Growth and toxigenesis of C. botulinum type E in fishes packaged under modified atmospheres

Modified atmosphere packaging of fresh fish is used to market high quality products in some European countries. The potential risk of C. botulinum growth in these extended shelf-life foods is still a concern; especially since toxigenesis may precede organoleptic spoilage. This paper will present toxigenic data from rockfish, salmon and sole muscle tissues which were inoculated with a pool of non-proteolytic C. botulinum type E at seven levels (10⁻² −10⁴ spores/sample), and stored under vacuum and 100% CO₂, at incubation temperatures between 30 and 4°C, for up to 60 days. Factorial experimental design allowed predictive formulae to be developed able to describe the lag time prior to C. botulinum toxigenesis and the probability of one spore to initiate toxigenesis based upon the storage conditions. Accurate characterization of the microbial ecology of C. botulinum in modified atmosphere-packaged fish, will support safe exploitation of these packaging systems in the market place, and identify critical control points for potential product or process abuses.     

Probabilistic representation of the exposure of consumers to Clostridium botulinum neurotoxin in a minimally processed potato product

We have examined the potential of a well-specified, minimally processed potato product as a vehicle for the exposure of consumers to Clostridium botulinum neurotoxin. The product is a relatively simple combination of raw potato flakes, flour, starch and other minor ingredients and has an extended lifetime under refrigeration conditions. A combination of information and data, from a variety of sources that includes the manufacturer, has shown that the product is particularly safe with respect to non-proteolytic C. botulinum hazards. The model concentrates on a simple end point, the toxicity of an individual retail unit of the product at the point of consumer preparation, which is related to an individual risk. The probabilistic analysis was built using Bayesian Belief Network (BBN) techniques.     

Effects of electron-beam irradiation on the shelf life, microbial populations and sensory characteristics of summer truffles (Tuber aestivum) packaged under modified atmospheres

The effects of two doses of electron-beam irradiation (1.5 kGy and 2.5 kGy) on the microbial populations (total mesophilic aerobes, Pseudomonas genus, Enterobacteriaceae family, molds and yeasts) and sensory characteristics of Tuber aestivum packaged under modified atmospheres were monitored immediately after treatment, and weekly during 42 days of storage at 4 °C. Treatment with 1.5 and 2.5 kGy reduced the pseudomonads populations by 4.3 and 5.5 logs, respectively. Enterobacteriaceae counts decreased by 5.4 logs with the 1.5 kGy dose and counts below the detection limit (<1.0 log cfu/g) were obtained with the 2.5 kGy dose. Lactic acid bacteria and yeasts were less affected by the ionizing radiation treatments and they became the dominant microbial populations throughout storage with microbial counts up to 7.1 log cfu/g. The carbon dioxide levels inside the packages containing irradiated truffles were lower than those of the non-irradiated ones, suggesting a decrease in the respiration rate of the treated ascocarps. The treatments with 1.5 and 2.5 kGy e-beam did not negatively affect the sensory characteristics of truffles, but a visible superficial yeast growth was detected in truffles irradiated with 1.5 kGy at the end of their shelf life (day 28). Treatment with 2.5 kGy e-beam has prolonged the shelf life to 42 days, compared with 21 days for the untreated samples.     

Effects of electron-beam and gamma irradiation treatments on the microbial populations, respiratory activity and sensory characteristics of Tuber melanosporum truffles packaged under modified atmospheres

The effects of electron-beam or gamma irradiation (doses of 1.5 kGy and 2.5 kGy of either one) on the microbial populations, respiratory activity and sensory characteristics of Tuber melanosporum packaged under modified atmospheres were monitored immediately after treatment, and subsequently every seven days during 35 days of storage at 4 °C. Treatments with 1.5 and 2.5 kGy reduced the total mesophilic aerobes counts respectively by 4.3 and 5.6 log cfu/g for electron-beam treatment, and by 6.4 and 6.6 log cfu/g for gamma irradiation. Other microbial groups studied (Pseudomonas genus, Enterobacteriaceae family, lactic acid bacteria, mesophilic aerobic spores, molds and yeasts) were not detected after the treatments. A decrease in the respiratory activity was detected in all the irradiated batches, indicating that the carbon dioxide levels were lower and the oxygen levels higher than those of the non-irradiated ones. Two species of yeasts, Candida sake and Candida membranifaciens var. santamariae, survived the irradiation treatments and became the dominant microbial populations with counts of up to 7.0 log cfu/g. The growth of these microorganisms was visible on the surface of irradiated truffles from day 21 onwards, affecting the flavor and the general acceptability of the ascocarps. Moreover, a watery exudate was detected in the treated truffles from the third week onwards, so the application of irradiation treatments in doses equal to or above 1.5 kGy did not preserve the quality characteristics of T. melanosporum truffles beyond 28 days.     

