Gut-on-a-Chip for the Analysis of Bacteria-Bacteria Interactions in Gut Microbial Community: What Would Be Needed for Bacterial Co-Culture Study to Explore the Diet-Microbiota Relationship?

Bacterial co-culture studies using synthetic gut microbiomes have reported novel research designs to understand the underlying role of bacterial interaction in the metabolism of dietary resources and community assembly of complex microflora. Since lab-on-a-chip mimicking the gut (hereafter "gut-on-a-chip") is one of the most advanced platforms for the simulative research regarding the correlation between host health and microbiota, the co-culture of the synthetic bacterial community in gut-on-a-chip is expected to reveal the diet-microbiota relationship. This critical review analyzed recent research on bacterial co-culture with perspectives on the ecological niche of commensals, probiotics, and pathogens to categorize the experimental approaches for diet-mediated management of gut health as the compositional and/or metabolic modulation of the microbiota and the control of pathogens. Meanwhile, the aim of previous research on bacterial culture in gut-on-a-chip has been mainly limited to the maintenance of the viability of host cells. Thus, the integration of study designs established for the co-culture of synthetic gut consortia with various nutritional resources into gut-on-a-chip is expected to reveal bacterial interspecies interactions related to specific dietary patterns. This critical review suggests novel research topics for co-culturing bacterial communities in gut-on-a-chip to realize an ideal experimental platform mimicking a complex intestinal environment.

Efficacy of combined caprylic acid and thymol treatments for inactivation of Listeria monocytogenes on enoki mushrooms in household and food-service establishments.

Raw enoki mushroom is a high-risk vector for listeriosis, which led to foodborne outbreaks resulting in four deaths in the United States in 2020. This study aimed to investigate the washing method for the inactivation of L. monocytogenes in enoki mushrooms for household and food service establishments. Five methods of washing fresh agricultural products without using disinfectants were selected: (1) rinsing under running water (2 L/min, 10 min), (2-3) dipping in water (200 ml/20 g) at 22 or 40 °C for 10 min, and using (4) 10% NaCl or (5) 5% vinegar at 22 °C for 10 min. The antibacterial efficacy of each washing method along with the final rinse was tested with enoki mushrooms inoculated with a 3-strain cocktail of L. monocytogenes (ATCC 19111, 19115, 19117; ca. 6 log CFU/g). The 5% vinegar showed a significant difference in antibacterial effect compared to the other treatments except 10% NaCl (P < 0.05), with the maximum elimination of L. monocytogenes by 1.23 log CFU/g. Therefore, a disinfectant for enoki mushrooms that can complement the commonly used washing method was developed using antimicrobials (caprylic acid, CA: 0, 0.20, 0.40%; thymol, TM: 0, 0.075, 0.15%). By combined treatment of 0.40% CA and 0.15% TM at 22 °C for 10 min, L. monocytogenes was completely inactivated (>5.55 log reduction CFU/g) and did not recover after enrichment, although individual treatments of antimicrobials showed low bactericidal effects of <1.50 log reduction CFU/g. The bacterial membrane disintegration induced by the disinfectant was analyzed through flow cytometry. Additionally, the sensory scores (odor and appearance) and color parameters (L*, a*, and b*) of enoki mushrooms treated with the disinfectant were not significantly different from those of enoki mushrooms washed with water (P > 0.05). Our findings suggest a washing disinfectant consisting of low concentrations of CA and TM with synergistic antibacterial effects without quality deterioration that can ensure the safe consumption of raw enoki mushrooms in homes and food service establishments.

Hypoxia-Induced Suppression of Antiapoptotic Bcl-2 Expression in Human Bladder Tumor Cells Is Regulated by Caveolin-1-Dependent Adenosine Monophosphate-Activated Protein Kinase Activity.

