Hygienic status and biogenic amine content of mung bean sprouts

 Microbiological analyses of commercial mung bean sprouts showed the total, viable microbiological population to exceed 10⁸ cfu/g. Enterobacter cloacae, Klebsiella pneumoniae and Enterobacter agglomerans were found to be the dominant and most frequently isolated microbial species. Putrescine, cadaverine, spermidine and spermine were detected in all samples investigated. Formation of biogenic amines by pure culture isolates was studied in a modified decarboxylase medium at different temperatures, pH values and atmospheres. Highest activities were found under aerobic conditions at 20  °C. K. pneumoniae 861 produced 1.2 mg cadaverine/ml after an incubation period of 24 h and E. cloacae 862 produced 2 mg putrescine/ml after 48 h of incubation. For E. agglomerans 863, no biogenic amines were detected under these conditions. Production of cadaverine by E. cloacae 862 and K. pneumoniae 861 under aerobic conditions is presumably related to lysine decarboxylase activities. Although highest decarboxylase activities have usually been found at acidic pH values, amine production reached a maximum at pH 7. Under anaerobic conditions, E. cloacae 862 produced only about half the amount of putrescine as under aerobic conditions, whilst K. pneumoniae 861 produced significantly less cadaverine but was able to produce putrescine. Received: 10 October 1997 / Revised version: 21 January 1998     

Stimulation of Cadaverine Production by Foodborne Pathogens in the Presence of Lactobacillus,Lactococcus, and Streptococcus spp.

Abstract: The effect of Lactobacillus plantarum (FI8595), Lactococcus lactis subsp. cremoris MG 1363), Lactococcus lactis subsp. lactis (IL 1403), and Streptococcus thermophilus on cadaverine and other biogenic amine production by foodborne pathogens was investigated lysine decarboxylase broth. Both of lactic acid bacteria and foodborne pathogens used (especially Staphylococcus aureus, E. coli, Lc. lactis subsp. lactis and Lb. plantarum) had an ability to convert aminoacids into biogenic amine. The conversion of lysine into cadaverine was the highest (167.11 mg/L) by Lactobacillus spp. Gram‐positive bacteria generally had a greater ability to produce cadaverine with corresponding value of 46.26, 53.76, and 154.54 mg/L for Enterococcus faecalis, S. aureus, and Listeria monocytogenes, respectively. Significant variations on biogenic amine production were observed in the presence of lactic acid bacteria strains (P < 0.05). The role of lactic acid bacteria on biogenic amine production by foodborne pathogens varied depending on strains and specific amine. Cadaverine accumulation by Enterobactericeae was increased in the presence of lactic acid bacteria strains except for St. thermophilus, which induced 2‐fold lower cadaverine production by S. Paratyphi A. Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris induced 10‐fold higher increases in histamine for E. coli and K. pneumoniae, respectively. Lactic acid bacteria resulted in strong increases in cadaverine production by P. aeruginosa, although remarkable decreases were observed for histamine, spermidine, dopamine, agmatine, and TMA in the presence of lactic acid bacteria in lysine decarboxylase broth . The result of the study showed that amine positive lactic acid bacteria strains in fermented food led to significant amine accumulation by contaminant bacteria and their accumulation in food product may be controlled by the use of proper starters with amine‐negative activity. Practical Application: Foodborne pathogens and certain lactic acid bacteria are particularly active in the production of biogenic amines. Most of the strains of bacteria possess more than 1 amino acid decarboxylase activity under lysine enrichment culture conditions. Lactic acid bacteria strains had a significant role on increase putrescine accumulation by foodborne pathogens. The increased production of biogenic amines in mixed culture is the result of presence of amine positive lactic acid bacteria strains. The addition of a proper selected starter culture with amine‐negative activity is advisable to produce safer fermented food with low contents of biogenic amines.     

Biogenic amines and their producers in Akawi white cheese

The aim of this work was to study the biogenic amine content of brine‐ripened cheeses after one year of storage and then to investigate possible contaminating micro‐organisms with decarboxylase activity. The biogenic amine production of isolates was tested in vitro. The most frequent biogenic amines were putrescine, histamine and tyramine. The biogenic amine content detected in one cheese sample was above 120 mg/kg; this can be considered toxicologically relevant. Decarboxylase activity was found for 33 contaminating micro‐organisms. Isolates belonging to Bacillus licheniformis, Debaryomyces hansenii, Staphylococcus equorum and Serratia marcescens produced significant amounts of putrescine and cadaverine.     

Contribution of contaminant enterobacteria and lactic acid bacteria to biogenic amine accumulation in spontaneous fermentation of pork sausages.

