Dynamics of microbiota in the imported beef primal cuts during storage at different chilled temperatures.

Primal cuts of Australian beef transported by sea were stored under different chilled temperatures (0, 2, and 4 °C) for 6 weeks in different packaging conditions (aerobic or anaerobic packaging). The number of microorganisms and the transition of the microbiota were investigated using culture methods and amplicon sequencing. After 6 weeks of storage, the beef tended to show a high total viable count under aerobic packaging conditions and a high lactic acid bacteria count under anaerobic packaging conditions. The result of amplicon sequencing analysis showed that different beef samples had different predominant bacterial groups. Moreover, at high storage temperatures, Serratia sp. having high putrefactive activity showed increased abundance, while at low storage temperatures, Lactobacillus sp. showed increased abundance. Thus, differences in the packaging conditions and distribution temperatures after import affect the number of bacteria and the type of microorganisms in the Australian beef primal cuts, which may affect their quality.

Dynamics of microbiota in Japanese Black beef stored for a long time under chilled conditions.

In this study, changes in the microbiota of Japanese Black beef carcasses, which are expected to be transported for a long time in chilled temperatures, were investigated. Three Japanese Black beef samples (carcasses A, B, and C) immediately after slaughter were stored at 0 °C for 15 weeks under aerobic and vacuum conditions. The initial bacterial counts were 50 CFU/g for carcass A and less than the reliable quantitative detection limit for carcasses B and C. Under aerobic storage conditions, the bacterial count increased to 8.0 log CFU/g or higher, which is a measure of putrefaction, at 6-9 weeks. Under anaerobic storage conditions, the bacterial counts of carcasses A and C reached 3.5-6.5 log CFU/g, but carcass B showed no bacterial growth during the 15-week storage period. The predominant group was Pseudomonas spp. under aerobic conditions and Serratia spp. under anaerobic conditions. To the best of our knowledge, there is no previous study investigating the transition of microbiota when Japanese Black beef is stored at low temperatures for a long period of time, and the results of this study are considered very important findings for the expansion of international trade of Japanese Beef in the future.

In vitro antioxidant, anti-glycation and immunomodulation activities of fermented blue-green algae Aphanizomenon flos-aquae.

To clarify the antioxidant, anti-glycation and immunomodulatory capacities of fermented blue-green algae Aphanizomenon flos-aquae (AFA), hot aqueous extract suspensions made from 10% AFA were fermented by Lactobacillus plantarum AN7 and Lactococcus lactis subsp. lactis Kushiro-L2 strains isolated from a coastal region of Japan. The DPPH and O2- radical scavenging capacities and Fe-reducing power were increased in the fermented AFA. The increased DPPH radical scavenging capacity of the fermented AFA was fractionated to mainly < 3 kDa and 30-100 kDa. The increased O2- radical scavenging capacities were fractionated to mainly < 3 kDa. Anti-glycation activity in BSA-fructose model rather than BSA-methylglyoxal model was increased by the fermentation. The increased anti-glycation activity was fractionated to mainly 30-100 kDa. The NO concentration in the murine macrophage RAW264.7 culture media was high with the fermented AFA. The increased immunomodulation capacity was also fractionated to mainly 30-100 kDa. These results suggest that the fermented AFA is a more useful material for health foods and supplements.

The use of next generation sequencing for improving food safety: Translation into practice.

Next Generation Sequencing (NGS) combined with powerful bioinformatic approaches are revolutionising food microbiology. Whole genome sequencing (WGS) of single isolates allows the most detailed comparison possible hitherto of individual strains. The two principle approaches for strain discrimination, single nucleotide polymorphism (SNP) analysis and genomic multi-locus sequence typing (MLST) are showing concordant results for phylogenetic clustering and are complementary to each other. Metabarcoding and metagenomics, applied to total DNA isolated from either food materials or the production environment, allows the identification of complete microbial populations. Metagenomics identifies the entire gene content and when coupled to transcriptomics or proteomics, allows the identification of functional capacity and biochemical activity of microbial populations. The focus of this review is on the recent use and future potential of NGS in food microbiology and on current challenges. Guidance is provided for new users, such as public health departments and the food industry, on the implementation of NGS and how to critically interpret results and place them in a broader context. The review aims to promote the broader application of NGS technologies within the food industry as well as highlight knowledge gaps and novel applications of NGS with the aim of driving future research and increasing food safety outputs from its wider use.

