Histamine level and species identification of billfish meats implicated in two food-borne poisonings

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.     

Histamine and other biogenic amines and histamine-forming bacteria in miso 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.     

Histamine content and histamine-forming bacteria in dried milkfish (Chanos chanos) products

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), Staphylococcusxylosus (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.     

Histamine production by Enterobacter aerogenes in tuna dumpling stuffing at various storage temperatures

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.     

The function of lactic acid bacteria and brine solutions on biogenic amine formation by foodborne pathogens in trout fillets

The influences of lactic acid bacteria and brine solutions on the biogenic amine formation by Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enterococus faecalis, Pseudomonas aeruginosa, Listeria monocytogenes, Aeromonas hydrophila and Salmonella paratyphi A in fermented trout fillets were investigated. Fish fillets were divided into four groups, group 1 without any lactic acid bacteria inoculation, group 2 and group 3 with different salt concentration inoculated with lactic acid bacteria and food-borne pathogens, and group 4 inoculated with lactic acid bacteria and food-borne pathogens without a salt solution. The histamine content in trout fillets in group 4 was found to be more than 10 mg/100 g, while the other groups contained less than 7.5 mg/100 g. The highest tyramine production was found for group 1 and group 3, ranging from 3 to 18 mg/100 g. Lactic acid bacteria did not seem to play an important role on biogenic amine production by food borne pathogens, while adding brine solution on fillets has inhibitory effects on some of the biogenic amines.     

Histamine levels in seventeen species of fresh and processed South African seafood

Histamine levels were determined in fresh and processed seafood from a representative range of 10 outlets after several incidents of scombroid seafood poisoning occurred. Species included seventeen fresh and processed scombroid- and non-scombroid fish, marine mollusks and crustaceans. Histamine levels in fresh seafood were generally low (0–9 ppm) with the exception of one sample of snoek (scombroid fish; >50 ppm) and one sample of yellowtail (non-scombroid fish; >50 ppm). Both species are rich in free histidine (1.5–5.3 ppb), a precursor of histamine. Processed seafood had, in general, low histamine concentrations (0–3 ppm) with the exception of fish meal (76 ppm), salted herring (47 ppm), one sample of smoked snoek (>50 ppm) and dried tuna (8000 ppm). In total, 5 of 80 examined samples (6%) contained histamine concentrations above the legal limit of 50 ppm. Experimental formation of histamine was demonstrated to be strongly temperature- and time-dependent. Samples were not contaminated with Vibrio spp., Pseudomonas spp., Klebsiellas spp. or Enterobacteria.     

Occurrence of histamine and histamine-forming bacteria in kimchi products in Taiwan

Twenty kimchi products sold in supermarkets and 17 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 and salt content in all samples ranged from 3.6 to 5.1 and 1.5–16.0%, respectively. The supermarket kimchi products had 1–7.2 log CFU/g of APC and <3–600 MPN/g of total coliform (TC), and the retail market kimchi products had 4–8.03 log CFU/g of APC and <3 to >2400 MPN/g of TC. Only one of the retail market kimchi products contained 20 MPN/g Escherichia coli. Although, supermarket kimchi products had an average histamine content of 49.8 mg/100 g, 15 of them had histamine content greater than 5 mg/100 g, the allowable level set by the US Food and Drug Administration (FDA) for scombroid fish and/or product. In contrast, only eight retail market kimchi products had histamine levels greater than 5 mg/100 g. Among the supermarket samples, three contained histamine at 50.2, 273 and 535 mg/100 g, that are more than the 50 mg/100 g hazard action level. Four histamine-producing bacteria capable of producing 13.6–43.1 ppm of histamine in MRS broth supplemented with 0.25% l-histidine were identified as Lactobacillus para. paracasei (one strain), Lb. brevis (one strain), and Brevibacillus brevis (two strains). To our knowledge, this is the first report to demonstrate the occurrence of high contents of histamine and histamine-forming bacteria in kimchi products.     

