Isolation and characterization of acid-sensitive Lactobacillus plantarum with application as starter culture for Nham production

The aim of this study was to derive new starter culture variants that are unable to grow below pH 4.6, the desirable pH of the Thai fermented pork sausage, Nham, specified by Thailand Food Standard, and apply them in Nham fermentation. Several acid-sensitive mutants of one of the commercial Nham starter cultures, Lactobacillus plantarum BCC 9546, were isolated as spontaneous neomycin-resistant mutants. The growth of three representative mutants was characterized in MRS broth, which revealed that their cell numbers and acid production were lower than that of the wild-type. The H⁺-ATPase activities of the three mutants were found significantly lower than that of the wild-type under either neutral or acidic conditions. Consequently, internal pH values of the mutants appeared to be lower, especially in acidic environment (pH 5). The most acid-sensitive mutant was applied in experimental Nham production and the pH of Nham fermented with the mutant had significantly higher pH at the end of fermentation (3 days) and after an additional 4 days of storage at 30 °C. These results indicate that the use of acid-sensitive L. plantarum as starter culture can reduce the severity of post-acidification and increase the shelf life of Nham at ambient temperature.     

Isolation and characterisation of a novel Podoviridae-phage infecting Weissella cibaria N 22 from Nham, a Thai fermented pork sausage

A novel Podoviridae lactic acid bacteria (LAB) phage from Nham, a Thai fermented pork sausage, is reported. From a total of 36 samples, 41 isolates of LAB were obtained and employed as hosts for the isolation of phages. From these LAB, only one phage, designated Φ 22, was isolated. The lactic acid bacterial isolate named N 22, sensitive to phage Φ 22 infection was identified by an API 50 CHL kit and N 22’s complete sequence of the 16S rDNA sequence. BLASTN analysis of the 16S rDNA sequence revealed a 99% similarity to the 16S rDNA sequence of Weissella cibaria in the GenBank database. Electron micrographs indicated that the phage head was icosahedral with head size and tail length of 92 × 50 nm and 27 nm, respectively. On the basis of the morphology, this phage belongs to the family Podoviridae. Host-range determination revealed that the phage Φ 22 was not capable of infecting the other 40 isolates of LAB and referenced Weissella strains used. A one-step growth experiment showed that the latent period and burst size were estimated at 110 min and 55 phage particles/infected cell, respectively. Furthermore, the phage was infective over a wide range of pH (pH 5.0-8.0) and the D time of Φ 22 was calculated as 88 s at 70 °C and 15 s at 80 °C. Phage titers decreased below the detection limit (20 PFU/ml) after heating for more than 60 s at 80 °C, or 20 s at 90 °C or less than 10 s at 100 °C. The results from the study of Nham revealed that Φ 22 was active against the potential starter culture (W. cibaria N 22) for Nham fermentation. Phage infection could adversely affect the fermentation process of Nham by delaying acidification when using W. cibaria N 22 as a starter. However, the results from a sensory test revealed that the panelists did not detect any defects in the final products. This is the first report on the isolation of W. cibaria phage.     

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.     

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.     

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.     

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.     

Changes in biogenic amine levels during storage of Mexican-style soft and Spanish-style dry-ripened sausages with different aw values under modified atmosphere

Two raw sausages were prepared: a soft and a dry-ripened one, both by local traditional and industrial manufacturing practices. Sausages were packaged under a CO₂/N₂ atmosphere at different targeted activity water (a_w) values: 0.96 and 0.92 (soft sausages) and 0.88 and 0.82 (dry-ripened sausages). Sausages were then stored at 5 °C for 42 days or at 12 °C for 240 days (soft and a dry-ripened sausages, respectively). The time-related changes in dominant microbiota, pH and biogenic amine contents during storage were determined. Tyramine was the most abundant biogenic amine in all the sausages. Biogenic amine levels were higher in dry-ripened sausages than in soft sausages at packaging. However, during refrigerated storage soft sausages were fermented and the levels of biogenic amines increased (P < 0.05). At the end of storage, traditional soft sausages with 0.96 a_w presented comparable levels of biogenic amines to traditional dry-ripened sausages.     

