Exploring the Relationships Between Bacterial Community, Taste-Enhancing Peptides and Aroma in Thai Fermented Fish (Pla-ra).

As a traditional Thai condiment, Pla-ra is used to add flavor and richness to dishes. Nine treatment combinations of Pla-ra formulations created from 3 types of fish (Mor fish, Kradee fish, and Mor + Kradee fish) and 4 different carbohydrate sources (none, rice bran, roasted rice, and rice bran─roasted rice mixture) were studied through a 12 month fermentation period (1, 3, 5, 7, 8, 9, 10, 11, and 12 months). 16S rRNA Next Generation Sequencing (NGS) and LC-MS/MS techniques were used to analyze the microbial diversity and identify taste-enhancing peptides. Descriptive sensory analysis was performed on the extracts of the 108 Pla-ra samples mixed in a model broth. Koku perception and saltiness-enhancing attributes were clearly perceived and dominant in all samples, even though glutamyl peptides, including γ-Glu-Val-Gly, were found at subthreshold levels. The samples from mixed fish and Mor fish fermented with roasted ground rice and rice bran for 12 months had the most typical Pla-ra odors and tastes and had high taste-enhancing activities. NGS analysis revealed the presence of bacteria containing a large number of protease and aminopeptidase genes in the samples. Bacillus spp., Gallicola spp., and Proteiniclasticum spp. correlated well with the generation of glutamyl and arginyl peptides and typical odors in the samples. These results confirmed the typical sensory quality of Pla-ra depended on protein sources, carbohydrate sources, and bacteria communities. Further optimization of the microbial composition found could lead to the development of starter cultures to control and promote flavor development in fermented fish products.

Chicken foot broth byproduct: A new source for highly effective peptide-calcium chelate.

As a means of adding value, chicken foot broth byproduct can be processed to obtain calcium and bioactive peptides from the separated bones and meat residues. In this study, cleaned, dried, and powdered bones yielded 31.4 ± 0.6% calcium content. The meat residues were hydrolyzed to obtain over a hundred distinctive peptides, which were analyzed using LC-MS/MS and the SpirPep web-based tool. The peptides were rich in Glu, Asp, Lys, Gly and Leu, and also exhibited diverse bioactivities, among them primarily inhibition of dipeptidyl peptidase IV and angiotensin-converting enzyme. Calcium chelation assay determined the peptides to bind calcium at 235.7 ± 20.0 mg/g peptide-calcium chelate. Caco-2 cells treated with the chelate at calcium concentrations of 0-10 mM exhibited enhanced absorption relative to CaCl2. This demonstrates that calcium and chelating peptides generated from the same byproduct can produce peptide-calcium chelate, a potential ingredient in functional foods.

Investigation of Kokumi Substances and Bacteria in Thai Fermented Freshwater Fish (Pla-ra).

A 16S rRNA next-generation sequencing technique was applied to investigate the microbial diversity and liquid chromatography-tandem mass spectrometry was used to identify glutamyl peptide profiles of 10 Thai fermented freshwater fish (Pla-ra) samples. A total of 12 genera of bacteria were able to be detected, with Tetragenococcus spp., Staphylococcus spp., and Lactobacillus spp. dominating. Of the 18 glutamyl peptides analyzed, 17 were found, even though the amounts detected were lower than the taste threshold. Despite this, an increase in mouthfulness sensation, reflecting kokumi activity, was clearly identified in most of the samples, which might be because of a synergistic effect of different sub-threshold compounds present in the samples. In principle component analysis, the relationship between microorganisms and glutamyl peptide generation was observed, especially between Tetragenococcus spp. and Lentibacillus spp. and the generation of γ-Glu-Val-Gly. Correlations between microbial diversity and the generation of taste enhancers were identified in this study.

Adaptation of Campylobacter jejuni to biocides used in the food industry affects biofilm structure, adhesion strength, and cross-resistance to clinical antimicrobial compounds.

The emergence of biocide-adapted Campylobacter jejuni strains that developed into biofilms and their potential to develop clinical resistance to antimicrobial compounds was studied. C. jejuni was grown in sub-lethal concentrations of five biocides used in the food industry. C. jejuni exhibited adaptation to these biocides with increased minimum inhibitory concentrations. The 3-D structures of the biofilms produced by the biocide-adapted cells were investigated by atomic force microscopy (AFM). The results revealed marked variability in biofilm architecture, including ice-crystal-like structures. Adaptation to the biocides enhanced biofilm formation, with significant increases in biovolume, surface coverage, roughness, and the surface adhesion force of the biofilms. Adaptation to commercial biocides induced resistance to kanamycin and streptomycin. This study suggests that the inappropriate use of biocides may lead to cells being exposed to them at sub-lethal concentrations, which can result in adaptation of the pathogens to the biocides and a subsequent risk to public health.

