Bacterial community succession and biogenic amine changes during fermentation of fish-chili paste inoculated with different commercial starter cultures

The microbial dynamics, diversity and succession characterisation during the fermentation of fish-chili paste, with and without starter cultures by high-throughput sequencing, were investigated to identify the relationship between microbial composition and the accumulation of biogenic amines. Results showed that Firmicutes was the predominant phylum, and Lactobacillus and Weissella were the main genera during fermentation process, regardless of the inclusion of starter cultures. Compared with naturally fermented samples (NS; no starter cultures), Pediococcus and Staphylococcus flourished in samples inoculated with SBM-52 starter culture (Staphylococcus xylosus + Staphylococcus carnosus + Pediococcus pentosaceus + Pediococcus acidilactici). Staphylococcus also accounted for a larger proportion in samples inoculated with WBX-43 starter culture (Staphylococcus xylosus + Staphylococcus carnosus). Furthermore, Enterobacter was inhibited in inoculated samples. Unweighted pair-group method with arithmetic means and principal component analyses revealed that microbiota structures were different among NS, SBM and WBX samples. In case of biogenic amines, the concentration of putrescine, cadaverine and tyramine increased to higher levels in NS samples compared with inoculated samples. Our work also investigated the relationship between core bacterial communities and biogenic amines. This study provided an improved understanding for the effects of starter cultures on the microbial community and the quality of fish-chili paste.     

Biogenic amine formation in Nham,a Thai fermented sausage,and the reduction by commercial starter culture, Lactobacillus plantarum BCC 9546

Biogenic amines are of concern for sausage due to their toxicological effects on nervous, blood pressure, gastric and intestinal systems. In this study, the influence of raw pork meat quality and starter culture inoculation on biogenic amines accumulation in Nham, a Thai traditional fermented pork, were studied. Before Nham processing, pork meat was stored at 30 °C for 6 h, and at 4 and −20 °C for 2 days. Formation of biogenic amines (cadaverine, putrescine, histamine and tyramine) was significantly higher in Nham processed from stored meat. Accumulation of these biogenic amines in Nham reduced significantly by the addition of Lactobacillus plantarum BCC 9546, a commercial Nham starter culture. The results highlight the importance of using fresh meat products and the inclusion of an appropriate starter culture to minimise the formation of biogenic amines during the process of Nham fermentation.     

Potential of wine-associated lactic acid bacteria to degrade biogenic amines

Some lactic acid bacteria (LAB) isolated from fermented foods have been proven to degrade biogenic amines through the production of amine oxidase enzymes. Since little is known about this in relation to wine micro-organisms, this work examined the ability of LAB strains (n = 85) isolated from wines and other related enological sources, as well as commercial malolactic starter cultures (n = 3) and type strains (n = 2), to degrade histamine, tyramine and putrescine. The biogenic amine-degrading ability of the strains was evaluated by RP-HPLC in culture media and wine malolactic fermentation laboratory experiments. Although at different extent, 25% of the LAB isolates were able to degrade histamine, 18% tyramine and 18% putrescine, whereas none of the commercial malolactic starter cultures or type strains were able to degrade any of the tested amines. The greatest biogenic amine-degrading ability was exhibited by 9 strains belonging to the Lactobacillus and Pediococcus groups, and most of them were able to simultaneously degrade at least two of the three studied biogenic amines. Further experiments with one of the strains that showed high biogenic amine-degrading ability (L. casei IFI-CA 52) revealed that cell-free extracts maintained this ability in comparison to the cell suspensions at pH 4.6, indicating that amine-degrading enzymes were effectively extracted from the cells and their action was optimal in the degradation of biogenic amines. In addition, it was confirmed that wine components such as ethanol (12%) and polyphenols (75 mg/L), and wine additives such as SO₂ (30 mg/L), reduced the histamine-degrading ability of L. casei IFI-CA 52 at pH 4.6 by 80%, 85% and 11%, respectively, in cell suspensions, whereas the reduction was 91%, 67% and 50%, respectively, in cell-free extracts. In spite of this adverse influence of the wine matrix, our results proved the potential of wine-associated LAB as a promising strategy to reduce biogenic amines in wine.     

