The development of indigenous starter cultures for producing fermented foods that meet the expectations of Cambodians is necessary to preserve the country’s food supply. In this study, the safety of 46 lactic acid bacteria strains based on the phenotypic antibiotic susceptibility to clinically relevant antibiotics was assessed. The antibiotic susceptibility of 39 lactobacilli and seven pediococci isolated from Cambodian fermented foods to 16 antibiotics was studied according to ISO 10932/IDF 233. The results were interpreted based on the minimal inhibition concentrations obtained, using differently defined breakpoints and concentration distributions as well as data from the scientific literature. Applying only breakpoints, the results demonstrated two Lactiplantibacillus pentosus, three Companilactobacillus futsaii, three Levilactobacillus namurensis and seven Pediococcus pentosaceus strains with acquired resistance. However, considering further information, one Companilactobacillus futsaii, one Limosilactobacillus fermentum and respectively three Lactiplantibacillus pentosus and Levilactobacillus namurensis strains would possess an acquired resistance. The genetic background for the absence of transmissible antibiotic resistances in lactic acid bacteria strains intended for food application must be confirmed by molecular methods for potential starter cultures.

Background: Probiotics are generally considered as safe, but infections may rarely occur in vulnerable patients. Alternatives to live microorganisms to manage dysbiosis may be of interest in these patients. Reuterin is a complex component system exhibiting broad spectrum antimicrobial activity and a possible candidate substance in these cases. Methods: Reuterin supernatant was cultured from Lentilactobacillus diolivorans in a bioreactor in a two-step process. Storage stability at −20°C and effect of repeated freeze-thaw cycles were assessed by high performance liquid chromatography (HPLC). Antimicrobial activity was tested against Clostridium difficile , Listeria monocytogenes , Escherichia coli , Enterococcus faecium , Staphylococcus (S.) aureus , Staphylococcus epidermidis , Streptococcus (S.) agalactiae , Propionibacterium acnes , and Pseudomonas aeruginosae . Male BALBc mice were gavage fed with reuterin supernatant ( n = 10) or culture medium ( n = 10). Fecal volatile organic compounds (VOC) were assessed by gas chromatography mass spectroscopy; the microbiome was examined by 16S rRNA gene sequencing. Results: The supernatant contained 13.4 g/L reuterin (3-hydroxypropionaldehyde; 3-HPA). 3-HPA content remained stable at −20°C for 35 days followed by a slow decrease of its concentration. Repeated freezing/thawing caused a slow 3-HPA decrease. Antimicrobial activity was encountered against S. aureus , S. epidermidis , and S. agalactiae . Microbiome analysis showed no differences in alpha and beta diversity markers. Linear discriminant effect size (LEfSe) analysis identified Lachnospiraceae_bacterium_COE1 and Ruminoclostridium_5_uncultured_Clostridiales_ bacterium (in the reuterin medium group) and Desulfovibrio_uncultured_ bacterium , Candidatus Arthromitus , Ruminococcae_NK4A214_group , and Eubacterium_xylanophilum_group (in the reuterin group) as markers for group differentiation. VOC analysis showed a significant decrease of heptane and increase of 3-methylbutanal in the reuterin group. Conclusion: The supernatant produced in this study contained acceptable amounts of 3-HPA remaining stable for 35 days at −20°C and exhibiting an antimicrobial effect against S. aureus , S. agalactiae , and S. epidermidis . Under in vivo conditions, the reuterin supernatant caused alterations of the fecal microbiome. In the fecal, VOC analysis decreased heptane and increased 3-methylbutanal were encountered. These findings suggest the high potential of the reuterin system to influence the intestinal microbiome in health and disease, which needs to be examined in detail in future projects.