Development of Multiple-Locus Variable-Number Tandem-Repeat Analysis for Molecular Subtyping of Campylobacter jejuni by Using Capillary Electrophoresis.

Campylobacter jejuni is a common cause of the frequently reported food-borne diseases in developed and developing nations. This study describes the development of multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) using capillary electrophoresis as a novel typing method for microbial source tracking and epidemiological investigation of C. jejuni. Among 36 tandem repeat loci detected by the Tandem Repeat Finder program, 7 VNTR loci were selected and used for characterizing 60 isolates recovered from chicken meat samples from retail shops, samples from chicken meat processing factory, and stool samples. The discrimination ability of MLVA was compared with that of multilocus sequence typing (MLST). MLVA (diversity index of 0.97 with 31 MLVA types) provided slightly higher discrimination than MLST (diversity index of 0.95 with 25 MLST types). The overall concordance between MLVA and MLST was estimated at 63% by adjusted Rand coefficient. MLVA predicted MLST type better than MLST predicted MLVA type, as reflected by Wallace coefficient (Wallace coefficient for MLVA to MLST versus MLST to MLVA, 86% versus 51%). MLVA is a useful tool and can be used for effective monitoring of C. jejuni and investigation of epidemics caused by C. jejuni.

Genotyping of Salmonella enterica serovar Typhimurium isolates by multilocus variable number of tandem repeat high-resolution melting analysis (MLV-HRMA).

Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) is one of the most important virulent foodborne pathogens in industrialized countries. The ability to type bacterial strains is essential for surveillance, investigation of outbreaks, and epidemiological studies. Multilocus variable number tandem repeat combined with high-resolution melting analysis (MLV-HRMA) is a fast, cost-efficient, and easy sample genotyping method. In this study, MLV-HRMA and multilocus variable number tandem repeat analysis (MLVA) were used to differentiate between the allelic variants in 5 tandem repeat (TR) loci in 117 Salmonella Typhimurium isolates derived from various farms, slaughterhouses, market, and humans in Thailand. Both MLV-HRMA and MLVA analyses resulted in the identification of a total of 43 different genotypes, but slight differences were observed in cluster analysis results between the 2 methods. The unweighted pair-group method with arithmetic mean-based cluster analysis showed the same core clades; some small differences in the placement of sister-clades and subgrouping were observed due to the inability to reliably type the polymorphic STTR3 locus in the MLV-HRMA. The results of this study show that the MLV-HRMA, following the selection of suitable TR loci, is a relatively reliable and rapid screening method capable of differentiating between Salmonella Typhimurium isolates on the basis of allelic diversity at TR loci. As such, MLV-HRMA can be potentially used to investigate and track sources of contamination in order to effectively control Salmonella contamination in the food supply chain.

Development of new multilocus variable number of tandem repeat analysis (MLVA) for Listeria innocua and its application in a food processing plant.

Listeria innocua is an important hygiene indicator bacterium in food industries because it behaves similar to Listeria monocytogenes, which is pathogenic to humans. PFGE is often used to characterize bacterial strains and to track contamination source. However, because PFGE is an expensive, complicated, time-consuming protocol, and poses difficulty in data sharing, development of a new typing method is necessary. MLVA is a technique that identifies bacterial strains on the basis of the number of tandem repeats present in the genome varies depending on the strains. MLVA has gained attention due to its high reproducibility and ease of data sharing. In this study, we developed a MLVA protocol to assess L. innocua and evaluated it by tracking the contamination source of L. innocua in an actual food manufacturing factory by typing the bacterial strains isolated from the factory. Three VNTR regions of the L. innocua genome were chosen for use in the MLVA. The number of repeat units in each VNTR region was calculated based on the results of PCR product analysis using capillary electrophoresis (CE). The calculated number of repetitions was compared with the results of the gene sequence analysis to demonstrate the accuracy of the CE repeat number analysis. The developed technique was evaluated using 60 L. innocua strains isolated from a food factory. These 60 strains were classified into 11 patterns using MLVA. Many of the strains were classified into ST-6, revealing that this MLVA strain type can contaminate each manufacturing process in the factory. The MLVA protocol developed in this study for L. innocua allowed rapid and easy analysis through the use of CE. This technique was found to be very useful in hygiene control in factories because it allowed us to track contamination sources and provided information regarding whether the bacteria were present in the factories.