Oxidation in HiOx-packaged pork Longissimus muscle predisposes myofibrillar and sarcoplasmic proteins to N-nitrosamine formation in nitrite-curing solution

The effect of meat protein in situ oxidation on the formation of N-nitrosodiethylamine (NDEA) was investigated. Fresh minced pork was untreated (Con) or treated with 700 mg/kg α-tocopherol (Toc) or 300 mg/kg tea polyphenols (PPE), packaged in a HiOx atmosphere (78.8% O₂, 18.8% CO₂, 2.4% N₂), then stored at 2 ± 1 °C for up to 10 days. Crude myofibrillar (MP) or sarcoplasmic (SP) protein (20 mg/mL) extracted from stored meat was reacted with 43 μM sodium nitrite at 80 °C for 1 h. Lipid oxidation was totally inhibited in PPE pork but increased in Con and Toc samples after 10 days. There was significant protein oxidation (losses of sulfhydryls, formation of protein carbonyls) in both MP and SP in all samples during storage. However, the Con group suffered more extensive protein oxidation than Toc and PPE and produced more NDEA (P < 0.05), indicating that protein oxidation promoted nitrosation.     

Influence of storage temperature and duration on lipid and protein oxidation and flavour changes in frozen pork dumpling filler

This study was conducted to evaluate the effect of storage temperature and duration on oxidation and flavour changes in frozen pork dumpling filler. Freshly prepared dumplings were stored for 0, 30, 60, 90, and 180 d at − 7 °C, − 18 °C, and an oscillation between − 7 °C and − 18 °C. The samples stored at − 7 °C for 180 d had significantly higher levels of TBARS and protein carbonyls than those stored at − 18 °C and the fluctuating − 7 °C/− 18 °C (P < 0.05). The percentage of unsaturated fatty acids in total lipids decreased with extended storage times. The volatile compounds with pleasant odours decreased with time, while the compounds with pungent tastes and smells increased (P < 0.05). The sensory results showed that the dumplings stored at higher frozen temperatures for long periods of time had significantly lower acceptability scores (P < 0.05). The results suggest that oxidation is a primary cause of quality deterioration in pork dumpling filler during frozen storage.     

Dabsyl derivatisation as an alternative for dansylation in the detection of biogenic amines in fermented meat products by reversed phase high performance liquid chromatography

The commonly applied HPLC method to determine biogenic amines in dry fermented meat after dansylation was compared with an alternative dabsylation procedure. The use of dabsyl chloride at 70 °C resulted in a 25-min reduction of the derivatisation time, in comparison with the dansylation at 40 °C. Furthermore, the use of irritating ammonia to remove the excess of dansyl chloride can be avoided. Introduction of the SPE cleaning procedure on the C18 cartridge resulted in a reliable and sensitive method of biogenic amines determination in a complex protein–fat matrix, which is typical of dry fermented sausages.     

Determination of biogenic amines in foods using ultra-performance liquid chromatography (UPLC)

A rapid ultra-performance liquid chromatographic (UPLC) method for the determination of biogenic amines (putrescine, cadaverine, spermidine, spermine, phenylethylamine, histamine, tyramine and tryptamine) in selected food samples is described. The eight biogenic amines, which are the most important to be determined in food samples, were derivatized with dansyl chloride prior to UPLC separation. The dansylated amines were separated on an Agilent Zorbax Eclipse XDB – C18 column (50 × 4.6 mm ID, 1.8 μm) using gradient elution with a binary system of acetonitrile–water, a flow rate of 1.0 ml/min and UV detection at 225 nm. The analysis is very fast, all amines are well resolved and are eluted from the column in less than 6 min. The average repeatability of the method ranged between 1.02% and 2.14%. Limits of detection (LODs) for considered amines ranged between 0.032 and 0.098 μg/l; calibration curves showed very good linearity (r = 0.9994–1.0000). The method was applied to the analysis of amines in pork, beef, chicken and fish meat, cheese and edible mushrooms.     

