Thai Fermented Pork Sausage Research Articles

Influence of sucrose level and inoculation of Lactobacillus plantarum on the physicochemical, textural, microbiological, and sensory characteristics of Isan sausage (Thai fermented pork sausage).

The aim of this study was to evaluate the influence of sucrose levels (0.3% and 1.2%) and inoculation of Lactobacillus plantarum on the quality of Isan sausage. The results show that the inoculation accelerated the pH reduction in fermentation. The inoculated samples had significantly (p<.05) lower thiobarbituric acid reactive substances and volatile basic nitrogen values. The total microbial counts and lactic acid bacteria (LAB) counts increased rapidly during fermentation and then decreased during storage. The inoculated samples had significantly higher total microflora and LAB counts. The inoculated samples had a higher hardness texture profile as well as more intense flavor and darker color in sensorial evaluation. Less influence on the quality parameters of the samples due to the different sucrose levels was observed. In conclusion, a combination of 0.3% sucrose and inoculation of L.plantarum (7logcfu/g) is recommended for producing Isan sausages.

Identification and in vitro assessment of potential probiotic characteristics and antibacterial effects of Lactobacillus plantarum subsp. plantarum SKI19, a bacteriocinogenic strain isolated from Thai fermented pork sausage.

A total of 2257 lactic acid bacteria were preliminarily screened for antagonistic activity against Lactobacillus sakei subsp. sakei JCM 1157. Strain SKI19 was selected and identified at the subspecies level as Lactobacillus plantarum subsp. plantarum SKI19, using 16S rRNA gene sequence analysis combined with recA and dnaK genes' amplification. Antibacterial activity of SKI19 was completely lost after treatment of neutralized cell free culture supernatant with proteolytic enzymes, suggesting that SKI19 produced a bacteriocin-like substance that inhibited not only closely related species, but was also effective against Listeria monocytogenes DMST 17303. Viewed under scanning electron microscope, cell membranes of the indicator strain appeared to collapse after exposure to the bacteriocin-like substance. In vitro tests concerning probiotic properties, SKI19 survived under simulated gastrointestinal tract conditions, and adhesion of its cell surface to xylene and chloroform was 90.14 and 89.85%, respectively. Complete inhibition by SKI19 against pathogenic bacteria (Escherichia coli DMST 4212, L. monocytogenes DMST 17303, and Staphylococcus aureus DMST 8840) was observed in co-cultivation under anaerobic conditions. A safety assessment showed that SKI19 was susceptible to several antibiotics and had no haemolytic activity. PCR amplification of virulence factors with the specific primers for ace, asa1, cylLS , efaAfs , hyl, and gelE genes were negative for SKI19. Also, SKI19 did not harbor any hdc, tdc, odc or ldc genes involved in biogenic amine production. The results reveal that SKI19 has probiotic potential and antibacterial activity, and is safe for further application in certain food products.

Use of γ-aminobutyric acid producing lactic acid bacteria as starters to reduce biogenic amines and cholesterol in Thai fermented pork sausage (Nham) and their distribution during fermentation

The objectives of this study were to reduce the amounts of biogenic amines (BAs) and cholesterol in Thai fermented pork sausage (Nham) using the γ-aminobutyric acid (GABA) producers, Pediococcus pentosaceus HN8 and Lactobacillus namurensis NH2, as a mixed starter (6 log CFU/g for each strain). The distribution of lactic acid bacteria (LAB) was also monitored to follow the prevalence of the mixed starter during fermentations. The starter cultures in the inoculated Nham sets (TSM, starter + 5 g/kg monosodium glutamate, MSG; and TSN, starter + no MSG) reduced the amounts of 7 BAs to meet the safety regulation requirements in the order of β-phenylethylamine > histamine > tyramine > putrescine > spermine > spermidine > cadaverine. In contrast, with the control fermentations (TNN, no starter + no MSG and TNM, no starter + MSG, 5 g/kg) only histamine was higher than the recommended level. Cholesterol content was also reduced by 35% in the GABA Nham set (TSM, GABA 3962 mg/kg) when compared with the TNN; and this was significantly lower than the popular commercial Nham brands (114–125 mg cholesterol/100 g). Determination of the LAB succession confirmed that the starter cultures played the biggest roles in reducing both the BAs and cholesterol levels.

