Inactivation of Tulane virus and feline calicivirus by aqueous ozone.

Ongoing challenges with reproducible human norovirus cultivable assays necessitate the use of surrogates, such as feline calicivirus (FCV-F9) and Tulane virus (TV), during inactivation studies. Chlorine alternates used as control strategies include aqueous and gaseous ozone. This study aimed at determining the inactivation of FCV-F9 and TV by a portable ozone-generating device. FCV-F9 (∼8 log PFU/mL) or TV (∼6 log PFU/mL) in sterile-low-organic matter-containing-water was treated for 0-5 min, or in sterile-water containing newborn calf serum (high-organic matter/protein) for 0-38 min with ∼1 ppm ozone (pH 7-6). Infectivity was determined from triplicate treatments using plaque assays. FCV-F9 titers significantly decreased by 6.07 log PFU/mL after 5 min in ozonated low-organic-matter-containing-water and was non-detectable (≤2 log PFU/mL) after 36 min treatments in high-organic-matter-containing water (p < 0.05). TV titers decreased by 4.18 log PFU/mL after 4 min in ozonated low-organic-matter water (non-detectable after 4.5 min) and were non-detectable after 22.5 min treatments of high-organic-matter-containing water (p < 0.05). Overall, ∼1 ppm aqueous ozone significantly decreased FCV-F9 by >6 log PFU/mL after 5 min, TV to non-detectable levels (≤2 log PFU/mL) after 4.5 min and required longer treatments (>32 and >20 min, respectively) for ≥4 log reduction in high-organic-matter-containing water (p < 0.05). For ozone treatment of both viruses, the linear and Weibull models were similar for low-organic-load water, though the Weibull model was better for the high-organic load water. Prior filtration or organic load removal is recommended before ozonation for increased viral inactivation with decreased treatment-time.

Assessment of antimicrobial activity of melittin encapsulated in bicontinuous microemulsions prepared using renewable oils.

The objective of this study is to demonstrate that melittin, a well-studied antimicrobial peptide (AMP), can be solubilized in an active form in bicontinuous microemulsions (BMEs) that employ biocompatible oils. The systems investigated consisted of Winsor-III and -IV BME phases composed of Water/Aerosol-OT (AOT)/Polysorbate 85/isopropyl myristate and a Winsor-IV BME employing Polysorbate 80 and limonene. We found that melittin resided in an α-helix-rich configuration and was in an apolar environment for the AOT/Polysorbate 85 Winsor-III system, suggesting that melittin interacted with the surfactant monolayer and was in an active conformation. An apolar environment was also detected for melittin in the two Winsor-IV systems, but to a lesser extent than the Winsor-III system. Small-angle X-ray scattering analysis indicated that melittin at a concentration of 1.0 g/Laq in the aqueous subphase of the Winsor-IV systems led to the greatest impact on the BME structure (e.g., decrease of quasi-periodic repeat distance and correlation length and induction of interfacial fluidity). The antimicrobial activity of the Polysorbate 80 Winsor-IV system was evaluated against several bacteria prominent in chronic wounds and surgical site infections (SSIs). Melittin-free BMEs inhibited the growth of all tested bacteria due to its oil, limonene, while the inclusion of 1.0 g/Laq of melittin in the BMEs enhanced the activity against several bacteria. A further increase of melittin concentration in the BMEs had no further enhancement. These results demonstrate the potential utility of BMEs as a delivery platform for AMPs and other hydrophilic and lipophilic drugs to inhibit antibiotic-resistant microorganisms in chronic wounds and SSIs.