Biogenic amine content and biogenic amine quality indices of sardines (Sardina pilchardus) stored in modified atmosphere packaging and vacuum packaging

A comparative study of the effects of packaging on the formation of biogenic amines during storage of sardines (Sardina pilchardus) at 4 °C in air, modified atmosphere pack (MAP) and vacuum pack (VP) was carried out. Sardines were organoleptically acceptable for up to 3 days in air, 12 days in MAP and 9 days in VP. The biogenic amine content generally increased in all treatments with increasing storage time. The concentrations of putrescine and/cadaverine in fish stored in air reached maximum levels of 12.2 mg/100g at 12 days and 10.0 mg/l00 g at 15 days. Significant differences were found (P < 0.05) in the levels of cadaverine and putrescine among the three treatments. Spermidine and spermine levels increased slightly and did not change much throughout the storage period for all experimental conditions. The amine contents of sardine were highest in sardine stored in air, followed by VP and MAP. Quality indices related to the contents of the major biogenic amines were calculated and they correlated well with organoleptic qualities.     

Antioxidant potential of curry (Murraya koenigii L.) and mint (Mentha spicata) leaf extracts and their effect on colour and oxidative stability of raw ground pork meat during refrigeration storage

The aim of this study was to investigate the antioxidant activity of different solvent extracts of curry and mint leaf and their effect on colour and oxidative stability of raw ground pork meat stored at 4 ± 1 °C. The results indicated that among the two individual leaf categories, the ethanol extract of curry leaf (EHEC) and the water extract of mint leaf (WEM) showed higher DPPH and ABTS⁺ activity. EHEC also exhibited the highest total phenolic contents while these were the lowest for WEM. WEM showed the highest superoxide anionic scavenging activity (%). The pork meat samples treated with EHEC and WEM showed a decrease in the Hunter L- and a-values and a increase in b-value during storage at 4 °C. However, the pH and TBARS values were higher in control samples irrespective of storage periods. In conclusion, EHEC and WEM have the potential to be used as natural antioxidants to minimise lipid oxidation of pork products.     

Manufacture of Turkish Beyaz cheese added with probiotic strains

The survival of the probiotic strains Lactobacillus fermentum (AB5-18 and AK4-120) and Lactobacillus plantarum (AB16-65 and AC18-82), all derived from human faces, was investigated in Turkish Beyaz cheese production. Three batches of Turkish Beyaz cheese were produced: one with the test probiotic culture mix (P), another with a commercial starter culture mix including Lactoccocus lactis subsp. cremoris, Lactococcus lactis subsp. lactis (C) and the third with equal parts of the commercial starter culture mix and test probiotic culture mix (CP). The cheeses were ripened at 4 °C for 120 days and the viability of cultures was determined monthly. Cheese samples were analyzed for total solids, fat in solids, titratable acidity, pH, salt in total solids, proteolysis, sensory evaluation, aroma compounds and biogenic amines. While initial lactic acid bacteria load in P cheese was 2.7 × 10⁹ at the beginning, it was 7.42 × 10⁷ cfu/g at the end of 120 days of ripening. The results showed that test probiotic culture mix was successfully used in cheese production without adversely affecting the cheese quality during ripening. The chemical composition and sensory quality of P cheeses were also comparable with C cheeses. The present study indicates that probiotic cultures of human origin are feasible for Turkish Beyaz cheese production.     

Changes in microbiological, biochemical and physico-chemical properties of Nham inoculated with different inoculum levels of Lactobacillus curvatus

