Identification of bacteria crucial to histamine accumulation in pacific mackerel during storage

Bacterial growth and histamine formation in Pacific mackerel during storage at 0, 4, 15, and 25 degrees C were monitored. To identify bacterial species contributing to histamine formation, several groups of bacteria were isolated by using selective media under temperatures corresponding to the various storage conditions. Initially, low counts of bacteria were found in the gill, skin, and intestine of fresh fish, and only weak histamine formers were found in the gill. Histamine was found in the muscle when fish were stored above 4 degrees C, and aerobic plate counts reached 10(6) CFU/g. When fish became unsuitable for human consumption by abusive storage, toxicological levels of histamine were always found. The highest level of histamine formed was 283 mg/100 g in 2 days. The optimum temperature for supporting growth of prolific histamine formers was 25 degrees C. The most prolific and prevalent histamine former was Morganella morganii, followed by Proteus vulgaris, both of which were isolated on violet red bile glucose (VRBG) agar. At 15 degrees C, a significant level of histamine was still produced in fish muscle, although prolific histamine formers were less frequently detected than at 25 degrees C. The isolates on thiosulfate citrate bile salts sucrose (TCBS) agar were weak histamine formers and identified as Vibrio parahaemolyticus and Vibrio alginolyticus. At 4 degrees C, less than 57.4 mg/100 g of histamine was found in fish stored for 14 days. Most isolates were natural bacterial flora in the marine environment and identified as weak histamine formers. At 0 degrees C, neither histamine former nor histamine production was detected up to 14 days of storage.     

Source and identification of histamine-producing bacteria from fresh and temperature-abused albacore.

Histamine-producing bacteria were isolated from fresh and temperature-abused albacore using two different isolation procedures. Typically, the bacterial isolates on Niven's or modified Niven's medium produced negligible or low levels of histamine (<300 ppm) in histamine enumeration broth. The most frequently found species using this approach was Hafnia alvei. By prescreening on selective media (eosin methylene blue [EMB] agar for enteric bacteria; deMan Rogosa Sharpe agar for lactic acid bacteria: KF streptococcus agar for streptococci; pseudomonas isolation [PI] agar for pseudomonads; and staphylococcus medium 110 agar for staphylococci) prior to plating on histidine decarboxylase differential media, detection rate of true histamine formers increased. Prolific histamine producers capable of forming >1,000 ppm histamine in culture broth were isolated when PI and EMB agars were used for prescreening. Among the selective media tested, EMB agar was most effective in selecting high histamine producers, as demonstrated by the highest rate of true positives based on histamine analysis. Histamine-producing isolates were mostly enteric bacteria, including Morganella morganii, H. alvei, Klebsiella spp., Citrobacter freundii, Enterobacter spp., and Serratia spp. M. morganii isolated on PI agar from temperature-abused albacore muscle was found to be the highest histamine former. This species was not isolated from fresh albacore. while other enteric bacteria were frequently detected on the gills. However, only a few species isolated from both fresh and temperature-abused muscles were identified as high histamine formers.     

Histamine Formation in Albacore Muscle Analyzed by AOAC and Enzymatic Methods

Histamine formation in fresh albacore was studied. Whole albacore left on deck up to 1 d contained negligible levels of histamine (<0.4 mg/1 00g muscle). When they were abused at ambient temperatures for 1–4 days, 2 of 9 samples showed histamine levels >5 mg/100g. Histamine level was not affected by on-board handling techniques studied, i.e., bleeding and spiking. Enzymatic assay was compared to the AOAC fluorometric method for histamine analysis. Enzymatic results showed a good correlation with those of the AOAC method (r²=0.829). Although the enzymatic method tended to overestimate histamine at levels <1 mg/1 00g, it was rapid, simple, and suitable as a screening method for routine analysis.     