Effect of media,additives, and incubation conditions on the recovery of high pressure and heat-injured Clostridium botulinum spores

The effect of additives and post-treatment incubation conditions on the recovery of high pressure and heat-injured (i.e., processed at 620 MPa and 95 and 100 °C for 5 min) spores of Clostridium botulinum strains, 62-A (proteolytic type A) and 17-B (nonproteolytic type B) was studied. High pressure and heat-injured spores were inoculated into TPGY (Trypticase–Peptone–Glucose–Yeast extract) anaerobic broth media containing additives (lysozyme, l-alanine, l-aspartic acid, dipicolonic acid, sodium bicarbonate, and sodium lactate) at various concentrations (0–10 μg/ml) individually or in combination. The spore counts of high pressure and heat-injured 62-A and 17-B recovered from TPGY broth containing lysozyme (10 μg/ml) incubated for 4 months versus that recovered from peptone–yeast extract–glucose–starch (PYGS) plating agar containing lysozyme (10 μg/ml) incubated under anaerobic conditions for 5 days were also compared. None of the additives either individually or in combination in TPGY broth improved recovery of injured spore enumeration compared to processed controls without additives. Addition of lysozyme at concentrations of 5 and 10 μg/ml in TPGY broth improved initial recovery of injured spores of 17-B during the first 4 days of incubation but did not result in additional recovery at the end of the 4 month incubation compared to the processed control without lysozyme. Adding lysozyme at a concentration of 10 μg/ml to PYGS plating agar resulted in no effect on the recovery of high pressure and heat-injured 62-A and 17-B spores. The recovery counts of high pressure and heat-injured spores of 62-A and 17-B were lower (i.e., <1.0 log units) with PYGS plating agar compared to the MPN method using TPGY broth as the growth medium.     

The two-component system CBO2306/CBO2307 is important for cold adaptation of Clostridium botulinum ATCC 3502

Clostridium botulinum is a notorious foodborne pathogen. Its ability to adapt to and grow at low temperatures is of interest for food safety. Two-component systems (TCSs) have been reported to be involved in cold-shock and growth at low temperatures. Here we show the importance of TCS CBO2306/CBO2307 in the cold-shock response of C. botulinum ATCC 3502. The relative expression levels of the cbo2306 and cbo2307 were up to 4.4-fold induced in the cold-shocked cultures but negatively regulated in the late-log and stationary growth phase in relation to early logarithmic growth phase in non-shocked cultures. Importance of the CBO2306/CBO2307 in the cold stress was further demonstrated by impaired growth of insertional cbo2306 or cbo2307 knockout mutants in relation to the wild-type strain ATCC 3502. The results suggest that the TCS CBO2306/CBO2307 is important for cold-shock response and adaptation of C. botulinum ATCC 3502 to low temperature.     

某企业婴儿肉毒中毒乳粉相关样品中肉毒梭菌的分离与分型

目的 对从某企业获取的30批次婴儿配方乳粉样品进行肉毒毒素和肉毒梭菌检测,对分离到的1株B型肉毒梭菌进行全基因组测序分析。方法 参照GB 4789.12—2016《食品安全国家标准 食品微生物学检验 肉毒梭菌及肉毒毒素检验》对样品进行肉毒梭菌分离及肉毒毒素分型实验;对分离到的菌株进行全基因组测序,并分析菌株的遗传特征。结果 30批次样品中均未检出肉毒毒素;将增菌液进行小鼠腹腔注射后,4批次乳粉样品出现了典型小鼠肉毒中毒症状,但仅从1批次乳粉样品中分离到肉毒梭菌。基因组测序分析显示,该菌为Ⅰ群B型肉毒梭菌,毒素基因簇为Ha型,毒素基因为B2亚型。结论 针对背景微生物复杂的婴儿配方乳粉中肉毒梭菌检测,不应以菌种分离作为金标准,而应以增菌液小鼠毒性实验结合肉毒抗血清保护实验作为确认方法。全基因组测序可对分离菌种进行精准鉴定和相关遗传特征分析,为中毒事件处理提供可靠的技术支撑。     