Susceptibility of gut indigenous lactic acid bacteria in BALB/c mice to oral administered Lactobacillus plantarum.

Cells of Lactobacillus plantarum strains AN1 and Tennozu-SU2 exert anti-inflammatory responses in ICR mouse models of inflammatory bowel disease and protective effects against S. Typhimurium infection in BALB/c mice, respectively. To clarify the existence of L. plantarum-susceptible gut indigenous bacteria, AN1 and Tennozu-SU2 cells were administered to BALB/c mice via drinking water. Gene amplicon sequencing of 16S rRNA of caecal content revealed that the AN1 and Tennozu-SU2 cells affected the abundance of caecal indigenous lactobacilli, but the effect on the dominant Clostridiales and Bacteroidales was not clear. With Blood and Liver (BL) agar containing 5% v/v horse blood, six typical colonies from faecal samples were detected as the principal lactobacilli. Among them, two typical colonies were isolated and identified to be AN1 and Tennozu-SU2. Two and one typical colonies detected in all mice were identified to be L. reuteri and L. murinus, respectively. The other one was identified and estimated to be indigenous L. plantarum detected in the Tennozu-SU2 group.

[Development of the Genus and Species Determination Method for Histamine Producing Bacteria Isolated from Fishery Product with High-Resolution Melting Analysis].

Histamine in foods with a high histidine content may be produced by bacteria with histidine decarboxylase activity. Consumption of food enriched in histamine can produce symptoms of histamine poisoning that include flushing, headache, and urticaria. The number of histamine poisoning cases in Japan has decreased with developments in food hygiene management technology. However, approximately 10 cases are still reported each year. In addition, there have been cases where histamine was detected in the end products, prompting large product recalls. To prevent and identify causes of histamine toxicity, manufacturers must identify the bacteria causing the illness. A simple method of identification is needed, since sequence-based identification is complicated to perform and the analysis takes a long time. High-Resolution Melting Analysis (HRMA) is a method that detects differences in the base sequences of PCR products manifested as varied melting temperatures of double-stranded DNA. The present study was intended to develop a rapid identification method for major histamine-producing bacteria using HRMA. Species-specific HRMA primers were designed that specifically targeted the hdcA gene of 20 Gram-negative histamine-producing bacterial strains. The designed primers were used for HRM analysis of the 20 histamine-producing bacterial strains. The strains were divided into three groups (A, B, and C) based on differences in melting temperature values obtained by Tm Calling analysis program. Group A comprised terrestrial bacteria, such as Morganella, Enterobacter, and Raoultella, while Groups B and C comprised marine bacteria, such as those belonging to the genera Vibrio and Photobacterium. The melting profiles obtained in Group A by HRMA were used to identify the aforementioned terrestrial bacteria. The findings indicated that HRMA can easily identify the major gram-negative histamine-producing bacteria. A flow chart was created to identify histamine-producing bacterial species. This method enables the identification of histamine-producing bacterial species more quickly and easily than conventional sequence-based methods. Therefore, the method could be valuable for food companies to screen raw materials and products and track the source of contamination, which will in turn contribute to the prevention of histamine-food poisoning and investigation of its causes.

Effects of fermented green-loofah and green-papaya on nitric oxide secretion from murine macrophage raw 264.7 cells.

To clarify the effect of lactic acid bacteria (LAB) fermentation on the immunomodulation capacity of green-loofah and green-papaya, aqueous suspensions prepared from the fresh and dry-powdered vegetables were fermented by Lactococcus lactis subsp. lactis Uruma-SU1 and Lactobacillus plantarum Uruma-SU4. Fermented and non-fermented suspensions were added to murine macrophage RAW264.7 culture with and without Escherichia coli O111 lipopolysaccharide (LPS). In the absence of LPS, nitric oxide (NO) secretion was elevated significantly in LAB fermented suspensions compared to that in non-fermented suspensions. NO production in fermented suspensions was observed even at low sample concentrations, but it was attenuated in the centrifuged supernatant. With LPS treatment, inhibition of NO secretion was shown with the high concentration of the non-fermented and also fermented samples. These results suggest that fermented green-loofah and green-papaya suspensions can play both immunostimulatory and anti-inflammatory roles at low and high doses, respectively.

Will the emergence of core genome MLST end the role of in silico MLST?