Histamine Production by Morganella morganii in Mackerel, Albacore, Mahi-mahi, and Salmon at Various Storage Temperatures

Morganella morganii was studied for its growth and histamine formation in mackerel, albacore, mahi‐mahi, and salmon stored at various temperatures from ‐30 °C to 37 °C. The optimal temperature for histamine formation was 25 °C. Mackerel, albacore, and mahi‐mahi were shown as good substrates for histidine decarboxylation by M. morganii at elevated temperatures (> 15 °C). M. morganii inoculated in all fish species including salmon formed histamine above the FDA guideline. Their growth was controlled by cold storage of the fish at 4 °C or below, but histamine formation was controlled only by frozen storage. Although histamine was not detected in any frozen samples, it accumulated rapidly in the previously frozen fish stored at 25 °C.     

Histamine Formation and Bacterial Spoilage of Albacore Harvested off the U.S. Northwest Coast

Iced and previously frozen albacore were monitored for histamine formation and bacterial growth during storage at 0–37°C. The optimum temperature for histamine formation in albacore tuna (Thunnus alalunga) was 25°C, and whole fish were more susceptible to histamine formation than dressed fish at that temperature. Storage at 25°C resulted in the highest histamine level, 60.4 mg/100g in whole fish stored for 7 days. When albacore were frozen prior to storage, reduced amount of histamine was found at 7.14 mg/100 g after 7 day storage at 25°C, only after decomposition became obvious. No histamine was found in any of the albacore samples stored in ice for 18 days.     

Determination of trans‐ and cis‐Urocanic Acid in Relation to Histamine,Putrescine, and Cadaverine Contents in Tuna (Auxis Thazard) at Different Storage Temperatures

Scombroid fish poisoning is usually associated with consumption of fish containing high levels of histamine. However, reports indicate that some cases have responded to antihistamine therapy while ingested histamine levels in these cases were low. Potentiation of histamine toxicity by some biogenic amines, and release of endogenous histamine by other compounds such as cis‐urocanic acid (UCA) are some hypotheses that have been put forth to explain this anomaly. Very little is known about the effects of storage conditions on the production of both UCA isomers and biogenic amines in tuna. Thus, the production of trans‐ and cis‐UCA, histamine, putrescine, and cadaverine in tuna during 15 d of storage at 0, 3, and 10 °C and 2 d storage at ambient temperature were monitored. The initial trans‐ and cis‐UCA contents in fresh tuna were 2.90 and 1.47 mg/kg, respectively, whereas the levels of putrescine and cadaverine were less than 2 mg/kg, and histamine was not detected. The highest levels of trans‐ and cis‐UCA were obtained during 15 d storage at 3 °C (23.74 and 21.79 mg/kg, respectively) while the highest concentrations of histamine (2796 mg/kg), putrescine (220.32 mg/kg) and cadaverine (1045.20 mg/kg) were obtained during storage at room temperature, 10 and 10 °C, respectively. Histamine content increased considerably during storage at 10 °C whereas trans‐ and cis‐UCA contents changed slightly. The initial trans‐UCA content decreased during storage at ambient temperature. Thus, unlike histamine, concentrations of trans‐ and cis‐UCA did not result in elevated levels during storage of tuna.     

QUALITY CHANGES IN ICE-STORED TROPICAL WIRE-NETTING REEF COD (EPINEPHELUS MERRA)

The sensory, bacteriological and biochemical qualities of tropical wire‐netting reef cod (Epinephelus merra) stored in melting ice were studied. The total bacterial load in immediately iced (II) fish was 10 ⁶ cfu /g on the 17th day of storage. The histamine formers were 10 ¹ cfu /g on the initial day, whereas the total lactics was 10 ¹ cfu /g on the third day. The total coliforms decreased from MPN 35 to 0 per gram, while the anaerobic sulfite reducers did not exhibit any consistent trend. The fish delayed for 6 h before icing or delayed iced (DI) fish exhibited bacterial loads of 10 ⁶ cfu /g on the third day, while the total lactics ranged from 10 ¹to 10 ² cfu /g and the histamine formers ranged from 10 ²to 10 ⁴ cfu /g. The total coliforms declined and the anaerobic sulfite reducers increased over a period of 5 days. The pH and hypoxanthine increased during the storage. Both the total volatile base nitrogen and trimethylamine nitrogen attained the maximum limit of acceptability at the end of the storage. The fatty acid content was 0.17% as oleic acid even on the 17th day in II fish. The II fish exhibited a longer shelf life (17 days) when compared to the shorter shelf life (3 days) exhibited by the DI fish.     