Lunasin reduces the susceptibility of IL-10 deficient mice to inflammatory bowel disease and modulates the activation of the NLRP3 inflammasome.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition that can cause severe damage to the gastrointestinal tract leading to lower quality of life and productivity. Our goal was to investigate the protective effect of the soy peptide lunasin in an in vivo model of susceptibility to IBD and to identify the potential mechanism of action in vitro. In IL-10 deficient mice, oral administration of lunasin reduced the number and frequency of mice exhibiting macroscopic signs of susceptibility to inflammation and significantly decreased levels of the proinflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-18 by up to 95%, 90%, 90%, and 47%, respectively, in different sections of the small and large intestines. Dose-dependent decrease of caspase-1, IL-1ß, and IL-18 in LPS-primed and ATP-activated THP-1 human macrophages demonstrated the ability of lunasin to modulate the NLRP3 inflammasome. We demonstrated that lunasin can decrease susceptibility to IBD in genetically susceptible mice by exerting anti-inflammatory properties.

Antiviral Effects of Quillaja saponaria Extracts Against Human Noroviral Surrogates.

Aqueous extracts of Quillaja saponaria Molina are US FDA approved as food additives in beverages with known antiviral activity. Due to lack of commercially available vaccines against human noroviruses (HNoVs), alternate methods to prevent their spread and the subsequent emergence of variant strains are being researched. Furthermore, HNoVs are not yet culturable at high enough titers to determine inactivation, therefore surrogates continue to be used. This research analyzed the effect of aqueous Quillaja saponaria extracts (QE) against HNoV surrogates, Tulane virus (TV), murine norovirus (MNV-1), and feline calicivirus (FCV-F9) at room temperature (RT) and 37 °C. Viruses (~ 5 log PFU/mL) were individually treated with 1:1 or 1:5 (v/v) diluted QE (pH ~ 3.75), malic acid control (pH 3.0) or phosphate-buffered saline (pH 7.2, as control) at 37 °C or RT for up to 6 h. Individual treatments were replicated three times using duplicate plaque assays for each treatment. FCV-F9 at ~ 5 log PFU/mL was not detectable after 15 min by 1:1 QE at 37 °C and RT. At RT, 1:5 QE lowered FCV-F9 titers by 2.05, 2.14 and 2.74 log PFU/mL after 0.5 h, 1 h and 2 h, respectively. MNV-1 showed marginal reduction of < 1 log PFU/mL after 15 min with 1:1 or 1:5 QE at 37 °C without any significant reduction at RT, while TV titers decreased by 2.2 log PFU/mL after 30 min and were undetectable after 3 h at 37 °C. Longer incubation with higher QE concentrations may be required for improved antiviral activity against MNV-1 and TV.

Switchgrass extractives to mitigate Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium contamination of romaine lettuce at pre- and postharvest.

The antimicrobial potential of switchgrass extractives (SE) was evaluated on cut lettuce leaves and romaine lettuce in planta, using rifampicin-resistant Escherichia coli O157:H7 and Salmonella Typhimurium strain LT2 as model pathogens. Cut lettuce leaves were swabbed with E. coli O157:H7 or S. Typhimurium followed by surface treatment with 0.8% SE, 0.6% sodium hypochlorite, or water for 1 to 45 min. For in planta studies, SE was swabbed on demarcated leaf surfaces either prior to or after inoculation of greenhouse-grown lettuce with E. coli O157:H7 or S. Typhimurium; the leaf samples were collected after 0, 24, and 48 h of treatment. Bacteria from inoculated leaves were enumerated on tryptic soy agar plates (and also on MacConkey's and XLT4 agar plates), and the recovered counts were statistically analyzed. Cut lettuce leaves showed E. coli O157:H7 reduction between 3.25 and 6.17 log CFU/leaf, whereas S. Typhimurium reductions were between 2.94 log CFU/leaf and 5.47 log CFU/leaf depending on the SE treatment durations, from initial levels of ∼7 log CFU/leaf. SE treatment of lettuce in planta, before bacterial inoculation, reduced E. coli O157:H7 and S. Typhimurium populations by 1.88 and 2.49 log CFU after 24 h and 3 h, respectively. However, SE treatment after bacterial inoculation of lettuce plants decreased E. coli O157:H7 populations by 3.04 log CFU (after 0 h) with negligible reduction of S. Typhimurium populations. Our findings demonstrate the potential of SE as a plant-based method for decontaminating E. coli O157:H7 on lettuce during pre- and postharvest stages in hurdle approaches.

