Diversity of plasmids encoding histidine decarboxylase gene in Tetragenococcus spp. isolated from Japanese fish sauce

Nineteen isolates of histamine producing halophilic bacteria were isolated from four fish sauce mashes, each mash accumulating over 1000 ppm of histamine. The complete sequences of the plasmids encoding the pyruvoyl dependent histidine decarboxylase gene (hdcA), which is harbored in histamine producing bacteria, were determined. In conjunction, the sequence regions adjacent to hdcA were analyzed to provide information regarding its genetic origin. As reference strains, Tetragenococcus halophilus H and T. muriaticus JCM10006^T were also studied. Phenotypic and 16S rRNA gene sequence analyses identified all isolates as T. halophilus, a predominant histamine producing bacteria present during fish sauce fermentation. Genetic analyses (PCR, Southern blot, and complete plasmid sequencing) of the histamine producing isolates confirmed that all the isolates harbored approximately 21-37 kbp plasmids encoding a single copy of the hdc cluster consisting of four genes related to histamine production. Analysis of hdc clusters, including spacer regions, indicated > 99% sequence similarity among the isolates. All of the plasmids sequenced encoded traA, however genes related to plasmid conjugation, namely mob genes and oriT, were not identified. Two putative mobile genetic elements, ISLP1-like and IS200-like, respectively, were identified in the up- and downstream region of the hdc cluster of all plasmids. Most of the sequences, except hdc cluster and two adjacent IS elements, were diverse among plasmids, suggesting that each histamine producers harbored a different histamine-related plasmid. These results suggested that the hdc cluster was not spread by clonal dissemination depending on the specific plasmid and that the hdc cluster in tetragenococcal plasmid was likely encoded on transformable elements.     

Histamine contents of fermented fish products in Taiwan and isolation of histamine-forming bacteria

Twenty-seven imported fermented fish products from Southeast Asian countries and sold in the supermarkets in Taiwan, including fish sauce, fish paste and shrimp paste, were tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, total volatile basic nitrogen, trimethylamine, and aerobic plate count in all samples ranged from 4.8% to 6.5%, 16.2% to 45.3%, 51 to 275 mg/100 g, 5.4 to 53.9 mg/100 g and 1.0 to 4.2 log CFU/g, respectively. The average content for each of eight different biogenic amines in all samples was less than 90 ppm, except for histamine which has an average content of 394 ppm in fish sauce, 263 ppm in fish paste, and 382 ppm in shrimp paste. Most of the tested fermented fish products (92.6%) had histamine levels greater than the FDA guideline of 50 ppm, while seven of them (25.9%) contained >500 ppm of histamine. Although Bacillus coagulans and Bacillus megaterium were identified as the two histamine-producing bacteria capable of producing 13.7 and 8.1 ppm of histamine, respectively, in trypticase soy broth broth supplemented with 1.0% l-histidine, they were not determined to be the main contributors to histamine accumulation in these fermented fish products.     

Characterization of Ca-activated cell-bound proteinase from Virgibacillus sp. SK37 isolated from fish sauce fermentation

Cell-bound proteinase from Virgibacillus sp. SK37 isolated from the first month of fish sauce fermentation was characterized. The enzyme showed the maximum activity at 65 °C, pH 7.0 and 9.5, using azocasein as a substrate. The enzyme required at least 10 mmol/l Ca²⁺ to effectively hydrolyze casein and the extent of casein degradation increased with Ca²⁺ concentration. Ethylenediaminetetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF) largely inhibited the activity, indicating a characteristic of Ca²⁺-activated serine proteinase. Among six synthetic substrates tested, the enzyme preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, indicating a subtilisin-like proteinase. Although activity towards actomyosin at 20 g/100 ml NaCl decreased to 63% compared to at 5 g/100 ml, the enzyme showed high stability at 25 g/100 ml NaCl, 30 °C. This was the first study to report biochemical characteristics of cell-bound proteinase from a moderately halophilic bacterium isolated from fish sauce.     