Establishment of a simple and rapid identification method for Listeria spp. by using high-resolution melting analysis, and its application in food industry.

Listeria monocytogenes is the causative bacteria of listeriosis, which has a higher mortality rate than that of other causes of food poisoning. Listeria spp., of which L. monocytogenes is a member, have been isolated from food and manufacturing environments. Several methods have been published for identifying Listeria spp.; however, many of the methods cannot identify newly categorized Listeria spp. Additionally, they are often not suitable for the food industry, owing to their complexity, cost, or time consumption. Recently, high-resolution melting analysis (HRMA), which exploits DNA-sequence differences, has received attention as a simple and quick genomic typing method. In the present study, a new method for the simple, rapid, and low-cost identification of Listeria spp. has been presented using the genes rarA and ldh as targets for HRMA. DNA sequences of 9 Listeria species were first compared, and polymorphisms were identified for each species for primer design. Species specificity of each HRM curve pattern was estimated using type strains of all the species. Among the 9 species, 7 were identified by HRMA using rarA gene, including 3 new species. The remaining 2 species were identified by HRMA of ldh gene. The newly developed HRMA method was then used to assess Listeria isolates from the food industry, and the method efficiency was compared to that of identification by 16S rDNA sequence analysis. The 2 methods were in coherence for 92.6% of the samples, demonstrating the high accuracy of HRMA. The time required for identifying Listeria spp. was substantially low, and the process was considerably simplified, providing a useful and precise method for processing multiple samples per day. Our newly developed method for identifying Listeria spp. is highly valuable; its use is not limited to the food industry, and it can be used for the isolates from the natural environment.

Microbiological evaluation of water during the 2011 flood crisis in Thailand.

In 2011, a severe flood occurred in Thailand, covering nearly half the country in water for several months. The contamination of floodwater and subsequent contamination of water for human consumption could have potentially led to a widespread health crisis. However, to date, no study has been conducted to determine the safety of the waters used for human consumption in Thailand during the severe flood. Therefore, we conducted microbiological analysis of 4 kinds of water (floodwater, river water, tap water, and filtered tap water) collected from industrial and residential areas that were damaged due to flooding. Higher net levels of bacteria were found in water with a higher turbidity. No clear trend was observed in the pH value of all 4 water samples. The level of total bacterial contamination in the water samples was estimated by real-time quantitative polymerase chain reaction (PCR). Eleven of the 12 tap water samples and all of the filtered tap water samples had a total bacterial load that exceeded the Thai water quality standards. One of the tap water samples and one of the filtered tap water samples were found to be positive for Shigella sp., although none of the floodwater samples showed detectable levels of this pathogen as determined by PCR analysis. One of the samples of floodwater was also found to be positive for Leptospira sp., but none of the tap water or filtered tap water samples were positive. Most of the tap water samples and all filtered tap water samples were found to be contaminated with Vibrio cholerae. Bacterial contamination in water samples was also analyzed by denaturing gradient gel electrophoresis (DGGE) analysis. These results revealed that several microorganisms were transferred via floodwater to different areas in the central part of Thailand and cross-contaminated between floodwater and water for human consumption.

Enhancing gamma-aminobutyric acid content in germinated brown rice by repeated treatment of soaking and incubation.

Gamma-aminobutyric acid (GABA), commonly produced by germination of brown rice grain, is a free amino acid which could help relieving or preventing non-communicable diseases in human. Several research works have been conducted on GABA production from germinated brown rice. However, the yielded GABA (10.1-69.2 mg/100 g germinated brown rice) was comparatively low; thus the amount was insufficient to be used as active ingredients in functional foods. The objective of this study was to explore alternative methods in order to gain higher yield of GABA. A new process of repeated soaking (in tap water at 35 °C, 3 h) and incubation (at 37 °C, 21 h) during germination was developed. The amount of GABA produced was highest at 116.88 ± 9.24 mg/100 g germinated brown rice (dry basis). However, an unpleasant odour was generated by some microorganisms during long germination. Lactic acid was applied at soaking step to overcome this problem; whereby 0.5% lactic acid solution (vol./vol.) could effectively control the microorganisms without impairing GABA producing ability and sensory qualities.