PURPOSE: Adenosine monophosphate-activated protein kinase (AMPK) is thought to inhibit cell proliferation or promote cell death, but the details remain unclear. In this study, we propose that AMPK inhibits the expression of anti-apoptotic B-cell lymphoma 2 (Bcl-2) by relying on the hypoxia-inducible factor 1 alpha (HIF-1α)-induced caveolin-1 (Cav-1) expression pathway in noninvasive human bladder tumor (RT4) cells. METHODS: In cells exposed to a hypoxic environment (0.5% oxygen), the levels of expression and phospho-activity of the relevant signaling enzymes were examined via Western blots and reverse transcription-polymerase chain reaction. Cell proliferation was assessed using a Cell Counting Kit-8 assay. RESULTS: The level of expression of Cav-1 was very low or undetectable in RT4 cells. Hypoxia was associated with significantly decreased cell growth, along with marked induction of HIF-1α and Cav-1 expression; additionally, it suppressed the expression of the antiapoptotic marker Bcl-2 while leaving AMPK activity unchanged. Under hypoxic conditions, HIF-1α acts as a transcription factor for Cav-1 mRNA gene expression. The cell growth and Bcl-2 expression suppressed under hypoxia were reversed along with decreases in the induced HIF-1α and Cav-1 levels by AMPK activation with metformin (1mM) or phenformin (0.1mM). In addition, pretreatment with AMPK small interfering RNA not only increased the hypoxia-induced expression of HIF-1α and Cav-1, but also reversed the suppression of Bcl-2 expression. These results suggest that HIF-1α and Cav-1 expression in hypoxic environments is regulated by basal AMPK activity; therefore, the inhibition of Bcl-2 expression cannot be expected when AMPK activity is suppressed, even if Cav-1 expression is elevated. CONCLUSION: For the first time, we find that AMPK activation can regulate HIF-1α induction as well as HIF-1α-induced Cav1 expression, and the hypoxia-induced inhibitory effect on the antiapoptotic pathway in RT4 cells is due to Cav-1-dependent AMPK activity.

Toxic potential of Bacillus cereus isolated from fermented alcoholic beverages.

Pathogenicity of Bacillus cereus is associated with the production of various toxins. This study investigated the distribution of toxin genes encoding haemolysin BL (hblA, hblC, and hblD), nonhemolytic enterotoxin (nheA, nheB, and nheC), enterotoxin FM (entFM), cytotoxin K (cytK), and cereulide (ces) in 185 B. cereus strains isolated from draft beer, microbrewed beer, pasteurized beer, grape wine, other fruit wine, refined rice wine, traditional Korean pure liquor, and traditional Korean turbid rice wines. A total of 93.0% (172 isolates) of these isolates possessed at least one toxin gene. The nonhemolytic enterotoxin-encoding genes were highly prevalent in the isolates; the detection rate of enterotoxins was 91.4% for nheC, 81.6% for entFM, 62.7% for nheB, 57.3% for nheA, 53.0% for hblC, 48.6% for cytK, 36.8% for hblA, and 36.2% for hblD. Overall, 54.6% and 33.0% of strains carried the integrated Nhe-encoding gene cluster (nheA, nheB, nheC at the same time) and had the Hbl-encoding gene cluster, respectively. The cereulide synthetase gene was detected in only 2.2% of isolates. Toxin gene distribution patterns could be classified into 8 major profile clusters, and the most prevalent profile was the presence of enterotoxin genes only and no emetic toxin genes. Therefore, B. cereus in fermented alcoholic beverages was predominantly of the diarrhoeagenic type. Our results may provide important basic information when considering microbial standards and regulations for B. cereus in related products.

A Closer Look at Changes in High-Risk Food-Handling Behaviors and Perceptions of Primary Food Handlers at Home in South Korea across Time.

