Prospects for Food Fermentation in South-East Asia, Topics From the Tropical Fermentation and Biotechnology Network at the End of the AsiFood Erasmus+Project

Fermentation has been used for centuries to produce food in South-East Asia and some foods of this region are famous in the whole world. However, in the twenty first century, issues like food safety and quality must be addressed in a world changing from local business to globalization. In Western countries, the answer to these questions has been made through hygienisation, generalization of the use of starters, specialization of agriculture and use of long-distance transportation. This may have resulted in a loss in the taste and typicity of the products, in an extensive use of antibiotics and other chemicals and eventually, in a loss in the confidence of consumers to the products. The challenges awaiting fermentation in South-East Asia are thus to improve safety and quality in a sustainable system producing tasty and typical fermented products and valorising by-products. At the end of the “AsiFood Erasmus+ project” (www.asifood.org), the goal of this paper is to present and discuss these challenges as addressed by the Tropical Fermentation Network, a group of researchers from universities, research centers and companies in Asia and Europe. This paper presents current actions and prospects on hygienic, environmental, sensorial and nutritional qualities of traditional fermented food including screening of functional bacteria and starters, food safety strategies, research for new antimicrobial compounds, development of more sustainable fermentations and valorisation of by-products. A specificity of this network is also the multidisciplinary approach dealing with microbiology, food, chemical, sensorial, and genetic analyses, biotechnology, food supply chain, consumers and ethnology

Phenotypic Characterization And Comparative Genomics Analysis Of Listeriaphages

Listeria monocytogenes is a foodborne pathogen that is widespread in natural and urban environments. L. monocytogenes is also commonly found in food processing facilities where specific strains or subtypes have been shown to persist over time. Listeriaphages are phages that can specifically infect L. monocytogenes and other Listeria spp. Phages have been approved for use as a biocontrol agent for this pathogen; however knowledge on its efficacy against diverse L. monocytogenes, particularly from food associated environments, is still limited. We developed a collection of diverse phages for further studies which included (i) phenotypic and genotypic characterization of listeriaphages isolated from dairy farms, (ii) evaluation of phage susceptibility and development of phage cocktails against persistent subtypes from food-associated environments, and (iii) whole genome sequencing and comparative genomics of selected phages. Over 100 listeriaphages were isolated from silage samples collected over the course of 1.5 years on two dairy farms. Initial phenotypic and genotypic characterization of phages from this collection revealed considerable host range (9 lysis groups) and genomic diversity (genome sizes of 25- 140 kb). Among the 9 major Listeria serotypes used to determine the host range, the serotype 3c strain was found to be highly resistant to phages while serotype 4 strains were the most susceptible to phages. Variation in phage susceptibility (4.6-95.4%) was observed among different persistent isolates of multiple ribotypes from a food processing facility. While phage cocktails could temporally reduce the bacterial populations of some subtypes, others were unaffected by phage treatment. L. monocytogenes isolates also rapidly developed phage-resistance characteristics in laboratory challenge studies. Whole genome sequencing of 10 listeriaphages revealed considerable genomic diversity of listeriaphages on dairy farms. The phage genome sizes could be classified into 3 ranges: small (36-38 kb; n=3), mid-sized (64-67 kb; n=4), and large (133-135 kb; n=3). All genomes were found to be organized into 3 functional modules: (i) DNA packaging and structural proteins; (ii) cell lysis; (iii) DNA replication, modification, and metabolism. Genomes of six newly sequenced phages appear to resemble three previously described listeriaphages. However, four phages showed no sequence homology to any bacteriophages in the NCBI databases, suggesting they are novel listeriaphages. Our data provide valuable information for further development of effective and suitable phage-based biocontrol agents and other applications

Control of <i>Salmonella</i> in Chicken Meat by a Phage Cocktail in Combination with Propionic Acid and Modified Atmosphere Packaging

Salmonella contamination in poultry meat is an important food safety issue as this pathogen can lead to serious illness and economic losses worldwide. In poultry meat processing, a variety of strong bacteriostatic agents has been introduced for controlling Salmonella including bacteriophages (phages), organic acids, and modified atmosphere packaging (MAP). In our study, two selected phages including vB_SenM_P7 and vB_SenP_P32 were used in combination with propionic acid (PA) and MAP for controlling Salmonella of multiple serovars on chicken meat under storage at 4 °C. The two phages showed strong lytic activity against over 72 serovars of Salmonella tested (25.0 to 80.6%). Phages, vB_SenM_P7 and vB_SenP_P32 showed 40% and 60% survival rates, respectively, after the exposure to temperatures up to 70 °C. Both phages remained active, with nearly 100% survival at a wide range of pH (2 to 12) and 15% NaCl (w/v). The available chlorine up to 0.3% (v/v) led to a phage survival rate of 80–100%. A combination of Salmonella phage cocktail and 0.5% PA could reduce Salmonella counts in vitro by 4 log CFU/mL on day 3 whereas a phage cocktail and 0.25% PA showed a 4-log reduction on day 5 during storage at 4 °C. For the phage treatment alone, a 0.3-log reduction of Salmonella was observed on day 1 of storage at 4 °C. In the chicken meat model, treatment by a phage cocktail and PA at both concentrations in MAP conditions resulted in a complete reduction of Salmonella cells (4–5 log unit/g) on day 2 of storage whereas each single treatment under MAP conditions showed a complete cell reduction on day 4. For the meat sensory evaluation, chicken meat treated with a phage cocktail-PA (0.5%) in MAP condition showed the highest preference scores, suggesting highly acceptability and satisfactory. These findings suggest that a combined treatment using a phage cocktail and PA in MAP conditions effectively control Salmonella in poultry meat during storage at low temperature to improve the quality and safety of food