The contribution of selected contaminant bacteria to the qualitative and quantitative profile of biogenic amine formed was examined during the spontaneous fermentation of pork sausages (without starter culture). The study mainly dealt with the evolution of the amine profile and microbial development during spoilage of meat, but also focused on the biogenic amine build-up during sausage fermentation using pork meat contaminated by a tyramine-positive Lactobacillus brevis strain and a cadaverine-positive Enterobacter cloacae strain. Amines and microbial counts correlated well during meat spoilage and sausage fermentation. The high accumulation of cadaverine was associated with the undesirably high number of Enterobacteriaceae. Tyramine production during sausage fermentation was not entirely attributable to contaminant lactic acid bacteria. On the contrary, the spontaneous fermenting flora needed to produce these types of product are also involved. The contribution of contaminant bacteria to the putrescine and other biogenic amines during meat fermentation remains unclear.     

The ability of biogenic amines and ammonia production by single bacterial cultures

The amino acid decarboxylating activity and production of biogenic amines, trimethylamine and ammonia by Morganella morganii (two strains), Klebsiella pneumoniae (three strains), Hafnia alvei (two strains), Enterococcus faecalis, Photobacterium phosphoreum, Micrococcus sp., Psychrobacter immobilis, Corynebacterium sp., Vibrio fischeri, Vibrio harveyi and Pseudomonas putida were investigated using a rapid HPLC method. In a laboratory medium containing amino acid (histidine, ornithine, lysine, tyrosine and arginine), not all bacterial strains produced the biogenic amines but most of them produced histamine, putrescine, cadaverine and ammonia. Cadaverine production by Klebsiella pneumoniae (8152), Klebsiella pneumoniae (673), Klebsiella pneumoniae (2122), Hafnia alvei (6578), Hafnia alvei (11999), Vibrio fischeri (25) Vibrio harveyi (42) and Pseudomonas putida (10936) was 531, 422, 532, 485, 472, 343, 547 and 343 mg/l, respectively in lysine decarboxylase broth. Tyramine was produced in highest concentration (526 mg/l) by Enterococcus faecalis (775). Agmatine was not produced apart from Psychrobacter immobilis (100) in an arginine decarboxylase broth.     

Production of biogenic amines by lactic acid bacteria and enterobacteria isolated from fresh pork sausages packaged in different atmospheres and kept under refrigeration

The occurrence of in vitro amino acid activity in bacterial strains associated with fresh pork sausages packaged in different atmospheres and kept in refrigeration was studied. The presence of biogenic amines in decarboxylase broth was confirmed by ion-exchange chromatography and by the presence of the corresponding decarboxylase genes by PCR. From the 93 lactic acid bacteria and 100 enterobacteria strains analysed, the decarboxylase medium underestimates the number of biogenic amine-producer strains. 28% of the lactic acid bacteria produced tyramine and presented the tdc gene. All the tyramine-producer strains were molecularly identified as Carnobacterium divergens. Differences on the relative abundance of C. divergens were observed among the different packaging atmospheres assayed. After 28 days of storage, the presence of argon seems to inhibit C. divergens growth, while packing under vacuum seems to favour it. Among enterobacteria, putrescine was the amine more frequently produced (87%), followed by cadaverine (85%); agmatine and tyramine were only produced by 13 and 1%, respectively, of the strains analysed. Packing under vacuum or in an atmosphere containing nitrogen seems to inhibit the growth of enterobacteria which produce simultaneously putrescine, cadaverine, and agmatine. Contrarily, over-wrapping or packing in an atmosphere containing argon seems to favour the growth of agmatine producer-enterobacteria. The production of putrescine and cadaverine was associated with the presence of the corresponding amino acid decarboxylase genes. The biogenic amine-producer strains were included in a wide range of enterobacterial species, including Kluyvera intermedia, Enterobacter aerogenes, Yersinia kristensenii, Serratia grimesii, Serratia ficaria, Yersinia rodhei, Providencia vermicola and Obesumbacterium proteus.     

