Synergistic effects of ε-poly-l-lysine and lysozyme against Pseudomonas aeruginosa and Listeria monocytogenes biofilms on beef and food contact surfaces.

This study investigated the synergistic effects of ε-poly- L -lysine (ε-PL) and lysozyme against P. aeruginosa and L. monocytogenes biofilms. Single-culture biofilms of two bacteria were formed on silicone rubber (SR), stainless steel (SS), and beef surfaces and then treated with lysozyme (0.05-5 mg/mL) and ε-PL at minimum inhibitory concentrations (MICs) of 1 to 4 separately or in combination. On the SR surface, P. aeruginosa biofilm was reduced by 1.4 and 1.9 log CFU/cm2 within 2 h when treated with lysozyme (5 mg/mL) and ε-PL (4 MIC), respectively, but this reduction increased significantly to 4.1 log CFU/cm2 (P < 0.05) with the combined treatment. On beef surface, P. aeruginosa and L. monocytogenes biofilm was reduced by 4.2-5.0, and 3.3-4.2 log CFU/g when lysozyme was combined with 1, 2, and 4 MIC of ε-PL at 25 °C, respectively. Compared to 5 mg/mL lysozyme alone, the combined treatment with 1, 2, and 4 MIC of ε-PL on beef surface achieved additional reduction against P. aeruginosa biofilm of 0.5, 0.8, and 0.7 log CFU/g, respectively, at 25 °C. In addition, 0.25 mg/mL lysozyme and 0.5 MIC of ε-PL significantly (P < 0.05) suppressed the quorum-sensing (agrA) and virulence-associated (hlyA and prfA) genes of L. monocytogenes.

Synergistic effects of sequential treatment using disinfectant and e-beam for inactivation of hepatitis a virus on fresh vegetables.

Hepatitis A virus (HAV) has adversely affected public health worldwide, causing an economic burden on many countries. Fresh vegetables are reported as a source of HAV infections during production, harvesting, and distribution, which cause the emergence of foodborne illnesses. Therefore, in this study, the synergistic effects of chemical (sodium hypochlorite [NaOCl] and chlorine dioxide [ClO2]) and physical (electron-beam [e-beam] irradiation) sequential treatment for HAV inactivation on fresh vegetables were investigated, and the physicochemical quality changes of vegetables were evaluated after each treatment. On bell pepper and cucumber sequentially treated with NaOCl (50-500 ppm) and e-beam (1-5 kGy), the HAV titer was reduced by 0.19-4.69 and 0.28-4.78 log10 TCID50/mL, respectively. Sequential treatment with ClO2 (10-250 ppm) and e-beam (1-5 kGy) reduced the HAV titer on bell pepper and cucumber by 0.41-4.78 and 0.26-4.80 log10 TCID50/mL, respectively. The sequential treatments steadily decreased the HAV titers on each food by a significant difference (p < 0.05) compared to the controls. The treatment combinations of 500 ppm NaOCl and 3 kGy (e-beam) on bell pepper and 150 ppm NaOCl and 1 kGy (e-beam) on cucumber provided maximum synergistic effects. It was also found that sequential treatment with 50 ppm ClO2 and 5 kGy (e-beam) on bell pepper and 10 ppm ClO2 and 5 kGy (e-beam) on cucumber most efficiently inactivated HAV. Additionally, bell pepper and cucumber showed no significant quality changes (p < 0.05) after the treatment. Therefore, the sequential treatment with NaOCl or ClO2 and e-beam is expected to effectively control HAV on fresh vegetables without changing the food quality compared to either treatment alone.

Development and efficacy assessment of hand sanitizers and polylactic acid films incorporating caffeic acid and vanillin for enhanced antiviral properties against HCoV-229E.