Food-handling behaviors and risk perceptions among primary food handlers were investigated by consumer surveys from different subjects in 2010 (N = 609; 1st survey will be called here "Year 2010") and 2019 (N = 605; 2nd survey will be called here "Year 2019"). Year 2010 was characterized by consumers' risk perception-behavior gap (i.e., consumers knew safe methods for food-handling, but responses regarding the behaviors did not support their confidence in food safety): they 1) did not wash/trim foods before storage, 2) thawed frozen foods at room temperature, and 3) exposed leftovers to danger zone temperatures. These trends were not improved and the gaps in Year 2010 remained in Year 2019. Year 2010 was also characterized by other common high-risk behaviors improved during 8 years for the following aspects: 1) 70.0% of consumers divided a large portion of food into smaller pieces for storage, but few consumers (12.5%) labeled divided foods with relevant information, and 2) they excessively reused kitchen utensils. Whereas in Year 2019, more consumers (25.7%) labeled food and usage periods for kitchen utensils were shortened. Consumers usually conformed to food safety rules in both Year 2010 and 2019: 1) separate storage of foods, 2) storage of foods in the proper places/periods, 3) washing fruits/vegetables before eating, 4) washing hands after handling potentially hazardous foods, and 5) cooking foods and reheating leftovers to eat. Our findings provided resources for understanding consumers' high-risk behaviors/perceptions at home, highlighting the importance of behavioral control.

Changes in Consumers' Food Purchase and Transport Behaviors over a Decade (2010 to 2019) Following Health and Convenience Food Trends.

Although consumers' food purchase/transport have been reported as causes of food safety risks, there is a lack of empirical data that are feasible to identify persistent and emerging risky behaviors of consumers. This longitudinal trend study consists of individual consumer surveys in 2010 (n = 609) and 2019 (n = 605) to analyze changes in risky behaviors linked to food purchase/transport over a decade. Overall, the results identified purchase/transport time and purchase order as the emerging and unchanged risk factors, respectively. Consumers' preferences into channels for purchase (large discount stores rather than small/traditional markets) and transport (using cars or delivery) implied the convenience as the noticeable trend. Whereas, unexpected increases in purchase/transport time highlighted the underestimated risks in long-term exposure of foods under inadequate temperature. Food should not be exposed to danger zones > 1-2 h, but consumers might be unaware of the risk especially for preferred channels (e.g., 77 and 36 min. are required for purchase and transport from large discount stores, respectively). In the case of unchanged risky behavior, more than half of consumers in both surveys did not follow proper purchasing orders. Our findings highlight the necessity for novel countermeasures and the improvement of current consumer guidelines against emerging and unchanged risky behaviors, respectively.

Author Correction: Risk factors influencing contamination of customized cosmetics made on-the-spot: Evidence from the national pilot project for public health.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Labchip-based diagnosis system for on-site application: Sensitive and easy-to-implement detection of single recoverable Cronobacter in infant formula without post-enrichment treatment.

Microfluidic labchips have achieved much advancement in the molecular diagnosis of foodborne pathogens. Whereas difficulties in the flow control during the transportation of liquid fluids can occur and should be overcome. Manipulations of reaction temperature and the complex procedures from sample pre-treatment to analysis in a single chip device are major obstacles for the on-site application. Thus, the efficient temperature control of samples without any flow of reaction fluids in microfluidic channels of plastic chip and the simplest protocol omitting post-enrichment processing steps may overcome these limitations represented by the stability and the complexity, respectively. This study aims to develop a novel type of labchip and thermocycler specialized for the gene amplification in microfluidic channels and to evaluate the detectability by sensing the minimum recoverable level of Cronobacter in powdered infant formula (PIF). We developed a thermocycling device accelerating reactions through dual heating-blocks optimized to control temperatures of samples in microfluidic-channels by direct contact with labchip sequentially and repetitively. The structural design of microfluidic channels was to eliminate interference factors associated with the optical detection of fluorescent signals (without distortion due to air bubbles in the reaction chamber). To improve the applicability, a portable device and simplified operation to allow direct loading of samples in the chip without post-enrichment procedures were also adopted. Detection performance was evaluated by a sensitivity/specificity tests using 50 isolates of Cronobacter. Cross-reactivity tests for non-Cronobacter organisms and gDNA [human, raw materials of PIF (cow, soybean)] showed that there was no interference-factor causing false-positive results. In terms of the applied research conducted by using PIF, the enrichment of samples without broth medium (distilled water) displayed outstanding performance and 12 h of incubation facilitated detecting target at concentration as low as 1 CFU/300 g PIF (as initial contamination level) without post-enrichment treatment. Validation of the operation conditions using 30 commercial PIF products was also consistent. The present study presents a novel approach of microfluidic technology with perspective to not only the performance and the practicability [easy-to-implement protocol, portable materials, cost-effectiveness (the use of a miniaturized plastic chip requires a minimum level of materials)] for on-site diagnosis.

