index level (30 mg/100 g) for fish decomposition at 25°C within 48 hours and 37°C within 24 hours. However, bacterial growth and histamine formation were controlled by cold storage of the samples at 4°C or below. Once the frozen mahi-mahi samples stored at −20°C for 2 months were thawed and stored at 25°C after 24 hours, histamine started to accumulate rapidly (>50 mg/100 g of fish).

An incident of food-borne poisoning causing illness in 37 victims due to ingestion of fried fish sticks occurred in September 2014, in Tainan city, southern Taiwan. Leftovers of the victims' fried fish sticks and 16 other raw fish stick samples from retail stores were collected and tested to determine the occurrence of histamine and histamine-forming bacteria. Two suspected fried fish samples contained 86.6 mg/100 g and 235.0 mg/100 g histamine; levels that are greater than the potential hazard action level (50 mg/100 g) in most illness cases. Given the allergy-like symptoms of the victims and the high histamine content in the suspected fried fish samples, this food-borne poisoning was strongly suspected to be caused by histamine intoxication. Moreover, the fish species of suspected samples was identified as milkfish ( Chanos chanos ), using polymerase chain reaction direct sequence analysis. In addition, four of the 16 commercial raw milkfish stick samples (25%) had histamine levels greater than the US Food & Drug Administration guideline of 5.0 mg/100 g for scombroid fish and/or products. Ten histamine-producing bacterial strains, capable of producing 373–1261 ppm of histamine in trypticase soy broth supplemented with 1.0% L-histidine, were identified as Enterobacter aerogenes (4 strains), Enterobacter cloacae (1 strain), Morganella morganii (2 strains), Serratia marcescens (1 strain), Hafnia alvei (1 strain), and Raoultella orithinolytica (1 strain), by 16S ribosomal DNA sequencing with polymerase chain reaction amplification.

Bacillus polymyxa D05-1, isolated from salted fish product and possessing amine degrading activity, was used as a starter culture in salted fish fermentation in this study. Fermentation was held at 35°C for 120 days. The water activity in control samples (without starter culture) and inoculated samples (inoculated with B. polymyxa D05-1) remained constant throughout fermentation, whereas the pH value rose slightly during fermentation. Salt contents in both samples were constant in the range of 17.5–17.8% during the first 60 days of fermentation and thereafter increased slowly. The inoculated samples had considerably lower levels of total volatile basic nitrogen (p

Bacillus polymyxa D05-1 isolated from salted fish product which possess histamine degrading activity was added to the salted fish products and held at 30C for 6 weeks in this study. Aerobic plate counts (APCs) in low spiked samples (4.0 log CFU/g) were remained constant during storage time, whereas, those of control samples gradually increased during first 3 weeks of storage and thereafter remained constant. However, the aerobic plate counts (APCs) of high spiked samples (6.0 log CFU/g) rapidly decreased to 4.0 log CFU/g at first week of storage, and thereafter increased slightly. The both spiked samples had considerably lower levels of total volatile basic nitrogen (P

Histamine is the causative agent of scombroid poisoning, a foodborne chemical hazard. Histamine is degraded by the oxidative deamination activity of certain microorganisms. In this study, eight histamine-degrading bacteria isolated from salted fish products were identified as Rummeliibacillus stabekisii (1 isolate), Agrobacterium tumefaciens (1 isolate), Bacillus cereus (2 isolates), Bacillus polymyxa (1 isolate), Bacillus licheniformis (1 isolate), Bacillus amyloliquefaciens (1 isolate), and Bacillus subtilis (1 isolate). Among them, B. polymyxa exhibited the highest activity in degrading histamine than the other isolates. The ranges of temperature, pH, and salt concentration for growth and histamine degradation of B. polymyxa were 25–37°C, pH 5–9, and 0.5–5% NaCl, respectively. B. polymyxa exhibited optimal growth and histamine-degrading activity at 30°C, pH 7, and 0.5% NaCl in histamine broth for 24 hours of incubation. The histamine-degrading isolate, B. polymyxa, might be used as a starter culture in inhibiting histamine accumulation during salted fish product fermentation.