BACKGROUND: Although three years after the outbreak of SARS-CoV-2, the virus is still having a significant impact on human health and the global economy. Infection through respiratory droplets is the main transmission route, but the transmission of the virus by surface contact cannot be ignored. Hand sanitizers and antiviral films can be applied to control SARS-CoV-2, but sanitizers and films show drawbacks such as resistance of the virus against ethanol and environmental problems including the overuse of plastics. Therefore, this study suggested applying natural substrates to hand sanitizers and antiviral films made of biodegradable plastic (PLA). This approach is expected to provide advantages for the easy control of SARS-CoV-2 through the application of natural substances. METHODS: Antiviral disinfectants and films were manufactured by adding caffeic acid and vanillin to ethanol, isopropyl alcohol, benzalkonium chloride, and PLA. Antiviral efficacies were evaluated with slightly modified international standard testing methods EN 14,476 and ISO 21,702. RESULTS: In suspension, all the hand sanitizers evaluated in this study showed a reduction of more than 4 log within 2 min against HCoV-229E. After natural substances were added to the hand sanitizers, the time needed to reach the detection limit of the viral titer was shortened both in suspension and porcine skin. However, no difference in the time needed to reach the detection limit of the viral titer was observed in benzalkonium chloride. In the case of antiviral films, those made using both PLA and natural substances showed a 1 log reduction of HCoV-229E compared to the neat PLA film for all treatment groups. Furthermore, the influence of the organic load was evaluated according to the number of contacts of the antiviral products with porcine skin. Ten rubs on the skin resulted in slightly higher antiviral activity than 50 rubs. CONCLUSION: This study revealed that caffeic acid and vanillin can be effectively used to control HCoV-229E for hand sanitizers and antiviral films. In addition, it is recommended to remove organic matter from the skin for maintaining the antiviral activity of hand sanitizer and antiviral film as the antiviral activity decreased as the organic load increased in this study.

Innovative nonthermal technologies for inactivation of emerging foodborne viruses.

Various foodborne viruses have been associated with human health during the last decade, causing gastroenteritis and a huge economic burden worldwide. Furthermore, the emergence of new variants of infectious viruses is growing continuously. Inactivation of foodborne viruses in the food industry is a formidable task because although viruses cannot grow in foods, they can survive in the food matrix during food processing and storage environments. Conventional inactivation methods pose various drawbacks, necessitating more effective and environmentally friendly techniques for controlling foodborne viruses during food production and processing. Various inactivation approaches for controlling foodborne viruses have been attempted in the food industry. However, some traditionally used techniques, such as disinfectant-based or heat treatment, are not always efficient. Nonthermal techniques are considered a new platform for effective and safe treatment to inactivate foodborne viruses. This review focuses on foodborne viruses commonly associated with human gastroenteritis, including newly emerged viruses, such as sapovirus and Aichi virus. It also investigates the use of chemical and nonthermal physical treatments as effective technologies to inactivate foodborne viruses.

Survival of human coronavirus 229E at different temperatures on various food-contact surfaces and food and under simulated digestive conditions.

The COVID-19 pandemic caused by SARS-CoV-2 has had a major impact on human health and the global economy. Various transmission possibilities of SARS-CoV-2 have been proposed, such as the surface of food in the cold chain and food packaging, as well as the fecal-oral route, although person-to-person contact via droplets and aerosols has been confirmed as the main route of transmission. This study evaluated the survivability of HCoV-229E, a SARS-CoV-2 surrogate, in suspension, on food-contact surfaces and on food at various temperatures, and in simulated digestive fluids by TCID50 assay. In suspension, HCoV-229E survived after 5 days at 20 °C with a 3.69 log reduction, after 28 days at 4 °C with a 3.07 log reduction, and after 12 weeks at -20 °C with a 1.18 log reduction. On food-contact surfaces, HCoV-229E was not detected on day 3 on stainless steel (SS), plastic (LDPE), and silicone rubber (SR) at 20 °C with a 3.28, 3.24 and 3.28 log reduction, respectively, and survived after 28 days on SS and LDPE at 4 °C with a 3.13 and 2.88 log reduction, respectively, and survived after 12 weeks on SS, LDPE, and SR at -20 °C with a 1.92, 1.32 and 1.99 log reduction, respectively. On food, HCoV-229E was not detected on day 3 on lettuce and day 4 on chicken breast and salmon at 20 °C with a 3.61, 3.26 and 3.08 log reduction, respectively, and on day 14 on lettuce and day 21 on chicken breast and salmon at 4 °C with a 3.88, 3.44 and 3.56 log reduction, respectively. The virus remained viable for 12 weeks in all foods at -20 °C with 2-2.47 log reduction. In addition, in simulated digestive fluid experiments, HCoV-229E was relatively resistant in simulated salivary fluid (SSF; pH 7, 5), fed state simulated gastric fluid (FeSSGF; pH 3, 5, 7), and fasted state simulated intestinal fluid (FaSSIF; pH 7). However, the virus was less tolerant in fasted state simulated gastric fluid (FaSSGF; pH 1.6) and fed state simulated intestinal fluid (FeSSIF; pH 5). Therefore, this study suggested that HCoV-229E remained infectious on various food-contact surfaces and foods; in particular, it survived longer at lower temperatures and survived depending on the pH of the simulated digestive fluid.