Two putatively novel bacteriocins active against Gram-negative food borne pathogens produced by Weissella hellenica BCC 7293

Weissella hellenica BCC 7239, isolated from Thai fermented pork sausage called Nham, produced two putatively novel bacteriocins, 7293A and 7293B. Both bacteriocins had broad antimicrobial spectra and exceptionally inhibited several important Gram-negative food-borne pathogens (Pseudomonas aeruginosa, Aeromonas hydrophila, Salmonella Typhimurium and Escherichia coli). The highest amount of bacteriocin was produced in MRS and APT media at 30 °C without agitation. Bacteriocin 7293A showed relatively higher antimicrobial activity than bacteriocin 7293B. However, pH and thermal stability of bacteriocin 7293A was lower. These bacteriocins were of proteinaceous nature, in which the complete inactivation of their antimicrobial activity after treatment by proteolytic enzymes, including trypsin, α-chymotrypsin, pepsin and protease K was observed, whilst lipase and α-amylase exhibited no effect. Antimicrobial activity of both peptides was also not inactivated by organic solvents (ethanol, isopropanol, acetone, acetonitrile) and surfactants (Tween 20, Tween 80 and Triton X-100). Bacteriocin 7293A and B exhibited bactericidal effect against both Gram-positive and Gram-negative indicators without cell-lysis. According to ESI/MS analysis, the molecular masses of bacteriocin 7293A and B were determined to be 6249.302 and 6489.716 Da, respectively. Because their molecular masses were not similar to those of other known bacteriocins, both bacteriocin 7293A and B could be novel bacteriocins. Thus, both novel bacteriocins hold promise for applications in the prevention or treatment of pathogenic infections as food and feed additives to replace antibiotics for enhancing the productivity and sustainability of food animals.

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 6logCFU/g of each of the two strains produced a maximal amounts of GABA (4051mg/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 (TSM) 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 (TNN), or only the addition of MSG (TNM), or with only the starter (TSN). 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 (TNN). 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.

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.

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.

Monitoring Lactobacillus plantarum BCC 9546 starter culture during fermentation of Nham, a traditional Thai pork sausage

The use of Lactobacillus plantarum BCC 9546 (LpBCC9546) as a starter culture for Nham, a traditional Thai fermented pork sausage ensures product quality and consistency. However, no direct evidence has confirmed the growth of this starter during Nham fermentation. In order to investigate its role during Nham fermentation, LpBCC9546 was genetically modified to distinguish it from the natural microflora in Nham. LpBCC9546 was transformed with a recombinant plasmid pRV85 to produce the recombinant strain LpG11, which is resistant to erythromycin and emits green fluorescence. LpG11 was used as a starter culture for Nham fermentation, and its growth was monitored by plating on a selective medium and assay of fluorescent activity. During Nham fermentation the numbers of LpG11 increased ten fold during the first 12 h of fermentation, reaching maximum numbers of between 10 7 and 10 8 cfu g − 1 after 24 h, and then declining after 60 h to 10 5 cfu g − 1 at 168 h. The growth of LpG11 starter culture during Nham fermentation was very similar to that of the untransformed LpBCC9546, although after a prolonged period of fermentation the recombinant LpG11 bacteria appeared to lose the plasmid, or were outgrown by naturally present L. plantarum. The acidity, texture and color of fermented Nham inoculated with recombinant LpG11 or untransformed LpBCC9546 were similar. These results indicated that the recombinant L. plantarum strain LpG11 is a suitable starter culture for Nham fermentation, and that the ability to monitor its growth directly during Nham fermentation could be exploited to further improve Nham production.

Identification of a glutaminase-producing bacterial strain isolated from Thai fermented pork sausage and characterisation of glutaminase production

Glutaminase is one of the most important flavour-enhancing enzymes used in the food industry. Among 108 glutaminase producing bacteria isolated from fermented foods in Thailand, one isolate (JRT-7) that exhibited the highest glutaminase activity was selected for further study. Morphological and physiological characteristics, and 16S rDNA sequence-based phylogenetic analysis identified strain JRT-7 asKurthia gibsonii. Moreover, the highest specific activity of glutaminase (0.057 U/mg) was obtained when JRT-7 was grown in a medium containing 2.5% succinate and 2.5% L-glutamine.