Prevalence of Antimicrobial Resistant and Extended-Spectrum Beta-Lactamase-producing Escherichia coli in Dairy Cattle Farms in East Tennessee.

Antimicrobials have been widely used in dairy farms to prevent and control dairy cattle diseases since 1960s. This led to the emergence of antimicrobial resistant bacteria (ARB) that, along with their antimicrobial resistance genes (ARGs), can spread from dairy farms to humans. Therefore, regular antimicrobial resistance (AMR) monitoring is important to implement proper mitigation measures. The objective of this study was to determine the prevalence of AMR and extended-spectrum beta-lactamases (ESBLs)-producing Escherichia coli in dairy cattle. A cross-sectional study was conducted in four dairy cattle farms (A-D) in East Tennessee. A total of 80 samples consisting of 20 samples each of bulk tank milk, feces, dairy cattle manure-amended soil, and prairie soil adjacent to the farms were collected and cultured for the isolation of E. coli. Tetracycline (TETr)-, third-generation cephalosporin (TGCr)- and nalidixic acid (NALr)-resistant E. coli (n = 88) were isolated and identified on agar media supplemented with TET, cefotaxime, and NAL, respectively. TGCr E. coli were tested for ESBLs and other coselected ARGs. TETr (74%, n = 88) was the most common, followed by TGCr (20%) and NALr (8%). Farms had significant (p < 0.001) differences: the highest prevalence of TGCr (55%) and TETr (100%) were observed in farm D, while all NALr isolates were from farm C. Over 83% of TGCr isolates (n = 18) harbored ESBL gene blaCTX-M. Majority (78%) of the E. coli isolates were multidrug-resistant (MDR), being positive for beta-lactams (blaCTX-M), TETs tet(A), tet(B), tet(M)), sulfonamides (sul2), aminoglycosides (strA), and phenicols (floR). This study indicated the widespread occurrence of MDR ESBLs-E. coli in dairy cattle farms. AMR surveillance of more dairy farms and identification of farm-level risk factors are important to mitigate the occurrence and spread of ARB of significant public health importance, such as ESBLs-E. coli.

Utilization of tofu processing wastewater as a source of the bioactive peptide lunasin.

The goal of our study was to design a simple and feasible method to obtain lunasin, a naturally-occurring bioactive peptide, from tofu whey wastewater. A combination of alcoholic precipitation of high-molecular weight proteins from the whey, isoelectric precipitation of lunasin enriched material, and purification via gel filtration chromatography was selected as the best approach using tofu whey prepared at the laboratory scale. This process was applied to tofu whey produced by a local tofu factory and 773 mg of 80% purity lunasin was obtained per kg of dry tofu whey. Significant reduction of nitric oxide, and pro-inflammatory cytokines TNF-α and IL-6 over lipopolysaccharide activated murine macrophages demonstrate its biological activity. Our three-step process is not only simpler and faster than the previously reported methods to obtain lunasin but provides a sustainable approach for the valorization of a waste product, promoting the better utilization of soybean nutrients and active compounds.

Prevalence of Mastitis and Antibiotic Resistance of Bacterial Isolates from CMT Positive Milk Samples Obtained from Dairy Cows, Camels, and Goats in Two Pastoral Districts in Southern Ethiopia.