Inactivation methods for human coronavirus 229E on various food-contact surfaces and foods.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of the COVID-19 outbreaks, is transmitted by respiratory droplets and has become a life-threatening viral pandemic worldwide. The aim of this study was to evaluate the effects of different chemical (chlorine dioxide [ClO2] and peroxyacetic acid [PAA]) and physical (ultraviolet [UV]-C irradiation) inactivation methods on various food-contact surfaces (stainless steel [SS] and polypropylene [PP]) and foods (lettuce, chicken breast, and salmon) contaminated with human coronavirus 229E (HCoV-229E). Treatments with the maximum concentration of ClO2 (500 ppm) and PAA (200 ppm) for 5 min achieved >99.9% inactivation on SS and PP. At 200 ppm ClO2 for 1 min on lettuce, chicken breast, and salmon, the HCoV-229E titers were 1.19, 3.54, and 3.97 log10 TCID50/mL, respectively. Exposure (5 min) to 80 ppm PAA achieved 1.68 log10 reduction on lettuce, and 2.03 and 1.43 log10 reductions on chicken breast and salmon, respectively, treated with 1500 ppm PAA. In the carrier tests, HCoV-229E titers on food-contact surfaces were significantly decreased (p < 0.05) with increased doses of UV-C (0-60 mJ/cm2) and not detected at the maximum UV-C dose (Detection limit: 1.0 log10 TCID50/coupon). The UV-C dose of 900 mJ/cm2 proved more effective on chicken breast (>2 log10 reduction) than on lettuce and salmon (>1 log10 reduction). However, there were no quality changes (p > 0.05) in food samples after inactivation treatments except the maximum PAA concentration (5 min) and the UV-C dose (1800 mJ/cm2).

Growth characterization of Propionibacterium and propionic acid production capabilities at different temperatures and pH levels.

Bacteria from the Propionibacterium genus were cocktailed to investigate growth and production of propionic acid at different temperatures and pH levels. A gas chromatograph with a flame ionization detector was also used for instrumental analysis. The Propionibacterium cocktails did not produce propionic acid at 10 and 20 °C for 10 days, but produced propionic acid at concentrations of 3265.32, 3670.76, and 1926.04 µg/mL at 25, 30, and 40 °C for 18 days, respectively. In pH tests, the cocktails did not produce propionic acid at pH 3 and 9 for 14 and 7 days, respectively. However, they produced propionic acid at concentrations of 2596.66, 2952.66, 3321.35, and 3586.95 µg/mL at pH 4, 5, 6, and 7 for 18 days, respectively. Growth characteristics of Propionibacterium cocktails by temperature and pH were set so that they reached the extinction stage after four days in the logarithmic phase.

Efficacy of ficin and peroxyacetic acid against Salmonella enterica serovar Thompson biofilm on plastic, eggshell, and chicken skin.

Salmonella is the leading cause of zoonotic foodborne illnesses worldwide and a prevalent threat to the poultry industry. For controlling contamination, the use of chemical sanitizers in combination with biological compounds (e.g., enzymes) offers a solution to reduce the chemical residues. The current study investigated the biofilm reduction effects of a food-grade enzyme-ficin-and a common sanitizer-peroxyacetic acid (PAA)-against an emerging pathogen, Salmonella enterica ser. Thompson, on plastic, eggshell, and chicken skin surfaces. Results showed that PAA could kill S. Thompson, but ficin cannot. Maximum biofilm reduction was 3.7 log CFU/cm2 from plastic after individual treatment with PAA. However, sequential treatment of ficin and PAA led to biofilm reductions of 3.2, 5.0, and 6.5 log CFU/cm2 from chicken skin, eggshell, and plastic, respectively. Fourier-transform infrared spectroscopy and microscopic analysis confirmed that ficin increased PAA action, causing biofilm matrix destruction. Moreover, the quality of the food surfaces was only altered by 12.5 U/mL ficin and was not altered by PAA. This combined use of enzyme and sanitizer solved major safety issues and proved promising against S. Thompson-associated contaminations in poultry and poultry processing lines.

Impact of chlorine dioxide and electron-beam irradiation for the reduction of murine norovirus in low-salted "jogaejeotgal", a traditional Korean salted and fermented clam.

