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.     

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.     

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.     

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.     

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.     

Histamine and biogenic amine production by Morganella morganii isolated from temperature-abused albacore

Histamine-producing bacteria were isolated from albacore stored at 0, 25, 30, and 37 degrees C. They were screened using Niven's differential medium, and their histamine production was confirmed by high-pressure liquid chromatography analysis. The optimum temperature for growth of histamine-producing bacteria was 25 degrees C. The bacterium producing the highest level of histamine was isolated from fish abused at 25 degrees C. It was identified as Morganella morganii by morphological, cultural, biochemical, and antimicrobial characteristics and by the Vitek microbial identification system. The M. morganii isolate was inoculated into tuna fish infusion broth medium, and the effect of temperature was determined for microbial growth and formation of histamine and other biogenic amines. The isolate produced the highest level of histamine, 5,253 ppm, at 25 degrees C in the stationary phase. At 15 degrees C, histamine production was reduced to 2,769 ppm. Neither microbial growth nor histamine formation was detected at 4 degrees C. To determine whether the isolate can also produce other biogenic amines that can potentiate histamine toxicity, production of cadaverine, putrescine, serotonin, tryptamine, tyramine, phenylethylamine, spermidine, and spermine by the isolate was also monitored. Cadaverine, putrescine, and phenylethylamine were detected with microbial growth in the tuna fish infusion broth medium. The optimum temperature for cadaverine, putrescine, and phenylethylamine formation was found to be 25 degrees C, as it was for histamine.     

Growth, inactivation and histamine formation of Morganella psychrotolerans and Morganella morganii - development and evaluation of predictive models

Mathematical models for growth, heat inactivation and histamine formation by Morganella psychrotolerans and Morganella morganii were studied to evaluate the importance of these bacteria in seafood. Curves for growth and histamine formation by M. psychrotolerans in broth and seafood were generated at constant and changing storage temperatures (n=12). Observed and predicted times to formation of 100, 500 and 2000 ppm histamine were used for evaluation of an existing M. psychrotolerans histamine formation model [Emborg, J., Dalgaard, P., 2008-this issue-this issue. Modelling and predicting the growth and histamine formation by Morganella psychrotolerans. International Journal of Food Microbiology. doi:10.1016/j.ijfoodmicro.2008.08.016] Growth rates for M. psychrotolerans and M. morganii were determined at different constant temperatures from 0 degrees C to 42.5 degrees C whereas heat inactivation was studied between 37.5 degrees C and 60 degrees C. A M. morganii growth and histamine formation model was developed by combining these new data (growth rate model) and data from the existing literature (maximum population density and yield factor for histamine formation). The developed M. morganii model was evaluated by comparison of predicted growth and histamine formation with data from the existing literature. Observed and predicted growth rates for M. psychrotolerans, at constant temperatures, were similar with bias- and accuracy factor values of 1.15 and 1.45, respectively (n=11). On average times to formation of critical concentrations of histamine by M. psychrotolerans were acceptably predicted but the model was not highly accurate. Nevertheless, predictions seemed useful to support decisions concerning safe shelf-life in relation to formulation, storage and distribution of chilled seafood. Parameters for the effect of temperature on growth and inactivation of M. psychrotolerans and M. morganii differed markedly with Tmin of -8.3 to -5.9 degrees C vs. 0.3 to 2.8 degrees C, Topt of 26.0 to 27.0 degrees C vs. 35.9 to 37.2 degrees C and Tmax 32.0 to 33.3 degrees C vs. 44.0 to 47.4 degrees C, D(50 degrees C) of 5.3 min vs. 13.1 min and z-values of 6.8 degrees C and 7.2 degrees C. At temperatures above approximately 15 degrees C M. morganii grew faster than M. psychrotolerans. Bias- and accuracy factor-values of 1.41 and 2.44 (n=93) showed the predicted growth of M. morganii to be faster than previously observed in fresh fish and broth. In agreement with this, predicted times to formation of critical histamine concentrations by M. morganii were on average shorter than observed in fresh fish. A combined model was suggested to predict histamine formation by both psychrotolerant and mesophilic Morganella during storage of fresh fish between 0 degrees C and 37 degrees C.     

Bacterial Growth and Histamine Production on Vacuum Packaged Tuna

Vacuum and nonvacuum packaged samples of tuna or tuna spiked with Klebsiella oxytoca T₂, Morganella morganii JM or Hafnia alvei T₈ were stored at 2°C and 10°C and examined for bacterial growth and histamine production on day 3, 6, 10 and 15. Unlike those stored at 2°C, tuna samples stored at 100°C showed significant bacterial growth over time and had high levels of histamine (>200 mg/100g tuna on day 15). Vacuum packaging did not show any beneficial effect in controlling bacterial growth and histamine production on tuna samples at these two temperatures. Low temperature storage was more effective than vacuum packaging in controlling histamine production by the bacteria tested.     