We aimed to assess the in vitro antimicrobial activity and the in vivo effect on the murine fecal microbiome and volatile organic compound (VOC) profile of (S)-reutericyclin. The antimicrobial activity of (S)-reutericyclin was tested against Clostridium difficile, Listeria monocytogenes, Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Staphylococcus (S.) epidermidis, Streptococcus agalactiae, Pseudomonas aeruginosa and Propionibacterium acnes. Reutericyclin or water were gavage fed to male BALBc mice for 7 weeks. Thereafter stool samples underwent 16S based microbiome analysis and VOC analysis by gas chromatography mass spectrometry (GC-MS). (S)-reutericyclin inhibited growth of S. epidermidis only. Oral (S)-reutericyclin treatment caused a trend towards reduced alpha diversity. Beta diversity was significantly influenced by reutericyclin. Linear discriminant analysis Effect Size (LEfSe) analysis showed an increase of Streptococcus and Muribaculum as well as a decrease of butyrate producing Ruminoclostridium, Roseburia and Eubacterium in the reutericyclin group. VOC analysis revealed significant increases of pentane and heptane and decreases of 2,3-butanedione and 2-heptanone in reutericyclin animals. The antimicrobial activity of (S)-reutericyclin differs from reports of (R)-reutericyclin with inhibitory effects on a multitude of Gram-positive bacteria reported in the literature. In vivo (S)-reutericyclin treatment led to a microbiome shift towards dysbiosis and distinct alterations of the fecal VOC profile.

Naturally fermented foods are an important part of the typical diet in Cambodia. However, the food safety status of these products has not been widely studied. The aim of this study was, therefore, to provide an overview of the quality of these foods in relation to microbiology and biogenic amines. Additionally, the obtained results were compared to the habits and practices of Cambodians in handling this type of food. A total of 57 fermented foods (42 fishery and 15 vegetable products) were collected from different retail markets in the capital of Cambodia. Pathogenic Salmonella spp., Listeria spp., and Listeria monocytogenes were not detected in 25 g samples. Generally, less than 102 cfu/g of Staphylococcus aureus, Escherichia coli, Pseudomonas spp., Enterobacteriaceae, and molds were present in the fermented foods. Bacillus cereus group members (<102 to 2.3 × 104 cfu/g), lactic acid bacteria (<102 to 1.1 × 107 cfu/g), halophilic and halotolerant bacteria (<102 to 8.9 × 106 cfu/g), sulfite-reducing Clostridium spp. (<102 to 3.5 × 106 cfu/g), and yeasts (<102 to 1.1 × 106 cfu/g) were detected in this study. Still, the presence of pathogenic and spoilage microorganisms in these fermented foods was within the acceptable ranges. Putrescine, cadaverine, tyramine, and histamine were detected in 100%, 89%, 81%, and 75% of the tested products, respectively. The concentrations of histamine (>500 ppm) and tyramine (>600 ppm) were higher than the recommended maximum levels in respectively four and one of 57 fermented foods, which represents a potential health risk. The results suggest that the production process, distribution, and domestic handling of fermented foods should be re-evaluated. Further research is needed for the establishment of applicable preservation techniques in Cambodia.

The increase in antibiotic resistance is a global concern for human and animal health. Resistant microorganisms can spread between food-producing animals and humans. The objective of this review was to identify the type and amount of antibiotics used in poultry production and the level of antibiotic resistance in Escherichia coli isolated from broilers. Isolate information was obtained from national monitoring programs and research studies conducted in large poultry-producing regions: US, China, Brazil, and countries of EU-Poland, United Kingdom, Germany, France, and Spain. The survey results clearly display the absence of a harmonized approach in the monitoring of antibiotics per animal species and the evaluation of resistances using the same methodology. There is no public long-term quantitative data available targeting the amount of antibiotics used in poultry, with the exception of France. Data on antibiotic-resistant E. coli are available for most regions but detection of resistance and number of isolates in each study differs among regions; therefore, statistical evaluation was not possible. Data from France indicate that the decreased use of tetracyclines leads to a reduction in the detected resistance rates. The fluoroquinolones, third-generation cephalosporins, macrolides, and polymyxins ("highest priority critically important" antibiotics for human medicine according to WHO) are approved for use in large poultry-producing regions, with the exception of fluoroquinolones in the US and cephalosporins in the EU. The approval of cephalosporins in China could not be evaluated. Tetracyclines, aminoglycosides, sulfonamides, and penicillins are registered for use in poultry in all evaluated countries. The average resistance rates in E. coli to representatives of these antibiotic classes are higher than 40% in all countries, with the exception of ampicillin in the US. The resistance rates to fluoroquinolones and quinolones in the US, where fluoroquinolones are not registered for use, are below 5%, while the average of resistant E. coli is above 40% in Brazil, China, and EU, where use of fluoroquinolones is legalized. However, banning of fluoroquinolones and quinolones has not totally eliminated the occurrence of resistant populations.