Histamine formation by histamine-forming bacteria in douchi,a Chinese traditional fermented soybean product

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.     

Lipid components and water soluble metabolites in salted and dried tuna (Thunnus thynnus L.) roes

The salted and dried product of tuna roe (bottarga) is a seafood characteristic of the Mediterranean area and exported all over the world. Samples of bottarga from bluefin tunas (Thunnus thynnus, L.) caught in the southwest Mediterranean sea were analysed. The samples were characterised by high content of marine wax esters (55–67 mol% of lipid classes), of docosahexaenoic (22:6 n-3, 25 w%) and oleic (18:1 n-9, 19 w%) fatty acids. Cholesterol was detected as 7–9 w% of lipids. Free fatty acids, index of lipid hydrolysis, represented 32–39 mol% over total fatty acids. Among metabolites, nutrients as taurine, nicotinamide and β-alanine, were found. The microflora comprised staphylococci, enterococci (2.2 log₁₀ CFU/g) and lactic acid bacteria (3 log₁₀ CFU/g). The food-borne pathogens Staphylococcus aureus, Listeria monocytogenes and Salmonella spp. were not detected. These findings indicate tuna bottarga as valuable source of nutrients.     

Occurrence and diversity of free-living protozoa on butterhead lettuce

The occurrence and diversity of free-living protozoa (FLP) on butterhead lettuce (Lactuca sativa L.) was investigated using four different sampling techniques (washing, swabbing, homogenization, and excising). FLP were recovered from all leaf samples (n = 64), and cultures were FLP-positive after 1 week. Identification of FLP was performed by light microscopy and sequencing of denaturing gradient gel electrophoresis (DGGE)-separated 18S rRNA gene fragments. Bodo saltans, Spumella (-like) spp. and Cercozoa were the most common heterotrophic nanoflagellates. Amoebae belonged mainly to the Vannellida and Tubulinida. Colpoda steinii and Cyclidium glaucoma were the most common ciliates. The total number of FLP on middle leaves estimated by the Most Probable Number method ranged from 9.3 × 10² MPN/g to 2.4 × 10⁵ MPN/g leaf, with flagellates (92 MPN/g to 2.4 × 10⁵ MPN/g) being more abundant than amoebae (< 3 MPN/g to 9.3 × 10³ MPN/g) and ciliates (< 3 MPN/g to 9.3 × 10² MPN/g). Washing or rinsing leaves followed by spin-drying in a household salad spinner reduced the protozoan number with maximum one log unit. Our survey shows that FLP on lettuce leaves are a common and diverse but largely unexplored group of microorganisms.     

Histamine formation by histamine-forming bacteria and yeast in mustard pickle products in Taiwan

The occurrence of histamine, histamine-forming bacteria and yeast were tested in 37 mustard pickle products sold in both retail markets and supermarkets in southern Taiwan. Aerobic plate count (APC), total coliform, and Escherichia coli were also tested for microbiological quality. Salt content, pH value, titratable acidity and sulphite content were determined for quality of mustard pickle products. Only one retail market sample and one supermarket sample had 8.9 and 7.4 mg histamine per 100 g products, although the average content for each of the nine biogenic amines was less than 2 mg/100 g. Ten histamine-forming bacterial strains and 6 histamine-producing yeast strains capable of producing 8.7 to 1260 ppm of histamine in trypticase soy broth (TSB) supplemented with 1% l-histidine (TSBH) were identified as Staphylococcus capitis (four strains),Staphylococcus pasteuri (two strains), Enterobacter cloacae (four strains), Candida glabrata (two strains) and Candida rugosa (four strains). S. capitis, which was previously reported to be halotolerant, was a potent histamine-former, capable of producing more than 1000 ppm of histamine in TSBH in the presence of 0.5–10% NaCl. The numbers of the aerobic plate count (APC) in all samples were below the Taiwanese regulatory level of 5 log CFU/g. None of the samples contained total coliform or E. coli. The values of pH, salt content, titratable acidity and sulphite content in all samples ranged from 3.8% to 5.0%, 2.0% to 10.0%, 0.21% to 1.18% and <2.0–1876 ppm, respectively.     

