Distribution of psychrophilic microorganisms in a beef slaughterhouse in Japan after cleaning.

The purpose of this study was to investigate the abundance and distribution of psychrophilic microorganisms associated with spoilage in beef slaughterhouse environments after cleaning. The processing lines and equipment used in slaughtering and boning were swabbed, and the microbial count was determined using a TSA and MRS medium and Chromocult® Coliform agar incubated at 15ºC and 37ºC, respectively. As a result, the brisket saw (handle side) and trolley hook were the most heavily contaminated with microorganisms, with each having a microbial adhesion rate of 66.7%. The microbial adhesion rates of the apron and milling cutter (edge side) were 50%, respectively, and those of the foot cutter (edge and handle side), splitting saw (edge side), and knife (handle side) were 33.3%, respectively. Next, four colonies were randomly isolated from the petri dish used for the bacterial count measurement to identify the predominant microbial species of the microorganisms attached to each equipment. As a result of Sanger sequencing analysis, yeasts such as Candida zeylanoides and Rhodotorula sp. and bacteria including Pseudomonas sp. and Rhodococcus sp. were identified from the equipment used in the slaughtering line, and it was assumed that these microorganisms were of environmental origin. In contrast, only Pseudomonas sp. and Candida zeylanoides were isolated from the boning line. Despite the use of cleaning operations, this study identified some equipment was contaminated with microorganisms. Since this equipment frequently comes into direct contact with the carcass, it is critical to thoroughly remove the microorganisms through accurate cleaning to prevent the spread of microbial contamination on the carcasses.

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.

Detection and isolation of typical gut indigenous bacteria in ICR mice fed wheat bran and wheat straw fibre.

Wheat bran (WB) and wheat straw fibre (WSF) are by-products of the wheat flour industry. To prove the existence of indigenous gut bacteria responsible for WB and WSF, the Institute of Cancer Research (ICR) mice were fed a diet containing no fibre (CS), 10% WB, or 5% WSF for 14 d. The caecal microbiome was analysed by 16S rDNA (V4 region) amplicon sequencing. Typical colonies were isolated and estimated by 16S rRNA gene BLASTn analysis. The predominant amplicon sequence variants in all diet groups belonged to Bifidobacterium pseudolongum- and Faecalibaculum rodentium-like bacteria. Lactobacillus johnsonii- and Limosilactobacillus reuteri-like bacteria were high in the WB group compared with those in the CS group. Lactobacillus johnsonii Wheat-1 and L. reuteri Wheat-12 strains could be isolated. Lactobacillus johnsonii Wheat-1 exhibited good fermentation activity in 10% (w/v) WB suspension. Superoxide anion radical scavenging capacity of the WB suspension was significantly increased by the fermentation.

Comparison between the Antimicrobial Activity of Essential Oils and Their Components in the Vapor Phase against Food-related Bacteria.

In this study, the antibacterial properties of the volatile components of four essential oils (cinnamon, clove, origanum, and peppermint oil) and five of their components (allyl isothiocyanate (AITC), carvacrol, citral, eugenol, and (+)-limonene) against five food-related bacteria (Escherichia coli, Listeria monocytogenes, Salmonella Typhimurium, Pseudomonas fluorescens, and Enterococcus faecalis) were evaluated. The results of disc volatilization method revealed that AITC exhibited antibacterial activity against the five tested strains at the lowest concentration, as did cinnamon oil and carvacrol. Moreover, the total aerobic bacterial count in coleslaw salad was suppressed in all test groups treated with AITC compared to that in control.

Detection of low-mineral- and high-salt responsible caecal indigenous bacteria in ICR mice.

Both deficiency and overdose of minerals and salts negatively affect health. Changes in the dietary composition have immediate effects on the gut microbiota. This study was performed to clarify the presence of indigenous gut bacteria responsible for minerals and/or salts (MS-RIB). ICR mice were fed a diet containing 3.5% (w/w) mineral mix (control), 1% mineral mix (LM), or 3.5% mineral mix and 4% NaCl (HS) for 14 days. The caecal microbiota was examined using 16S rRNA gene (V4) amplicon sequencing. Consumption of drinking water was 2.5-fold higher in the HS group than in the other groups. Body weight gain was 55% lower in the HS group than in the other groups. At the family level, the relative abundance of Eryspelotrichaceae and Clostridiaceae was lower in the HS group than in the other groups. In contrast, the abundance of Bacteroidaceae was higher in the HS group. At the operational taxonomic unit level, Desulfovibrionaceaer-, Turicibacter sanguinis-, belonging to Eryspelotrichaceae, and Clostridium disporicum-like bacteria were dominant in the control group. Among these bacteria, T. sanguinis- and C. disporicum-like bacteria were markedly suppressed by HS. In the LM group, Bacteroides acidifaciens-like bacteria were suppressed. Suppression of C. disporicum and Turicibacter following consumption of the HS diet was the most notable effect, contrasting the results of previous studies.