Bacteriocinogenic Lactobacillus plantarum ST16Pa isolated from papaya (Carica papaya) — From isolation to application: Characterization of a bacteriocin

Strain ST16PA, isolated from papaya was identified as Lactobacillus plantarum based on biochemical tests, PCR with species-specific primers and 16S rDNA sequencing. L. plantarum ST16PA produces a 6.5 kDa bacteriocin, active against different species from genera Enterobacter, Enterococcus, Lactobacillus, Pseudomonas, Streptococcus and Staphylococcus and different serotypes of Listeria spp. The peptide is inactivated by proteolytic enzymes, but not when treated with ??-amylase, catalase, lipase, Triton X-100, SDS, Tween 20, Tween 80, urea, NaCl and EDTA. However, presence of 1% Triton X-114 deactivates the bacteriocin. No change in activity was recorded after 2 h at pH values between 2.0 and 12.0, and after treatment at 100 °C for 120 min or 121 °C for 20 min. The mode of activity against Lactobacillus sakei ATCC 15521, Enterococcus faecalis ATCC 19443 and Listeria innocua 2030C was bactericidal, resulting in cell lysis and enzyme-leakage. No significant differences in cell growth and bacteriocin production were observed when strain ST16Pa was cultured in MRS broth at 26 °C and 30 °C for 24 h (25 600 AU/ml). However, even though strain ST16PA grows well in MRS broth at 15 °C and 37 °C, a reduction of bacteriocin production was observed (400 AU/ml and 1600 AU/ml, respectively). In addition, effect of MRS medium components, different initial pH and additions of glycerol or vitamins to the media on bacteriocin ST16Pa production was studied.Peptide ST16PA adsorbs (400 AU/ml) to producer cells. However, bacteriocin ST16Pa was adsorbed at 50% to cells of L. innocua 2030C and at 75% to L. sakei ATCC 15521 and E. faecalis ATCC 19433 when experiments were conducted at 30 °C and pH 6.5. Adsorption of bacteriocin ST16Pa to target cells at different temperatures, pH and in presence of potassium sorbate, sodium nitrate, sodium chloride, ascorbic acid, Tween 80 and Tween 20 were also studied. To the best of our knowledge, this is the first report on detection of L. plantarum in papaya.     

A NaCl-stable serine proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce

An extracellular proteinase from Virgibacillus sp. SK33, isolated from 1 month-old fish sauce, was purified to electrophoretic homogeneity, using hydrophobic interaction chromatography and hydroxyapatite with purification fold of 2.5 and 7% yield. The anomalous molecular weight (MW) of 19 kDa was obtained from SDS–PAGE, whereas a MW of 33.7 kDa was determined by MALDI-TOF. Optimum conditions for catalytic activity were 55 °C and pH 7.5. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferentially hydrolysed Suc-Ala-Ala-Pro-Phe-AMC, indicating a serine proteinase with subtilisin-like characteristics. K_m and k_cat of the purified proteinase were 27 μM and 12 s⁻¹, respectively. Proteinase activity, toward both synthetic and anchovy substrates, increased with NaCl up to 25%. The proteinase exhibited high stability in both the absence and presence of NaCl up to 25%. Approximately 2.5-fold increase in activity was observed in the presence of divalent cations, including Ca²⁺, Mg²⁺ and Sr²⁺ at 100 mM. MALDI-TOF MS and LC–ESI-MS/MS analyses, as well as N-terminal sequences, revealed that the purified enzyme did not match microbial proteinases in the database, indicating it to be a novel proteinase.     

Microbiota during fermentation of chum salmon (Oncorhynchus keta) sauce mash inoculated with halotolerant microbial starters: Analyses using the plate count method and PCR-denaturing gradient gel electrophoresis (DGGE)

Nine different combinations of mugi koji (barley steamed and molded with Aspergillus oryzae) and halotolerant microorganisms (HTMs), Zygosaccharomyces rouxii, Candida versatilis, and Tetragenococcus halophilus, were inoculated into chum salmon sauce mash under a non-aseptic condition used in industrial fish sauce production and fermented at 35 ± 2.5 °C for 84 days to elucidate the microbial dynamics (i.e., microbial count and microbiota) during fermentation. The viable count of halotolerant yeast (HTY) in fermented chum salmon sauce (FCSS) mash showed various time courses dependent on the combination of the starter microorganisms. Halotolerant lactic acid bacteria (HTL) were detected morphologically and physiologically only from FCSS mash inoculated with T. halophilus alone or with T. halophilus and C. versatilis during the first 28 days of fermentation. Only four fungal species, Z. rouxii, C. versatilis, Pichia guilliermondii, and A. oryzae, were detected throughout the fermentation by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). In FCSS mash, dominant HTMs, especially eumycetes, were nonexistent. However, under the non-aseptic conditions, undesirable wild yeast such as P. guilliermondii grew fortuitously. Therefore, HTY inoculation into FCSS mash at the beginning of fermentation is effective in preventing the growth of wild yeast and the resultant unfavorable flavor.     