Prediction of the amount and rate of histamine degradation by diamine oxidase (DAO)

Histamine is a biogenic amine that forms in a variety of foods and can cause food poisoning at high concentrations (>500 ppm). In situations where the formation of histamine in food cannot be prevented through refrigeration, diamine oxidase (DAO) enzyme may be used to degrade histamine to safe levels. The aims of this work were to apply DAO in model (buffer) and real (cooked tuna soup used in the manufacture of a fish paste product, Rihaakuru) systems, in order to obtain predictions for the rates and amounts of histamine degradation. The two systems were set up with a constant concentration of histamine (500 mg/L) and the DAO enzyme (2534 units/L) at a temperature of 37 °C, agitation at 100 rpm and an incubation time of 10 h with variable pH (5–7) and salt concentrations (1–5%). A total of 15 experiments were designed for each system using central composite design (CCD). The data from these experiments were fitted into regression models; initially the data were used to generate an exponential decline model and then the data from this were fitted into a secondary response surface model (RSM) to predict the rate and amount of histamine degradation by DAO. The model system results indicated that DAO activity was not significantly affected by salt (p > 0.05), and that activity reached a maximum within the pH range of 6–6.5 with an optimum at pH 6.3. However, the results obtained with the tuna soup model showed that the optimum oxidation of histamine using DAO occurred between pH 6–7 and salt 1–3%. This study defined the conditions for the use of DAO to degrade 500 mg/L of histamine in tuna soup used to manufacture Rihaakuru. The models generated could also be used to predict the rate and amount of histamine degradation in other foods that have similar characteristics to tuna soup.     

Toxicity of 4′-O-methylpyridoxine-5′-glucoside in Ginkgo biloba seeds

A vitamin B₆ derivative, 4′-O-methylpyridoxine (MPN) is responsible for food poisoning by Ginkgo biloba seeds. MPN-5′-glucoside is also contained in Ginkgo biloba seeds. In this study, we examined the toxicity of MPN-5′-glucoside in mice and MPN-5′-glucoside content in Ginkgo biloba seeds in order to estimate its contribution to poisoning by Ginkgo biloba seeds. The MPN-5′-glucoside content was comparable to that of MPN in row seeds but was much higher than that of MPN in heated Ginkgo biloba seeds. However, the total MPN content showed no significant differences in heated seeds compared with the content in non-treated seeds. The MPN-5′-glucoside (0.8 mmol/kg b.w.) had a lethal effect in mice, as well as MPN. Additionally, the onset time of convulsions after administration of MPN-5′-glucoside was longer than that after administration of MPN. Ginkgo biloba seeds are generally eaten after heating in Asia. Therefore, the intake of MPN-5′-glucoside, rather than MPN, might cause food poisoning by Ginkgo biloba seeds.     

A SURVEY OF HISTAMINE LEVELS IN COMMERCIALLY PROCESSED SCOMBROID FISH PRODUCTS12

Analysis of 100 samples of each of 3 types of commercially processed scombroid fish products obtained at the retail level showed mean histamine concentrations (mg/100 g) of 3.58 for chunk light tuna, 1.50 for albacore tuna, and 2.56 for mackerel. Only 2 samples of chunk light tuna and 2 of mackerel contained greater than 10 mg% histamine. The highest level of histamine found in albacore tuna was 5.35 mg/100 g, while 99% ofthe samples had less than 5 mg% histamine. Chunk light tuna samples contained up to 28.0 mg/100 g, and mackerel samples contained up to 31.5 mg/100 g; 92% of the chunk light tuna and 95% of the mackerel had less than 5% histamine. The histamine levels found in these products were far below any level associated with food poisoning incidents.     