Recent Advances in the Application of Antibacterial Complexes Using Essential Oils.

Although antibacterial spectrum of essential oils (EOs) has been analyzed along with consumers' needs on natural biocides, singular treatments generally require high concentration of EOs and long-term exposures to eliminate target bacteria. To overcome these limitations, antibacterial complex has been developed and this review analyzed previous reports regarding the combined antibacterial effects of EOs. Since unexpectable combined effects (synergism or antagonism) can be derived from the treatment of antibacterial complex, synergistic and antagonistic combinations have been identified to improve the treatment efficiency and to avoid the overestimation of bactericidal efficacy, respectively. Although antibacterial mechanism of EOs is not yet clearly revealed, mode of action regarding synergistic effects especially for the elimination of pathogens by using low quantity of EOs with short-term exposure was reported. Whereas comprehensive analysis on previous literatures for EO-based disinfectant products implies that the composition of constituents in antibacterial complexes is variable and thus analyzing the impact of constituting substances (e.g., surfactant, emulsifier) on antibacterial effects is further needed. This review provides practical information regarding advances in the EO-based combined treatment technologies and highlights the importance of following researches on the interaction of constituents in antibacterial complex to clarify the mechanisms of antibacterial synergism and/or antagonism.

Risk factors influencing contamination of customized cosmetics made on-the-spot: Evidence from the national pilot project for public health.

Customized cosmetics made by consumers or sellers on-the-spot have several safety issues, and therefore require a preventative approach to their safety management. The present study aimed to identify potential factors affecting the safety of customized cosmetics made on-the-spot. Heavy metals and microbial contaminants in customized cosmetics were analyzed in 120 samples. It was revealed that the transfer of cosmetics to new containers during the production process is a significant risk factor for cross-contamination and that heat treatment is crucial for reducing the number of microorganisms in the products. For instance, cosmetics made with heat and with no transfer showed relatively low microbial counts ranging from not detected to 440 CFU/ml. The high pH (>pH 10) of samples did not guarantee the microbial safety of the freshly made cosmetics (with a rinse-off product having 2,830 CFU/ml and a pH of 11.2). There was no significant difference in microbial counts among cosmetic types (P > 0.05); however, semisolid types, especially creams and rinse-off products, were susceptible to contamination (maximum 2,710 and 2,830 CFU/ml, respectively). Most microorganisms in the customized cosmetics (40.8%) decreased to non-detectable levels during 60 days of storage. None of the samples harbored heavy metals. Sequencing analysis of isolates revealed some bacteria and mold that could cause human infections. The results of this study suggest that the regulation of customized cosmetics should consider the risk factors revealed in this study, as the products made on-the-spot are also final products sent directly to consumers.

Clinical study on concurrent use of electro-acupuncture or Chuna manual therapy with pregabalin for chemotherapy-induced peripheral neuropathy: safety and effectiveness (open-labeled, parallel, randomized controlled trial, assessor-blinded): A study protocol.