Molecular subtyping for source tracking of Escherichia coli using core genome multilocus sequence typing at a food manufacturing plant.

When harmful bacteria are detected in the final product at a food manufacturing plant, it is necessary to identify and eliminate the source of contamination so that it does not occur again. In the current study, the source of contamination was tracked using core genome multilocus sequence typing (cgMLST) analysis in cases where Escherichia coli was detected in the final product at a food manufacturing plant. cgMLST analysis was performed on 40 strains of E. coli collected from the environment [floor (26 strains), drainage ditch (5 strains), container (4 strains), post-heating production line (1 strain)] and products [final product (3 strains) and intermediate product (1 strain)]. In total, 40 E. coli isolates were classified into 17 genogroups by cgMLST analysis. The 4 E. coli strains isolated from the intermediate and final products were classified into two genogroups (I and II). Certain isolates collected from the environment also belonged to those genogroups, it was possible to estimate the transmission of E. coli in the manufacturing plant. Thus, the dynamics of E. coli in the food manufacturing location were clarified by using cgMLST analysis. In conclusion, our results indicate that cgMLST analysis can be effectively used for hygiene management at food manufacturing locations.

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.

Typical gut indigenous bacteria in ICR mice fed a soy protein-based normal or low-protein diet.

For patients with inflammatory bowel disease, cow's milk allergy, and lactose intolerance, soymilk is a potential alternative to cow's milk. In this study, we aimed to identify the effects of a soy protein-based low-protein diet on the body and organ weights and the gut microbiome of six-week-old mice fed a diet containing 20% (SP) or 5% (LP) soy protein for 14 days via 16S rRNA (V4) amplicon sequencing. Body weight gain (growth) and liver, spleen, and fat tissue weight were significantly suppressed by the LP diet. Operational taxonomic unit numbers and α-diversity were lower in the LP group than in the SP group. A principal coordinate analysis revealed differences in the gut microbiome compositions of SP and LP mice. The abundances of caecal Roseburia sp., Alistipes sp., and bacteria from the family Muribaculaceae were lower in the LP group than in the SP group. In contrast, the abundance of Desulfovibrionaceae, which is positively correlated with inflammation, was higher in the LP group than in the SP group. These results differed from the effects of a milk casein-based low-protein diet (reported previously). Based on these findings, we conclude that the undesirable effects of a low-protein diet and/or protein deficiency are related to changes in the gut microbiome composition and may differ depending on the kind of proteins used.

Impact of pH and protein hydrophobicity on norovirus inactivation by heat-denatured lysozyme.

Norovirus, the leading cause of non-bacterial food poisoning, is responsible for several outbreaks associated with bivalves and ready-to-eat food products worldwide. As norovirus is resistant to alcohol, which is commonly used in food manufacturing processes, sodium hypochlorite is used for its inactivation. However, sodium hypochlorite has two disadvantages: it cannot be added to foods, and its effect is significantly reduced in the presence of organic compounds. Thus, a novel disinfectant against norovirus is urgently required for food hygiene. Thermally denatured egg white lysozyme inactivates norovirus; however, the optimal inactivating conditions and the underlying mechanism are unclear. In the present study, the inactivating mechanism of heat-denatured lysozyme against norovirus was analyzed using murine norovirus strain 1 (MNV-1). We found that the inactivating effect was enhanced by adjusting the pH of the lysozyme solution before thermal denaturation to 6.5 or higher. The reaction of heat-denatured lysozyme and MNV-1 was irreversible, and norovirus was completely inactivated after exposure to heat-denatured lysozyme. Furthermore, it was found that lysozyme residues 5-39 contributed to the norovirus-inactivating effect. Notably, the hydrophobicity and positive charges in this region contributed to the norovirus-inactivating effect, as evidenced by the norovirus inactivation test using mutated residues 5-39. These findings are novel and highlight the possible application of heat-denatured lysozyme as a disinfectant against norovirus in a wide range of food processes.

Detection and isolation of the typical gut indigenous bacteria from ddY mice fed a casein-beef tallow-based or egg yolk-based diet.