Growth and histamine formation of Morganella morganii in determining the safety and quality of inoculated and uninoculated bluefish (Pomatomus saltatrix).

The objective of this study was to determine the effect of normal microflora and Morganella morganii on histamine formation and olfactory acceptability in raw bluefish under controlled storage conditions. Fillets inoculated with and without M. morganii were stored at 5, 10, and 15 degrees C for 7 days. Microbial isolates from surface swabs were identified and screened for histidine decarboxylase activity. Olfactory acceptance was performed by an informal sensory panel. Histamine levels were quantified using high-performance liquid chromatography and fluorescence detection. While olfactory acceptance decreased, histamine concentration and bacterial counts increased. Storage temperature had a significant effect on histamine levels, bacterial counts, and olfactory acceptance of the bluefish. Inoculation with M. morganii had a positive significant effect on histamine formation for bluefish held at 10 and 15 degrees C (P < 0.0001). The results of the study will serve in supporting U.S. Food and Drug Administration (FDA) regulations regarding guidance and hazard levels of histamine in fresh bluefish.     

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.     

Biogenic Amine Formation and Microbiological Quality of Anchovy (Engraulis encrasicolus) Treated with Lavender and Lemon Balm Ethanol Extracts

Biogenic amine formation and bacterial growth of vacuum‐packed European anchovy treated with 1% lemon balm (LB) or lavender (LD) ethanol extracts was investigated. The treated samples were stored at 2 ± 1 °C for 11 d and analyzed for biogenic amines, total viable count, fecal coliform count, and lactic acid bacterial count. The analyses were done on 0, 4, 7, 9, and 11 d of storage. Both LB and LD extracts proved effective in reducing most biogenic amines, with histamine reduced by 3‐fold compared to control. The calculated biogenic amine index showed that LB extract was the most effective in inhibiting the biogenic amine formation. The reduction in total viable count was low and the bacterial contamination reached more than 7 log cfu/g at the end of the storage period for all the samples. Moreover, the reduction in fecal coliform and lactic acid bacterial count by LB and LD extracts was below 1 log cfu/g. Treatment of European anchovy with LB and LD extracts proved to be a very effective preservation technique to reduce the biogenic amine formation and lower the risk of histamine fish poisoning but is not effective in reduction of bacterial load. Consequently, LB and LD extracts could be used to reduce the biogenic amine formation which provides additional safety prior to spoilage for histamine sensitive fish species.     

Significant histamine formation in tuna (Thunnus albacares) at 2 degrees C--effect of vacuum- and modified atmosphere-packaging on psychrotolerant bacteria

Occurrence and importance of psychrotolerant histamine producing bacteria in chilled fresh tuna were demonstrated in the present study. The objective was to evaluate microbial formation of histamine and biogenic amines in chilled fresh tuna from the Indian Ocean and stored either vacuum-packed (VP) or modified atmosphere-packed (MAP). Firstly, biogenic amines and the dominating microbiota were determined in VP tuna involved in an outbreak of histamine fish poisoning in Denmark. Secondly, the microbiota of fresh MAP tuna was evaluated at the time of processing in Sri Lanka and chemical, microbial and sensory changes were evaluated during storage at 1-3 degrees C. To explain the results obtained with naturally contaminated tuna the effect of VP and MAP on biogenic amine formation by psychrotolerant bacteria was evaluated in challenge tests at 2 degrees C and 10 degrees C. The VP tuna that caused histamine fish poisoning had a histamine concentration of >7000 mg/kg and this high concentration was most likely produced by psychrotolerant Morganella morganii-like bacteria or by Photobacterium phosphoreum. Similar psychrotolerant M. morganii-like bacteria dominated the spoilage microbiota of fresh MAP tuna with 60% CO2/40% N2 and formed >5000 mg/kg of histamine after 24 days at 1.7 degrees C. These psychrotolerant bacteria were biochemically similar to M. morganii subsp. morganii and their 16S rDNA (1495 bp) showed >98% sequence similarity to the type strain of this species. Toxic concentrations of histamine were produced at 2.1 degrees C in inoculated VP tuna by both the psychrotolerant M. morganii-like bacteria (7400+/-1050 mg/kg) and P. phosphoreum (4250+/-2050 mg/kg). Interestingly, MAP with 40% CO2/60% O2, in challenge tests, had a strong inhibitory effect on growth and histamine formation by both the psychrotolerant M. morganii-like bacteria and P. phosphoreum. In agreement with this, no formation of histamine was found in naturally contaminated fresh MAP tuna with 40% CO2/60% O2 during 28 days of storage at 1.0 degrees C. To reduce current problems with histamine fish poisoning due to VP tuna it is suggested, for lean tuna loins, to replace vacuum packaging with MAP containing approximately 40% CO2 and approximately 60% O2.     