Biogenic amines formation in Streptococcus thermophilus isolated from home-made natural yogurt

Twelve different biogenic amines formation in 58 isolates of Streptococcus thermophilus from home-made natural yogurt were investigated in histidine (HDB) and lysine decarboxylase broth (LDB). All S. thermophilus isolates had an ability to produce twelve different biogenic amines in HDB and LDB. Most of the S. thermophilus isolates formed low amounts of histamine (1–50 mg/L) from histidine. Apart from one isolate, S. thermophilus produced tyramine at low (47 isolates) and medium (10 isolates) levels. The amount of each specific biogenic amine produced by S. thermophilus was generally lower than 100 mg L⁻¹. Also, the presence of hdcA gene was investigated using PCR technique and relation between gene and histamine production was conducted in S. thermophilus isolates. This study showed that most of the S. thermophilus isolates have the ability to form biogenic amines, especially histamine, and tyramine, which is an important consideration when selecting strains as starter cultures.     

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.     

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.     

Biogenic amines and potential histamine – Forming bacteria in Rihaakuru (a cooked fish paste)

The biogenic amine concentration in Rihaakuru (a fish paste) (n = 28), obtained from different parts of the Maldives (North, South, and Central), was determined by HPLC. Ten biogenic amines were detected; agmatine, not detected (ND) – 161 ppm; cadaverine, ND – 387 ppm; histamine, ND – 5487 ppm; putrescine, ND – 290 ppm; phenylethylamine, ND – 23 ppm; serotonin, ND – 91 ppm; spermine, ND – 329 ppm; spermidine, ND – 79 ppm; tryptamine, ND – <5 ppm; and tyramine, ND – 50 ppm. Nine biogenic amines were found in 3 samples, 8 in 10 samples, 7 in 6 samples, 6 in 3 samples, 4 in 5 samples, and 1 was found in 1 sample. Histamine was detected at levels that are regarded as a risk to human health. Fourteen isolates were selected from two randomly selected samples out of the 28 samples of Rihaakuru and screened for histamine production. Twelve of the 14 isolates produced histamine, with the highest histamine producers being Bacillus massiliensis Nai5 (6.65 ppm) and Bacillus polyfermenticus (5.58 ppm); while Bacillus malacitensis produced the least (<0.5 ppm).     

The effect of ripening and storage conditions on the distribution of tyramine,putrescine and cadaverine in Edam-cheese

The aim of the work was to describe the development of selected biogenic amines (histamine, tyramine, putrescine and cadaverine) in 4 layers of Dutch-type cheese (Edam-cheese) depending on 3 ripening/storage regimes during a 98-day period. Biogenic amines were analysed by means of ion-exchange chromatography. A further goal was to identify microbial sources of biogenic amines in the material analysed. Phenotype characterization and repetitive sequence-based PCR fingerprinting were used to identify the isolated bacteria. The highest content of tyramine, putrescine and cadaverine was determined in cheeses stored in a ripening cellar at a temperature of 10 °C during the whole observation period. Lower biogenic amines content was determined in samples which were moved into a cold storage device (5 °C) after 38 days of storage in a ripening cellar (10 °C). The lowest concentrations of biogenic amines were detected in cheeses which were moved into a cold storage device (5 °C) after 23 days of storage in a ripening cellar (10 °C). During the 98-day period, histamine was not detected in any of the regimes. Within the cheeses analysed, non-starter lactic acid bacteria Lactobacillus curvatus, Lactobacillus casei/paracasei and Lactobacillus plantarum were detected as the main producers of the biogenic amines tested. In starter bacteria Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris the decarboxylase activity tested was not detected.     

Effects of starter cultures and nitrite levels on formation of biogenic amines in sucuk