Fucoidans constitute a family of fucose-rich sulfated polysaccharides, which possess multiple characteristics, including antioxidant, antitumor, antivirus, anticoagulant, and anti-inflammatory properties. In addition, the incidence of colon cancer has risen rapidly worldwide. In the present study, fucoidan extracts were extracted from the Sargassum glaucescens (SG) pretreated by compressional-puffing, and four fucoidans (SG1-SG4) were obtained with different puffing conditions. It was found that SG4 possessed the highest extraction yield, relatively high cytotoxicity against human colon carcinoma HT-29 cells, and relatively low cytotoxicity to normal cells, as compared to the other extracted fucoidans. Moreover, SG4 caused cell cycle arrest of HT-29 cells at sub-G1, S, and G2/M phases. SG4 also induced HT-29 cellular apoptosis, as evidenced by the loss of mitochondrial membrane potential (MMP), increased cytochrome c release, activation of caspase-9 and -3, increased DNA fragmentation, and increased early and late apoptotic cells visualized by annexin V/propidium iodide (PI) assay. Additional biological experiments revealed that the Akt/mammalian target of rapamycin (mTOR)/S6 pathway is involved in SG4-induced apoptosis of HT-29 cells. These results clearly indicate that SG4 showed anti-colon cancer potential via the induction of cell cycle arrest and apoptosis, and thus may have a possible application as an adjuvant therapeutic agent in colon cancer treatment.

Our previous study suggested that extrusion can enhance the soluble dietary fiber (SDF) and improve the physicochemical properties of fiber-rich orange pomace (OP). The aim of this study was to determine the predictive indices for the hypoglycemic, hypocholesterolemic, and fermentation capacities of extruded orange pomace (EOP) with several in vitro assays. The results revealed that EOP could effectively retard glucose diffusion and inhibit α-amylase activity relative to OP. Moreover, EOP had the binding capacities of cholesterol micelles and bile acids. During the in vitro fermentation of EOP, a high content of short chain fatty acid was produced. Scanning electron microscope images showed a more porous and irregular shaped structure of the EOP, which may influence physiological activities, relative to OP. The morphological alterations in the EOP were caused by mechanical shear. Thus, EOP has potential to become a functional food additive for glycemic control and the attenuation of blood cholesterol.

Traditional edible barbecue products use with lemon juice not only make the barbecue more delicious but also reduce the risk of PAHs in the barbecue products. One of the major economics crops in Taiwan, the waste from citrus fruits was very tremendous mass. However, the peelings of citrus fruits are rich in essential oil, especially, the limonene is the major. Whether the anti-carcinogenesis activities of terpene, such as limonene, in citrus fruits essential oil extraction. This study to demonstrate the PAHs content in fish skin increased markedly after being roasted at 210℃ for 20 minutes and greater mutagenicity risk of roasted fish skin was observed by Ame's test. The reduction of mutagenicity risk of roasted fish skin, which the antimutagenic abilities of substances in descending order were limonene > cold pressure oil > lemon >grapefruit. The antimutagenicity rate and ability of the three extracts were limonene: 18–23%; cold-pressed lemon oil: 18–22%; and steam distilled lemon essential oil: 8–16%. The obvious anti- mutagenicity effects against the PAHs mutagenicity of roasted fish skins can be found in citrus fruits essential oil extraction.

Thirty dried flying fish products were purchased from fishing village stores in Taiwan and tested to detect the presence of histamine and histamine-forming bacteria. Except for histamine and cadaverine, the average content of various biogenic amines in the tested samples was less than 3.5 mg/100 g. Eight (26.6%) dried flying fish samples had histamine levels greater than the United States Food and Drug Administration guideline of 5 mg/100 g for scombroid fish and/or scombroid products, whereas four (13.3%) samples contained more than the hazard action level of 50 mg/100 g. One histamine-producing bacterial isolate was identified as Staphylococcus xylosus by 16S rDNA sequencing with polymerase chain reaction amplification. This isolate was capable of producing 507.8 ppm of histamine in trypticase soy broth supplemented with 1.0% l-histidine (TSBH). The S. xylosus isolate was a halotolerant bacterium that had a consistent ability to produce more than 300 ppm of histamine at 3% sodium chloride concentration in TSBH medium after 72 hours.