Biogenic and volatile amines in Chinese mitten crab (Eriocheir sinensis) stored at different temperatures

Biogenic amines, total volatile base nitrogen (TVB‐N) and trimethylamine nitrogen (TMA‐N) concentrations in the muscle of postmortem Chinese mitten crab stored at 4 and 20 °C were studied. Among five biogenic amines, histamine, tyramine, tryptamine, putrescine and cadaverine, histamine was the main biogenic amine formed during the storage time and reached highest levels of 91.22 mg kg^?1 after 72 h at 4 °C and 181.23 mg kg^?1 after 24 h at 20 °C. Histamine could be used a safety index for crab. The levels of other biogenic amines were less than 10 mg kg^?1 and fluctuated with storage time. TVB‐N and TMA‐N levels did not change greatly throughout 72 h storage at refrigerated temperature. TVB‐N and TMA‐N were not considered to be reliable indicators of the freshness of crab.     

Biogenic amine survey and organoleptic changes in fresh, stored, and temperature-abused bluefish (Pomatomus saltatrix).

Changes in histamine, putrescine, and cadaverine concentrations in bluefish filets (Pomatomus saltatrix) stored at 5, 10, and 15 degrees C were determined using high-performance liquid chromatography. An organoleptic assessment was conducted simultaneously with the biogenic amine analyses. The histamine levels found in fresh bluefish obtained from wholesale seafood distributors ranged between <1 ppm and 99 with an average of 39 ppm. Putrescine and cadaverine were not found in fresh bluefish. Fish fillets stored at each of the three temperatures developed histamine. The greatest accumulation of histamine was observed in fish stored at 15 degrees C, which developed histamine levels as high as 2,200 ppm. Putrescine levels increased at each temperature during storage. Cadaverine was present only in uninoculated bluefish stored at 15 degrees C. Histamine achieved higher levels in bluefish pieces inoculated with Morganella morganii, which demonstrates that bluefish support bacterial histamine formation. Histamine levels at each temperature exceeded the 50-ppm advisory level established by the Food and Drug Administration before 100% sensory rejection. Standard plate counts increased during storage of fish at all temperatures, but the correlation between histamine levels and standard plate count was not significant.     

Effects of aluminium foil and cling film on biogenic amines and nucleotide degradation products in gutted sea bream stored at 2±1 °C

Biogenic amines and nucleotide degradation products of sea bream stored in ice, wrapped in aluminium foil (WAF) and in cling film (WCF) at 2±1 °C were investigated by using a rapid HPLC method. Results obtained from this study showed that for household purposes packing fish in different materials has a little effect on the biogenic amines formation and nucleotide degradation products. The highest decrease of IMP content was observed for sea bream in WAF, followed by WCF. INO values showed a fluctuation and remained below the levels of 5.5 μmol/g for all storage conditions. Hx value constantly increased with the storage time during chilled storage. For all of the storage condition, K and Ki value increased linearly with storage time. At the end of the storage period, K, Ki, H and G value reached 60–76%, 65–81%, 30–54% and 89–173%, respectively. Among biogenic amines, (trimetylamine) TMA, putrescine, cadaverine, spermidine, spermine, tryptamine, tyramine, β-phenylalanine and histamine were detected during storage period. TMA and putrescine were observed to increase linearly during storage period. Histamine production was only found at the end of storage period. The highest histamine values for fish wrapped in aluminium foil were 6.4 mg/100 g and fish wrapped in cling film was 4.6 mg/100 g.     