Chemical characterisation and histamine-forming bacteria in salted mullet roe products

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 <1.0 to 7.1 log CFU/g, respectively. None of these samples contained total coliform and Escherichia coli. The average content of each of the nine biogenic amines in all samples was less than 4 mg/100 g, and only one mullet roe sample had the histamine content (8.18 mg/100 g) greater than the 5.0 mg/100 g allowable limit suggested by the US Food and Drug Administration. Two histamine-producing bacterial strains capable of producing 10.7 ppm and 9.6 ppm of histamine in trypticase soy broth (TSB) supplemented with 1.0% l-histidine (TSBH) were identified as Staphylococcus carnosus by 16S rDNA sequencing with PCR amplification, and they were isolated from the sample with higher histamine content (8.18 mg/100 g).     

Thermal inactivation kinetics of Shiga toxin-producing Escherichia coli in buffalo Mozzarella curd

The use of raw milk in the processing of buffalo Mozzarella cheese is permitted, but the heat treatment used for stretching the curd must ensure that the final product does not contain pathogens such as Shiga toxin-producing Escherichia coli (STEC) that may be present on buffalo dairy farms. This study carried out challenge tests at temperatures between 68°C and 80°C for 2 to 10 min to simulate curd temperatures during the stretching phase. Curd samples were inoculated with 2 STEC strains (serotypes O157 and O26), and their inactivation rates were assessed in the different challenge tests. The curd samples were digested with papain to ensure a homogeneous dispersion of bacteria. The STEC cells were counted after inoculation (range 7.1–8.7 log cfu/g) and after heat treatments using the most probable number (MPN) technique. A plot of log MPN/g versus time was created for each separate experiment. The log linear model with tail was used to provide a reasonable fit to observed data. Maximum inactivation rate (k_max, min⁻¹), residual population (log MPN/g), decimal reduction time (min), and time for a 4D (4-log₁₀) reduction (min) were estimated at each temperature tested. A 4D reduction of the O26 STEC strain was achieved when curd was heated at 68°C for 2.6 to 6.3 min or at 80°C for 2.1 to 2.3 min. Greater resistance was observed for the O157 strain at 68°C because k_max was 1.48 min⁻¹. The model estimates can support cheesemakers in defining appropriate process criteria needed to control possible STEC contamination in raw milk intended for the production of Mozzarella.     

Fish oil encapsulation with chitosan using ultrasonic atomizer

An encapsulation technique was developed using an ultrasonic atomizer and three processing steps: emulsification, ultrasonic atomization, and freeze drying. Emulsion preparation variables such as concentration of wall materials [chitosan (CS), maltodextrin (MD) and whey protein isolate (WPI)] and tuna oil were optimized. The size and stability of the emulsion droplet and the properties of the encapsulated powders after freeze drying were characterized. At 20 g/100 g tuna oil, the optimum ratios of CS to MD and of CS to WPI were 1:10 and 1:1, respectively. There was a significant difference (P<0.05) in the emulsion particle sizes when the preparation conditions were varied. The combination of CS and MD giving the smallest particle size had the highest emulsion stability. The EPA and DHA content (240 mg/g) of the encapsulated powder were slightly higher than commercial specification (100 mg/g) and they had low moisture content and water activity, acceptable appearance and encapsulation efficiency. The ultrasonic technology used in this study could lead to application in the food industry improving the stability of tuna and other oils.     