Histamine is a toxic chemical and is the causative agent of food poisoning. This foodborne toxin may be degraded by the oxidative deamination activity of certain microorganisms. In this study, we isolated four histamine-degrading Lactobacillus plantarum bacteria from miso products. Among them, L. plantarum D-103 exhibited 100% degradation of histamine in de Man Rogosa Sharpe (MRS) broth containing 50 ppm of histamine after 24 h of incubation at 30°C. The optimal growth, histamine oxidase, and histamine-degrading activity of L. plantarum D-103 were observed in histamine MRS broth at pH 7.0, 3% NaCl, and 30°C. It also exhibited tolerance to broad ranges of pH (4 to 10) and salt concentrations (0 to 12%) in histamine MRS broth. Therefore, the histamine-degrading L. plantarum D-103 might be used as an additive culture to prevent histamine accumulation in miso products during fermentation.

The effects of polyethylene packaging (PEP) (in air) and vacuum packaging (VP) on the histamine related quality of milkfish sticks stored at different temperatures (-20°C, 4°C, 15°C, and 25°C) were studied. The results showed that the aerobic plate count (APC), pH, total volatile basic nitrogen (TVBN), and histamine contents increased as storage time increased when the PEP and VP samples were stored at 25°C. At below 15°C, the APC, TVBN, pH, and histamine levels in PEP and VP samples were retarded, but the VP samples had considerably lower levels of APC, TVBN, and histamine than PEP samples. Once the frozen fish samples stored at -20°C for 2 months were thawed and stored at 25°C, VP retarded the increase of histamine in milkfish sticks as compared to PEP. In summary, this result suggested the milkfish sticks packed with VP and stored below 4°C could prevent deterioration of product quality and extend shelf-life.

Forty-two mahi-mahi fillets and 17 dried products sold in retail markets in Taiwan were tested for histamine and histamine-forming bacteria. The levels of pH, salt content, water content, Aw, TVBN, APC, TC and Escherichia coli in mahi-mahi fillet samples ranged from 5.6 to 6.5, 0.05 to 2.44%, 70.9 to 82.8%, 0.95 to 0.99, 5.9 to 23.5 mg/100 g, 3.1 to 7.0 log CFU/g

Pineapple peel, a byproduct of agricultural processing, contains high levels of water-insoluble fiber-rich fraction (WIFF) (~42%, wt/wt). Our previous work has demonstrated that cellulose, hemicellulose (xylan and xyloglucan), and pectic substances are the major polysaccharides of pineapple-peel WIFF. Based on its chemical composition and unique characteristics, we hypothesized that daily consumption of WIFF would improve intestinal function in hamsters. Male Golden Syrian hamsters were fed a diet supplemented with either 5% cellulose or various amounts of WIFF (2.5%, 5%, or 10%). Activities of fecal bacterial enzymes, short-chain fatty acid concentrations, and microbial number in the cecal content, and also biochemical indicators in the cecal and feces of hamsters, were evaluated in all groups. The supplementation of WIFF in a diet at a level of 2.5% significantly (P < .05) decreased the daily fecal ammonia output; shortened the gastrointestinal transit time; reduced the activities of β-D-glucosidase, β-D-glucuronidase, mucinase, and urease in feces; and also enhanced the total amounts of short-chain fatty acid in the cecal content and the growth of gut microflora such as Lactobacillus spp and Bifidobacterium spp. These results indicate that WIFF could improve cecal ecosystem function of hamsters by reducing the toxic compounds excreted by intestinal microflora. Therefore, pineapple-peel WIFF could be a promising candidate for a functional ingredient beneficial to human intestinal function and health.