GROWTH OF KLEBSIELLA PNEUMONIAE UH-2 AND PROPERTIES OF ITS HISTIDINE DECARBOXYLASE SYSTEM IN RESTING CELLS1

Klebsiella pneumoniae UH-2, previously isolated from spoiled skipjack tuna grew rapidly at 37C, 25C and 10C (generation times of 0.64, 0.79 and 4.57 h, respectively) and produced large amounts of histamine at these temperatures. Histamine was found during storage of resting cells at 2C, but growth did not occur. The K. pneumoniae UH-2 histidine decarboxylase system has in situ properties that may contribute to rapid histamine formation in fish tissue. The conversion of histidine to histamine in situ had an apparent Km = 0.98 mM at the optimum pH, 4.0, with ca. 70% of this activity at pH 6. These properties and recent reports of its prominence in fish spoilage suggest that K. pneumoniae could play a major role in the formation of histamine in susceptible species.     

Histamine accumulation and histamine-forming bacteria in Indian anchovy (Stolephorus indicus)

Accumulation of histamine, trimethylamine (TMA), and total volatile base nitrogen (TVB-N), as well as microbial population incidence in Indian anchovy (Stolephorus indicus) during storage in ice and at 15 and 35 °C were investigated. Histamine was as low as 1.9 mg/100 g in 15 days at ice storage, but it increased to 19.0 mg/100 g after 32 h at 15 °C. Histamine rapidly increased to 25.4 mg/100 g when stored at 35 °C for 8 h. TVB-N and TMA began to sharply increase after 11 days in ice storage, but abruptly increased after 16 and 8 h of storage at 15 and 35 °C, respectively. A high number of Enterobacteriaceae (10¹⁰–10¹¹ cfu/g) was detected and shown to be the dominant group of microbial flora during spoilage of Indian anchovy at both 15 and 35 °C. A total of 153 bacterial strains were selected from the prescreening step using various selective media. Only 75.8% of these selected isolates showed a positive reaction in Niven's differential medium, and 27.6% of the positive isolates were true histamine formers when confirmed by the enzymatic method. Prolific histamine formers were identified as Morganella morganii, Proteus vulgaris, and Enterobacter aerogenes, and produced high histamine content of 104.1–203.0 mg/100 ml. Optimum growth and histamine production of selected strains of these three species was at 35 °C in histamine evaluation broth (HEB) containing 0.5% NaCl, pH 5. E. aerogenes produced the highest histamine of 500 mg/100 ml at the optimum condition. All studied strains did not produce histamine at ⩾10% NaCl.     

CHEMICAL AND MICROBIOLOGICAL QUALITIES OF DRY-SALTED (LAKERDA) BONITO (SARDA SARDA, BLOCH 1793)

In this study, changes in the chemical and microbiological properties of bonito (Sarda sarda, Bloch 1793) salted by dry salting (lakerda) and stored for 6 months at 4 ± 1C were investigated. The fresh gutted and sliced bonitos were used as raw material for salting by using the ratio of fish to salt as 5:1 by weight. Salted fish remained in the liquid pickle formed by salt and the liquid extracted from the fish. This solution was not drained during the storage period. The product is described as “lakerda” in Turkey. In the fresh fish, total volatile basic nitrogen (TVB‐N) and trimethylamine nitrogen (TMA‐N) values were 11.21 mg/100 g and 1.19 mg/100 g, respectively. During the storage period, TVB‐N and TMA values increased slowly to 27.67 mg/100 g and 4.99 mg/100 g, respectively, at the end of the storage period. In the first month after salt processing, the total mesophilic bacteria count was 4.6 × 10² cfu/g. In the later months, mesophilic, psychotropic, coliform bacteria and yeast‐mold did not reproduce. According to the results, bonito salted by dry salting and stored at 4 ± 1C was in good quality in terms of chemical and microbiological properties during the storage period of 6 months.     