The effects of starter cultures, starter A (Lactobacillus sakei+Staphylococcus carnosus) and starter B (Pediococcus acidilactici+Staphylococcus xylosus+Lactobacillus curvatus), nitrite levels (0, 75, and 150ppm) and ripening period on biogenic amine production were investigated in sucuk (Turkish dry-fermented sausage) production. Changes in biogenic amines, pH, water activity, moisture, non-protein nitrogen, nitrite and counts of lactic acid bacteria, Micrococcus/Staphylococcus, Enterobacteriaceae, and moulds-yeasts were determined. Ripening period had a very significant effect (P<0.01) on the all biogenic amines. Sausages produced by fermentation with starter cultures, as compared to natural fermentation (control), had lower amounts of putrescine, cadaverine and tyramine (P<0.05). Starter cultures A and B were found to have almost the same effect on the formation of biogenic amines. Use of nitrite in sucuk production affected the formation of biogenic amines except for spermidine and spermine (P<0.01). Lactic acid bacteria counts in the control group were lower than that of starter culture groups. Lactic acid bacteria, Micrococcus/Staphylococcus, and moulds-yeasts counts increased during ripening period while levels of species of Enterobacteriaceae decreased, which was also found to be under the detectable level (<100cfug(-1)), by day 3 of ripening.     

Tyrosine- and histidine-decarboxylase positive lactic acid bacteria and enterococci in dry fermented sausages

Lactic acid bacteria (LAB) and enterococci were isolated immediately after stuffing (day 0), at the end of ripening (28th day) and at the end of storage (112th day) from dry fermented sausages produced by two different producers (K; R) in two diameters (4.5 and 7 cm) using either of two spice mixtures (P; H) and either of two starter cultures (Pediococcus pentosaceus, C; Lactobacillus curvatus + Staphylococcus carnosus, F), resulting in a total of 16 different combinations. Tyrosine-decarboxylase DNA sequence (tyrdc) was identified on average in 88% and 44% of enterococci and LAB isolates, respectively at the end of ripening, the corresponding figures regarding histidine-decarboxylase gene sequence (hisdc) was 71% and 16%, respectively. Lactobacillus plantarum, L. brevis and L. casei/paracasei, and Enterococcus faecium and Enterococcus faecalis were identified as tyramine/histamine producers in the sausages.     

Biogenic and volatile amine-related qualities of three popular fish species sold at Kuwait fish markets

Three fish species commonly consumed in Kuwait were studied for their total volatile base nitrogen (TVBN) and biogenic amine composition during normal commercial activities at the fish markets. Samples of the fish species Zubaidi or silver pomfret (Pampus argenteus), Hammour or orange–spotted grouper (Epinephelus coioides), and Negrule or grunt (Pomadasys kaakan) were analyzed using steam distillation and high performance liquid chromatography (HPLC) procedures. Fish samples collected in the morning had lower levels of TVBN, ranging from 25.9 mg/100 g to 30.2 mg/100 g compared with the range of 31.2 mg/100 g to 40.9 mg/100 g for samples collected in the evening. Among all three fish species, Zubaidi (Pampus argenteus) showed the lowest TVBN content while Negrule (Pomadasys kaakan) had the highest. Biogenic amine levels were also low in the fish species. The data suggest that fish handling practices at the fish markets in Kuwait do not result in significant abuse of fish temperatures, and that the amounts of volatile and biogenic amines produced during normal activities did not rise to levels indicating fish decomposition.     

Proteinase-producing halophilic lactic acid bacteria isolated from fish sauce fermentation and their ability to produce volatile compounds

Halophilic lactic acid bacteria were isolated from fish sauce mashes fermented at 1 to 12 months. Seven out of sixty-four isolates were selected according to their proteolytic activity and growth at 25% NaCl for characterization and investigation of volatile compound production. All selected isolates were Gram-positive cocci with pairs/tetrads and grew at 0-25% NaCl, pH 4.5-9.0. Results of 16S rRNA gene sequence analysis showed 99% homology to Tetragenococcus halophilus ATCC 33315. The restriction fragment length polymorphism (RFLP) patterns of all isolates were also similar to those of T. halophilus ATCC 33315. These isolates were, thus, identified as T. halophilus. All isolates hydrolyzed fish protein in the medium containing 25% NaCl. Intracellular aminopeptidase of 7 isolates exhibited the highest activity of 2.85-3.67 U/ml toward Ala-p-nitroanilide (Ala-pNA). T.halophilus strains MS33 and M11 showed the highest alanyl aminopeptidase activity (P < 0.05), and produced histamine in mGYP broth containing 5 and 25% NaCl in the level of 6.62-22.55 and 13.14-20.39 mg/100 ml, respectively. Predominant volatile compounds of fish broth containing 25% NaCl inoculated with T. halophilus MS33 and MRC5-5-2 were 1-propanol, 2-methylpropanal, and benzaldehyde, corresponding to major volatile compounds in fish sauce. T.halophilus appeared to play an important role in volatile compound formation during fish sauce fermentation.     