Effect of modified atmosphere and temperature abuse on the growth from spores and cereulide production of Bacillus weihenstephanensis in a cooked chilled meat sausage

The effect of modified atmosphere packaging (MAP) on the germination and growth of toxin producing psychrotolerant Bacillus spp is not well described. A model agar system mimicking a cooked meat product was used in initial experiments. Incubation at refrigeration temperature of 8 °C for 5 weeks of 26 Bacillus weihenstephanensis including two emetic toxin (cereulide) producing strains showed that B. weihenstephanensis is sensitive to MAP containing CO₂. The sensitivity to 20% CO₂ was dependent on strain and oxygen level, being increased when oxygen was excluded from the MAP. Growth from spores was observed at the earliest within 2 weeks when 20% CO₂ was combined with 2% O₂ and in 3 weeks when combined with “0”% O₂ (the remaining atmosphere was made up from N₂). Results were validated in a cooked meat sausage model for two non-emetic and one emetic B. weihenstephanensis strain. The packaging film oxygen transfer rates (OTR) were 1.3 and 40 ml/m²/24 h and the atmospheres were 2% O₂/20% CO₂ and “0”% O₂/20% CO₂. Oxygen availability had a large impact on the growth from spores in the MAP meat sausage, only the most oxygen restricted condition (OTR of 1.3 ml/m²/24 h and “0”% O₂/20 % CO₂) inhibited growth of the three strains during 4 weeks storage at 8 °C. Cereulide production was undetectable during storage at 8 °C irrespective of choice of the MAP (quantified by liquid chromatography mass spectrometry/mass spectrometry). MAP storage at 8 °C for 1 and 3 weeks followed by opening of packages and temperature abuse for 1.5 h daily at 20 °C during 1 week resulted in increased cell counts and variable cereulide production in the meat sausage. A pre-history at 8 °C for 1 week in MAP with OTR of 1.3 or 40 ml/m²/24 h and 2% O₂ resulted in cereulide concentrations of 0.816 – 1.353 µg/g meat sausage, while a pre-history under the most oxygen restricted condition (OTR of 1.3 ml/m²/24 h, “0”% O₂/20 % CO₂) resulted in minimal cereulide production (0.004 µg/g meat sausage) at abuse condition. Extension of MAP storage at 8 °C for 3 weeks followed by abuse resulted in a substantially reduced cereulide production.Data demonstrates that MAP can be used to inhibit growth of a psychrotolerant toxin producing Bacillus spp. during chill storage at 8 °C, and substantially reduce the risk of emetic food poisoning at abuse condition. Results are of relevance for improving safety of ready to eat processed chilled foods of extended durability.     

Quantitative risk assessment for hazards that arise from non-proteolytic Clostridium botulinum in minimally processed chilled dairy-based foods

A modular process risk model has been constructed that describes the manufacture of dairy dessert products and hazards that arise from non-proteolytic Clostridium botulinum. The model describes batch manufacture and consumer storage of a family size generic dairy dessert but includes a realistic quantification that could apply to a specific food product. The dairy dessert sector is an expanding part of the UK market. The model includes modules that describe spore loads in raw materials, spore inactivation during thermal processing, volume partition and the population kinetics for non-proteolytic C. botulinum during sequential isothermal storage regimes. Where possible elements of uncertainty and variability are identified explicitly. The model is constructed as a belief network from published data and expert opinions. The model provides marginal probabilities, and associated sensitivities, for a range of endpoint measures centred on the toxicity of a single retail unit after an extended period of storage. The decimal reduction time for non-proteolytic C. botulinum spore populations at the highest (hold) temperature of the primary thermal process and the highest temperature experienced during poorly controlled (consumer) storage are dominant factors determining risks. Priorities for additional information to support risk assessments have been identified.     