Control of Histamine‐Producing Bacteria and Histamine Formation in Fish Muscle by Trisodium Phosphate

Scombrotoxin fish poisoning remains the primary cause of seafood poisoning outbreaks despite preventive guidelines. The purpose of this study was to investigate the use of pH for the control of growth and histamine formation by histamine‐producing bacteria in fish muscle. We examined pH effects on growth and histamine formation in tuna fish infusion broth and in inoculated tuna and mahi‐mahi fish muscle. Histamine production was significantly less for all bacterial strains at pH 8.5 compared to pH 5.5 in tuna fish infusion broth with no significant difference in growth. Elevated pH due to phosphate treatment of fish muscle tissues significantly reduced histamine formation with no effect on the growth of histamine‐producing bacteria. This study revealed that phosphate treatment of mahi‐mahi and tuna fish muscle resulted in significantly lower histamine production over 4 d of storage at 10 °C. Phosphate treatment of fish muscle may serve as a secondary barrier in addition to FDA recommended time and temperature controls for reducing public health concerns of scombrotoxin fish poisoning.     

Endogenous level of acetic acid in yellowfin tuna (Thunnus albacares): a pilot study about a possible controversy on its residue nature

ABSTRACT

A method based on headspace solid-phase microextraction (HS-SPME) followed by GC-MS analysis was developed for the determination of underivatised acetic acid in fresh tuna fish muscle. Parameters such as the fibre selected and the extraction time and temperature were optimised and the linearity, detection limits and precision of the whole analytical procedure were assessed. The method was then applied to determine the acetic acid concentration in fresh yellowfin tuna muscles (Thunnus albacares) in order to evaluate the endogenous level and its variations during the shelf life under different storage conditions. A qualitative comparison was also made with variations in histamine levels to evaluate the possibility of the joint monitoring of acetic acid and histamine to identify fish stored in poor conditions. The caudal area always had a lower content of acetic acid than the ventral area, independent of the storage time and temperature. A difference was found between the 6- and 3-day time points and day 0 at a storage temperature of 8°C and between the 6-day time point and day 0 at a storage temperature of 0°C, independent of the anatomical area of the sampled tissue. The evaluation of acetic acid could represent an important approach in the field of food safety to detect the illicit use of acetic acid as an antibacterial preservative treatment or to eliminate the unpleasant smell of trimethylamine.     

Influence of the in vivo addition of alpha-tocopheryl acetate with three lipid sources on the lipid oxidation and fatty acid composition of Beluga sturgeon, Huso huso, during frozen storage

This study was conducted to investigate the effect of dietary alpha-tocopheryl acetate (vitamin E) and oil sources on fish flesh quality characteristics of Huso huso during frozen storage. Practical-type diets containing 0 or 250 mg vitamin E kg⁻¹ with three lipid sources, fish oil (FO), soybean oil (SO) and canola oil (CO), were fed to H. huso for 120 days. Fillet samples were analysed fresh or after storage at −18 ± 1 °C for 12 months. Replacement of FO by SO and CO in diets for H. huso significantly altered the fatty acid (FA) profile, which also influenced the FA composition during frozen storage. Dietary vitamin E had a significant effect on muscle vitamin E content and lipid oxidation during storage (P > 0.05). Oxidation was reduced for fish fed vitamin E and results showed that dietary vitamin E supplementation can slow down the level of lipid oxidation in H. huso muscles during frozen storage.     

Microbiological Properties and Biogenic Amines of Whole Pike‐Perch (Sander Lucioperca,Linnaeus 1758): A Perspective on Fish Safety During Postharvest Handling Practices and Frozen Storage

Abstract: The biogenic amines (tyramine, histamine, cadaverine, and puterscine) and microbiological properties (mesophilic, psychrotrophic, and Pseudomonas spp.) of whole pike‐perch (Sander lucioperca) was investigated during 2 d prestorage icing and 90 d frozen storage (−24 °C). At the end of ice storage, a noticeable increase only was found for puterscine level (P < 0.05), and microbial loads of fish increased in comparison with fresh fish (P < 0.05). During the frozen storage, as time passed, a continuous increase of biogenic amines and decrease of bacterial load (except for Pseudomonas spp. at the last 30 d) was detected (P < 0.05). The total contents of biogenic amines ranged from 6.24 to 91.76 μg/g during the investigated period. Puterscine was the major amine detected in pike‐perch and its concentration varied between 1.75 and 56.95 μg/g; due to a more step‐wise increase it was a good quality indicator. At the end of storage, all of the obtained values are below the tolerable maximum amounts based on available regulations. Based on biogenic amines content and microbial load, it could be concluded that pike‐perch can be consumed without any health risks after 2 d icing condition and 90 d frozen storage. Practical Application: Biogenic amines as one of the commonest forms of food intoxication occur in protein‐rich food such as fish. Short‐time icing during transportation is the simplest method to fish preserving for processing or long‐term storage. In this study formation of biogenic amines and bacterial changes in ungutted pike‐perch as highly demanded fish species for human consumption, during transportation and frozen storage was investigated. The results of the research can be advantageously used by fish industry. These findings suggest that the production and storage practices of fish in the retails condition could have acceptable food quality level.     