Isolation and characterization of acid-sensitive mutants of Pediococcus acidilactici

Acid-sensitive mutants of Pediococcus acidilactici BCC 9545, a starter culture of the Thai fermented pork sausage nham, were isolated as spontaneous neomycin resistant mutants. The mutants generally produced less acid and acidified the culture media less than the parent strain in a 72 h culturing period. Interestingly, the ATPase activities of the mutants did not differ considerably from that of the parent strain in acidic conditions. It was also found that the internal pH values of the mutant strains were somewhat lower in neutral environment, while at pH 5.0 their internal pHs were significantly lower compared to the parent's. Inhibiting the H +-ATPase activities in energized cells by N, N′-dicyclohexyl carbodiimide also revealed that protons were leaking from the mutants at neutral pH, which increased under acidic conditions. In contrast, the parent strain exhibited a smaller proton leak and only under acidic conditions. The membrane fatty acid analysis of the mutants indicated that under acidic conditions the mutants had a significantly smaller major unsaturated/saturated fatty acids ratio ((C 18:1 + C 18:3n6)/(C 16:0 + C 18:0)) compared to the parent strain's membrane. Taken together, these observations suggest there is a reasonable possibility that the membrane fatty acid profile differences in the mutants resulted in their acid-sensitivity.

Changes in lipid composition and fatty acid profile of Nham, a Thai fermented pork sausage, during fermentation

Changes in lipid composition and fatty acid profile of Nham during fermentation were investigated. Total lipids of Nham were in the range 2–3%. The extracted lipid of initial Nham mix consisted mainly of triglycerides (TG), accounting for more than 75% of the total lipid, followed by phospholipids (PL) and a trace amount of diglycerides (DG) and free fatty acid (FFA). During fermentation, TG, DG and PL decreased with a concomitant increase in FFA, indicating lipolysis of Nham lipids during fermentation. Changes in fatty acids of the total lipids, non-polar and polar lipid fractions were observed during fermentation. In both total and non-polar lipid fractions, the major fatty acids found in a descending order were oleic (C18:1), linoleic (C18:2) and palmitic (C16:0) acids, which together accounted for 90% of the total fatty acids. Increases in fatty acid contents in both total and non-polar lipid fractions, were observed with a corresponding decrease in the quantity of fatty acids of phospholipids. As the fermentation proceeded, peroxide value generally increased while TBARS values decreased. Overall, lipid oxidation in Nham occurred during fermentation but did not cause the objectionable odour and taste in any Nham tested.

Influence of minced pork and rind ratios on physico-chemical and sensory quality of Nham – a Thai fermented pork sausage

The effects of incorporating varying levels of minced pork and rind on physico-chemical and sensory quality of Nham were studied. An increase in cooked pork rind resulted in higher moisture, lipid, and initial pH values of Nham ( P < 0.05). However, no significant effects were observed on fermentation characteristics of Nham ( P > 0.05). At the end of fermentation, Nham with a higher meat component exhibited higher texture profile analysis force, hardness, and cohesiveness ( P < 0.05). The results suggested the importance of meat on the restructuring effect, which contributes to the texture formation of Nham. Incorporation of a higher amount of cooked pork rind improved water-binding properties, leading to decreased weight loss and released water. Based on the results of sensory evaluation, up to 43% pork rind can be used in the formulation with no adverse effect on texture and overall liking of Nham. However, the ratio of 5:5 was the most appropriate for minimising the cost of production.

An overview of small-scale food fermentation technologies in developing countries with special reference to Thailand: scope for their improvement

Small-scale food fermentation technologies in developing countries have evolved through years of experience rather than through scientific breakthroughs. Many small-scale manufacturers are, therefore, reluctant to accept change and modify fermentation processes. Upgrading the quality and safety of fermented foods, while reducing their production cost and maintaining their authenticity and uniqueness, is of utmost importance. One strategic approach, which has been successfully implemented for the improvement of small-scale soy sauce fermentation in Thailand, is the consortium approach. This approach has allowed industry to work closely in sharing knowledge and common problems, which in turn has provided scientists with the research direction that would best benefit the industry. This consortium approach has brought about changes in the methodologies used in the production of soy sauce, by shortening processing times, introducing fiberglass tanks as bioreactors instead of the traditionally used small earthenware containers and introducing cost-effective waste management systems. One barrier to the application of starter cultures in the small-scale fermentation industry is the loss of uniqueness of fermented products. However, the advent of molecular biology techniques has allowed science to tailor starter cultures to the specific requirements of the manufacturer. Using the techniques of molecular biology, it has been shown that microflora of a specific product vary according to origin and sensory quality. A cell bank is being developed to serve as a resource base for Thai fermented pork sausage in order to facilitate the application of starter cultures in the manufacture of that product.