一起由食用家庭自制辣椒酱引起肉毒中毒的实验室诊断

目的 对1例疑似肉毒中毒病例的相关样品进行实验室检测。方法 参照GB 4789.12—2016对病例暴露食品样品（自制辣椒酱、咸菜、芝麻酱、卤猪蹄）和粪便标本进行前处理,应用实时荧光定量聚合酶链式反应（PCR）检测样品/标本中肉毒毒素基因,通过动物试验进行毒素检测和型别确认,经接种疱肉培养基和TPGYT培养基增菌培养,采用血琼脂平板进行分离、纯化,并做菌种鉴定。结果 5份样品/标本经实时荧光定量PCR法检测,仅在自制辣椒酱样品中检测到A型肉毒毒素基因;通过动物试验进行毒素检测,在自制辣椒酱样品中检测到A型肉毒毒素,其他样品/标本中未检测到肉毒毒素;5份样品/标本中,从自制辣椒酱和卤猪蹄样品中均分离到A型肉毒梭菌。结论 该起中毒事件是由食用肉毒梭菌污染的家庭自制辣椒酱引起。     

Effect of chemical and environmental factors on Aspergillus ochraceus growth and toxigenesis in green coffee

Post-harvest processing (traditional or ecological wet method, and dry method) and coffee pH did not play a significant role in Aspergillus ochraceus growth and OTA production. However, A_w did play a key role: the optimum for growth and toxigenesis was 0.95; below 0.80, coffee was protected. Temperature affected the rate of toxin production, when A_w was compatible: toxigenesis occurred from 10°C with an optimum at 35°C. The critical stage in the process was drying, where conditions propitious to A. ochraceus (A_w of 0.99–0.80) could be found for 2 days or more. Caffeine and chlorogenic acids had an inhibiting effect on OTA production.     

Synergistic antimicrobial effect of pyrophosphate on Listeria monocytogenes and Escherichia coli O157 in modified atmosphere packaged and refrigerated seabass slices

Effect of pyrophosphate (PP) in combination with modified atmosphere (MAP) (80% CO₂, 10% O₂ and 10% N₂) on the survival of Listeria monocytogenes and Escherichia coli O157 inoculated on seabass slices stored at 4 °C was investigated. PP pretreatment showed the synergistic effect on microbiological inhibition with MAP as evidenced by the lowered TVC and LAB counts, compared with samples stored in air and those kept under MAP. Microbiological changes of seabass slices inoculated with different levels of L. monocytogenes or E.coli O157 (10³ and 10⁵ cfu/g) were monitored during storage. PP pretreatment reduced colony count of E. coli O157 and extended the lag phase of L. monocytogenes. Therefore, MAP in combination with PP pretreatment not only retarded microbiological deterioration of seabass slices but also reduced or inactivated some pathogenic bacteria to some extent.     

Predictive model of Vibrio parahaemolyticus growth and survival on salmon meat as a function of temperature

The growth and survival curves of a strain of pandemic Vibrio parahaemolyticus TGqx01 (serotype O3:K6) on salmon meat at different storage temperatures (range from 0 °C to 35 °C) were determined. In order to model the growth or inactivation kinetics of this pathogen during storage, the modified Gompertz and Weibull equations were chosen to regress growth and survival curves, respectively, and both equations produced good fit to the observed data (the average R² value equals to 0.990 for modified Gompertz and 0.920 for Weibull equation). The effect of storage temperature on the specific growth rate (μ) was modeled by square root type equation, and the relationship between μ and lag time (λ) was described by a rule of μ × λ = constant. The shape factor (n) and scale factor (b) values of the Weibull equations versus the temperature (°C) were plotted and the temperature effects on these parameters were described by two linear empirical equations. The predicted growth and survival curves from the model were compared to real enumeration results, using the correlation coefficient (R²), bias factor (B_f) and accuracy factor (A_f), to assess the performance of the established model. The results showed that the overall predictions for V. parahaemolyticus TGqx01 growth or inactivation on salmon at tested temperatures agreed well with observed plate counts, and the average R², B_f and A_f values were 0.958, 1.019 and 1.035, respectively.     