A study was carried out from August 2017 to February 2018 on lactating dairy cows, one-humped dromedary camels, and goats to determine mastitis in the Bule Hora and Dugda Dawa districts of in Southern Ethiopia. Milk samples from 564 udder quarters and udder halves from 171 animals consisting of 60 dairy cows, 51 camels, and 60 goats were tested for mastitis. Sixty-four positive udder milk samples were cultured, and bacterial mastitis pathogens were isolated and identified. The antibiotic resistance of bacterial isolates from milk with mastitis was tested against nine antimicrobials commonly used in the study area. Cow- and quarter-level prevalence of mastitis in dairy cows, camels, and goats was 33.3%, 26.3%, and 25% and 17.6%, 14.5%, and 20%, respectively. In cattle, the prevalence was significantly higher in Dugda Dawa than in Bule Hora. Major bacterial isolates were coagulase-negative Staphylococcus species (39.1%), S. aureus (17.2%), S. hyicus (14.1%), and S. intermedius and Escherichia coli (9.4% each). In camels, udder abnormality and mastitis were significantly higher in late lactation than in early lactation. Mastitis tends to increase with parity in camels. E. coli isolates were highly resistant to spectinomycin, vancomycin, and doxycycline, whereas most S. aureus isolates were multidrug-resistant. Most of the rural and periurban communities in this area consume raw milk, which indicates a high risk of infection with multidrug-resistant bacteria. We recommend a community-focused training program to improve community awareness of the need to boil milk and the risk of raw milk consumption.

Production and Characterization of High Value Prebiotics From Biorefinery-Relevant Feedstocks.

Hemicellulose, a structural polysaccharide and often underutilized co-product stream of biorefineries, could be used to produce prebiotic ingredients with novel functionalities. Since hot water pre-extraction is a cost-effective strategy for integrated biorefineries to partially fractionate hemicellulose and improve feedstock quality and performance for downstream operations, the approach was applied to process switchgrass (SG), hybrid poplar (HP), and southern pine (SP) biomass at 160°C for 60 min. As a result, different hemicellulose-rich fractions were generated and the chemical characterization studies showed that they were composed of 76-91% of glucan, xylan, galactan, arabinan, and mannan oligosaccharides. The hot water extracts also contained minor concentrations of monomeric sugars (≤18%), phenolic components (≤1%), and other degradation products (≤3%), but were tested for probiotic activity without any purification. When subjected to batch fermentations by individual cultures of Lactobacillus casei, Bifidobacterium bifidum, and Bacteroides fragilis, the hemicellulosic hydrolysates elicited varied responses. SG hydrolysates induced the highest cell count in L. casei at 8.6 log10 cells/ml, whereas the highest cell counts for B. fragilis and B. bifidum were obtained with southern pine (5.8 log10 cells/ml) and HP hydrolysates (6.4 log10 cells/ml), respectively. The observed differences were attributed to the preferential consumption of mannooligosaccharides in SP hydrolysates by B. fragilis. Lactobacillus casei preferentially consumed xylooligosaccharides in the switchgrass and southern pine hydrolysates, whereas B. bifidum consumed galactose in the hybrid poplar hydrolysates. Thus, this study (1) reveals the potential to produce prebiotic ingredients from biorefinery-relevant lignocellulosic biomass, and (2) demonstrates how the chemical composition of hemicellulose-derived sources could regulate the viability and selective proliferation of probiotic microorganisms.

Aichi virus inactivation by heat in 2-ml glass vials.

