Enterotoxin C2 Production by S. aureus in Entree Salads

Staphylococcus aureus strain 361 was inoculated into salads containing chicken, macaroni, potato, or artificially flavored soy product. Egg was added to some. The pH averaged 6.2 in the control made with water dressing rather than vinegar, 4.7 with home-style cooked salad dressing, and 4.9 with commercial salad dressing. Following incubation at 37°C for 8 or 24 hr, pH, total counts, and enterotoxin C₂ were determined. Enterotoxin was detected at 8 hr in all macaroni and potato dressing types. At 8 hr, enterotoxin was not detected in most soy product or chicken salads. By 24 hr the lowest levels were in the soy product salads but all products contained toxin. When the inoculum level was reduced below the 10⁵ CFU per g used in the main study, enterotoxin production was delayed but occurred at 8 hr if the inoculum level was 10³.     

Bacterial Histamine Production as a Function of Temperature and Time of Incubation

Optimal temperature, lower temperature limit, extent, and rate of histamine production in a tuna fish infusion broth (TFIB) varied for the strains of Proteus morganii, Klebsiella pneumoniae, Hafnia alvei, Citrobacter freundii, and Escherichia coli studied. P. morganii and K. pneumoniae produced large quantities of histamine in a relatively short incubation period (<24 hr) at 15°C, 30°C, and 37°C; production was fastest at 37°C. H. alvei, C. freundii, and E. coli produced toxicologically significant levels of histamine (>2500 nmoles/ml) only at 30°C and 37°C on prolonged incubation (≥48 hr). At 72 hr of incubation, optimal temperature for histamine production was 37°C for E. coli and C freundii; 30°C for P. morganii strain 110SC2, K. pneumoniae, and H. alvei; and 15°C for P. morganii strain JM. The lower temperature limits for production of toxicologically significant levels of histamine in TFIB were 7°C for K. pneumoniae; 15°C for both P. morganii strains; and 30°C for H. alvei, C. freundii, and E. coli.     

Detection and identification of histamine-producing bacteria associated with harvesting and processing mahimahi and yellowfin tuna

Histamine poisoning is one of the most common chemically induced seafoodborne illnesses reported in the United States today. The causative agents are biogenic amines, commonly produced by gram-negative bacteria. The purpose of this study was to detect and identify histamine-producing bacteria associated with standard industry practices during the harvesting, receiving, and processing of mahimahi and yellowfin tuna in North Carolina. Twenty-nine composite samples were obtained from 18 mahimahi and 11 yellowfin tuna and analyzed for their histamine content. No sample analyzed exceeded 2 ppm histamine, the lower detection limit. Composite fish muscle and environmental samples were screened (n = 386) for the presence of histamine-producing bacteria. Twenty-six percent (145) of 549 isolates selected on the basis of their morphological characteristics tested positive on Niven's media. Sixty-three Niven-positive isolates were gram negative, and 58 were gram positive. Of the 43 isolates tested further, 5 were confirmed as histamine producers, and all 5 produced at low levels (< 250 ppm in 48 h at > 15 degrees C). Three gram-negative and two gram-positive isolates were identified as Enterobacter cloacae and Staphylococcus kloosii, respectively. This study revealed that gram-negative bacteria might not be solely responsible for histamine production in at-risk fish. The confirmation of histamine-producing bacteria demonstrates the potential risk for histamine production. However, no detectable levels were found in the composite fish muscle samples analyzed even though 60% of the yellowfin tuna harvested did not meet the U.S. Food and Drug Administration's regulatory hazard analysis critical control point guidelines for temperature reduction. Therefore, no seafood safety risks were found under the standard industry practices observed in this study.     

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.     

Factors Affecting Amine Production by a Selected Strain of Lactobacillus bulgaricus

Lactobacillus bulgaricus-52 showed maximum growth and amine production in MRS broth in 24 hr at 37°C. The optimum pH for the production of histamine, tyramine, and tryptamine by L. bulgaricus-52 was 5.0. Highest yields of the different amines were obtained in the absence of NaCI in the growth medium. A concentration of even 0.5% NaCI had a slight inhibitory affect on the synthesis of amines. A similar trend was observed in terms of amine production by L bulgaricus-52 in milk samples held at 37°C for different time intervals.     

Antibacterial activity of essential oil vapor for histamine-producing bacteria

In this study, we evaluated the antibacterial activity of essential oil vapors against histamine-producing bacteria Morganella morganii NBRC3848 and Raultella planticola NBRC3317. We measured the minimum inhibitory dose (MID) of 14 essential oils towards these two strains. Allyl isothiocyanate (AIT) and salicylaldehyde (SA) vapors showed higher antibacterial activity than the other 12 essential oil vapors. Both AIT and SA vapors suppressed growth of total aerobic bacteria and histamine-producing bacteria in bigeye tuna and mackerel meat during storage at 12°C. These vapors also inhibited histamine accumulation in bigeye tuna meat and mackerel meat. Thus, application of AIT and SA vapors is effective for preventing increase of histamine-producing bacteria and histamine formation in fish meat.     