Synergistic and antagonistic effect of lactic acid bacteria on tyramine production by food-borne pathogenic bacteria in tyrosine decarboxylase broth

The effect of lactic acid bacteria (LAB) strains on tyramine (TYR) and also other biogenic amines (BA) production by eight common food-borne pathogen (FBP) in tyrosine decarboxylase broth (TDB) was investigated by using a rapid HPLC method. Significant differences were observed among the FBP strains in ammonia (AMN) and BA production apart from tryptamine, histamine (HIS) and spermine formation (p < 0.05). Salmonella paratyphi A was characterised as the main amine producer. LAB had an important synergetic role in some BA production by food-borne pathogenic bacteria, although the effect of some LAB strains on BA production was strain-dependent. Lactococcus spp. and Streptococcus spp. resulted in significantly higher TYR accumulation by Aeromonas hydrophila and Enterococcus faecalis in TDB. The presence of Lactococcus and/or Lactobacillus in TDB significantly increased HIS production by A. hydrophila, Escherichia coli, Ent. faecalis, Klebsiella pneumoniae and Pseudomonas aeruginosa, whereas HIS accumulation was significantly reduced by Staphylococcus aureus, S. paratyphi A and Listeria monocytogenes.     

Behavior of Salmonella during fermentation,drying and storage of cocoa beans

Due to cocoa being considered a possible source of Salmonella contamination in chocolate, the behavior of Salmonella during some cocoa pre-processing stages (fermentation, drying and storage) was investigated. The fermentation process was carried out on a pilot scale (2 kg beans/box) for 7 days. Every day a fermentation box was inoculated with a Salmonella pool (ca. 4 log MPN/g). The results showed that Salmonella did not affect (P > 0.05) the growth of the main microorganism groups involved in cocoa fermentation. On the other hand, the pathogen was influenced (P < 0.05) by yeast, acetic acid bacteria and pH. In spite of Salmonella showing counts ≤ 1 log MPN/g in the first days, at the end of fermentation it grew in all samples, reaching counts as high as 7.49 log MPN/g. For drying and storage, cocoa beans were inoculated during the fermentation (experiment A) or during the drying (experiment B). In these stages the decline of the water activity affected the pathogen behavior. In experiment A during the drying, Salmonella count increased in most of the samples. In experiment B either a slight growth or no growth in the samples inoculated up to 48 h was observed, whereas the other samples showed reductions from the initial count. After 30 days of storage at room temperature, the water activity decreased to 0.68, and reductions of Salmonella ranged from 0.93 to 2.52 log MPN/g. Despite the reductions observed during the storage, the pathogen was detected even after 120 days. Therefore, the results showed that Salmonella growth or survival depends on when the contamination occurs.     

Study on chemical composition and biological activities of essential oil and extract from Salvia pisidica

In this study, total contents of phenolic, flavanol and flavonol, antioxidant activities and antimicrobial activities of the Turkish endemic Salvia pisidica Boiss. & Heldr. ex Bentham (Lamiaceae) extract and essential oil were assessed in vitro. Total phenolic, flavanol and flavonol contents in the extract were 54.57 mg gallic acid equivalents (GAE)/g, 16.70 mg catechine equivalents (CE)/g and 18.19 mg rutin equivalents (RE)/g, respectively. Antioxidant activities (IC₅₀ value) of the extract and essential oil were determined as 4.88 and 6.41 mg/mL by DPPH assay, respectively. 31 compounds were determined in the essential oil using GC-MS and the major compounds (%) were camphor (23.76), sabinol (19.2), α-thujone (14.2) and eucalyptol (1.8-cineole) (5.8).The antimicrobial activity of the methanolic extract and the essential oil against 13 bacterial and two yeast strains was determined. The extract (concentration 5 g/100 ml or 10 g/100 ml) was effective against most of the strains tested, yet not against Bacillus cereus, Staphylococcus aureus, Aeromonas hydrophila and the two yeast strains tested. The essential oil (2 g/100 ml) showed an antimicrobial effect against all the gram (+) bacteria tested, against Saccharomyces cerevisiae, but was not effective against all gram (−) bacteria and Candida albicans. These results show that S.piscidica essential oil and extract could be considered as a natural alternative to traditional food preservatives and be used to enhance food safety and shelf life.