Novel starter cultures to inhibit biogenic amines accumulation during fish sauce fermentation

Bacteria with amine oxidase activity have become a particular interest to reduce biogenic amines concentration in food products such as meat and fish sausages. However, little information is available regarding the application of these bacteria in fish sauce. Hence, our study was aimed to investigate the effect of such starter cultures in reducing biogenic amines accumulation during fish sauce fermentation. Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05 isolated from fish sauce which possess amine oxidase activity were used as starter cultures in this study. Fermentation was held for 120 days at 35 °C. The pH value increased in all samples, while salt concentration remained constant throughout fermentation. Aerobic bacteria count was significantly lower (p < 0.05) in the control than in inoculated samples as a result of starter cultures addition. However, it decreased during fermentation due to the growth inhibition by high salt concentration. Proteolytic bacterial count decreased during fermentation with no significant difference (p > 0.05) among samples. These bacteria hydrolyzed protein in anchovy to produce free amino acid precursors for amines formation by decarboxylase bacteria. The presence of biogenic amines producing bacteria in this study was considered to be indigenous from raw material or contamination during fermentation, since our cultures were negative histamine producers. Amino acid histidine, arginine, lysine and tyrosine concentration decreased at different rates during fermentation as they were converted into their respective amines. In general, biogenic amines concentration namely histamine, putrescine, cadaverine and tyramine increased throughout fermentation. However, their concentrations were markedly higher (p < 0.05) in the control (without starter cultures) as compared to the samples treated with starter cultures. Histamine concentration was reduced by 27.7% and 15.4% by Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05, respectively. Both cultures could also reduce other amines during fermentation. After 120 days of fermentation, the overall biogenic amines concentration was 15.9% and 12.5% less in samples inoculated with Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05, respectively, as compared to control samples. These findings emphasized that application of starter cultures with amines oxidase activity in fish sauce fermentation was found to be effective in reducing biogenic amines accumulation.     

Histamine contents of salted seafood products in Taiwan and isolation of halotolerant histamine-forming bacteria

Fifty-seven salted seafood products sold in the fishing village stores in Taiwan, including salted fish product, salted mollusc product and salted shrimp product, were tested to determine the occurrence of histamine and histamine-forming bacteria. Although the average content of each of nine different biogenic amines in all samples was less than 5.0 mg/100 g, 10.5% (6/57) of tested samples had the histamine content greater than the 5.0 mg/100 g allowable limit suggested by the US Food and Drug Administration. One histamine-producing bacterial strain, capable of producing 78.5 ppm of histamine in trypticase soy broth supplemented with 1.0% l-histidine (TSBH), was identified as Bacillus megaterium. The B. megaterium isolate was a halotolerant bacterium which grew well to an elevated NaCl concentration of 15% in TSBH medium. Besides, it had a consistent ability to produce >300 ppm of histamine at 10% NaCl concentration in TSBH medium after 72 h.     

Incidence, diversity and toxin gene characteristics of Bacillus cereus group strains isolated from food products marketed in Belgium

The major objectives of this study were to determine the incidence, diversity and characteristics of Bacillus cereus group spp. isolated from food products marketed in Belgium. The food products investigated in this study included cooked pasta, lasagna, béchamel sauce, bolognaise sauce, fresh minced beef, fresh-cut vegetables and raw basmati rice. B. cereus group spp. were detected in 56.3% (324 of 575) of the samples giving rise to 380 strains. The highest incidence (100%) occurred in the raw basmati rice. Although only 10 (2.6%) of the 380 isolates were determined to be psychrotolerant (able to grow at ≤ 7 °C), 25 (6.2%), 189 (49.7%) and 334 (87.9%) isolates were able to grow at mild temperature abuse conditions of 8 °C, 9 °C and 10 °C, respectively. The large diversity of the isolates obtained (overall and between isolates obtained from the same product type) was highlighted by the results of the (GTG)₅ PCR fingerprinting of 80 selected isolates. Sixty-one of these 80 isolates belonged to 15 distinct clusters (≥ 85% Pearson correlation) whereas the remaining 19 were each clustered separately. Further diversity was also found in the distribution of toxin genes as 16 different profiles were observed in the 80 selected isolates. Whilst none of 80 selected strains harboured the ces gene required for the production of the emetic toxin cereulide, 42 strains (52.5%) carried all seven genes required for the production of the diarrhoeal enterotoxins: haemolytic BL, non-haemolytic enterotoxin and cytotoxin K. The results of this study highlight not only the omnipresence but also the highly diverse ecology of B. cereus spp. within and across several food product types available on the retail market in Belgium. They should also provide the impetus for more studies to enable detailed risk assessment studies to be performed.     