Antimicrobial activity of lauric arginate-coated polylactic acid films against Listeria monocytogenes and Salmonella typhimurium on cooked sliced ham.

UNLABELLED: A novel type of environmentally friendly packaging with antibacterial activity was developed from lauric arginate (LAE)-coating of polylactic acid (PLA) films after surface activation using a corona discharge. Scanning electron microscopy (SEM)-based analysis of the LAE/PLA films confirmed the successful coating of LAE on the PLA surface. The mechanical properties of the LAE/PLA films with different levels of LAE-coating (0% to 2.6%[w/w]) were essentially the same as those of the neat PLA film. The antibacterial activity of the LAE/PLA films against Listeria monocytogenes and Salmonella enterica Serovar Typhimurium (S. Typhimurium) was confirmed by a qualitative modified agar diffusion assay and quantitative JIS Z 2801:2000 method. Using the LAE/PLA film as a food-contact antimicrobial packaging for cooked cured ham, as a model system, suggested a potential application to inhibit L. monocytogenes and S. Typhimurium on ham with a 0.07% (w/w) LAE coating on the PLA when high transparency is required, as evidenced from the 2 to 3 log CFU/tested film lower pathogen growth after 7 d storage but even greater antibacterial activity is obtained with a LAE coating level of 2.6% (w/w) but at the cost of a reduced transparency of the finished product. This article shows how we can simply develop functional green packaging of PLA for food with effective and efficient antimicrobial activity by use of LAE coating on the surface via corona discharge. PRACTICAL APPLICATION: The effectiveness of an innovative antimicrobial LAE-coated PLA film against foodborne pathogens was demonstrated. Importantly, the application of the LAE to form the LAE-coated PLA film can be customized within current film manufacturing lines.

Application of the Chinese steamed bun starter dough (CSB-SD) in breadmaking.

The application of Chinese steamed bun starter dough (CSB-SD) in breadmaking was investigated. The activation of CSB-SD to activate the growth of lactic acid bacteria (LAB) and to increase the number of yeast, prior to making bread, was conducted by mixing CSB-SD with wheat flour and water and then incubating for 24 h. Wheat flour was then substituted by this activated CSB-SD (aCSB-SD) at 10%, 30%, and 50% (w/w) to make bread. Dough and bread properties were studied comparing to the control (without aCSB-SD). From the farinograph results, a high aCSB-SD substitution level resulted in a less stability in dough with a higher degree of softening. Extensigraph results suggested that after aging, all the substituted dough yielded a greater resistance to extension with lower extensibility values than the control. Substitutions with 30% and 50% (w/w) aCSB-SD significantly increased the total CO(2) gas generation. Scanning electron microscopy SEM images of the 30% and 50% (w/w) substituted dough showed a well-developed gluten matrix. The 50% (w/w) substituted breads obtained a greater risen volume, finer crumb grain, and retained more softness after 5-d storage than the control. In addition, both the 30% and 50% (w/w) substituted breads showed a slightly increased mold stability, as compared to the 0% and 10% (w/w) substituted breads.

Contamination profiles of Escherichia coli and enterococci in steamed chicken meat products.

This study determined the sources of contamination by Escherichia coli and enterococci in frozen ready-to-eat chicken products. The efficiency of the heat treatment process was sufficient to eliminate E. coli or enterococci. However, the prevalence of E. coli and enterococci in cooked chicken after chilling was 2.7%, and after slicing and dicing it was 1.3 and 9.3%, respectively. These results indicated that contamination occurred after cooking. In the finished product, E. coli was absent, while enterococcus prevalence was reduced to 1.3%. The environment at each production step, such as the machine surfaces, workers' gloves, and the condensate, was sampled to determine the correlation with the contamination in products. E. coli and enterococci were found on the machine surfaces in all production steps, but E. coli contamination was mostly from the infeed transfer belt at the chilling step, while the enterococcus contamination arose mostly from the slicing or dicing steps, especially from the dicing machine belt, which directly contacts the products. Indeed, E. coli and enterococci were detected on food contact surfaces throughout the production period, including prior to its commencement. These results indicated that the cleaning before and during the production process was ineffective. In addition, cleaning and sanitizing food contact surfaces followed by nonfood contact surfaces (floor and drains) by use of a high-pressure water hose created aerosol with microbes from the floors and drains and spread such microbes onto already cleaned food contact surfaces.

Risk analysis of Listeria spp. contamination in two types of ready-to-eat chicken meat products.