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.     

Biogenic amine changes in barramundi (Lates calcarifer) slices stored at 0 °C and 4 °C

The biogenic amines formation in barramundi (Lates calcarifer) slices kept for 15 days at 0 °C and 4 °C were investigated using nine biogenic amines, total plate counts and biogenic amines formers. Significant differences in biogenic amines concentrations of barramundi slices stored at 4 °C and at 0 °C after 3 days of storage were observed. All amines, except tryptamine, 2-phenylethylamine, tyramine and agmatine in the slices increased with time during storage at both temperatures. At the end of the storage period, histamine concentrations were 82 mg/kg and 275 mg/kg for samples kept at 0 °C and 4 °C, respectively. At day 15, the total plate count was approximately 8.6 log CFU/g for sample kept at 0 °C and 9.7 log CFU/g for samples kept at 4 °C. Histamine-forming bacteria (HFB) in all samples ranged from 5.4 to 6.1 log CFU/g at 0 °C and 4 °C, respectively. The observed shelf-life of barramundi slices were 6–9 days.     

Studies on the low-salt Chinese potherb mustard (Brassica juncea, Coss.) pickle. I—The effect of a homofermentative l(+)-lactic acid producer Bacillus coagulans on starter culture in the low-salt Chinese potherb mustard pickle fermentation

A homofermentative l(+)-lactic acid producer Bacillus coagulans B179 has been evaluated as starter cultures for low-salt potherb mustard pickle process. This study examined the impact of inoculation by B. coagulans B179 as starter culture on the spontaneous lab-scale fermentation process of potherb mustard pickle. Three different salt concentrations trials, i.e., 5 g/100 g, 8 g/100 g and 10 g/100 g, respectively, were carried out. As a result, the addition of starter culture of B. coagulans B179 to 5 g/100 g salinity fermentation showed an inhibiting effect upon the population of non-lactic acid bacteria (LAB) and marked increase in population of LAB and production of titratable acidity but did not significantly influence the pickle end products quantity of acid produced by fermentation as compared with a treatment without inoculation. However, in the higher salt concentrations, i.e., 8 g/100 g and 10 g/100 g NaCl trials, inoculation had no impact on the fermentation of the pickle. From the standpoint of fermentation, as a functional starter culture, inoculated B. coagulans B179 can effectively improve desired native LAB growth and inhibit the growth of undesired fungi, which will thereby allow it to shorten fermentation period and enhance the pickle quality in the low-salt potherb mustard pickle production.     

Irradiation and organic acid treatment for microbial control and the production of biogenic amines in beef and pork

The effect of irradiation and organic acid treatment in controlling the growth of microorganisms and the formation of biogenic amines (BAs) in ground beef and pork was studied. Bacillus cereus, Enterobacter cloacae, and Alcaligenes faecalis were inoculated into the ground beef and pork with approximately 10⁷ CFU/g. A gamma irradiation was used with absorbed doses of 0, 0.5, 1, and 2 kGy, as irradiation treatment and 2 M solutions of acetic, citric, and lactic acid were used as organic acid treatment. Irradiation was effective in reducing the inoculated bacteria and achieved about 3 decimal reductions by 2 kGy. The levels of putrescine, tyramine, spermine, and total amount of biogenic amines were significantly reduced by irradiation of ground beef and pork inoculated with different microorganisms tested. On the other hand, organic acid treatment showed only 2 decimal reductions or less. The reduction of the BAs content was also limited and variable by organic acid treatment. Therefore, the results indicate that, for the controlling of microorganisms and production of biogenic amines in ground beef and pork, irradiation was more effective than organic acid treatment.     

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.     