Canned tuna meat was inoculated with Raoultella ornithinolytica at a level of 2.0 log CFU/g (low) or 5.0 log CFU/g (high) and stored at 4, 15, 25, or 37 °C to investigate bacterial growth and formation of total volatile base nitrogen (TVBN) and histamine in canned tuna meat. R. ornithinolytica grew rapidly in all samples stored at temperature above 15 °C regardless of the inoculation levels. The histamine contents quickly increased to higher than 50 mg/100 g within 12 h in the low inoculated samples and stored at 37 °C as well as in those inoculated with the high level and stored at 25 °C or higher. However, growth of R. ornithinolytica and its histamine production were inhibited when samples were stored at 4 °C. The TVBN contents in all samples were all below 30 mg/100 g during storage even when the levels of histamine in some samples increased to greater than 50 mg/100 g. Therefore, canned tuna meat was a good substrate for histamine formation by bacterial histidine decarboxylation at elevated temperatures (> 15 °C) when it is contaminated with R. ornithinolytica.

Enterobacter aerogenes was studied for its growth, and promoting the formation of total volatile base nitrogen (TVBN) and histamine in tuna dumpling stuffing stored at various temperatures from −20 °C to 37 °C. The bacterial number rapidly increased in low (2.0 log CFU/g) or high (5.0 log CFU/g) inoculated concentrations at temperature above 15 °C and reached the highest bacterial count at 37 °C. In addition, the low spiked sample stored at 37 °C for 12 h and the high spiked sample stored at 25 and 37 °C for 12 h, formed histamine at above 50 mg/100 g of the potential hazard level in most illness cases. However, bacterial growth was controlled by cold storage of the samples at 4 °C or below, but histamine formation was stopped only by frozen storage. Once the frozen stuffing samples were thawed and stored at 25 °C, histamine started to accumulate rapidly.

Fifty-seven salted seafood products sold in the fishing village stores in Taiwan, including salted fish product, salted mollusc product and salted shrimp product, were tested to determine the occurrence of histamine and histamine-forming bacteria. Although the average content of each of nine different biogenic amines in all samples was less than 5.0 mg/100 g, 10.5% (6/57) of tested samples had the histamine content greater than the 5.0 mg/100 g allowable limit suggested by the US Food and Drug Administration. One histamine-producing bacterial strain, capable of producing 78.5 ppm of histamine in trypticase soy broth supplemented with 1.0% l -histidine (TSBH), was identified as Bacillus megaterium. The B. megaterium isolate was a halotolerant bacterium which grew well to an elevated NaCl concentration of 15% in TSBH medium. Besides, it had a consistent ability to produce >300 ppm of histamine at 10% NaCl concentration in TSBH medium after 72 h.

Forty fish meals and five fish soluble concentrates (FSC) imported to Taiwan were sampled to determine biogenic amines and histamine-forming bacteria. Average levels of water activity (0.8), water content (39.9%), salt content (7.32%), and total volatile basic nitrogen (TVBN) (150.7 mg/100 g) in FSC samples were significantly higher than those of fish meal samples, whereas the average levels of crude fat (3.94%) and crude protein (40.7%) in FSC samples were lower than those of fish meal samples. Thirty-five (87.5%) fish meal samples and 5 (100%) FSC samples had TVBN levels above the decomposition limit level of 30 mg/100 g in fresh fish meat. Average contents of putrescine, cadaverine, histamine, and tyramine in tested samples were higher than those of tryptamine, 2-phenylamine, spermidine, and spermine. Four fish meal samples (10%, 4/40) and 1 FSC sample (20%, 1/5) had histamine levels >20 mg/100 g. Five histamine-producing bacterial strains isolated from fish meal samples producing 1.31–6.21 ppm of histamine in trypticase soy broth supplemented with 1.0% l-histidine (TSBH) were identified as Bacillus licheniformis (three strains), B. amyloliquefaciens (one strain), and B. subtilis (one strain).