Analysis of bacterial diversity during the fermentation of inyu, a high-temperature fermented soy sauce,using nested PCR-denaturing gradient gel electrophoresis and the plate count method

The diversity of bacteria associated with the fermentation of inyu, also known as black soy sauce, was studied through the nested PCR-denaturing gradient gel electrophoresis (DGGE) of samples collected from the fermentation stages of the inyu production process. The DGGE profiles targeted the bacterial 16S rDNA and revealed the presence of Citrobacter farmeri, Enterobacter cloacae, Enterobacter hormaechei, Enterococcus faecium, Klebsiella pneumoniae, Pantoea agglomerans, Salmonella enterica, Serratia marcescens, Staphylococcus sciuri and Weissella confusa. The bacterial compositions of 4 fermented samples were further elucidated using the plate count method. The bacteria isolated from the koji-making stage exhibited the highest diversity; Brachybacterium rhamnosum, E. hormaechei, K. pneumoniae, Kurthia gibsonii, Pantoea dispersa, Staphylococcus gallinarum, Staphylococcus kloosii and S. sciuri were identified. Koji collected during the preincubation stage presented the largest cell counts, and E. hormaechei, K. pneumoniae, E. cloacae and Enterobacter pulveris were identified. In brine samples aged for 7 and 31 days, the majority of the bacteria isolated belonged to 4 Bacillus species, but 4 Staphylococcus species and Delftia tsuruhatensis were also detected. This study demonstrates the benefits of using a combined approach to obtain a more complete picture of microbial populations and provides useful information for the control or development of bacterial flora during inyu fermentation.     

Kinetics of green asparagus ascorbic acid heated in a high-temperature thermoresistometer

 Thermal degradation of green asparagus ascorbic acid in high-temperature short-time conditions was studied by heating in a five-channel computer-controlled thermoresistometer. Ascorbic acid was heated to between 110  °C and 140  °C and the degradation kinetics were analyzed assuming that two different inactivation mechanisms were occurring, one aerobic and the other anaerobic. The two reactions followed first-order kinetics, with E _a=12.3(2.0) kcal/mol and k _125  °C=47.0(3.0)×10^–3 s^–1 for the aerobic oxidation, and E _a=6.1(1.4) kcal/mol and k _125  °C=4.1(0.2)×10^–3 s^–1 for the anaerobic degradation. Received: 30 January 1998 / Revised version: 11 June 1998     

Formation of biogenic amines by Gram-negative rods isolated from fresh,spoiled, VP-packed and MAP-packed herring (<Emphasis Type="Italic">Clupea harengus</Emphasis>)

Bacterial strains (120) were isolated from fresh, spoiled, VP-packed and MAP-packed herring. Identified bacterial strains were investigated for their abilities to produce biogenic amines in histidine, lysine and ornithine decarboxylase broth by a rapid high-performance liquid chromatography (HPLC) method. The microflora of fresh herring was dominantly Pseudomonas (30%), Enterobacteriaceae (23.2%), Vibrio (13.3%) and Moraxella (13.3%) but, the microflora of herring stored in VP and MAP was dominated by species belonging to Vibrio (23.3%) and Moraxella (20%), which indicates that these packaging systems prevented the growth of Pseudomonas and Enterobacteriaceae. In a laboratory medium containing amino acid (histidine, ornithine and lysine), most of bacterial strains produced histamine, putrescine and cadaverine. The highest histidine decarboxylase activities were observed in Klebsiella oxytoca, Hafnia alvei and Proteus vulgaris which produced 396, 232 and 54 mg histamine/L, respectively in histidine-enriched broth. The accumulation of cadaverine by Klebsiella oxytoca and Hafnia alvei was 325 and 208 mg/l, respectively. All strains isolated produced putrescine in an ornithine-enriched broth, ranging from 3 to 249 mg/l. The production of putrescine by Klebsiella oxytoca and Hafnia alvei was 249 and 195 mg/l, respectively. Moraxella spp and Acinetobacter spp did not produce histamine which indicates they did not have histidine decarboxylase activity.     

Formation of biogenic amines by Gram-negative bacteria isolated from poultry skin

The aim of this study is to explore production of biogenic amines (histamine, tyramine, putrescine, cadaverine, agmatine, spermine and spermidine) by 88 Gram-negative bacteria isolated from poultry skin (41 isolates of family Enterobacteriaceae, 21 isolates of genus Aeromonas, 16 isolates of genus Pseudomonas, and 10 isolates of other Gram-negative rods). Ion-exchange chromatography was employed to analyse the above mentioned amines. Enterobacteria were found to be the largest producers of amines with proved presence of tyramine, agmatine, putrescine, and cadaverine in cultivation broth after incubation of bacteria. Putrescine and cadaverine were the most abundant products. Presence of at least two biogenic amines, i.e. mainly concurrent presence of putrescine and cadaverine, was revealed in 19 enterobacteria strains. Eleven isolates classified into Aeromonas genus produced putrescine and five of them also formed cadaverine. The other observed biogenic amines (histamine, spermine and spermidine) were not found among tested isolates. Production of biogenic amines by any Pseudomonas family isolates was not proved.     