Characterization of fermented silver carp sausages inoculated with mixed starter culture

To improve the characteristics and functionality, and increase the use of fish muscle, three groups mixed starter cultures (group one: Lactobacillus plantarum-15, Staphylococcus xylosus-12 and Pediococcus pentosaceus-ATCC33316 [S-PXP]; group two: Lactobacillus planatrum-15, Staphylococcus xylosus-12 and Lactobacillus casei subs casei-1.001 [S-PXC]; and group three: Staphylococcus xylosus-12, Lactobacillus casei subsp. casei-1.001 and P. pentosaceus-ATCC33316 [S-XCP]) were inoculated in minced silver carp muscle to produce a fermented fish product. During the 48 h fermentation at 30 °C, silver carp muscle inoculated with mixed starter cultures resulted in a rapid pH decrease, suppression in the increase of thiobarturic acid (TBARS) values, total volatile base nitrogen (TVB-N), trimethylamine (TMA), and the growth of spoilage bacteria and pathogens, and had higher whiteness than the control (without any starter) (P<0.05). The changes in SDS-PAGE indicated extensive hydrolysis of muscle protein occurred during fermentation. This study showed that the mixed starter cultures could substantially improve the flavor, digestibility, and nutritional value of the silver carp muscle.     

Changes of yolk biogenic amine concentrations during storage of shell hen eggs

The RP-HPLC/UV method, using dabsyl derivatization, optimised for the determination of biogenic amines in egg yolk, was appropriate for quantification of putrescine, cadaverine, histamine, ethylamine, propylamine, ethanolamine, tyramine, tryptamine, spermine, spermidine, phenylethylamine. Detection limits ranged between 0.05 and 0.06 mg of biogenic amine/kg of egg yolk. Two experiments using, respectively, farm and avian eggs were conducted to evaluate yolk biogenic amine concentrations of fresh and stored eggs, and to explain the effect of temperature and time of storage in the levels of biogenic amines during egg shelf-life. Only five of the 11 biogenic amines under study were detected: putrescine, cadaverine, propylamine, ethylamine and ethanolamine. Storage time during shelf-life presented a significant effect on the levels of the five amines (p < 0.01). On the contrary, storage temperature did not presented a significant effect on the levels of the mentioned amines, p > 0.01. The significant reduction of biogenic amine concentration during the shelf-life justified the application of a multiple linear regression using stepwise method to estimate the storage time. The regression equation was applied with success to confirm the storage time of farm eggs and avian eggs that were stored at two different temperatures.     

Histamine production by bacilli bacteria, acetic bacteria and yeast isolated from fruit wines

Eleven red wines imported from foreign country and 40 domestic fruit wines, including 15 red wines, 4 white wines, 7 plum wines, and 14 other fruit wines, sold in the supermarkets in Taiwan were purchased and tested to determine the occurrence of biogenic amines and histamine-forming bacteria. The levels of pH, total soluble solids (TSS), titratable acidity (TA), reducing sugar (RS), total sugar (TS), sulphites, methanol (milligram per liter of pure ethanol), ethanol and Pb in all samples ranged from 3.0 to 4.1, 6.8 to 24.4 °Brix, 0.3 to 1.7 g/100 mL, 0.2 to 17.6 g/100 mL, 1.6 to 28.4 g/100 mL, <2 to 260.5 mg/L, <1 to 2559 mg/L, 5.0 to 15.6 g/100 mL and <1 to 46.2 μg/L, respectively. The levels of TSS, TA, RS, and TS in plum wine samples were significantly higher than those of the other wines samples, whereas the pH value in plum wine samples was lower than that of the other wines samples. The average content for each of the nine biogenic amines in all samples was less than 5.2 mg/L. However, higher levels of histamine and spermine were detected in domestic fruit wine samples than the imported red wine samples. Five histamine-forming isolates isolated from domestic red wine and jackfruit wine, capable of producing 13.0 mg/L to 69.1 mg/L of histamine in trypticase soy broth (TSB) supplemented with 2 g/100 mL l-histidine (TSBH) or MRS broth supplemented with 2 g/100 mL l-histidine (MRSH), were identified as Bacillus pumilus (one strain), Bacillus sp. (two strains) and Acetobacter pasteurianus (one strain) by 16S rDNA sequencing with PCR amplification, and Zygoascus hellenicus var. hellenicus (one strain) by internal transcribed spacer sequencing with PCR amplification. To our knowledge, this is the first report to demonstrate the occurrence of histamine-forming bacilli bacteria, acetic bacteria and yeast in fruit wine.