An incident of foodborne poisoning causing illness in 53 victims due to ingestion of fish fillets occurred in January, 2009, in Kaohsiung city, southern Taiwan. The two suspected fish samples contained 11.3 and 37.7 mg/100 g of histamine, which is greater than the 5.0 mg/100 g allowable limit suggested by the United States Food and Drug Administration. Given the allergy-like symptoms of the victims and the high histamine content in the suspected fish samples, this foodborne poisoning was strongly suspected to be caused by histamine intoxication. Five histamine-producing bacterial strains capable of producing 1.23 to 36.48 ppm of histamine in trypticase soy broth (TSB) supplemented with 1.0% L-histidine (TSBH) were identified as Bacillus subtilis (four strains) and Enterobacter aerogenes (one strain) by 16S rDNA sequencing with polymerase chain reaction (PCR) amplification. Moreover, the fish species of suspected samples were identified as mahi-mahi (Coryphaena hippurus) by using PCR direct sequence analysis. PRACTICAL APPLICATIONS Based on the finding that high contents of histamine (>30 mg/100 g) were detected in the suspected mahi-mahi samples, we speculate the temperature abuse of the fillets before cooking contributed to the presence of high histamine levels in mahi-mahi fillets and resulted in foodborne poisoning. Although histamine-producing strains, Bacillus subtilis and Enterobacter aerogenes, were isolated from suspected fish samples, they might not be the main contributors to histamine accumulation because of low histamine production. These results re-emphasize proper handling temperature for seafoods and offer an important awareness that Coryphaena hippurus fillets could become a hazardous food item in causing histamine poisoning even though no quality deficiency was observed on the fillets.

Fish gelatin hydrolysates have been shown to possess various biological activities due to their unique Gly-Pro-Y and Gly-X-Hyp sequences. In the current study, fish gelatin was extracted from non-extruded milkfish scale (FSG1) or extrusion-pretreated milkfish scale (FSG2), extracted gelatins were hydrolyzed with different combinations of Flavourzyme and Alcalase to give four different hydrolysates, namely: FSGH1 (FSG1 hydrolyzed with Flavourzyme), FSGH2 (FSG1 hydrolyzed with Alcalase + Flavourzyme), FSGH3 (FSG2 hydrolyzed with Flavourzyme), and FSGH4 (FSG2 hydrolyzed with Alcalase + Flavourzyme). The extrusion-pretreatment process enhanced the extraction yield of gelatin from fish scale. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) analyses showed the extracts FSG1 and FSG2 possessed characteristics of gelatin. Moreover, the physicochemical characteristics of FSGH1&ndash, FSGH4 were examined by analyses of their degree of hydrolysis, amino acid composition, UV spectrum, FTIR spectrum, molecular weight, and RP-HPLC profile. Additional biological functional analyses showed that all of the studied gelatin hydrolysates FSGH1&ndash, FSGH4 possessed antioxidant activity dose-dependently as revealed by DPPH scavenging, ABTS scavenging, and reducing power analyses. In addition, FSGH2 and FSGH4 showed higher angiotensin-I-converting enzyme (ACE)-inhibitory activity as compared to FSGH1 and FSGH3. Taken together, FSGH2 and FSGH4 showed high antioxidant activity and potent anti-ACE activity. Due to the potential antioxidant and antihypertensive properties of FSGH2 and FSGH4, further research is needed to explore their possible use as natural supplementary raw materials in food and nutraceutical products.

Nine tuna dumpling products were purchased at nine retail markets in southern Taiwan. Occurrence of biogenic amines, histamine-forming bacteria, and adulteration of pork and poultry were determined. This study showed the high contents of aerobic plate count, total coliform and Escherichia coli in tested tuna dumpling products. Average content of various biogenic amines in all tested samples was less than 2.0 mg/100 g. Fifteen histamine-producing bacterial strains isolated from tested samples produced 8.7–466 ppm of histamine in trypticase soy broth supplemented with 1.0% l -histidine (TSBH). Assay of multiplex polymerase chain reaction (PCR) revealed adulteration rates were 88.9% (8/9) and 33.3% (3/9) for pork and poultry, respectively, in tuna dumpling. In addition, six samples of tuna dumpling meat were identified as Thunnus obesus , while other three samples were identified as Thunnus thynnus .