The authors gratefully acknowledge the COST Action Ca18101 SOURDOugh biotechnology network towards novel, healthier and sustainable food and bloproCasseS

In this study, the influence of meat batter composition and sausage diameter on the development of microbiota and sensory traits of traditional, spontaneously fermented wild boar meat sausages are evaluated. This research also demonstrates how principal component analysis (PCA) can be used to relate product sensory properties to particular microbial genotype and to select potential starter or adjunct culture. Generally, similar microbiological results were obtained in all types of products. The undesirable microbiota was either not detected at any sausage production stage or its number decreased below the detection limit in ripened sausages. The low growth rate of lactic acid bacteria (LAB) was consistent with the obtained pH and slow acidification rate. Although no differences in the composition of LAB species were noticed between sausage types (50S=50 % wild boar meat in small casing, 50L=50 % wild boar meat in large casing, 100S=100 % wild boar meat in small casing), a clear separation based on LAB genotypes could be observed. Upon quantitative descriptive analysis, significant differences in sensory attributes between sausage types were established. According to the PCA, the overall acceptability traits of sausages are closely linked to one Leuconostoc mesenteroides genotype (LM_4). Of all tested technological properties, LM_4 strains showed remarkable acidification ability, lowering the pH from pH=5.41 to 3.74, and pronounced proteolytic activity on skimmed milk as well as antagonistic activity against Staphylococcus aureus (DSM 20231) and Brochothrix thermosphacta (LMG 17208). Lipolytic and haemolytic activities were not detected, and all analyzed strains were susceptible to tested antibiotics and possessed no biogenic amine genes.

Screening for various types of lactic acid bacteria (LAB) that form the biological agent γ-amino-butyric acid (GABA) is important to produce different kinds of GABA-containing fermented foods. So far, no GABA-producing LAB have been reported from Cambodian fermented foods. Most small-scale fermentations and even some industrial processes in this country still rely on indigenous LAB. The application of GABA-producing autochthonous starters would allow the production of Cambodian fermented foods with an additional nutritional value that meet the population's dietary habits and that are also more attractive for the international food market. Matrix-assisted laser desorption/ionizing time-of-flight mass spectrometry (MALDI-TOF MS) and partial 16S rDNA sequencing were used to identify 68 LAB isolates from Cambodian fermented foods. These isolates were classified and grouped with (GTG)5 rep-PCR, resulting in 50 strains. Subsequently, all strains were investigated for their ability to produce GABA by thin layer chromatography. GABA-positive strains were further analyzed by the GABase assay. Of the six GABA-positive LAB strains-one Lactobacillus futsaii, two Lactobacillus namurensis, and three Lactobacillus plantarum strains-two Lactobacillus plantarum strains produced high amounts of GABA (20.34 mM, 16.47 mM). These strains should be further investigated for their potential application as GABA-producing starter cultures in the food applications.

Spontaneously fermented wild boar and deer meat sausages produced in Croatia were physicochemically and microbiologically investigated at different time points of their production. Final products resulted with pH and a(w) values between 5.04-5.53 and 0.83-0.87, respectively. The histamine concentration was below 5.0 mg/kg whereas tyramine content ranged from 47.3 to 219.0 mg/kg. A total of 917 isolated lactic acid bacteria (LAB) were genotyped by rep-PCR and identified by 16S rRNA gene sequencing. Overall, Leuconostoc mesenteroides was identified as the most frequently isolated species (n = 259), followed by Lactobacillus sakei (n = 190) and Enterococcus casseliflavus (n = 106). Cluster analysis revealed a remarkable intraspecies diversity and a strong sausage-specific clustering of the LAB genotypes, indicating meat as a probable source of microbial diversity in sausages. 14.38% of LAB were positive for tyramine encoding gene and no other target biogenic amines genes were detected. Due to the elevated number of presumptive pathogens (E. coil. Enterobacteriaceae, B. cereus group, coliforms) in ready-to-eat sausages, 33.33% of products can be considered as inappropriate for human consumption.