The technological advancement of molecular epidemiological analysis using next-generation sequencing (NGS) for foodborne pathogens has a groundbreaking impact over the past three years. In particular, the emergence of cg (core genome) multilocus sequence typing(MLST) has a significant impact. This is because this technology made it possible for many researchers to carry out molecular epidemiological analysis on foodborne pathogens in a common language, using common definitions. The resolution of core genome MLST (cgMLST) far surpasses that of MLST, which only uses seven (usually, in some cases five) housekeeping genes. Therefore, cgMLST would in no doubt terminate the role of conventional MLST as the molecular epidemiological tool. However, the role of MLST would probably not end all together. Rather, the sequence type (ST) of the conventional MLST is expected to be used as in silico MLST by a wider range of researchers than ever in the next 10 years. This is because, with the arrival of the NGS era, we have come to be able to obtain ST of conventional MLST by simply entering the NGS text file into one's own PC. In other words, acquisition of ST data is no longer limited to researchers aiming to conduct MLST for the first place. The impact of such a change is large. In silico MLST will continue to be used as a tool for understanding the broad characteristics of bacterial strains. This review aimed to summarize the main information on STs that have been accumulated for representative foodborne pathogens, in particular for potential NGS users in this new era who have been not familiar with MLST until now.

Bacterial and fungal microbiota of spontaneously fermented Chinese products, Rubing milk cake and Yan-cai vegetable pickles.

The Rubing milk cake from Yunnan and the Yan-cai vegetable pickles from Guangdong are traditional spontaneously fermented foods in China. We evaluated the microbial properties of these products with the analysis of their bacterial and fungal microbiota using classical culture-dependent and culture-independent methods, including a 16S rDNA gene (V4) and an internal transcribed spacer (ITS) region pyrosequencing method with MiSeq system. The viable lactic acid bacteria (LAB) count was 8 and 6 log colony-forming units (CFU)/g in Rubing and Yan-cai samples, respectively. The yeast count was approximately 100-1000 times less than the LAB count in most samples, except one Yan-cai sample. In addition, the gram-negative rod count in half of the samples was similar to the LAB count. Pyrosequencing results revealed the high abundance (10%-20%) of gram-negative Pseudomonas spp. and Enterobacteriaceae in these samples. These results suggest that some of these traditional foods are undesirable as ready-to-eat (RTE) foods, even when these are typical lactic acid fermented foods.

Approaches to empower the implementation of new tools to detect and prevent foodborne pathogens in food processing.

Foodborne pathogens cause an important public health burden, which is estimated in 600 million cases and more than 400,000 deaths, globally every year. The most susceptible populations, such as children under the age of five, the elderly and immunocompromised, account for the majority of the deaths. Food safety incidents, outbreaks, sporadic cases, and recalls have recognized economic impact, estimated at 7 billion every year in the US. Food safety has become a priority, and the implementation of preventive controls and monitoring systems has raised the development of new tools to detect and prevent pathogens in the food chain. Detection tools have evolved quickly, from rapid testing methods to application of genomics and metagenomics. Importantly, to reduce food safety hazards at food processing, the food chain needs to be seen from farm to fork. This review summarized the main findings discussed during the 2016 OECD-sponsored symposium on food safety. These include i) trends in food safety that embrace the need to implement new tools in detection and prevention, ii) the very rapid evolution of technologies to detect foodborne pathogens, iii) holistic approaches to prevent pathogens require a whole chain approach, and iv) key pillars to facilitate global implementations of new tools in food safety.

Effect of glucose on Listeria monocytogenes biofilm formation, and assessment of the biofilm's sanitation tolerance.

Listeria monocytogenes is an important cause of human foodborne infections and its ability to form biofilms is a serious concern to the food industry. To reveal the effect of glucose conditions on biofilm formation of L. monocytogenes, 20 strains were investigated under three glucose conditions (0.1, 1.0, and 2.0% w v(-1)) by quantifying the number of cells in the biofilm and observing the biofilm structure after incubation for 24, 72, and 168 h. In addition, the biofilms were examined for their sensitivity to sodium hypochlorite. It was found that high concentrations of glucose reduced the number of viable cells in the biofilms and increased extracellular polymeric substance production. Moreover, biofilms formed at a glucose concentration of 1.0 or 2.0% were more resistant to sodium hypochlorite than those formed at a glucose concentration of 0.1%. This knowledge can be used to help design the most appropriate sanitation strategy.

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.

Effect of quantity of food residues on resistance to desiccation of food-related pathogens adhered to a stainless steel surface.