Survival of amine-forming bacteria during the ice storage of fish and shrimp

Survival of amine-forming bacteria during the ice storage of fish and shrimp was investigated up to 14 days of storage. On iced storage the total bacterial load was reduced to one log from an initial load of 10⁵ cfu g⁻¹ in fresh fish/shrimp due to cold shock. The total incidence of biogenic amine-forming bacteria was found to be 74·63% in fish and the same was recorded as 56·05% in shrimp. The amine-forming bacteria recorded were cadaverine- and putrescine-forming bacteria in fish/shrimp, and no histamine former was detected. Gram-negative, non-fermentative rods, viz. Alcaligenes. Flavobacterium, Acinetobacter. Shewanella andPseudomonas , were the predominant amine-forming bacteria during the ice storage of fish and shrimp, in addition to the only Gram-positive genus Micrococcus. The genera Aeromonas and Photobacterium also survived ice storage to a certain extent and may also be responsible for the formation of amines in fish and shrimp.     

Histamine and cadaverine production by bacteria isolated from fresh and frozen albacore (Thunnus alalunga).

Two hundred twenty-seven bacterial strains were isolated from fresh and frozen albacore stored either at -18 or -25 degrees C and investigated for their abilities to produce biogenic amines. As a preliminary screening, all 227 strains were tested in either Niven or Niven modified medium, which allowed the selection of 25 presumptive histamine-producing strains. High-pressure liquid chromatography revealed that only 10 of the 25 strains selected were able to produce low histamine concentrations (<25 ppm) in tryptic soy broth medium supplemented with 2% histidine. None of the 25 strains tested produced putrescine or spermine, whereas 6 strains produced spermidine. Histamine production by Stenotrophomonas maltophilia strain 25MC6 was not prevented at 4 degrees C, and the levels of this amine reached concentrations of 25.8 ppm after 6 days. Three S. maltophilia strains showed strong lysine-decarboxylating activity. Their cadaverine formation capacity was determined by high-pressure liquid chromatography in tryptic soy broth supplemented with 1% lysine; this revealed that the three S. maltophilia strains tested produced more than 700 ppm of cadaverine during the first 24 h of incubation at 37 degrees C. S. maltophilia strain 15MF, initially obtained from fresh albacore tuna, produced up to 2,399 ppm and 4,820 ppm of cadaverine after 24 and 48 h of incubation at 37 degrees C, respectively. To our knowledge, this is the first report on histamine and cadaverine production by strains of the species S. maltophilia, previously known as Pseudomonas and Xanthomonas maltophilia, isolated from fresh and frozen albacore tuna.     

Determination of histamine in fish products by capillary electrophoresis and ion‐pair liquid chromatography with diode‐array detection

A method for histamine determination in fish and fish products by capillary electrophoresis and ion‐pair liquid chromatography has been developed. Histamine was extracted from different fish samples with 10% trichloroacetic acid, and the filtrate was directly applied to capillary electrophoresis apparatus. A preliminary purification through a solid phases extraction cartridge (Sep‐pak C₁₈) was necessary for HPLC analysis. Histamine was detected ‘on‐line’ at 210 nm by both approaches. HPLC procedure allowed accurate (±2.2%) and repeatable (±1.6%) determination of histamine in fish products at appropriate levels (20–100 mg kg⁻¹) to meet current regulations. © 1999 Society of Chemical Industry