Inhibition by Lactobacillus sakei of other species in the flora of vacuum packaged raw meats during prolonged storage

The abilities of five Lactobacillus sakei strains and one Lactococcus lactis strain to retain inhibitory activity against several target organisms in the flora of product during 12 weeks storage of vacuum-packaged lamb and beef was investigated. L. sakei strains were generally found capable of developing dominant populations on both beef and lamb. L. lactis 75 grew poorly on lamb did not inhibit co-inoculated Brochothrix thermosphacta. Lamb inoculated with the Sakacin-A producer L. sakei Lb706 had lower Listeria monocytogenes populations than lamb inoculated with a bacteriocin-negative variant. In beef packs inoculated with Clostridium estertheticum spores and L. sakei strain 27, 44 or 63, the development of blown-pack spoilage was delayed by up to one week. Campylobacter jejuni inoculated onto beef was recovered from fewer packs when it was co-inoculated with 3000 CFU cm⁻² of L. sakei strain 27, 44 or 63. Observed inhibition did not always correlate with inhibition observed in earlier media-based studies, supporting the view that functionality identified using simple media-based screening methods may not be replicated in the complex environment of stored foods, and vice-versa. These findings further define a set of L. sakei strains with potential for the extended bio-preservation of minimally processed fresh beef and lamb.     

Comparing predicting models for heat inactivation of Listeria monocytogenes and Pseudomonas aeruginosa at different pH

Under the same experimental conditions it has been demonstrated that whereas survival curves of Listeria monocytogenes in the range of temperatures from 54 to 62 °C followed a first-order kinetic, those of Pseudomonas aeruginosa in the range of temperatures from 50 to 56 °C were not linear showing a shoulder followed by a linear region. The first order kinetic model did not describe survival curves of P. aeruginosa. A model based on the Weibull distribution (Log₁₀(N_t/N₀)=(1/−2.303)*(t/b)ⁿ)) accurately described the inactivation kinetics of both microorganisms at the three pHs of 4, 5.5, 7.4 investigated. For both microorganisms, the b value depended on the treatment temperature and the pH of the treatment medium. Whereas for L. monocytogenes the n value was independent of the treatment conditions, for P. aeruginosa the n value depended on the pH of the treatment medium.The model based on the Weibull distribution was capable of accurately predicting the treatment time to inactivate five Log₁₀ cycles of both microorganisms at the three pHs investigated.     

1959—2022年青海省食源性肉毒中毒事件流行病学分析

目的 分析青海省食源性肉毒中毒事件流行特征及影响因素,为防控提供科学依据。方法 收集1959—2022年青海省肉毒中毒数据,应用Excel 2021进行流行病学特征分析,利用R 4.2.3对事件数和病死率进行Mann-Kendall趋势检验,运用ArcGis 10.2软件制作空间分布图。结果 1959—2022年发生食源性肉毒中毒事件86起,发病348人,死亡195人,病死率为56.03%。中毒主要发生在第二、三季度,牧民为主要的发病人群,肉毒毒素型别主要为E型,“风干肉”为主要的中毒食品,生食和开锅即食为主要的食用方式。结论 青海省食源性肉毒中毒病死率整体呈下降趋势,发病地区也逐渐缩小,但作为我国高发地区之一,病死率仍处于较高水平,应进一步加强对高危人群食品安全教育,提高基层医疗机构对肉毒中毒的救治能力,减少中毒事件的发生和死亡。     

Growth of Clostridium perfringens from spore inocula in cooked cured beef: development of a predictive model

The objective of this study was to develop a model to predict the growth of C. perfringens from spores at temperatures applicable to the cooling of cooked cured meat products. C. perfringens growth from spores was not observed at a temperature of 12 °C for up to 3 weeks. The two parameters: germination, outgrowth, and lag (GOL) time and exponential growth rate, EGR, were determined using a function derived from mechanistic and stochastic considerations and the observed relative growths at specified times. A general model to predict the amount of relative growth for arbitrary temperature was determined by fitting the exponential growth rates to a square root Ratkowsky function, and assuming a constant ratio of GOL and generation times. The predicted relative growth is sensitive to the value of this ratio. A closed form equation was developed that can be used to estimate the relative growth for a general cooling scenario and determine a standard error of the estimate. The equation depends upon microbiological assumptions of the effect of history of the GOL times for gradual changes in temperature. Applying multivariate statistical procedures, a confidence interval was computed on the prediction of the amount of growth for a given temperature. The model predicts, for example, a relative growth of 3.17 with an upper 95% confidence limit of 8.50 when cooling the product from 51 to 11 °C in 8 h, assuming a log linear decline in temperature with time.     