Potential Bio-Control Agent from Rhodomyrtus tomentosa against Listeria monocytogenes

Listeria monocytogenes is an important foodborne pathogen implicated in many outbreaks of listeriosis. This study aimed at screening for the potential use of Rhodomyrtus tomentosa ethanolic leaf extract as a bio-control agent against L. monocytogenes. Twenty-two L. monocytogenes isolates were checked with 16 commercial antibiotics and isolates displayed resistance to 10 antibiotics. All the tested isolates were sensitive to the extract with inhibition zones ranging from 14 to 16 mm. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ranged from 16 to 32 µg/mL and 128 to 512 µg/mL, respectively. Time-kill assay showed that the extract had remarkable bactericidal effects on L. monocytogenes. The extract at a concentration of 16 µg/mL reduced tolerance to 10% NaCl in L. monocytogenes in 4 h. Stationary phase L. monocytogenes cells were rapidly inactivated by greater than 3-log units within 30 min of contact time with R. tomentosa extract at 128 µg/mL. Electron microscopy revealed fragmentary bacteria with changes in the physical and morphological properties. Our study demonstrates the potential of the extract for further development into a bio-control agent in food to prevent the incidence of L. monocytogenes contamination

Characterization of Listeria prophages in lysogenic isolates from foods and food processing environments.

Prophages are commonly found in Listeria genomes, potentially enhancing survival or fitness of Listeria spp. Currently, there is still limited information on the distribution of prophages among Listeria isolates of different allelic types and from various sources. In this study, by using mitomycin C induction, prophages were found in 23/144 isolates (16.0%), including 13 L. monocytogenes and 10 Listeria spp. isolates, resulting in 28 and 11 induced phages, respectively. These prophage-carrying isolates (lysogens) were obtained from foods and food-related environments presenting 3 common allelic types (ATs) of L. monocytogenes (lineage I, II and IV), 4 ATs of L. innocua and 1 AT of L. welshimeri. The likelihood of prophage-carrying isolates of L. monocytogenes was 14.4 (95% CI: 4.9-35.4), and 18.5 (95% CI: 4.8-50.2) for Listeria spp. The 39 induced phages were classified into 3 lysis groups by the host range test against 9 major serotypes of L. monocytogenes and 5 species of Listeria. Most phages were host-specific with higher ability to lyse L. monocytogenes serotype 4 than other serotypes. The genome size of phages ranged from 35±2 kb to 50±2 kb and belonged to two common phage families, Myoviridae and Siphoviridae. Restriction analysis classified 19 selected phages into 16 restriction profiles, suggesting highly diverse prophages with at least 16 types. This may contribute to the variation in the genomes of Listeria. Information obtained here provides basic knowledge for further study to understand the overall role of prophages in Listeria, including roles in survival or fitness in foods and food processing environments

Effectiveness of the Organic Acid-Based Antimicrobial Agent to Prevent Bacterial Contamination in Fish Meal

Animal feed production is an important step of the food animal production chain in a farm-to-table model. The contamination of raw ingredients with foodborne pathogens in feed production remains as an important safety issue where pathogens may spread into food animals to cause illnesses in humans when affected food animals are consumed. In the present study, we aimed to examine the quality and microbial contamination of fish meal and to investigate the effectiveness of the organic acid-based antimicrobial agent SALTEC 514TM against Salmonella to prevent bacterial contamination in fish meal. Fish meal samples (n = 4) collected from feed mills at different locations were analyzed for protein and total volatile basic nitrogen (TVBN) content to assess their nutritional value and freshness, and its microbiological quality. The protein and TVBN content ranged from 53.2 ± 3.1 to 67.5 ± 2.3 g/100 g and 73.8 ± 4.5 to 100.4 ± 11.2 mg/100 g meal, respectively. Total plate count of the fish meal samples ranged from 2.0 ± 0.3 to 4.5 ± 0.5 log units, whereas suspected foodborne bacteria, Escherichia coli and Salmonella, were not detected in all samples. Fish meal samples were artificially contaminated (day 0) and re-challenged (day 30 and 90) with Salmonella Enteritidis (3 log CFU/g) to test for the effectiveness of SALTEC 514TM, an organic acid-based antimicrobial formulation, in preventing Salmonella contamination and recontamination during storage. SALTEC 514TM, when applied at three different doses, was found to reduce the number of Salmonella in monitored samples after one day of storage. A low dose of 0.5 kg/ton SALTEC 514TM prevented Salmonella recontamination from occurring in fish meal samples stored for 37 days. In medium (1.0 kg/ton) and high doses (3.0 kg/ton), applications of SALTEC 514TM prevented the Salmonella recontamination for a maximum storage duration of 97 days. The application of SALTEC 514TM in fish meal and/or other feed ingredients may prove to be a safe alternative to reduce the microbial load, especially of foodborne-related microorganisms, to contribute to feed and food safety