The effect of inoculum level of Lactobacillus curvatus on Nham fermentation was studied. Nham inoculated with L. curvatus at 10⁴ (LC104) and 10⁶ cfu/g (LC106) exhibited a higher rate of fermentation than naturally fermented Nham (control) as indicated by greater rate of pH drop, lactic acid production, and changes in protein compositions. Based on the pH, the fermentation was completed within 72, 48 and 36 h for control, LC104, and LC106, respectively. Higher extent of proteolysis and lipolysis were observed during fermentation of Nham inoculated with starter bacteria (P<0.05). Due to higher acid production rate and extent, texture development of inoculated Nham was more rapid. Inoculated Nham exhibited higher TPA hardness and adhesiveness but lower fracturability than naturally fermented Nham (P<0.05). In terms of acceptability, control and LC104 had higher ratings on the overall liking than LC106 (P<0.05). However, LC104 was better accepted than control in terms of flavour, sourness, saltiness, and texture. Unusual smell was detected only in Nham inoculated at 10⁶ cfu/g. Based on physico-chemical properties and consumer acceptability, L. curvatus is a potential starter for Nham fermentation. However, inoculation of L. curvatus at high level may cause off-flavour in the product.     

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.     

Occurrence of heterocyclic amines in cooked meat products

Heterocyclic amines (HCAs), potent mutagens and a risk factor for human cancers, are produced in meats cooked at high temperature. The aim of this study was to determine the HCA content in cooked meat products (beef, chicken, pork, fish) prepared by various cooking methods (pan frying, oven broiling, and oven baking at 170 to 230 °C) that are preferred by U.S. meat consumers. The primary HCAs in these samples were PhIP (2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine) (1.49-10.89 ng/g), MeIQx (2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline) (not detected-4.0 ng/g), and DiMeIQx (2-amino-3,4,8-trimethyl-imidazo [4,5-f]quinoxaline) (not detected-3.57 ng/g). Type and content of HCAs in cooked meat samples were highly dependent on cooking conditions. The total HCA content in well-done meat was 3.5 times higher than that of medium-rare meat. Fried pork (13.91 ng/g) had higher levels of total HCAs than fried beef (8.92 ng/g) and fried chicken (7.00 ng/g). Among the samples, fried bacon contained the highest total HCA content (17.59 ng/g).     

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.     

Amine concentrations in wine stored in bottles at different temperatures

Excessive concentrations of amines are undesirable in wine because they could be toxic or have a negative effect on aroma. Wine storage temperature has a decisive influence on product quality because a rise in temperature augments reactions within the wine. The aim of this study was to examine the evolution of biogenic and volatile amines during storage of wines in bottle at different temperatures. To do so, Chardonnay wine was stored in bottles at 4, 20 and 35 °C, during 105 days. The results showed that wine storage temperature had only a slight effect on the concentration of amines. The concentration of histamine stands out, as it was higher in the wine kept for 105 days at 20 °C than at the two more extreme temperatures of 4 and 35 °C. The formation or degradation of amines was mainly produced during the first 45 days of wine storage for all the temperatures under study.     

Enhancement of γ-aminobutyric acid (GABA) in Nham (Thai fermented pork sausage) using starter cultures of Lactobacillus namurensis NH2 and Pediococcus pentosaceus HN8

The aim was to produce Nham that was enriched with γ-aminobutyric acid (GABA); therefore two GABA producing lactic acid bacteria (Pediococcus pentosaceus HN8 and Lactobacillus namurensis NH2) were used as starter cultures. By using the central composite design (CCD) we showed that addition of 0.5% monosodium glutamate (MSG) together with an inoculum size of roughly 6 log CFU/g of each of the two strains produced a maximal amounts of GABA (4051 mg/kg) in the ‘GABA Nham’ product. This was higher than any current popular commercial Nham product by roughly 8 times. ‘GABA Nham’ with the additions of both starters and MSG (T_SM) supported maximum populations of lactic acid bacteria (LAB) with a minimum of yeasts and no staphylococci or molds when compared to the controls that had no addition of any starters or MSG (T_NN), or only the addition of MSG (T_NM), or with only the starter (T_SN). Based on proximate analysis among the Nham sets, ‘GABA Nham’ was low in fat, carbohydrate and energy although its texture and color were slightly different from the control (T_NN). However, sensory evaluations of ‘GABA Nham’ were more acceptable than the controls and commercial Nham products for all tested parameters. Hence, a unique novel ‘GABA Nham’ fermented pork sausage was successfully developed.