INTRODUCTION: Chemotherapy-induced peripheral neuropathy (CIPN) is one of the major side effects of chemotherapy. Its main symptoms are pain, paresthesia, and numbness. However, the mechanisms underlying the development of CIPN remain unclear and standard treatments have not been established. Recently, there has been a growing interest in various approaches to overcome the limitations of the existing treatments. This study aims to evaluate the efficacy and safety of the concurrent use of two complementary and alternative therapies: electroacupuncture (EA) and Chuna manual therapy (CMT), with pregabalin, which is the conventional pharmacotherapy for neuropathic pain. METHODS/DESIGN: This is an open-label, parallel, assessor-blinded randomized controlled trial, which includes 90 patients with colorectal and breast cancer, who developed CIPN. After a 2-week preparation period, the patients are divided into three groups (pregabalin administration group, pregabalin + EA treatment group, and pregabalin + CMT treatment group), treated for approximately 5 weeks and followed-up 4 weeks after treatment. The primary outcome is assessed using the Functional Assessment of Cancer Therapy/Gynecologic Oncology Group Neurotoxicity subscale score (version 4.0) and the secondary outcome is measured using the Quality of Life Questionnaire-CIPN 20-Item Scale (version 3.0) and the quality of life questionnaire (version 3.0) developed by the European Organisation for Research and Treatment of Cancer. Moreover, exploratory efficacy and safety evaluations will be conducted based on the chemotherapy-completion rate and nerve conduction studies.

Health Functionality and Quality Control of Laver (Porphyra, Pyropia): Current Issues and Future Perspectives as an Edible Seaweed.

The growing interest in laver as a food product and as a source of substances beneficial to health has led to global consumer demand for laver produced in a limited area of northeastern Asia. Here we review research into the benefits of laver consumption and discuss future perspectives on the improvement of laver product quality. Variation in nutritional/functional values among product types (raw and processed (dried, roasted, or seasoned) laver) makes product-specific nutritional analysis a prerequisite for accurate prediction of health benefits. The effects of drying, roasting, and seasoning on the contents of both beneficial and harmful substances highlight the importance of managing laver processing conditions. Most research into health benefits has focused on substances present at high concentrations in laver (porphyran, Vitamin B12, taurine), with assessment of the expected effects of laver consumption. Mitigation of chemical/microbiological risks and the adoption of novel technologies to exploit under-reported biochemical characteristics of lavers are suggested as key strategies for the further improvement of laver product quality. Comprehensive analysis of the literature regarding laver as a food product and as a source of biomedical compounds highlights the possibilities and challenges for application of laver products.

Underestimated Risks of Infantile Infectious Disease from the Caregiver's Typical Handling Practices of Infant Formula.

The impact on infant caregiver as a reservoir of pathogens has not been exploited with perspective to powdered infant formula (PIF). Here we reveal novel route of pathogen transfer through hand-spoon-PIF unexpectedly occurred by even typical practices of caregivers, handling of PIF and storage of feeding-spoon in PIF container. Hand-spoon-PIF contamination route was simulated to analyze the transfer and subsequent survival of pathogens. Major pathogens associated with infantile fatal diseases (Cronobacter sakazakii, Salmonella enterica, Staphylococcus aureus) were readily transmitted to PIF from skin (3-6 log CFU/hand) via spoons following long-term survival of transferred pathogens (3 weeks; use-by date of PIF) as the excessive level of infectious dose, highlighting direct onset of diseases. Low bacterial load on skin (ca. 1 log CFU/hand) could prevent cross-contamination of PIF, however, at least 72 h survival of transferred pathogen on spoons demonstrated the probability on re-contamination of PIF. To our knowledge, this is the first study to investigate the cross-contamination of utensils in contact with powdered-foods. Bacterial load on hands is the key determinant of pathogen transfer and the extent of risk are species-dependent. These evidential results redefine risk of caregivers' practices and facilitate incorporation of cross-contamination into risk-assessment as underestimated route of infection.

AMPK Alters Detrusor Contractility During Emptying in Normal Bladder and Hypertrophied Bladder with Partial Bladder Outlet Obstruction via CaMKKß.