Effects of the addition of spleen of skipjack tuna (Katsuwonus pelamis) on the liquefaction and characteristics of fish sauce made from sardine (Sardinella gibbosa)

The effects of the addition of spleen of skipjack tuna (Katsuwonus pelamis), at levels of 0%, 10% and 20%, on the liquefaction and characteristics of fish sauce produced from the sardine (Sardinella gibbosa) with different salt concentrations (15%, 20% and 25%) were monitored during fermentation for 180 days. Fish sauces prepared from sardine with spleen supplementation contained greater total nitrogen, amino nitrogen, formaldehyde nitrogen and ammonia nitrogen than did those without spleen addition throughout the fermentation. The rate of liquefaction was dependent on the amount of spleen added. Reduction of salt content accelerated the hydrolysis of fish protein during fermentation. The liquefaction rate of the lower salt-treated samples was generally faster than were those treated with higher salt content. Among all treatments, sardine with 25% spleen and 15% salt added exhibited the greatest protein hydrolysis, particularly at the early stages, suggesting the combined effects of autolysis and spleen proteinase. The greater liquefaction was coincidental with the development of browning as well as the increase in redness of liquid formed. An acceptability test revealed that the samples were different in colour, aroma, taste and overall acceptance (p < 0.05). Fish sauce samples containing 20% salt, without and with 10% spleen addition had similar acceptabilities to commercial fish sauce. Therefore, the addition of spleen, as well as salt reduction, can accelerate the liquefaction of sardine for fish sauce production.     

Seasonal effects on the physicochemical characteristics of fish sauce made from capelin (Mallotus villosus)

Fresh capelin (Mallotus villosus) was harvested from the North Atlantic during both summer and winter fishing seasons. Reaction conditions for fish sauce processing were optimized with respect to temperature, salt concentration and reaction time, using a response surface methodology (RSM) experimental design. Whole capelin was minced and samples were ground with increasing salt concentrations. RSM optimizations were conducted, ranging from 5% to 30% salt, and incubating at 5° intervals from 0 to 65 °C. Autolytic activity was estimated by extracting the liquid formed by the mixture with trichloroacetic acid and estimating protein content by the Lowry method. Samples for fish sauce production were then prepared under optimized conditions by mixing ground capelin with 10% salt and incubating at 50 °C for up to 270 days for the summer capelin and up to 360 days for the winter capelin. Samples were collected at regular intervals and analyzed for liquid yield, moisture, protein, soluble solids, specific gravity, pH, colour and amino acid content. Kjeldahl protein content in the fish sauce from summer capelin was 2.03% after 250 days of fermentation and twice as high as that in winter capelin fish sauce. Moisture content and pH were lower in the summer capelin fish sauce, but Brix and density were higher than those in fish sauce from winter capelin. Brown colour formation was very rapid in the summer capelin fish sauce but slow in the winter capelin fish sauce. Summer capelin may be successfully utilized for the production of fish sauce without added enzymes.     

Using Fish Sauce as a Substitute for Sodium Chloride in Culinary Sauces and Effects on Sensory Properties

Historically, fish sauce has been a standard condiment and ingredient in various Southeast Asian cuisines. Moreover, fish sauce imparts umami taste, which may enhance perceived saltiness in food. This quality suggests that fish sauce may be used as a partial substitute for sodium chloride (NaCl) in food preparation, which may present a valuable option for health‐conscious and salt‐restricted consumers. However, the degree to which NaCl can be decreased in food products without compromising taste and consumer acceptance has not been determined. We hypothesized that NaCl content in food may be reduced by partial replacement with fish sauce without diminishing palatability and consumer acceptance. Preparations of 3 types of food were assessed to test this hypothesis: chicken broth (n = 72); tomato sauce (n = 73); and coconut curry (n = 70). In the first session, the percentage of NaCl that could be replaced with fish sauce without a significant change in overall taste intensity was determined for each type of food using the 2‐Alternative Forced Choice method. In the second session, subjects rated 5 samples for each food with varying NaCl and/or fish sauce content on 3 sensory attributes: deliciousness; taste intensity; and saltiness. Our results demonstrate that NaCl reduction was possible in chicken broth, tomato sauce, and coconut curry at 25%, 16%, and 10%, respectively, without a significant loss (P < 0.05) in deliciousness and overall taste intensity. These results suggest that it is possible to replace NaCl in foods with fish sauce without reducing overall taste intensity and consumer acceptance.     