Thirty-four tuna candy products sold in retail markets and supermarkets in Taiwan were purchased and tested to determine the biogenic amine, histamine-forming bacteria, and adulteration of bonito meat. The levels of pH value, water activity (Aw), water content, total volatile basic nitrogen (TVBN), aerobic plate count (APC) and total coliform (TC) in all samples ranged from 5.3 to 6.1, 0.47 to 0.65, 7.37% to 17.32%, 12.1 to 54.6 mg/100 g, l -histidine (TSBH) after incubation at 35 °C for 24 h. Assay of multiplex polymerase chain reaction (PCR) revealed that adulteration rate of bonito meat was 26.5% (9/34) in tuna candy samples. Tuna species in tuna candy samples was identified as Thunnus albacares for 29 samples (85.3%), Thunnus alalunga for four samples (11.8%) and Thunnus obesus for one sample (2.9%) by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP).

An incident of food-borne poisoning causing illness in 347 victims due to ingestion of fried fish cubes occurred in June, 2007, in Kaohsiung city, southern Taiwan. A high-performance liquid chromatography (HPLC) was developed for determining nine biogenic amines in two suspected fish samples by dansylation with dansyl chloride. One sample has histamine content (52.3 mg/100 g) greater than the hazard action level of 50 mg/100 g. Given the allergy-like symptoms of the victims and the high histamine content in the suspected fried fish samples, this food-borne poisoning was strongly suspected to be due to histamine intoxication. In addition, two weak histamine-producing isolates were identified as Acinetobacter baumannii, by 16S rDNA sequencing with PCR amplification.

Forty-three tuna sandwiches sold in diners and convenience store in southern Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, water content, ash content, salt content, total volatile basic nitrogen (TVBN), aerobic plate count (APC), total coliform (TC), and Escherichia coli in all samples ranged from 4.5 to 6.9, 36.8% to 62.8%, 1.11% to 6.55%, 0.10% to 3.90%, 11.2 to 78.0 mg/100 g

Thirty-two dried milkfish products sold in five retail markets in southern Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. Except for histamine and cadaverine, the average content of various biogenic amines in tested samples was less than 8.5 mg/100 g. Most of the tested dried milkfish products (78.1%) had histamine levels greater than the FDA guideline of 5 mg/100 g for scombroid fish and/or product, while fourteen of them (43.7%) contained > 50 mg/100 g hazard action level. Thirty histamine-producing bacterial strains, capable of producing 5.4 ppm to 562 ppm of histamine in trypticase soy broth supplemented with 1.0% L-histidine (TSBH), were identified as Enterobacter aerogenes (seven strains), Citrobacter sp. (one strain), Staphylococcus xylosus (ten strains), S. sciuri (one strain), Bacillus thuringiensis (two strains), Citrobacter freundii (five strains), Klebsiella pneumoniae (one strain) and E. cloacae (three strains), by 16S rDNA sequencing with PCR amplification.

Sixteen salted mullet roe products sold in the retail markets in Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, water content, total volatile basic nitrogen (TVBN) and aerobic plate count (APC) in all samples ranged from 5.4 to 5.8, 5.1% to 7.2%, 15.4% to 27.3%, 32.0 to 69.6 mg/100 g and

An incident of food-borne poisoning causing illness in seven victims, due to ingestion of tuna dumpling, occurred in March 2006, in Chiayi Prefecture, southern Taiwan. The leftovers of the victims’ tuna dumpling and the five other tuna dumpling samples from five other retail stores were collected and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, aerobic plate count (APC), total volatile basic nitrogen (TVBN), total coliform (TC) and Escherichia coli in all samples ranged from 6.08 to 6.43, 0.46% to 0.81%, 5.90 to 8.95 log CFU/g, 6.38 to 21.29 mg/100 g, 750 to 8000 most probable number (MPN)/g, and l -histidine (TSBH), were identified as Enterobacter sp. (three strains), Pantoea agglomerans (two strains), Klebsiella variicola (four strains) and Serratia marcescens (four strains), by 16S rDNA sequencing with PCR amplification, they were not determined to be the main contributors to histamine accumulation in suspected tuna dumpling.