Phytate Reduction in Brown Beans (Phaseolus vulgaris L.)

Brown beans (Phaseolus vulgaris L.) were subjected to treatments to evaluate effects of pH, temperature, CaCl₂, tannase and fermentation on degradation of phytate. Soaking was performed at 21°C, 37°C and 55°C at pH 4.0, 6.0, 6.4, 7.0, and 8.0. Optimal conditions for phytate degradation were pH 7.0 and 55°C. After soaking 4, 8 or 17 hr at these conditions 79%, 87% and 98% of phytate was degraded, respectively. Addition of tannase enhanced reduction of phytate. Fermentation of presoaked whole beans resulted in reduction of 88% of phytate after 48 hr.     

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.     

Effects of Frozen Storage on Survival of Staphylococcus aureus and Enterotoxin Production in Precooked Tuna Meat

This study investigated the survival of Staphylococcus aureus in precooked tuna meat for producing canned products during frozen storage (?20 ± 2 °C) as well as its growth and enterotoxin production at 35 to 37 °C after the storage. Samples (50 ± 5 g) of precooked albacore (loin, chunk, and flake) and skipjack (chunk and flake) tuna were inoculated with 5 enterotoxin‐producing strains of S. aureus at a level of approximately 3.5 log CFU/g and individually packed in a vacuum bag after 3 h incubation at 35 to 37 °C. Vacuum‐packed samples were stored in a freezer (?20 ± 2 °C) for 4 wk. The frozen samples were then thawed in 37 °C circulating water for 2 h and incubated at 35 to 37 °C for 22 h. Populations of S. aureus in all precooked tuna samples decreased slightly (<0.7 log CFU/g) after 4 wk of storage at ?20 ± 2 °C, but increased rapidly once the samples were thawed and held at 35 to 37 °C. Total S. aureus counts in albacore and skipjack samples increased by greater than 3 log CFU/g after 6 and 8 h of exposure to 35 to 37 °C, respectively. All samples became spoiled after 10 h of exposure to 35 to 37 °C, while no enterotoxin was detected in any samples. However, enterotoxins were detected in albacore loin and other samples after 12 and 24 h of incubation at 35 to 37 °C, respectively. Frozen precooked tuna meat should be used for producing canned tuna within 6 to 8 h of thawing to avoid product spoilage and potential enterotoxin production by S. aureus in contaminated precooked tuna meat.     

Storage Temperature Effect on Histamine Formation in Big Eye Tuna and Skipjack

Notable histamine formation (>30 mg/100g) was detected in big eye tuna (Thunnus obesus) and skipjack (Katsuwonus pelamis) captured in Azorean waters and stored for 1,3 and 6 days at 22,10 and 4°C, respectively. Higher levels (p<0.05) of histamine were produced by skipjack reflecting its higher histidine content. Measurements of pH or volatile basic nitrogen were not adequate for estimating the extent of histamine-related health hazard. Counts of histamine-forming bacteria increased during storage at 4 and 10°C, and histamine formation was suppressed at 4°C. Storage temperature and histidine content were the main factors controlling histamine levels in tuna.     

Bacillus macerans—a new potent histamine producing micro-organism isolated from Italian cheese

We describe the histamine activity of Bacillus macerans 1(3), isolated from a sample of italian cheese during seasoning. Bacillus macerans 1(3) did not hydrolyse gelatine, and had a clear positive indol production. On the other hand we have observed growth with 5% NaCl concentration and with pH = 5. This is a halotolerant bacteria. Strain 1(3) has a positive reaction in Niven medium at 24 and 48 h. A histamine formation was detected at all temperatures studied (43, 37, 30, 22 and 4°C). The maximum histamine formation and the maximum bacterial growth were detected at 30°C, with 4285 μg of histamine ml^-1 of quantification broth.     

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.     

Changes in histamine and microbiological analyses in fresh and frozen tuna muscle during temperature abuse

Temperature abuse of tuna (Thunnus alalunga) was carried out in order to assess the histamine buildup in fish-processing facilities where fish can be exposed to high temperatures for short periods of time. Histamine production was studied in tuna loins under different storage and abuse conditions. Tuna was stored at 0-2°C, 3-4°C, and 6-7°C, and abused for 2 h daily at 20°C and 30°C for 7-12 days. Loins abused at 30°C for 2 h daily contained potentially toxic histamine concentrations (67-382 mg kg^-1) when stored at a low refrigeration temperature (0-2°C), whereas when stored at 6-7°C, the loins contained highly toxic histamine concentrations (544.5-4156.6 mg kg^-1). Lower histamine concentrations (23-48 mg kg^-1 in loins stored at 0-2°C and 124.7-2435.8 mg kg^-1 in loins stored at 6-7°C) were observed in temperature-abused loins that were initially frozen. An increase over time was observed in most microbial counts tested. Bacteria isolated from the temperature-abused loins showed a varied ability of histamine production, with Morganella morganii, Klebsiella oxytoca, Staphylococcus hominis, and Enterococcus hirae being the most active histamine-producing bacteria.     