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.     

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.     

Optimization of stepwise blanching of frozen-thawed potato tissues (cv. Monalisa)

 Response surface methodology was used to compare the effect of temperature and time of the first step of blanching on compression, shear, tension and stress-relaxation parameters of frozen-thawed potato tissues. A central, composite rotatable design was used to study the effects of variation in levels of temperature (52.93–67.07  °C) and time (15.86–44.14 min) on rheological parameters. Blanching temperature was the most important factor affecting the mechanical properties tested. The models fitted for the apparent modulus of elasticity in compression, maximum tension force, and relaxed force in the first cycle (F _r1); all had R ²>0.85 (P≤0.01) and were used for doing predictions. Optimum conditions were with in the ranges of temperature (60–65  °C) and time (25–35 min) used for each factor. In the experimental verification of the models at 65  °C during 30 min, the lowest percentage residual between experimental and predicted values was obtained for F _r1 (0.644), which was therefore the most appropiate parameter for detecting the firming effect that the pectinesterase activity produced on frozen potato tissues as a consequence of stepwise blanching under these conditions. Received: 3 February 1999 / Revised version: 12 April 1999     

Impact of fermentation conditions and refrigerated storage on microbial quality and biogenic amine content of sauerkraut

White cabbage (Brassica oleracea L. var. capitata cv. Bronco) was fermented, at 0.5% and 1.5% NaCl, using Lactobacillus plantarum or Leuconostoc mesenteroides as starter cultures and, subsequently, sauerkraut was stored at 4 °C for 3 months. Microbial populations and six biogenic amines (putrescine, cadaverine, histamine, tyramine, spermine and spermidine) were investigated. Fermentation and storage increased aerobic mesophilic bacteria and LAB populations in sauerkrauts, and this was accompanied by a rise in biogenic amine content. L. plantarum sauerkrauts produced with 0.5% NaCl had the highest microbial counts, whilst no differences between salt contents were found with L. mesenteroides. Total biogenic amine amount was lower at 0.5% NaCl than at 1.5% in both induced fermentations and L. mesenteroides produced a lower content than did L. plantarum. Spermidine was the major contributor to the total biogenic amine content, followed by putrescine, whilst histamine was present at the lowest level. The individual and total biogenic amine levels in the experimental sauerkrauts stored at 4 °C for 3 months were below the upper limits reported in the literature for fermented products, indicating good quality and safety of the sauerkrauts. L. mesenteroides starter and 0.5% NaCl were the optimal fermentation conditions for producing sauerkrauts with the lowest biogenic amine contents.     

Biogenic amine formation and bacterial contribution in fish,squid and shellfish

Forty-one species of fish, squid and shellfish were analyzed for biogenic amine (BA) contents. Most of the fish samples showed lower BA contents, whereas some samples showed higher contents than the allowable levels. Shellfish and squid samples had negligible BA levels. Four fish species containing high BA levels were analyzed for changes in histamine contents during storage. In the most samples, the histamine contents remarkably increased up to 36.6–2123.9 mg/kg after 24 h of storage at 25 °C, while the contents began to gradually increase after 2–3 days of storage at 4–10 °C. The dominant microbial group was enterobacteria throughout the storage period. Meanwhile, out of total 119 strains isolated from different fish species showing high BA levels, 23 strains identified as Enterobacter aerogenes produced large amounts of histamine, putrescine and cadaverine, and 33 strains identified as two different Enterobacter spp. produced less histamine but large amounts of putrescine and cadaverine.     

Biogenic amine production by<Emphasis Type="Italic"> Morganella morganii</Emphasis> and<Emphasis Type="Italic"> Klebsiella oxytoca</Emphasis> in tuna

Biogenic amine formation in tuna inoculated with two micro-organisms reported as powerful in vitro histamine producers, Morganella morganii and Klebsiella oxytoca, was investigated. Inoculated and non-inoculated tuna samples were stored in ice, under refrigeration at 8–10 °C and at 20±1 °C. Ten biogenic amines were studied and changes were found only in cadaverine and histamine in inoculated samples. Therefore, the in vitro ability of K. oxytoca to form cadaverine and that of both microorganisms to form histamine was confirmed in inoculated samples. Storage temperature had a crucial effect on the amount and intensity of amine production. On the other hand, the potential ability of M. morganii to form putrescine was not shown in the inoculated samples, regardless of the storage temperature.     