The effects of whole egg on the cecal microbiome of ddY mice has been reported. To investigate the existence of susceptible indigenous bacteria (SIB) to egg yolks (EY), mice were fed a diet containing either 20% (w/w) milk casein and 17% beef tallow (CT) or 12% milk casein and 27% EY for 14 days, and then, the cecal microbiome was analyzed by 16S rRNA (V4) amplicon sequencing. To isolate the typical species in each diet group, culture-dependent viable bacterial counts were determined on Blood Liver (BL) and Gifu Anaerobic Medium (GAM) agar plates. The amplicon sequencing analysis revealed typical CT-SIB, such as Lachnospiraceae-like bacteria, and EY-SIB, such as Allobaculum-, Lactobacillus murinus-, and Bacteroides vulgatus-like bacteria. Two of the detected SIB species, L. murinus- and B. vulgatus-like bacteria, were successfully isolated from the BL and GAM agar plates and defined using a 16S rDNA BLAST search. PRACTICAL APPLICATIONS: The SIB defined in the CT and EY groups might have some effects on the nutritional and functional chemical compounds in the milk casein, beef tallow, and/or EY. Analysis of its functional properties of the isolates might develop the new and unique probiotic strains.

Detection and isolation of low molecular weight alginate- and laminaran-susceptible gut indigenous bacteria from ICR mice.

Alginate and laminaran are the main water-soluble polysaccharides in edible brown algae such as arame Eisenia bicyclis. To determine the alginate- and/or laminaran-susceptible indigenous bacteria (SIB) in the gut, the caecal microbiomes of ICR mice fed a diet containing 2% low molecular weight (LMW ≒50 kDa) alginate or laminaran were analysed by 16S rRNA gene (V4) amplicon sequencing. At the phylum level abundances, compared to those in mice fed a no-fibre diet, Firmicutes was lower and Bacteroidetes was higher in both LMW alginate- or laminaran-fed mouse groups. At the operational taxonomic unit level, Bacteroides acidifaciens- and Bacteroides intestinalis-like bacteria were considered alginate- and laminaran-SIB, respectively. B. acidifaciens PS-4 isolated from the ICR mice fermented LMW alginate and laminaran and mainly produced succinate. B. intestinalis ALB-11 also isolated from these mice fermented laminaran and mainly produced lactate. These SIB might exert interactive effects with edible brown algal consumption and affect host health.

Detection and isolation of protein susceptible indigenous bacteria affected by dietary milk-casein, albumen and soy-protein in the caecum of ICR mice.

In this study, we fed ICR mice with a high-sucrose diet containing 20% w/w of milk-casein (MC), egg-white (EW), or soy-protein (SP) for 14 days in order to detect the presence of protein-susceptible gut indigenous bacteria (P-SIB). The caecal microbiome was examined by 16S rDNA amplicon sequencing using a next-generation MiSeq system. Principal coordinate analysis of the operational taxonomic units (OTUs) revealed that the microbiomes differed among the three groups. Typical SIB found in the MC-fed group were Bacteroides acidifaciens-, Bacteroides sartorii-, Eisenbergiella sp., and Lachnospiraceae sp.-like; in the EW-fed group were Lactobacillus murinus and Enterococcus faecium/avium-like; and in the SP-fed group were Muribaculaceae sp.-like bacteria. We also found that a few Lachnospiraceae sp.- and Clostridium disporicum-like bacteria were suppressed in the EW-fed mice. Out of the P-SIB detected, B. acidifaciens, L. murinus, E. faecium, and E. avium could be isolated and identified using BL agar and 16S rDNA BLAST search, respectively.

Evaluation of Inactivation of Murine Norovirus in Inoculated Shell Oysters by High Hydrostatic Pressure Treatment.

One of the major foods causing norovirus gastroenteritis is bivalve shellfish, such as oysters. Depuration and relaying methods have been used to control norovirus. However, these methods may be inadequate to control norovirus gastroenteritis. The present study aimed to investigate the effectiveness of high hydrostatic pressure (HHP) treatment in controlling norovirus in shelled oysters, by evaluating the inactivating effect of HHP on murine norovirus strain 1 (MNV-1) inoculated into a buffer, oyster homogenate, and shelled oysters. First, MNV-1 was inoculated (infectivity of 4.5 log PFU/mL) into the buffer and oyster homogenate, with a pH of 6.3 and salinity (NaCl) of 1.5%, mimicking the habitats of the Pacific oyster (Crassostrea gigas). HHP treatment at 100, 200, 275, and 300 MPa for 2 and 5 min was conducted at an initial temperature of 0 or 5°C. The infectivity of MNV-1 in both the buffer and the oyster homogenate was lower when the initial temperature was 0°C. In the buffer, the infectivity of MNV-1 decreased to 1.8 log PFU/mL after HHP treatment (200 MPa for 5 min at 0°C), and the inactivating effect was higher in the buffer than in the oyster homogenate. MNV-1 was inoculated into shelled oysters (4.8 log PFU per oyster), and HHP treatment was done at 275, 300, and 350 MPa for 5 min at the initial temperature of 0°C. The infectivity of MNV-1 decreased to 2.8 log PFU per oyster after HHP treatment at 275 MPa for 5 min. The results indicate that the inactivating effect of HHP treatment varies, depending on the medium surrounding the viral particles. Inactivation was best in buffer, followed by oyster homogenate and shelled oysters. The data could inform the development of methods to control norovirus in oysters.