鱼类腐败菌腐败能力测定方法

通过分析比较接种腐败菌的大黄鱼无菌鱼块和灭菌鱼汁,在贮藏中感官、挥发性盐基氮(total volatile base nitrogen,TVBN)、三甲胺(trimethylamine,TMA)和腐败菌的变化,以及腐败菌生长动力学参数和腐败代谢产物的产量因子(YTVBN/CFU 和YTMA/CFU),探讨无菌鱼块和灭菌鱼汁两种腐败能力的测定方法。结果表明：接种腐败希瓦氏菌的无菌鱼块和灭菌鱼汁的货架期分别为162h 和132h,此时的TVBN 含量分别为32.16mg/100g 和29.64mg/100mL,TMA含量为9.31mg/100g 和0.99mg/100mL,腐败希瓦氏菌菌数为8.67 lg(CFU/g)和8.56 lg(CFU/mL),产量因子YTVBN/CFU为2.58 × 10-10mg TVBN/CFU 和1.98 × 10-10mL TVBN/CFU,产量因子YTMA/CFU 为2.12 × 10-10mg TMA/CFU 和1.27× 10-11mL TMA/CFU。TMA 作为接种鱼汁的理化指标是不可靠的,而TVBN 可以作为接种鱼汁的理化指标。接种腐败希瓦氏菌的无菌鱼块和灭菌鱼汁产量因子YTVBN/CFU 的相对误差为23.64%,因此灭菌鱼汁作为腐败菌腐败能力测定方法具有一定的可靠性。     

Influence of storage temperature on the quality of salted mackerel (Rastrelliger kangurta) and pink perch (Nemipterus japonicus)

Dry-salted mackerel and pink perch were stored at two temperatures: ambient (26·8 ± 3·3°C) and 2·5 ± 1°C. Changes in moisture content, salt content, water activity (a_w), peroxide value (PV), free fatty acid content (FFA), total volatile base nitrogen (TVBN) content, halophilic bacterial count and sensory scores for overall acceptability were studied. Loss of moisture and absorption of salt were considerably higher in the products stored at ambient temperature. The decrease in a_w was more pronounced at ambient temperature than at the lower temperature. Although the chemical indices of freshness (PV, FFA and TVBN) and the halophilic counts showed increasing trends, they were considerably lower in the products stored at the lower temperature. Sensory evaluation for overall acceptability indicated that storage at the lower temperature could considerably extend the shelf-life of salted fish.     

Histamine-related hygienic qualities and bacteria found in popular commercial scombroid fish fillets in Taiwan

To determine the histamine-related hygienic qualities and bacteria of scombroid fish fillets sold in traditional retail markets, 61 samples were collected from northern and southern Taiwan. It was found that the content of volatile base nitrogen in most samples was below 25 mg/100 g, which is the regulatory level in Taiwan. The ratio of unacceptable samples/total samples for aerobic plate count and Escherichia coli was 100% and 15% in northern samples and 100% and 20% in southern samples, respectively, compared with the requirements of hygienic standards. The average content of various biogenic amines in all samples were lower than 3 mg/100 g, except for histamine average content (4.6 mg/100 g) in southern samples. Among southern samples, four samples contained 12.8 to 28.8 mg/100 g histamine, which is more than 5 mg/100 g that is the allowable limit suggested by the U.S. Food and Drug Administration. Furthermore, 14 bacterial strains were isolated from sailfish fillets on a selective medium for histamine-forming bacteria. These presumptive histamine-forming strains, such as Proteus, Enterobacter, Klebsiella, Rahnella, and Acinetobacter, have been identified and found to produce 20 to 2,000 ppm histamine after incubating at 37 degrees C for 24 h.     

HISTAMINE PRODUCTION BY FOOD-BORNE BACTERIAL SPECIES12

A total of 112 bacterial strains representing 38 species were tested for their potential to elicit food poisoning outbreaks via histamine formation in foods. Proteus morganii and Enterobacter aerogenes displayed a quantitative superiority in terms of histamine production on a trypticase-soy broth-histidine (TSBH) medium and a tuna fish infusion broth (TFIB). When bacteria were incubated under standardized conditions in TSBH medium, histamine accumulated to levels exceeding 50 nmoles/ml of media with a total of 23 strains, including 13 of 15 P. morganii strains, 3 of 3 E. aerogenes strains, 3 of 12 Hafnia alvei strains, 1 of 4 Providencia alcalifaciens strains, 1 of 5 Enterobacter cloacae strains, 1 of 1 Proteus rettgeri strains, and 1 of 1 Citrobacter diversus strains. However, only 8 of the 15 P. morganii strains and the 3 E. aerogenes strains were capable of generating histamine in excess of 200 nmoles/ml in the TSBH medium. Of the 23 strains capable of appreciable histamine production in TSBH medium, P. morganii and E. aerogenes were, by far, the most prolific histamine producers in TFIB. Of the organisms tested, only P. morganii and E. aerogenes would appear to have the capability of forming sufficient histamine in scombroid fish products to elicit food poisoning outbreaks.     