AMP-activated protein kinase (AMPK) has been implicated in contractility changes in bladders with partial bladder outlet obstruction (PBOO), but the role of AMPK in the contractile response of normal bladder remains unclear. We investigated the phosphorylation of AMPKα and expression of the involved upstream AMPK kinases (AMPKKs) in a model of bladders with PBOO and sought to determine whether the pharmacological inhibition of these two factors affected detrusor contractility in normal bladders, using female Sprague-Dawley rats. Cystometry and Western blot analysis were performed in rats that were subjected to PBOO induction or a sham operation. Cystometry was performed in normal rats that received selective inhibitors of AMPKα and Ca2+/calmodulin-dependent protein kinase kinase (CaMKKß) (compound C and STO-609, respectively) at doses determined in the experiments. In the PBOO bladders, bladder weight and micturition pressure (MP) were higher and AMPKα phosphorylation (T172) and CaMKKß expression was significantly reduced. Compound C and STO-609 increased MP. The increased contractile response in bladders with PBOO-induced hypertrophy was related to decreased CaMKKß/AMPK signaling activity, and the pharmacological inhibition of this pathway in normal bladders increased detrusor contractility, implying a role of CaMKKß/AMPK signaling in the bladder in the regulation of detrusor contractility.

Whole-Genome Sequences of Five Geobacillus stearothermophilus Strains Isolated from Processing Lines of Powdered Infant Formula.

Geobacillus stearothermophilus is the thermophile present in processing lines of powdered infant formula (PIF). We report the whole-genome sequences of G. stearothermophilus strains isolated from work-in-process products (sterilized and concentrated milk) of manufacturing plants. Understanding the genomic basis governing the metabolism of G. stearothermophilus can contribute to the safety management of PIF during its manufacture.

Underrecognized niche of spore-forming bacilli as a nitrite-producer isolated from the processing lines and end-products of powdered infant formula.

Although nitrite in powdered milk formula (PIF) is a recognized health risk for infants, the presence of nitrite in PIF has only been investigated as a chemical contaminant during the inspection of end-products. The risk posed by microbial sources of nitrite during the PIF manufacturing process has not been considered. This is the first study to report the taxonomy and physiological characteristics of nitrite-producing bacteria isolated from PIF processing environments. All isolates identified as nitrite-producers (133 out of 501 strains collected over four years) from work-in-process and end-products of PIF were spore-forming bacilli. Nitrite-producing metabolism under PIF processing conditions was found in not only thermophilic isolates (3 Bacillus, 60 Geobacillus from 63 strains; 100%) but also in mesophilic isolates (65 Bacillus, 1 Anoxybacillus from 70 strains; 65.7%). Geobacillus was the only highly heat-resistant sporeformer and vigorous nitrite-producer exhibiting dramatic increases in nitrite over short periods of incubation (a maximum value within 3 h). High conversions of nitrate to nitrite (up to 88.8%) was also observed, highlighting bacteria as a key source of nitrite in PIF processing lines. Further research into the diversity of metabolic activity observed in this study can facilitate specialized management of nitrite-producers in PIF processing lines.

Prevalence of pathogenic Arcobacter species in South Korea: Comparison of two protocols for isolating the bacteria from foods and examination of nine putative virulence genes.

Contamination of foodstuffs by potentially enteropathogenic Arcobacter spp. is becoming a concern worldwide. However, few studies have examined virulence-associated genes in isolates of Arcobacter spp. from food. Here, we investigated the prevalence of three pathogenic Arcobacter species, A. butzleri, A. cryaerophilus, and A. skirrowii, in chicken, pork, and leafy green vegetables (n = 323) in South Korea. Samples were examined using two different protocols selected from a literature review: Acrobacter selective broth (ASB) II + Arcobacter selective medium (ASM) II (protocol A), and ASB II + modified charcoal cefoperazone deoxycholate agar supplemented with CAT (protocol B). Overall, Arcobacter spp. were detected in 45.8% of food samples, and the recovery rate of protocol B (37.8%) was significantly higher than that of protocol A (30.7%) (p < 0.05). Refrigerated chicken gizzard samples showed the highest detection rate (100%), followed by refrigerated chicken wing (79.5%), intestine (77.3%), neck skin (63.3%), pork (55.6%), frozen chicken legs (5.0%), and leafy green vegetables (4.4%) (p < 0.05). All isolates from chicken and leafy green vegetables were identified as A. butzleri, whereas A. cryaerophilus and A. skirrowii were mainly detected in pork. Most samples (95.8%) harbored more than one of nine putative virulence factors (cadF, ciaB, cj1349, hecA, hecB, mviN, pldA, irgA, and tlyA), and 91.3% harbored more than two. Isolates harboring all nine putative virulence genes were obtained from 1.9% of samples: five pork and one chicken. This study provides comprehensive and de facto evidence regarding prevalence of an emerging pathogen, Arcobacter spp., in various foods, along with their virulence potential. The results justify further research with respect to their role in food safety.