Modelling the individual cell lag phase: effect of temperature and pH on the individual cell lag distribution of Listeria monocytogenes

The individual-based approach of the lag phase is gaining interest, especially for pathogens that initially contaminate food products in low amounts. In this paper, the effect of temperature (30, 10, 7, 4 and 2 °C) and pH (7.4, 6.1, 5.5, 5.0, 4.7 and 4.4) on the individual cell lag phase of Listeria monocytogenes was examined in a factorial design, using OD measurements. Individual lag phases of about 100 individual cells per condition were examined and calculated using a linear extrapolation method. Generation times were calculated out of the slope.The obtained data were analyzed at three different levels: in a first approach, the mean values were calculated for each set of environmental conditions and compared to predictions made by the USDA's Pathogen Modeling Program (PMP) for analogous growth conditions. The PMP predictions of the generation times were in the same order of magnitude as the obtained data, although a persistent underestimation could be observed. The observed individual cell lag data differed from lag phase predictions by PMP. Possible reasons for this discrepancy are discussed.Secondly, histograms of individual lag phase measurements were constructed for the different temperature–pH combinations. In this way, the influence of both factors on the variability of individual lag phases could be estimated. At low stress levels, most individual cells showed a short lag phase resulting in a compression of the histograms at the zero-lag level, while, at high stress levels, the histograms shifted to longer lag phases with a significant increase in variability.Thirdly, 37 different distribution types were fitted to the datasets to reveal the distributions that fitted best the obtained data. The gamma distribution was preferred at moderate stress levels, while the Weibull distribution was chosen for harsher growth conditions.     

Antagonistic effect of fat on the antibotulinal activity of food preservatives and fatty acids

The effect of fat on the antibotulinal activity of 11 food preservatives, 12 free fatty acids, and nine lots of enzyme-modified cheese (EMC) was evaluated in a media system. Anhydrous milkfat or soybean oil was added to tubes of Trypticase–peptone–glucose–yeast extract medium (TPGY) supplemented with the additives (final pH adjusted to 5.9). Treatments were inoculated with 3-log₁₀ proteolytic Clostridium botulinum spores/ml (10-strain mixture of serotypes A and B) and incubated anaerobically at 30°C for up to 14 days. For the preservative and fatty acids studies, growth of C. botulinum was determined by measuring optical changes at OD_{640 nm}. Botulinal toxin production was determined in EMC-treatments using the mouse bioassay. Data revealed that the antibotulinal effects of nisin, and free fatty acids caprylic, capric, lauric, myristic, oleic, and linoleic acids were each significantly reduced in treatments supplemented with 20% fat (P<0.05). Similar trends were observed in TPGY supplemented with 20% fat and potassium sorbate, sorbic acid, monolaurin, polyphosphate emulsifier, or EDTA–lysozyme, but the differences were reduced. Fat was also antagonistic to the antibotulinal activity of five EMC-treatments. This study suggests that fat may reduce the efficacy of some antimicrobials added to or found naturally in foods.     

The bacteriology of fresh and spoiling Lake Victorian Nile perch (Lates niloticus)

A total of 177 bacterial cultures isolated from Lake Victorian Nile Perch (Lates niloticus) were investigated. The flora on newly caught Nile perch consisted of organisms belonging to the genera Moraxella, Alcaligenes, Acinetobacter, Pseudomonas, Aeromonas, Micrococcus and other Gram-positive organisms. 39% were identified as Gram-positive species and 61% were negative in the Gram-reaction. Three cultures out of 53 investigated caused weak rotten off-odours in sterile fish broth and one culture, an Aeromonas spp. produced strong rotten, fishy, hydrogen sulphide off-odours. From Nile perch spoiled at ambient temperature, 15 of the 42 strains isolated caused rotten, fishy, hydrogen sulphide off-odours. These specific spoilage bacteria were all identified as Aeromonas and all reduced trimethylamine oxide to trimethylamine and produced hydrogen sulphide. From spoiled iced Nile perch, 74 out of 82 (90%) of the bacteria isolated were identified as Pseudomonas. A small proportion of these (13 out of 74) produced off-odours in sterile fish broth resembling the spoiling fish. These specific spoilers could not be separated from the non-spoilers based on biochemical activities used in classical taxonomy. While the Pseudomonas spp. isolated did not produce trimethylamine or H₂S, a few of the remaining isolates (two Shewanella putrefaciens and five Aeromonas spp.) did produce these compounds. The role of Shewanella putrefaciens in the iced spoilage of Nile perch was, however, insignificant, since they only very late in the storage reached numbers where their spoilage could be detected.