白鱼腐败菌产生物胺能力分析

为了探讨白鱼（Anabarilius）腐败菌产生物胺的能力,釆用聚合酶链式反应技术检测腐败菌的组氨酸、赖氨酸、鸟氨酸和酪氨酸的脱羧酶基因,应用反相高效液相色谱技术测定接种腐败菌培养基和无菌白鱼鱼块中生物胺含量,并对主要腐败菌的生物胺产量因子进行分析。结果表明：8 种腐败菌均含有至少1 种氨基酸脱羧酶基因;接种不同腐败菌的液体培养基和无菌白鱼鱼块均检测到多种生物胺,其中Morganella sp.wf-1和Morganella sp.wf-2具有很强的产腐胺、尸胺和组胺能力,使得接种这两种腐败菌的无菌白鱼鱼块中腐胺、尸胺和组胺含量、单胺、二胺、生物胺指数和总生物胺含量等指标在腐败过程中均显著高于对照组和接种其他腐败菌的无菌白鱼鱼块。同时,Morganella sp.wf-1和Morganella sp.wf-2的腐胺、尸胺、组胺、单胺、二胺、生物胺指数和总生物胺的产量因子均高于其他腐败菌。结果表明,Morganella sp.wf-1和Morganella sp.wf-2是白鱼腐败过程中产生物胺的主要腐败菌。     

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

Thirty-two dried milkfish products sold in five retail markets in southern Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. Except for histamine and cadaverine, the average content of various biogenic amines in tested samples was less than 8.5 mg/100 g. Most of the tested dried milkfish products (78.1%) had histamine levels greater than the FDA guideline of 5 mg/100 g for scombroid fish and/or product, while fourteen of them (43.7%) contained > 50 mg/100 g hazard action level. Thirty histamine-producing bacterial strains, capable of producing 5.4 ppm to 562 ppm of histamine in trypticase soy broth supplemented with 1.0% L-histidine (TSBH), were identified as Enterobacter aerogenes (seven strains), Citrobacter sp. (one strain), Staphylococcusxylosus (ten strains), S. sciuri (one strain), Bacillus thuringiensis (two strains), Citrobacter freundii (five strains), Klebsiella pneumoniae (one strain) and E. cloacae (three strains), by 16S rDNA sequencing with PCR amplification.     

Influence of ultrasound on mass transport during cheese brining

 The effect of ultrasound on mass transfer during cheese brining has been investigated. The rate of water removal and NaCl gain increased when ultrasound was applied in comparison with brining performed under static or dynamic conditions, suggesting that ultrasound improves both external and internal mass transfer. A simple diffusional model was developed to simulate mass transport during acoustic brining. Model parameters were estimated using experimental data from acoustic brining experiments carried out on cheese cylinders of 1.7×10^–2m radius and 3×10^–2 m height at different temperatures (5, 15 and 20  °C). Effective water (D _W) and NaCl (D _S) diffusivities estimated using the proposed model ranged from 5.0×10^–10m²/s and 8.0×10^–10 m²/s at 5  °C to 1.3×10^–9m²/s and 1.2×10^–9 m²/s at 20  °C. Both D _W and D _S varied with temperature according to the Arrhenius equation. Through the proposed model, water losses and NaCl gains of the experiments used in the parameter identification were accurately simulated (average %var=98.2%) and also of two additional acoustic experiments carried out under different conditions of temperature (10  °C) and sample size and geometry [parallelepiped of (6×2.5×1.25)×10^–2 m] to those used in the parameter identification (average %var=98.4%). Received: 22 September 1998 / Revised version: 20 November 1998     

The Impact of Carvacrol on Ammonia and Biogenic Amine Production by Common Foodborne Pathogens

The impact of carvacrol at different levels (0.1%, 0.5%, and 1%) on ammonia (AMN) and biogenic amines (BAs) production by 8 common foodborne pathogens (FBPs) (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, Listeria monocytogenes, Aeromonas hydrophila, and Salmonella Paratyphi A) was studied using a rapid high‐performance liquid chromatography method. Significant differences among bacteria (P < 0.05) in AMN and BA production were observed using a tyrosine decarboxylase broth. Tyramine, dopamine, agmatine, spermine, and putrescine were the main amines produced by the bacteria. Tyramine production by P. aeruginosa was the highest (967 mg/L), whereas K. pneumoniae was the poorest tyramine producer (6.42 mg/L). AMN and BA production varied significantly depending on carvacrol levels and the specific bacterial strains. Tyramine production for all bacterial strains was significantly suppressed by addition of carvacrol at levels of 0.5% and 1%, but not 0.1%. Consequently, the effect of carvacrol on BA and AMN formation by FBP was dependent on bacterial strain as well as carvacrol level.