Traditional production of fermented dairy products in Montenegro is carried out without adding defined starter cultures. This way of production involves lactic acid bacteria (LAB) that are normally present in the raw milk and production environment. This autochthonous ("wild") fermentation microbiota represents a reservoir of unknown strains. In order to study the LAB diversity, 25 indigenous dairy products in Montenegro have been tested. Isolation was performed on microbial media M17 and MRS agar with or without supplementations under aerobic and anaerobic conditions at temperatures of 30?C, 37 ?C and 44?C. Identification of these isolates at species level was done by species-specific PCR and gene regions sequencing of representatives of each RAPD-cluster. RAPD-PCR was used to characterize the isolates at strain level. Nine Lactobacillus species, five Leuconostoc species, four Enterococcus species as well as strains of the species Lactococcus lactis, Pediococcus pentosaceus and Streptococcus thermophilus were detected. It can be concluded that a rich lactic acid bacteria diversity existed in the analyzed Montenegrin dairy products. Further examination of the isolates could lead to the development of autochthonous starter cultures that would contribute to a product that is characteristic for the geographical area and to which the local population is accustomed.

Food fermentation technologies have developed through years of experience rather than scientific findings. Therefore, many small-scale manufacturers are unwilling to accept changes and to modify fermentation processes. Still, traditional fermented foods have had an essential role in human nutrition for thousands of years. A wide range of diverse cereals, legumes, vegetables, fruits, fish, seafood, and meat are used as raw materials for the production of these fermented foods by back-slopping (a process where material from a previous successful batch is added to facilitate the initiation of a new batch) or naturally occurring microorganisms. Various microorganisms that influence the quality, safety, sensory properties, acceptability, and consistency of these products are present. Lactic acid bacteria with their beneficial characteristics are generally a significant contributor. Identification and profiling of microorganisms in fermented foods are of particular interest. But so far such information has not been explored in detail for some fermented products from the lower Mekong subregion. Improving the safety, quality, and acceptability of fermented foods, while reducing their production costs and maintaining their authenticity and uniqueness, is important. This can be achieved through starter culture application and greater attention to food safety, such as HACCP approaches. Recent scientific advances have led to the development of defined microbial starters, leading to the transfer of artisanal food production to more controlled and industrialized fermentation. The aim of this review is to determine the gaps in the knowledge of traditional fermented foods of the lower Mekong subregion and to present current information on the diversity of involved microorganisms. These data will be helpful for defining future strategies toward the sustainable production of safe fermented foods particularly those from southeast Asia.

The aim of this study is to characterise and examine the biochemical properties of 40 Lactococcus lactis strains isolated from indigenous Montenegrin dairy products in order to explore their potential to be used as starter cultures for producing typical Montenegrin cheese, such as ‘bijeli sir’, ‘masni sir’ and ‘njeguški sir’. Their safety regarding the production of biogenic amines, the presence of antimicrobial resistance and the antibacterial activity against relevant pathogens and spoilage microorganisms has also been tested. Based on the characterisation, all strains belong to L. lactis ssp. lactis. Out of these 40 strains, 23 displayed rapid acidification ability and proteolysis. However, none of the strains exhibited the ability of lipid degradation. Most of the strains were not associated with any health risk investigated. Summing up, a large percentage (27.5 %) of the tested strains showed good properties. These strains should be further examined for their possible application as specific starter cultures in the production of indigenous cheese in Montenegro

Increasing antibiotic resistance is a major public health concern. Fluoroquinolones are used to treat and prevent poultry diseases worldwide. Fluoroquinolone resistance rates are high in their countries of use. The aim of this study was to evaluate the effect of an acids-based feed additive, as well as fluoroquinolone antibiotics, on the prevalence of antibiotic-resistant E. coli. A total of 480 broiler chickens (Ross 308) were randomly assigned to 3 treatments: a control group receiving a basal diet; a group receiving a feed additive (FA) based on formic acid, acetic acid and propionic acid; and an antibiotic enrofloxacin (AB) group given the same diet, but supplemented with enrofloxacin in water. A pooled fecal sample of one-day-old chicks was collected upon arrival at the experimental farm. On d 17 and d 38 of the trial, cecal samples from each of the 8 pens were taken, and the count of E. coli and antibiotic-resistant E. coli was determined.The results of the present study show a high prevalence of antibiotic-resistant E. coli in one-day-old chicks. Supplementation of the diet with FA and treatment of broilers with AB did not have a significant influence on the total number of E. coli in the cecal content on d 17 and d 38 of the trial. Supplementation with FA contributed to better growth performance and to a significant decrease (P ≤ 0.05) in E. coli resistant to ampicillin and tetracycline compared to the control and AB groups, as well as to a decrease (P ≤ 0.05) in sulfamethoxazole and ciprofloxacin-resistant E. coli compared to the AB group. Treatment with AB increased (P ≤ 0.05) the average daily weight compared to the control group and increased (P ≤ 0.05) the number of E. coli resistant to ciprofloxacin, streptomycin, sulfamethoxazole and tetracycline; it also decreased (P ≤ 0.05) the number of E. coli resistant to cefotaxime and extended spectrum beta-lactamase- (ESBL-) producing E. coli in the ceca of broilers.