An incident of food borne poisoning causing illness in 43 victims due to ingestion of swordfish fillets occurred in December, 2004, in Taichung Prefecture, central Taiwan. Eight frozen raw swordfish fillets were collected from the suspected restaurant and analyzed for bacterial content and histamine-related quality. The levels of aerobic plate count, total coliform, and total volatile basic nitrogen in all samples ranged from 5.39 to 6.71 log CFU/g

Thirty-six natto products imported from Japan and three domestic natto products sold in the supermarkets in southern Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH and aerobic plate count (APC) in all samples ranged from 4.9 to 7.3 and 7.8 to 11.2 log CFU/g, respectively. None of these samples contained total coliform and E. coli. Although the average content for each of the nine biogenic amines in all samples was less than 5 mg/100 g, seven of them (17.9%) had histamine content greater than 5 mg/100 g, allowable limit suggested by the US Food and Drug Administration for scombroid fish and/or product. Four histamine-producing bacterial strains capable of producing 13.4 ppm to 17.5 ppm of histamine in trypticase soy broth (TSB) supplemented with 1.0% l -histidine (TSBH) were identified as Bacillus subtilis (two strains) and Staphylococcus pasteuri (two strains) by 16S rDNA sequencing with PCR amplification. To our knowledge, this is the first report to demonstrate the occurrence of histamine-forming bacteria in natto products.

Twenty-seven miso products sold in supermarkets and 13 products sold in retail markets were purchased from southern Taiwan, and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, and aerobic plate count (APC) in all samples ranged from 5.1 to 5.8, 6.1% to 13.8%, and 2.1 to 9.1 log CFU/g, respectively. Only one of the supermarket miso products contained 100 MPN/g total coliform. None of these samples contained Escherichia coli . Although the average content for each of the nine biogenic amines in all samples was less than 5 mg/100 g, two supermarket samples (22.1 and 11.9 mg/100 g) and one retail market sample had histamine content (10.2 mg/100 g) greater than the 5.0 mg/100 g allowable limit suggested by the US Food and Drug Administration. Eight histamine-producing bacterial strains, capable of producing 10.4–39.4 ppm of histamine in trypticase soy broth (TSB) supplemented with 1.0% l -histidine (TSBH), were identified as Staphylococcus pasteuri (one strain), Bacillus sp. (one strain), B. amyloliquefaciens (two strains), B. subtilis (two strains) and B. megaterium (two strains), by 16S rDNA sequencing with PCR amplification.

Two incidents of food-borne poisonings, causing illness in 59 and 43 victims due to ingestion of billfish meats, occurred in May 2004, in Pingtung, southern Taiwan and in December 2004, Taichung, central Taiwan, respectively. One fried billfish fillet and five frozen billfish fillet samples collected, respectively, from the suspected restaurants in Pingtung and Taichung, respectively, were tested to determine the histamine levels and identify fish species. Analyses of histamine showed that the suspected billfish samples in two food poisonings contained more than 150 mg/100 g of histamine, which is higher than the hazard action level of 50 mg/100 g. Judging from the allergy-like symptoms of the victims and the high histamine levels in the suspected billfish samples, both food-borne poisonings were strongly suspected to be caused by histamine intoxication. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to identify the species of the suspected billfish samples in both food poisonings. The 348 bp amplified fragment of the mitochondrial cytochrome b gene by PCR was digested with BsaJI, Cac8I and HpaII enzymes to distinguish the species of the suspected billfish samples. Consequently, the species of Pingtung and Taichung billfish samples implicated in food poisonings were identified as Makaira nigricans and Xiphias gladius, respectively.