This study was conducted to determine the risk of Listeria contamination in frozen ready-to-eat roasted and steamed chicken meat in a chicken plant in Thailand. Environmental surfaces were divided into three zones. Zone 1 included surfaces in direct contact with products. Zones 2 and 3 included indirect contact surfaces; zone 2 was next to zone 1, and zone 3 was located next to zone 2 and relatively far from the product. A mathematical model for the probability of product contamination after contact with contaminated zone 1 surfaces was established. This model was augmented by an already established model for the probability of Listeria contamination on zone 1 surfaces. Sensitivity analysis revealed that the prevalence of Listeria on zone 1 surfaces before cleaning and sanitizing, production time, and concentration and contact time of sanitizer were correlated with contamination of both products. Alternative risk management measures for reducing the risk of Listeria contamination were developed using sanitizer concentrations of 0.25 to 1.25% (vol/vol), sanitizer contact times of 5 to 20 min, and production times of 5 to 20 h. The plant's risk manager chose a 0.25% (vol/vol) sanitizer concentration, a contact time of 20 min, and a production time of 20 h. After implementation of the selected risk management option, the prevalence of Listeria on roasted and steamed products was reduced by 2.19 and 2.01%, respectively. The prevalence of Listeria in zones 1, 2, and 3 was also reduced by 3.13, 11.24, and 25.66%, respectively.

Risk assessment of Listeria spp. contamination in the production line of ready-to-eat chicken meat products.

The risk of Listeria spp. contamination was assessed in frozen ready-to-eat chicken meat production lines by establishing a mathematical model for determining the probability of Listeria spp. prevalence on environmental surfaces directly in contact with the product at various times in a chicken plant in Thailand. Environmental surfaces were divided into three zones. Zone 1 included surfaces in direct contact with products. Both zones 2 and 3 included indirect contact surfaces; zone 2 was next to zone 1, and zone 3 was next to zone 2, relatively far from the product. The model for probability of Listeria spp. contamination on surfaces in zone 1 was derived from the probability of Listeria spp. on surfaces in zone 1 after the cleaning and sanitizing process multiplied by the probability of Listeria spp. transferred from zones 2 and 3 and the probability of Listeria spp. growth. The surfaces in zone 1 were cleaned with warm water, cleaned with detergent, and sanitized with a sanitizer. Factors affecting cleaning and sanitizing were water temperature, concentration, and contact time of detergent and sanitizer. The probability of Listeria spp. prevalence on surfaces in zone 1 was not affected by water temperatures of 50, 60, and 70 degrees C and detergent concentrations of 0.5, 1, and 2% (vol/vol) at contact times of 5, 10, and 15 min. However, it was affected by sanitizer concentrations of 0.25, 0.5, and 1.25% (vol/vol) at contact times of 5, 10, and 20 min. Sensitivity analyses were conducted using the Monte Carlo simulation. The sanitizer concentration had the most significant influence on the prevalence of Listeria spp. on surfaces in zone 1. The prevalence of Listeria spp. on surfaces after cleaning and sanitizing, the production time, and the contact time with the sanitizer were highly correlated with the prevalence of Listeria spp. in zone 1. This model could be used as a management tool for assessing the risk of Listeria spp. contamination in food products.

Contamination profile of Listeria spp. in three types of ready-to-eat chicken meat products.

This study investigated contamination sources of Listeria spp. in frozen, ready-to-eat, roasted, steamed, and fried chicken meat products from a plant in Thailand, as well as the correlation between Listeria contamination in the production environment and the finished product. The cooking processes used in this factory (with a product core temperature of 80 degrees C for 1 min) were confirmed as adequate for eliminating Listeria spp. However, Listeria spp. were detected at the packing stage of roasted and steamed chicken products. An environmental swab test was conducted by means of the zone concept, whereby surfaces in the production area were divided into three zones. Zone 1 was made up of the equipment surfaces that came into direct contact with the products. Zone 2 consisted of equipment surfaces that were not in direct contact with the products, including surfaces that were difficult to be cleaned. Zone 3 included surfaces that did not come in direct contact with the products and were located far from the products. The results showed that the prevalence of Listeria spp. in roasted and steamed products was affected by the prevalence of Listeria contamination in all zones, especially zone 1, which demonstrated the highest correlation. In addition, the prevalence of Listeria contamination in zones 2 and 3 affected the prevalence of Listeria in zone 1. A correlation between Listeria on roasted chicken products and the surfaces of zone 1 at the start of production was also established.