Clam jeotgal, called "jogaejeotgal," is a Korean fermented seafood product with, generally, a high amount of added salt to inhibit the growth of pathogenic microorganisms. This study aimed to evaluate the efficacy of chlorine dioxide (ClO2) and sodium hypochlorite (NaOCl) against murine norovirus 1 (MNV-1), a surrogate for human norovirus, in salt-fermented clam, jogaejeotgal. The sequential effect of ClO2 and electron-beam (e-beam) irradiation on the inactivation of MNV-1 was also investigated. Treatments of up to 300 ppm ClO2 and 1000 ppm NaOCl were used to determine the disinfectant concentrations at which more than 1 log (90%) MNV-1 inactivation occurred. The sequential treatment of ClO2 (50-300 ppm) and e-beam (1-5.5 kGy) was performed after storage at 4 °C for 7 days. There was a 1.9-log reduction of the virus in seasoned clams irradiated at 5.5 kGy after ClO2 treatment at 300 ppm. No significant change (p > 0.05) in physicochemical quality was observed after the combined treatment, suggesting the potential for the use of a combined treatment using ClO2 (300 ppm) and e-beam (5.5 kGy) in the jeotgal manufacturing industry for the reduction of norovirus.

COVID-19 pandemic crisis and food safety: Implications and inactivation strategies.

BACKGROUND: The COVID-19 pandemic that emerged in 2019 has imposed huge consequences, including economic losses and threats to human health, which are still affecting many aspects throughout the world. SCOPE AND APPROACH: This review provides an overview of SARS-CoV-2 infection, the cause of COVID-19, and explores its impact on the food supply system and food safety. This review examines the potential risk of transmission through food and environmental surfaces before discussing an effective inactivation strategy to control the COVID-19 pandemic in the aspect of food safety. This article also suggests effective food safety management post-COVID-19. KEY FINDINGS AND CONCLUSIONS: Respiratory viruses including SARS-CoV-2 are responsible for huge impacts on the global economy and human health. Although food and water are not currently considered priority transmission routes of SARS-CoV-2, infection through contaminated food and environmental surfaces where the virus can persist for several days cannot be ignored, particularly when the surrounding environment is unhygienic. This approach could help determine the exact transmission route of SARS-CoV-2 and prepare for the post-COVID-19 era in the food safety sector.

Genotypic Classification of Staphylococcus aureus and Bacillus cereus from Korean Slaughterhouses Using Semiautomated Repetitive Sequence-Based Polymerase Chain Reaction.

A repetitive sequence-based polymerase chain reaction (rep-PCR) technique utilizing a semiautomated system, namely DiversiLab, was applied to determine the genotypes of Staphylococcus aureus and Bacillus cereus obtained from slaughterhouses. Twenty-four S. aureus and 16 B. cereus isolates from pigs and Hanwoo cattle from three slaughterhouses were used to create a DNA fingerprint library with the system software. Scatterplots demonstrated that rep-PCR groupings of S. aureus isolates were in good agreement with their origins. Specifically, linked rep-PCR profiles were observed for S. aureus isolates recovered from the same slaughterhouse, and higher genetic similarities were found among strains isolated from adjacent regions. All S. aureus isolates except one (ID: A-Hanwoo-9) from slaughterhouse "A" clustered with the three S. aureus reference strains, Korea Culture Center of Microorganisms (KCCM) 41291, KCCM 12214, and Culture Collection of Antimicrobial Resistant Microbes (CCARM) 3A007 (similarity values >95%). Moreover, most isolates obtained from slaughterhouse "B" clustered with S. aureus KCCM 11335 and KCCM 41331, and two isolates from slaughterhouse "C" clustered with CCARM 0027. Therefore, for this species, genotypic characteristics of regional isolates can be used to track the pathway of contamination. In contrast, B. cereus isolates showed high genetic diversity and could not be clustered with any specific group. Collectively, this system is useful for analyzing genetic diversity and is a rapid and reproducible typing method; however, there is a need to develop rep-PCR libraries for its use as a rapid identification method.

Development of enhanced primer sets for detection of norovirus.