In order to study the effect of food residues on the survival of food-borne pathogens, Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes were subjected to drying conditions in the presence of small amounts of food such as carrot juice, aqueous solution of nori, milk, and soy-milk. After drying for 2 h at room temperature in the absence of food residue, cell counts of S. Typhimurium, S. aureus, and L. monocytogenes decreased from 8 to 3, 6, and 5 log cfu/dish, respectively. Five milligrams of fresh carrot, 0.05 mg dried nori, and 100 nL milk or soy milk per 10 mm φ surface were sufficient to demonstrate a protective effect on the adhered pathogens, as confirmed by atomic force microscopy. Results from this study suggest that small sediments of food, not only protein rich but also carbohydrate rich, increase the resistance of surface-adherent bacteria to desiccation, rendering sanitization processes ineffective and encouraging cross contamination.

Comparison of the major virulence-related genes of Listeria monocytogenes in internalin A truncated strain 36-25-1 and a clinical wild-type strain.

BACKGROUND: Internalin A (InlA) facilitates the invasion of Listeria monocytogenes into a host cell. Some strains of Listeria monocytogenes express truncated forms of InlA, which reduces invasiveness. However, few virulence-related genes other than inlA have been analyzed in InlA-truncated strains. In the present study, we sequenced the draft genome of strain 36-25-1, an InlA-truncated strain, with pyrosequencing and compared 36 major virulence-related genes in this strain and a clinical wild-type strain. RESULTS: Strain 36-25-1 possessed all of the virulence-related genes analyzed. Of the analyzed genes, only 4 genes (dltA, gtcA, iap, and inlA) differed when the nucleotide sequences of strain 36-25-1 and the clinical wild-type strain were compared. Analysis of the deduced amino acid sequences found no mutations that significantly influenced virulence in genes other than inlA. CONCLUSIONS: The virulence-associated genes in strain 36-25-1 differ little from those of the clinical wild-type strain, indicating that a slight mutation in the nucleotide sequence determines the virulence of the InlA-truncated strain. In addition, the results suggest that, aside from InlA-mediated cell invasiveness, there is almost no difference between the virulence of strain 36-25-1 and that of the clinical wild-type strain.

Genetic distance in the whole-genome perspective on Listeria monocytogenes strains F2-382 and NIHS-28 that show similar subtyping results.

BACKGROUND: Genome subtyping approaches could provide useful epidemiological information regarding food pathogens. However, the full genomic diversity of strains that show similar subtyping results has not yet been completely explored. Most subtyping methods are based on the differences of only a portion of the genome. We investigated two draft genome sequences of Listeria monocytogenes strain F2-382 and NIHS-28, which have been identified as closely related strains by subtyping (identical multi-virulence-locus sequence typing and multiple-locus variable number tandem repeat analysis sequence types and very similar pulsed-field gel electrophoresis patterns), despite their different sources. RESULTS: Two closely related strains were compared by genome structure analysis, recombination analysis, and single nucleotide polymorphism (SNP) analysis. Both genome structure analysis and recombination analysis showed that these two strains are more closely related than other strains, from a whole-genome perspective. However, the analysis of SNPs indicated that the two strains differ at the single nucleotide level. CONCLUSION: We show the relationship between the results of genome subtyping and whole-genome sequencing. It appears that the relationships among strains indicated by genome subtyping methods are in accord with the relationships indicated by whole-genome analysis. However, our results also indicate that the genetic distance between the closely related strains is greater than that between clonal strains. Our results demonstrate that subtyping methods using a part of the genome are reliable in assessing the genetic distance of the strains. Furthermore, the genetic differences in the same subtype strains may provide useful information to distinguish the bacterial strains.

Caecal environment of rats fed far East Asian-modelled diets.

To clarify the effect of type of foods on the intestinal environment, Far East Asian- (FEA; rich in rice starch, soy protein and soy oil) and Far East Asian marine- (FEAM; rich in rice starch, fish meal, fish oil and brown alga) modelled diets and sucrose, casein and beef tallow-rich (SCB) diet were prepared. After the 2-week administration of diets in rats, caecal organic acids and putrefactive compounds (ammonia, indole, phenol and H2S, which are regarded as putative risk factors for tumours) were determined. The caecal microbiota was also analyzed using denaturing gradient gel electrophoresis and pyrosequencing with bar-coded primers targeting the bacterial 16S rRNA gene. Levels of n-butyrate, acetate, indole and phenol were high in rats fed FEA. On the other hand, H2S was clearly suppressed by both FEA and FEAM comparing with SCB. These results suggest that FEAM is preferable to FEA for optimal intestinal environment and host health. Both microbial analyses showed that the diversity of microbiota in the FEAM group was lower than in the other diet groups. Ratio of Firmicutes, Bacteroidetes and Proteobacteria in the SCB group was about 5:4:1. Firmicutes, particularly Lachnospiraceae, was promoted by FEA and FEAM.