An incident of food-borne poisoning, causing illness in three victims due to ingestion of dried milkfish, occurred in February, 2006, in Tainan Prefecture, southern Taiwan. The leftovers of the victims’ dried milkfish and three other dried milkfish samples, from the same retail store as the leftover fish, were collected and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, aerobic plate count (APC) and total volatile basic nitrogen (TVBN) in the leftover dried milkfish sample were significantly higher than those of the other three milkfish samples. None of the tested milkfish samples contained total coliform and Escherichia coli . Although the histamine contents in the three other milkfish samples were less than 5 mg/100 g, the suspected milkfish sample contained 61.6 mg/100 g of histamine, which is greater than the hazard action level of 50 mg/100 g. Given the allergy-like symptoms of the victims and the high histamine content in the suspected milkfish sample, this food-borne poisoning was strongly suspected to be due to histamine intoxication. Four histamine-producing bacterial strains, capable of producing 11.9–1243 ppm of histamine in trypticase soy broth supplemented with 1.0% l -histidine (TSBH), were identified as Staphylococcus sciuri subsp. sciuri , Serratia grimesii , Bacillus cereus and Raoultella ornithinolytica , by 16S rDNA sequencing with PCR amplification. R. ornithinolytica was a potent histamine-former capable of producing more than 800 ppm of histamine in TSBH in the presence of 1.5% or 3.5% NaCl. Therefore, R. ornithinolytica could be the causative bacterium responsible for the production of high content of histamine that eventually led to this dried milkfish poisoning incident.

Seven soybean and 19 black bean douchi products sold in the supermarkets in southern Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, water content, yeast and mold, and aerobic plate count (APC) in all samples ranged from 4.7 to 5.9, 4.4% to 14.0%, 6.8% to 51.6%, 3.0 to 5.1 log CFU/g, and 5.2 to 9.2 log CFU/g, respectively. None of these samples contained total coliform and Escherichia coli . Although black bean douchi products had an average histamine content of 29.0 mg/100 g, 18 of them had histamine contents greater than 5 mg/100 g, the allowable level set by the US Food and Drug Administration (FDA) for scombroid fish and/or products. In contrast, only four soybean douchi products had histamine levels greater than 5 mg/100 g. Among the black bean samples, four contained histamine at 56.3, 62.1, 80.2 and 80.8 mg/100 g, that are above the 50 mg/100 g hazard action level. Eight histamine-forming bacterial strains, capable of producing 11.7–601 ppm of histamine in trypticase soy broth (TSB) supplemented with 1% l -histidine (TSBH), were identified as Bacillus subtilis (four strains) Staphylococcus pasteuri (one strain) and Staphylocuccus capitis (three strains) by 16S rDNA sequencing with PCR amplification. S. capitis , which was previously reported to be halotolerant, was a potent histamine-former capable of producing more than 500 ppm of histamine in TSBH in the presence of 0.5–10% NaCl.

Toxic gastropods Polinices didamy, Natica lineata, Oliva miniacea, O. mustelina and O. hirasei are tetrodotoxin (TTX) bearing animals, which accumulate TTX in their muscle and digestive gland. Analysis by gel filtration on Sepharose CL-6B revealed that 0.05 M NaCl extracts of the muscle of five species of toxic gastropods contained TTX-binding high molecular weight substances (HMWS) (1500–2000 kDa). The TTX-binding capacities of those HMWS were 0.12, 0.62, 0.45, 0.28 and 0.35 MU/mg protein, respectively, but those HMWS had no neutralising effect on TTX or paralytic shellfish poison. The HMWS of the five toxic gastropods could be hydrolyzed with HCl and protease at 37 °C, pH 7.4, but not with ribonuclease T2, deoxyribonuclease I or α-amylase. After purifying the TTX-binding protein of N. lineata by Q Fast-Flow strong anion exchanger and then BioSep-SEC-S 2000, the TTX-binding capacity increased to 3.5 MU/mg and 4.2 MU/mg protein, respectively. The TTX-binding capacity of N. lineata HMWS had no obvious seasonal variation. The molecular weight of TTX-binding protein of N. lineata was estimated to be about 434 kDa, while it comprised two subunits with molecular weights of about 272 kDa and 47 kDa, respectively, under SDS–PAGE.