Norovirus (NV) is a major viral pathogen that causes nonbacterial acute gastroenteritis and outbreaks of food-borne disease. The genotype of NV most frequently responsible for NV outbreaks is GII.4, which accounts for 60-80% of cases. Moreover, original and new NV variant types have been continuously emerging, and their emergence is related to the recent global increase in NV infection. In this study, we developed advanced primer sets (NKI-F/R/F2, NKII-F/R/R2) for the detection of NV, including the variant types. The new primer sets were compared with conventional primer sets (GI-F1/R1/F2, SRI-1/2/3, GII-F1/R1/F2, and SRII-1/2/3) to evaluate their efficiency when using clinical and environmental samples. Using reverse transcription polymerase chain reaction (RT-PCR) and seminested PCR, NV GI and GII were detected in 91.7% (NKI-F/R/F2), 89.3% (NKII-F/R/R2), 54.2% (GI-F1/R1/F2), 52.5% (GII-F1/R1/F2), 25.0% (SRI-1/2/3), and 32.2% (SRII-1/2/3) of clinical and environmental specimens. Therefore, our primer sets perform better than conventional primer sets in the detection of emerged types of NV and could be used in the future for epidemiological diagnosis of infection with the virus.

Evaluation of antimicrobial effects of commercial mouthwashes utilized in South Korea.

Streptococcus mutans is frequently associated with dental caries. Bacterial fermentation of food debris generates an acidic environment on the tooth surface, ultimately resulting in tooth deterioration. Therefore, various mouthwashes have been used to reduce and prevent Streptococcus mutans. The aim of this study was to evaluate the antimicrobial activities of 4 commercial mouthwashes and those of 10% and 20% ethanol solutions (formula A, B, C, D, E and F) against Streptococcus mutans using biofilm and planktonic methods. The range of reduction in the viable cell count of Streptococcus mutans as estimated by the biofilm and planktonic methods was 0.05-5.51 log (P ≤ 0.01) and 1.23-7.51 log (P ≤ 0.001) compared with the negative control, respectively, indicating that the planktonic method had a stronger antibacterial effect against S. mutans. Among the tested formulations, formula A (Garglin regular® mouthwash) was the most effective against Streptococcus mutans (P ≤ 0.001).

Genetic analysis of the capsid region of norovirus GII.4 variants isolated in South Korea.

Norovirus is one of the major causes of non-bacterial gastroenteritis in humans. The aim of this study was to analyze the amino acid variation of open reading frame 2 of GII.4 variants in South Korea during the period from November 2006 to December 2012. Sixty-nine complete nucleotide sequences of open reading frame 2 were obtained from 113 GII.4 strains. The GII.4 2006b variants were detected predominantly between 2006 and 2009; however, new GII.4 variants, which were termed the 2010 variant and the 2012 variant, emerged in 2010 and 2012, respectively. The number of GII.4 2006b variants steadily decreased until 2012, whereas the number of gastroenteritis cases caused by the new variants increased between 2010 and 2012. The amino acid sequence in the ORF2 region obtained in this study was compared with other GII.4 variants isolated in various countries. Amino acid variations were observed primarily at epitope sites and the surrounding regions. Amino acids 294, 359, 393, and 413 of the P2 subdomain were the most variable sites among the GII.4 variants. The information in this study can be useful in basic research to predict the emergence and determine the genetic functions of new GII.4 variants.

Full-genomic analysis of a human norovirus recombinant GII.12/13 novel strain isolated from South Korea.

Norovirus (NoV) genogroups I and II are frequently recognized as the main causes of acute gastroenteritis and outbreaks of non-bacterial foodborne diseases. Furthermore, variants and recombinant strains of this virus are continuously emerging worldwide. The aim of this study was to identify NoV strains and to investigate and characterize rare genotypes. Stool samples (n = 500) were collected from patients with symptoms of acute gastroenteritis in Korea between December 2004 and November 2007. For analysis of the samples, rapid genotype screening was performed using reverse transcriptase-polymerase chain reaction. Full sequencing, using a newly designed set of 12 primers, revealed GII-12/13 strain. The partial sequence of GII-12/13 strain was compared with published NoV (GII-1 - 14) sequences targeting RdRp and capsid regions using phylogenetic analysis with the SimPlot program, which could evaluate recombination breakpoints. SimPlot analysis was also performed with the strain GII-12/Gifu-96/JPN (AB045603) for the RdRp region and with GII-13/G5175B-83/AUS(DQ379714) for the capsid region. NoV was detected in 19 of the 500 stool samples (3.8%). Genogroup GII-4 was found most frequently (n = 9, 1.8%), followed by GII-3 (n = 4, 0.8%), GII-6 (n = 3, 0.6%), GI-6 (n = 2, 0.4%), and GII-12/13 (n = 1, 0.2%). Importantly, we identified a novel NoV recombinant strain, C9-439 (KF289337), indicating potential risks, which suggested that, recombination occurred in the region between open reading frames 1 and 2 of the GII-12/13 strain and that breakpoints occurred in the polymerase region.