Effect of salt concentrations and drying methods on the quality and formation of histamine in dried milkfish (Chanos chanos)

The effects of salt concentrations (0-15.0%) and drying methods on the quality of dried milkfish were studied. The results showed that the levels of aerobic plate counts, total coliform, water activity, moisture contents, total volatile basic nitrogen (TVBN) and thiobarbituric acid (TBA) of the dried milkfish samples prepared with the same drying method decreased with increased salt concentrations. The samples prepared with the cold-air drying method had better quality in term of lower TVBN and TBA values than those of samples prepared with other drying methods. The histamine contents in all samples, except two, prepared with various salt concentrations by different drying methods were less than 1.9 mg/100 g. Two unsalted samples prepared with hot-air drying at 35 °C and sun drying methods were found to contain histamine at levels of 249.7 and 67.4 mg/100 g, respectively, which were higher than the potential hazard level of 50 mg/100 g.     

温度和姜精油对金枪鱼品质影响及生物胺相关性

将分别贮藏于不同温度(0、4、15℃)条件下的金枪鱼样品,以及4℃条件下用姜精油作为生物抗氧化剂处理过的样品作为研究对象,定期测定金枪鱼生物胺(腐胺Put、尸胺Cad、组胺His、酪胺Tyr)、反式尿刊酸的含量、挥发性盐基氮(total volatile basic nitrogen,TVB-N)值、K值和菌落总数,研究温度和姜精油对金枪鱼生物胺含量的影响及组胺与反式尿刊酸的关系。结果表明,温度对金枪鱼生物胺生成有较大影响,0℃条件下生物胺含量显著低于其他贮藏温度,到第6天时组胺、酪胺、腐胺和尸胺含量分别为28.25、5.47、14.84 mg/kg和17.05 mg/kg,而此时15℃条件下金枪鱼组胺、酪胺、腐胺和尸胺含量分别为135.4、14.63、29.49 mg/kg和41.55 mg/kg;生物胺中组胺含量最高,而酪胺含量始终处于低水平状态,即使在15℃条件下到第7天时其含量仍为16.24 mg/kg;结果同时表明,生物抗氧化剂对生物胺的产生和微生物生长有一定的抑制作用,并能有效地延缓蛋白质和ATP降解。相关分析和回归分析表明,组胺、酪胺、尸胺、与TVB-N值、K值和菌落总数高度相关,其相关系数r在0.804~0.981之间,且生物胺和菌落总数、TVB-N值之间存在重要对应关系,反式尿刊酸对组胺的产生影响不大,两者之间相关性较弱,在4℃时其相关系数r为0.630。     

Formation of biogenic amines in herring and mackerel

The formation of biogenic amines (histamine, cadaverine, putrescine and spermidine) was followed during vacuum packed storage at 2 degrees C or 10 degrees C in the scombroid fish mackerel and in the non-scombroid fish herring. Also the changes in the content of free amino acids and in the organoleptic and microbiological qualities were followed. At 10 degrees C the amine contents were 2-20 times higher at the time of rejection as compared with samples stored at 2 degrees C. In herring and mackerel similar amounts of histamine were accumulated, whilst cadaverine was formed at much higher levels in mackerel compared with herring. The high contents of cadaverine in mackerel can possibly explain why mackerel and not herring are often implicated in incidents of scombrotoxic poisoning.     

Biogenic amines and sensory changes associated with the microbial flora of Mediterranean gilt-head sea bream (Sparus aurata) stored aerobically at 0, 8, and 15 degrees C.

Changes in the concentrations of tyramine, agmatine, putrescine, cadaverine, spermidine, tryptamine, spermine, histamine, and trimethylamine were studied in parallel with the development of the microbial population during the storage of Mediterranean gilt-head sea bream (Sparus aurata) at three temperatures (0, 8, 15 degrees C). Changes in sensory scores were also recorded. Pseudomonads and H2S-producing bacteria were the dominant microorganisms. Enterobacteriaceae and lactic acid bacteria were also present in the fish microflora. Among the biogenic amines, putrescine and cadaverine were detected when pseudomonads exceeded 10(6) to 10(7) CFU/g. Histamine was produced only in samples stored at 15 degrees C. Tyramine, tryptamine, agmatine, and trimethylamine were absent regardless of the storage temperature.