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: Its purification and characterization

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5 kDa according to SDS–PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40 °C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe²⁺ > Hg²⁺ > Mn²⁺ > K⁺ > Mg²⁺ > Li⁺ > Cu²⁺). Trypsin activity decreased continuously as NaCl concentration increased (0–30%). K_m and k_cat values were 0.13 mM and 1.46 s⁻¹, respectively. Results suggest the enzyme have a potential application where room processing temperatures (25–35 °C) or high salt (30%) concentration are needed, such as in fish sauce production.     

Comparison of culture media for enrichment and isolation of Salmonella spp. from frozen Channel catfish and Vietnamese basa fillets

Frozen fillets of Channel catfish and Vietnamese basa fish were used to compare Salmonella spp. recovery effectiveness of selective enrichment in Rappaport–Vassiliadis (RV) broth and tetrathionate broth (TT) and selective isolation on Hekteon enteric (HE) agar, xylose lysine deoxycholate (XLD) agar, and bismuth sulfite (BS) agar. Isolate confirmation was through fatty acid methyl ester analysis. Of 60 samples analyzed, 25 were found contaminated with Salmonella (42% incidence). Salmonella spp. recovery after enrichment in RV medium was 35% on HE agar, 30% on XLD agar, and 42% on BS agar. Similarly, after enrichment in TT broth, HE and XLD agars recovered 22% each and BS agar recovered 37%. No performance difference (p > 0.05) was observed in the recovery of Salmonella using the combinations of BS, HE, and XLD agars with RV broth and BS agar with TT broth. The combination of selective enrichment in RV and selective isolation on BS gave numerically greatest isolation of Salmonella from Channel catfish and Vietnamese basa fish compared to other isolation combinations.     

Listeria monocytogenes survival of UV-C radiation is enhanced by presence of sodium chloride, organic food material and by bacterial biofilm formation

The bactericidal effect on food processing surfaces of ceiling-mounted UV-C light (wavelength 254 nm) was determined in a fish smoke house after the routine cleaning and disinfection procedure. The total aerobic counts were reduced during UV-C light exposure (48 h) and the number of Listeria monocytogenes positive samples went from 30 (of 68) before exposure to 8 (of 68). We therefore in a laboratory model determined the L. monocytogenes reduction kinetics by UV-C light with the purpose of evaluating the influence of food production environmental variables, such as presence of NaCl, organic material and the time L. monocytogenes was allowed to adhere to steel before exposure. L. monocytogenes grown and attached in tryptone soy broth (TSB) with glucose were rapidly killed (after 2 min) by UV-C light. However, bacteria grown and adhered in TSB with glucose and 5% NaCl were more resistant and numbers declined with 4-5 log units during exposure of 8-10 min. Bacteria grown in juice prepared from cold-smoked salmon were protected and numbers were reduced with 2-3 log when UV-C light was used immediately after attachment whereas numbers did not change at all if bacteria had been allowed to form a biofilm for 7 days before exposure. It is not known if this enhanced survival is due to physiological changes in the attached bacterial cells, a physical protection of the cells in the food matrix or a combination. In conclusion, we demonstrate that UV-C light is a useful extra bacteriocidal step and that it, as all disinfecting procedures, is hampered by the presence of organic material.     

Antioxidant effects of soy sauce on color stability and lipid oxidation of raw beef patties during cold storage

This study was conducted to evaluate the antioxidant effects of soy sauce on lipid oxidation and color stability of raw beef patties. Raw beef patties were formulated with four solutions such as NaCl (sodium chloride solution), NaCl/SS (1:1 ratio of sodium chloride and soy sauce solution), SS (soy sauce solution), or SS/A (soy sauce solution combined with 0.05% ascorbic acid) in the same salt concentration. Addition of soy sauce resulted in the decreased pH, lightness, and increased yellowness. Treatment SS/A had the lowest percent of metmyoglobin during storage (P < 0.05). A reduction (P < 0.05) in the 2-thiobarbituric acid, peroxide, and conjugated diene concentration as result of soy sauce addition were observed in treatments SS and SS/A at the end of the storage period. There were no differences (P > 0.05) in free fatty acid concentration at the end of storage. The combined addition of soy sauce and ascorbic acid greatly improved (P < 0.05) color stability and retarded lipid oxidation.     