A Lactobacillus plantarum strain was used for the production of rye, wheat, barley, and oat sourdoughs, and the influence of different sourdoughs on mixed rye-wheat bread quality parameters and acrylamide formation was evaluated. L.plantarum LUHS135 demonstrated versatile carbohydrate metabolism, good growth and acidification rates, and the ability to excrete amylolytic and proteolytic enzymes in various cereal sourdoughs. The same starter and different cereal substrates allow to produce sourdoughs showing different characteristics. The type of sourdough and its quantity had significant influence on acrylamide content in bread (P0.0001), and using 5% or 10% of wheat sourdough, 5%, 15%, or 20% of barley sourdough, and 5% or 15% of oat sourdough, it is possible to reduce acrylamide content in bread. Thus, manufacturers need to take into account application of apparent technological approaches for acrylamide in bread reducing.

Lactic acid bacteria (LAB) from spontaneous rye sourdough were isolated, identified, and characterized by their growth, acidification rate, and carbohydrate metabolism. The isolated LAB were used for production of rye sourdough, and the influence of sourdough on mixed rye - wheat bread quality and acrylamide formation was evaluated. In addition, comparative studies by using acidification with L(+) -lactic acid for mixed rye wheat bread production were performed.Isolated LAB (P. acidilactici, L plantarum, L curvatus) demonstrated versatile carbohydrate metabolism, grown at 30 degrees C and 37 degrees C, and acidic tolerance. When the isolated strains were used for rye sourdough production, they showed good growth, acidification rates while excreting amylolytic and proteolytic enzymes. Rye sourdoughs delay bread staling, and there was a significant effect of type of dough acidification (with LAB or L -(+)-lactic acid) and the quantity of acidification agent used (5% or 15%) on most of the analyzed bread quality parameters. L. plantarum sourdough (added in the amount of 5% and 15%) decreased the acrylamide content in bread samples (p < 0.0001), unlike dough acidification with L(+) -lactic acid. Thus the selected LAB could be recommended for improving the quality of bread, including the reduction of acrylamide content. (C) 2017 Elsevier Ltd. All rights reserved.

Lactic acid bacteria (LAB) play a dual role in winemaking as they are the main effectors of malolactic fermentation, but some members can also cause wine spoilage. PCR-DGGE has proved to be a quick tool to study the LAB community and their fluctuation in wine. For detecting wine-associated LAB by PCR-DGGE, the primer sets WLAB1/WLAB2GC, WBAC1/WBAC2GC, Lac1/Lac1o/Lac2GC, 341fGC/518r and rpoB1/rpoB1o/rpoB2GC were tested and evaluated in this study. The primer systems were assessed by the separation of LAB reference strains on DGGE gels and by attributing the resulting amplicons to defined species. Subsequently, the detection of LAB in wine samples and enrichments thereof was compared. While the primer systems WBAC1/WBAC2GC and 341fGC/518r were not appropriate, the Lac1/Lac1o/Lac2GC primer set performed well. However, multiple bands complicated the evaluation. The rpoB1/rpoB1o/rpoB2GC set seemed to be promising for the detection of LAB in wine, although further improvements in terms of the detection limit need to be done. Due to the pronounced sensitivity and the sufficient discrimination of LAB at species level, the WLAB1/WLAB2GC primer system was found to be most suitable for studying the occurrence of LAB in wine.