Typical indigenous bacteria in the cecum of ddY mice fed a casein-beef tallow diet or whole-egg diet.

With the aim to prove the existence of food ingredient-susceptible indigenous bacteria (SIBs) in the gut, the gut microbiota of ddY mice fed a diet containing 20% milk casein and 17% beef tallow (CT diet) or 40% whole-egg powder (Egg diet) for 14 days were analyzed by 16S ribosomal RNA gene (V4) amplicon sequencing. At the genus level, abundance levels of Desulfovibrionaceae sp., Oscillospira, and Bacteroides were high in mice fed the CT diet. At the operational taxonomic unit level, Bacteroides acidifaciens- and Bacteroides caecimuris-like bacteria were highly abundant in mice fed the CT diet. Faecalibaculum rodentium- and Allobaculum stercoricanis-like bacteria were highly abundant in the mice fed the Egg diet. These sets of taxa were assumed to be estimates of casein-tallow SIBs and egg SIBs, respectively, in the gut microbiome. Among the SIBs, B. caecimuris and F. rodentium could be isolated using blood and liver agar. PRACTICAL APPLICATIONS: B. caecimuris and F. rodentium are regarded as casein-tallow and/or egg SIBs in mice. It is possible to demonstrate functionality with orally administered food ingredients. Isolation and application of human or livestock SIB is considered promising for the development of new probiotics and functional foods.

Whole-Genome Sequence of Listeria newyorkensis, Isolated from River Water in Japan.

Listeria newyorkensis is a bacterium that was recently classified into the genus Listeria So far, there are only two reports of isolation of L. newyorkensis from food. Here, we report the whole-genome sequence of L. newyorkensis strain 2-1, isolated from river water in Japan.

Draft Genome Sequence of the Histamine-Producing Bacterium Enterobacter kobei Strain 42-12.

Bacterially produced histamine in food can be a cause of food poisoning. The whole-genome sequence is described for one histamine-producing Enterobacter kobei 42-12 isolate from the edible portion of salted, dried fish.

Effects of Lactobacillus plantarum Uruma-SU4 fermented green loofah on plasma lipid levels and gut microbiome of high-fat diet fed mice.

To clarify the effect of loofah Luffa cylindrica and fermented loofah on hyperlipidemia, the in vitro bile acid lowering capacity and blood lipid levels of ddY mice fed high-fat diet supplemented with loofah were determined. Furthermore, the caecal microbiomes patterns were analysed using 16S rDNA amplicon sequencing with a next generation sequencer (MiSeq) system. Green loofah was homogenized and autoclaved (LH), and subsequently fermented with Lactobacillus plantarum Uruma-SU4 (FL). In vitro bile acid (taurocholic, glycocholic and deoxycholic acids (DCA)) lowering capacity was significantly high in FL. The levels of plasma triacylglyceride in mice which were fed a high-fat diet containing 17% beef tallow was lowered by 5% dried FL (FLD) and was unaffected by dried LH (LHD). Caecal Lactobacillus johnsonii and Clostridium disporicum known as predominant lactic acid bacteria in mice gut and urso-DCA producer, respectively, were increased by FLD. On the other hand, Flintibacter butyricus was lowered by both LHD and FLD. These results suggest that if green loofah cannot be consumed as a fresh vegetable, lactic acid fermentation may be useful in generating effective nutritional supplements and functional foods.

Whole-Genome Sequence of a Serotype 1/2b Listeria monocytogenes Strain Isolated from Raw Seafood in Japan.

Listeria monocytogenes is a pathogen typically acquired through the ingestion of foods. It has been specifically reported that the pathogen is widely distributed in raw seafood in Japan. Here, we report the whole-genome sequence and sequence type (ST) of a Listeria monocytogenes strain isolated from salmon roe sold in the Japanese retail market.

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.