Using lactic acid bacteria for developing novel fish food products

Lactic acid bacteria‐induced fermentation of fish was tested with the purpose of developing a novel fish food product with desired meat properties. Three selected lactic acid bacteria strains, isolated from lightly preserved fish products, were inoculated into minced frozen fillet of yellowfin tuna, and changes in fish properties were observed during 52 days of storage at 8 °C. Organoleptic, bacteriological and biochemical studies (pH, histamine, TVB‐N, malonaldehyde) and shelf‐life measurements suggest that Leuconostoc mesenteroides could be considered as a possible starter culture for fish fermentation, but additional experiments should be performed. © 2000 Society of Chemical Industry     

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.     

Effect of Sodium Erythorbate on Residual Nitrite, pH and Bacterial Growth in Liver Sausage Formulated with Different Levels of Mechanically Separated Pork

Some microbiological, chemical, and physical characteristics of liver sausage formulated with 0, 25, 50, and 75% mechanically separated pork (MSP), 156 ppm sodium nitrite and 0 or 550 ppm sodium erythorbate were studied during refrigerated storage (5°C, 6 days) and subsequent elevated-temperature holding (20–24°C, 48 hr). Growth of inoculated Clostridium sporogenes was inhibited by 156 ppm nitrite in all sausages even after 48 hr at 20–24°C, regardless of MSP level. Growth of aerobic mesophiles and facultative anaerobes at 20–24°C was also inhibited in sausage formulated with 156 ppm nitrite and 550 ppm erythorbate. Erythorbate addition and increased MSP levels resulted in higher Hunter a color values. Residual nitrite, pH values, and total iron content of liver sausage increased with increasing MSP content.     

Histamine, Cadaverine and Putrescine Forming Bacteria from Ripened Spanish Semipreserved Anchovies

Histidine decarboxylase activity and production of putrescine and cadaverine were assessed in 200 bacterial isolates from ripened semipreserved Spanish anchovies. Highest levels of histidine decarboxylase activity were observed in Morganella morganii, with 2336.74 ± 356.32 ppm of histamine, produced in laboratory media, after 24 hr incubation at 37°C. Microorganisms producing histamine also produced detectable putrescine and cadaverine. The Niven medium was used to evaluate the 200 isolates for histidine decarboxylase activity, after incubation 24 hr at 37°C. An enzymic technique was used to distinguish false positives and to quantify bacterial histamine levels. The number of microorganisms was the most important factor in the accumulation of histamine.     

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.     

A rapid method for detection of histamine-producing bacteria

A method for detecting histamine-producing bacteria is described. The method is based on automated conductance measurements in a histidine-containing medium (HDB) incubated at 25°C, with a large and distinct increase in the conductance being indicative of the presence of histamine-producing bacteria. An initial low pH (5.5) is optimal for measuring the histidine decarboxylating activity of Morganella morganii, but clear results are obtained at higher pH values (6.0–7.0) as well. It is shown that the histidine decarboxylating activity of M. morganii is unaffected by the presence of non-histamine producing bacteria. Using this new method in the examination of mackerel spoiled at high temperatures, the results obtained indicated the presence of large amounts of histamine-producing organisms. These were subsequently isolated and identified as belonging to the family Enterobacteriaceae.     

Inhibition of Clostridium sporogenes PA 3679 and Natural Bacterial Flora of Cooked Vacuum-Packaged Bratwurst by Sodium Acid Pyrophosphate and Sodium Tripolyphosphate With or Without Added Sodium Nitrite

Survival and growth of inoculated Clostridium sporogenes PA3679 and natural aerobic and anaerobic bacterial flora were studied in refrigerated (5°C) and subsequently temperature abused (24–25°C), cooked, vacuum-packaged bratwurst containing 0.5% sodium acid pyrophosphate (SAPP) or sodium tripolyphosphate (STPP) with or without sodium nitrite. Phosphates alone or combined with nitrite did not affect aerobic bacterial counts but resulted in reduced clostridial and anaerobic counts at 5°C. Upon temperature abuse, inhibition of all bacteria by SAPP was significant (P<0.05) for up to 48 hr and greatly enhanced by 100 ppm but not by 50 ppm sodium nitrite, whereas STPP lost its antimicrobial properties after 24 hr. Soluble orthophosphate levels had a positive correlation with bacterial inhibition in SAPP-treated bratwurst.