Evaluation of microbiological quality of sushi sold in restaurants and supermarkets in turkey

WOS: 000390294000003Sushi is a popular traditional Japanese food in worldwide. This study was conducted to investigate the physical, chemical, sensory and microbiological quality of sushi sold in supermarket and restaurants. 300 sushi samples were purchased from 5 different popular restaurants (A, B, D, E, and F) and 1 supermarket (C). Four main types of sushi (California roll (CR), sesame roll (SMR), sake roll (SR), take roll (TR)) were chosen from each station. pH values of sushi rice samples were found to be all below 4.6. According to sensory overall quality of sushi samples, CR and SMR from station C had significantly the lowest values (p<0.05). Among the sushi samples, pathogenic bacteria Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus were not detected in 25 g portion of any samples. Counts of total aerobic bacteria were high in stations A, B, D, E, and F (between 4.3-7 log CFU/g), except for station C (3.5 log CFU/g). Results for coliform counts showed similar trends as those of E. coli counts in samples. E. coli counts of all sushi types from all stations (except SMR, SR and TR from station D and SR from station E) exceeded the limit of acceptability (<100 MPN/g). Our finding may suggest a common presence of coliforms and E. coli in raw materials used in sushi preparation.Ege University, Izmir, Turkey, Scientific Research Project Programs [14SUF031]This study was supported by Ege University, Izmir, Turkey, Scientific Research Project Programs (Project No: 14SUF031)

Lysis Profiles of <i>Salmonella</i> Phages on <i>Salmonella</i> Isolates from Various Sources and Efficiency of a Phage Cocktail against <i>S.</i> Enteritidis and <i>S.</i> Typhimurium

Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium are major foodborne pathogens of concern worldwide. Bacteriophage applications have gained more interest for biocontrol in foods. This study isolated 36 Salmonella phages from several animal farms in Thailand and tested them on 47 Salmonella strains from several sources, including farms, seafood processing plant and humans in Thailand and USA. Phages were classified into three major groups. The estimated phage genome size showed the range from 50 &#177; 2 to 200 &#177; 2 kb. An effective phage cocktail consisting of three phages was developed. Approximately 4 log CFU/mL of S. Enteritidis and S. Typhimurium could be reduced. These phages revealed a burst size of up to 97.7 on S. Enteritidis and 173.7 PFU/cell on S. Typhimurium. Our phage cocktail could decrease S. Enteritidis on chicken meat and sunflower sprouts by 0.66 log CFU/cm2 and 1.27 log CFU/g, respectively. S. Typhimurium on chicken meat and sunflower sprouts were decreased by 1.73 log CFU/cm2 and 1.17 log CFU/g, respectively. Overall, animal farms in Thailand provided high abundance and diversity of Salmonella phages with the lysis ability on Salmonella hosts from various environments and continents. A developed phage cocktail suggests a potential biocontrol against Salmonella in fresh foods

Quality of <i>Kapi</i>, Salted Shrimp Paste of Thailand, Inoculated with <i>Bacillus</i> spp. K-C3

<p>The use of <i>Bacillus</i> spp. K-C3, isolated from commercial <i>Kapi</i>, salted shrimp paste of Thailand, as the starter culture for <i>Kapi</i> production, at different levels including 10², 10⁴, and 10⁶ CFU/g dry weight sample named <i>Kapi</i>-B2, <i>Kapi</i>-B4, and <i>Kapi</i>-B6, respectively, in comparison with naturally fermented <i>Kapi</i> (<i>Kapi</i>-C) was studied. All inoculated samples exhibited higher extent of proteolysis and lipolysis as indicated by higher trichloroacetic acid (TCA)-soluble peptide, degree of hydrolysis, and free fatty acid content in the final products. The greater rate of fermentation in the inoculated samples directly affected characteristics of <i>Kapi</i> and yielded <i>Kapi</i> with browner color and enhanced lipid oxidation as indicated by peroxide value (PV) and thiobarbituric acid reactive substance (TBARS) values. Increased antioxidative properties, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging activities, and ferric reducing antioxidant power (FRAP), were obtained in <i>Kapi</i> with added inoculum. Moreover, those inoculated samples also contained higher intensity of volatile compounds, in which <i>N</i>-containing compounds, mainly pyrazine derivatives, were dominant. <i>Kapi</i>-B4 and <i>Kapi</i>-B6 showed the highest extent of these aforementioned values compared with <i>Kapi</i>-C and <i>Kapi</i>-B2. Therefore, inoculation with <i>Bacillus</i> spp. K-C3 at the level higher than 10⁴ CFU/g dry weight sample is a potential means to accelerate the fermentation rate and yielded <i>Kapi</i> with preferable characteristics.</p