Caecal fermentation, putrefaction and microbiotas in rats fed milk casein, soy protein or fish meal.

To clarify the effect of soy protein (SP) and fish meal (FM), compared to milk casein (MC), on the intestinal environment, we examined caecal environment of rats fed the test diets. Four-week-old rats were fed AIN-76-based diet containing 20 %, w/w MC, SP or FM for 16 days. Caecal organic acids were analysed by HPLC. Caecal putrefactive compounds (indole, phenol, H2S and ammonia) were analysed by colorimetric assays. Caecal microflora was determined by 16S rRNA gene-DGGE and pyrosequencing with bar-coded primers targeting the bacterial 16S rRNA gene. n-Butyric and lactic acid levels were high in rats fed SP and FM, respectively. Butyrate-producing bacteria, such as Oscillibacter, and lactate-producing bacteria, such as Lactobacillus, were detected in each diet group. Also, the putrefactive compound contents were high in rats fed SP and FM. In this study, both DGGE and pyrosequencing analyses were able to evaluate the dynamics of the intestinal microbiota. The results indicate that dietary proteins can alter the intestinal environment, affecting fermentation by the intestinal microbiota and the generation of putrefactive compounds.

FLX pyrosequencing analysis of the effects of the brown-algal fermentable polysaccharides alginate and laminaran on rat cecal microbiotas.

Edible brown algae are used as major food material in Far East Asian countries, particularly in South Korea and Japan. They contain fermentable dietary fibers, alginic acid (uronic acid polymer) and laminaran (ß-1,3-glucan), that are fermented into organic acids by intestinal bacteria. To clarify the effect of edible algae on the intestinal environment, the cecal microbiotas of rats fed diets containing no dietary fiber (control) or 2% (wt/wt) sodium alginate or laminaran for 2 weeks were analyzed using FLX amplicon pyrosequencing with bar-coded primers targeting the bacterial 16S rRNA gene. The most abundant phylum in all groups was Firmicutes. Specifically, Allobaculum was dominant in all diet groups. In addition, Bacteroides capillosus (37.1%) was abundant in the alginate group, while Clostridium ramosum (3.14%) and Parabacteroides distasonis (1.36%) were only detected in the laminaran group. Furthermore, rats fed alginate showed simplified microbiota phylotypes compared with others. With respect to cecal chemical compounds, laminaran increased cecal organic acid levels, particularly propionic acid. Alginate increased total cecal organic acids. Cecal putrefactive compounds, such as indole, H(2)S, and phenol, were decreased by both alginate and laminaran. These results indicate that edible brown algae can alter the intestinal environment, with fermentation by intestinal microbiota.

Inhibitory effects of Leuconostoc mesenteroides 1RM3 isolated from narezushi, a fermented fish with rice, on Listeria monocytogenes infection to Caco-2 cells and A/J mice.

Listeria monocytogenes causes listeriosis in humans mainly through consumption of ready-to-eat foods. Immunocompromised persons, the elderly, and pregnant women and their fetuses or newborns are at highest risk for the infection. To isolate probiotic lactic acid bacteria (LAB) with inhibitory effects against L. monocytogenes, we screened for acid and bile resistant LABs from narezushi, a traditional salted and long-fermented fish with cooked rice. Then, inhibitory effects of the selected LABs on L. monocytogenes invasion and infection of human enterocyte Caco-2 cells and Listeria-susceptible A/J mice were determined. From a total of 231 LAB isolates, we selected five acid and bile resistant isolates (four were Lactobacillus plantarum and one was Leuconostoc mesenteroides). Among the five isolates, Ln. mesenteroides (Lnm-1RM3) showed the highest inhibition against L. monocytogenes invasion into Caco-2 cells. In the case of L. monocytogenes orally infected A/J mice, recovery of the pathogen from the spleen was suppressed by drinking water containing 9 log CFU/ml of Lnm-1RM3 cells. The inhibitory effects were also shown by heat-killed Lnm-1RM3 cells. These results suggest that live and also heat-killed Lnm-1RM3 cell intake might prevent L. monocytogenes entero-gastric invasion and infection.