Suppressive effect of Tetragenococcus halophilus,isolated from fish-nukazuke,on histamine accumulation in salted and fermented fish

Occasionally, quantities higher than 1000 mg/kg of histamine (Hm) accumulate in salted and fermented fish products by the histidine decarboxylase of halophilic lactococci Tetragenococcus sp. In a total of 200 isolates from fish nukazuke (salted and fermented fish with rice bran), 13 strains produced Hm more than 200 μg/ml in 0.5% histidine containing broth, whereas 130 isolates produced absolutely no Hm. Among the strains, 22 strains suppressed the Hm production of the Hm-forming (HmF) strains. Both the HmF and Hm-suppressing (HmS) strains were identified as Tetragenococcus halophilus. To observe the Hm-suppressing effect, a specified quantity of live cells was needed. In the case of 10% NaCl salted sardine, inoculation with 3 log cells/g of a strain HmF-131 resulted in a significant Hm accumulation, 2800 μg/g in 30 days at 20 °C. The increase in Hm was clearly suppressed by 9 log live cells/g of strain HmS-129. These results suggest that HmS-129 can be used as a starter for salted and fermented fish products, enhancing food safety.     

Improvement of Fish Sauce Quality by Strain CMC5‐3‐1: A Novel Species of Staphylococcus sp.

Staphylococcus sp. CMC5‐3‐1 and CMS5‐7‐5 isolated from fermented fish sauce at 3 to 7 mo, respectively, showed different characteristics on protein hydrolysis and volatile formation. These Gram‐positive cocci were able to grow in up to 15% NaCl with the optimum at 0.5% to 5% NaCl in tryptic soy broth. Based on ribosomal 16S rRNA gene sequences, Staphylococcus sp. CMC5‐3‐1 and CMS5‐7‐5 showed 99.0% similarity to that of Staphylococcus piscifermentans JCM 6057^T, but DNA–DNA relatedness was <30%, indicating that they were likely to be new species. DNA relatedness between these 2 strains was only 65%, suggesting that they also belonged to different species. The α‐amino group content of 6‐month‐old fish sauce inoculated with Staphylococcus sp. CMC5‐3‐1 was 740.5 mM, which was higher than that inoculated by the strain CMS5‐7‐5 (662.14 mM, P < 0.05). Histamine was not produced during fermentations with both strains. Fish sauce inoculated with Staphylococcus sp. CMC5‐3‐1 showed the highest content of total glutamic acid (P < 0.05). The major volatile compound detected in fish sauce inoculated with Staphylococcus sp. CMC5‐3‐1 was 2‐methypropanal, contributing to the desirable dark chocolate note. Staphylococcus sp. CMC5‐3‐1 could be applied as a starter culture to improve the umami and aroma of fish sauce.     

The effect of lactose, NaCl and an aero/anaerobic environment on the tyrosine decarboxylase activity of Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis

The aim of this work was to study, under model conditions, combined effects of the concentration of lactose (0-1% w/v), NaCl (0-2% w/v) and aero/anaerobiosis on the growth and tyramine production in 3 strains of Lactococcus lactis subsp. lactis and 2 strains of L. lactis subsp. cremoris. The levels of the factors tested were chosen with respect to the conditions which can occur during the real process of natural cheese production, including the culture temperature (10 ± 1 °C). In all strains tested, tyrosine decarboxylation was most influenced by NaCl concentration; the highest production of tyramine was obtained within the culture with the highest (2% w/v) salt concentration applied. Two of the strains L. lactis subsp. lactis produced tyramine only in broth with the highest NaCl concentration tested. In the remaining 3 strains of L. lactis, tyramine was detected under all conditions applied. The tested concentration of lactose and aero/anaerobiosis had a less significant effect on tyramine decarboxylation. However, it was also found that at the same concentrations of NaCl and lactose, a higher amount of tyramine was detected under anaerobic conditions. In all strains tested, tyramine decarboxylation started during the active growth phase of the cells.