Microbiological criteria and ecology of commercially available processed cheeses according to the product specification and physicochemical characteristics.

Although global cheese manufacturers release a variety of products onto the market, research on the microbiological quality and safety of cheese has focused mainly on conventional cheeses made from milk. Here, this study aimed to investigate commercially processed cheese products produced by mixing conventional cheeses after melting. Two approaches were used: a summary and comparison of legal definitions and standards/regulations regarding the microbiological criteria used by major cheese traders in the global market (Australia/New Zealand, China, European Union, Japan, Mexico, Republic of Korea, and the United States) and a comprehensive microbiological analysis of commercial products (n = 800), along with an assessment of salinity, pH, water activity, and heating conditions. The results of the literature search showed that major importing countries (China, Japan, Mexico, and the Republic of Korea) have stricter microbiological criteria for commercially available cheese products than major exporters (Australia/New Zealand, EU, and the USA). The former set limits with respect to the number of total coliforms in the product. Microbiological analyses were designed according to global standards and recommendations. No test sample contained detectable levels of Clostridium perfringens, enterohemorrhagic Escherichia coli, Listeria monocytogenes, Salmonella, or Staphylococcus aureus. In addition, no coliform bacteria (including E. coli) were detected. Overall, 79.9% of the samples contained detectable aerobic plate counts (1.0-7.8 log CFU/g); these levels varied significantly according to product type (grated cheese > chunks; cream cheese > portions or sliced) (p < .05). There was no significant association between microbe levels and salinity, water activity, pH, and heating conditions. The results can be used to develop a comprehensive database about commercially processed cheese products available in the global market and, as such, may be helpful for both national authorities and cheese manufacturers when considering novel strategic management plans for microbiological quality and safety.

Citrus fruit extracts with carvacrol and thymol eliminated 7-log acid-adapted Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes: A potential of effective natural antibacterial agents.

Despite the widespread belief that citrus fruit extracts (CFEs) are microbiologically safe due to their acidity, limited bactericidal effect results in low applicability as antibacterial agent and outbreaks occurred by acid-adapted pathogens. Here, we examined the antibacterial effects of CFEs [lime (Citrus medica), lemon (Citrus limon), calamansi (Citrus microcarpa)] combined with essential oil components (EOCs; carvacrol and thymol) against non-acid-adapted/acid-adapted Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes under 22 °C for 5 min. CFEs (<20%) alone or small amounts of EOCs (2.0 mM; 0.032%) alone could not inactivate the target bacteria effectively. However, combined treatments exhibited marked synergy: CFE + EOCs eliminated all the bacteria (>6.9 log CFU/ml). Among the CFEs tested, the highest synergism was shown by calamansi, an exotic citrus fruit previously unrecognized as an antibacterial agent. Although acid-adaptation improved bacterial survival, calamansi (<20%) + EOCs (<0.032%) completely inactivated even the most resistant pathogen (E. coli O157:H7). Validation test also showed that all tested commercial juice products also eliminated acid-adapted pathogens when used with EOCs. Physicochemical analysis of tested CFEs (pH measurement and HPLC analysis of components) revealed that low pH and flavanone (hesperidin) did not contribute to the synergistic bactericidal effects. Rather, the high citric acid content is likely to contribute to the strong synergistic effect with EOCs by damaging susceptible bacterial membranes. Sensory scores for CFEs were not altered by addition of EOCs at concentrations up to 1.5 mM. This study provides new insight into the utility of CFEs with EOCs to improve not only the microbiological safety of food products containing CFEs but also their applicability as natural antibacterial complex.