Aichi virus (AiV) that results in gastroenteritis worldwide, is spread through contaminated shellfish and water. The resistance/tolerance of AiV to common inactivation processes along with the absence of commercially available vaccines makes it necessary to study its thermal inactivation kinetics. This research evaluated the heat inactivation of AiV in cell-culture media using 2-ml sterile glass vials by the linear and Weibull models. Heat treatments of AiV titers of 7 log plaque forming units (PFU)/ml were conducted thrice in a water-bath at 50, 54, and 58 °C for up to 90 min. Plaque assays for each dilution in duplicate were used to determine infectious virus titers. Linear model D-values for AiV at 50 ± 1 °C (± = standard error) (come-up time = 68 s), 54 ± 0.7 °C (130 s), and 58 ± 0.6°C (251 s) were 43.3 ± 4.23 (R2 = 0.40, RMSE = 0.56), 5.69 ± 0.28 (R2 = 0.80, RMSE = 0.43), and 1.20 ± 0.63 min (R2 = 0.69, RMSE = 0.39), respectively, and the linear model z-value was 5.14 ± 0.39°C (R2 = 0.99, RMSE = 0.08). For the same temperatures, the Weibull model td = 1 values were 20.98 ± 8.8 (R2 = 0.62, RMSE = 0.46, α (scale parameter) = 2.30, ß (shape parameter) = 0.38), 3.84 ± 0.69 (R2 = 0.85, RMSE = 0.38, α = 1.08, ß = 0.66), and 0.87 ± 0.10 min (R2 = 0.80, RMSE = 0.32, α = 0.22, ß = 0.61), respectively and the z-value (using Td = 1 ) was 5.79 ± 0.22 °C (R2 = 1.0, RMSE = 0.03). A better fit was obtained with the Weibull model for log reductions versus time with higher R2 and lower RMSE values. Application of AiV inactivation parameters can help reduce the risk of AiV outbreaks.

Grape seed extract against Aichi virus infectivity in model foods and contact surfaces.

Aichi virus (AiV) is an enteric virus that affects humans and is prevalent in sewage waters. Effective strategies to control its spread need to be explored. This study evaluated grape seed extract (GSE) for: a) antiviral potential towards AiV infectivity at 37 °C and room temperature (RT); b) antiviral behavior in model foods (apple juice (AJ) and 2% fat milk) and also simulated gastric environments; and c) potential application as a wash solution on stainless steel surfaces. GSE at 0.5 mg/mL decreased AiV suspensions containing ~4.75 log PFU/mL to titer levels that were not detected after 30 s at both 37 °C and RT. Infectious AiV titers were not detected after 5 min treatment with 1 mg/mL GSE at 37 °C in AJ. GSE at 2 mg/mL and 4 mg/mL in 2% fat milk decreased AiV after 24 h by 1.18 and 1.57 log PFU/mL (4.75 log PFU/mL to 2.86 and 3.25 log PFU/mL), respectively. As a surface wash, GSE at 1 mg/mL after 30 s decreased AiV to undetectable levels under clean conditions. With organic load (mimicking unclean conditions), 2 and 4 mg/mL GSE reduced AiV after 5 min by 1.13 and 1.71 log PFU/mL, respectively. Overall, GSE seems to be a promising antiviral agent against AiV at low concentrations and short contact times.

Genomic Modeling as an Approach to Identify Surrogates for Use in Experimental Validation of SARS-CoV-2 and HuNoV Inactivation by UV-C Treatment.

Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) is responsible for the COVID-19 pandemic that continues to pose significant public health concerns. While research to deliver vaccines and antivirals are being pursued, various effective technologies to control its environmental spread are also being targeted. Ultraviolet light (UV-C) technologies are effective against a broad spectrum of microorganisms when used even on large surface areas. In this study, we developed a pyrimidine dinucleotide frequency based genomic model to predict the sensitivity of select enveloped and non-enveloped viruses to UV-C treatments in order to identify potential SARS-CoV-2 and human norovirus surrogates. The results revealed that this model was best fitted using linear regression with r 2 = 0.90. The predicted UV-C sensitivity (D 90 - dose for 90% inactivation) for SARS-CoV-2 and MERS-CoV was found to be 21.5 and 28 J/m2, respectively (with an estimated 18 J/m2 obtained from published experimental data for SARS-CoV-1), suggesting that coronaviruses are highly sensitive to UV-C light compared to other ssRNA viruses used in this modeling study. Murine hepatitis virus (MHV) A59 strain with a D 90 of 21 J/m2 close to that of SARS-CoV-2 was identified as a suitable surrogate to validate SARS-CoV-2 inactivation by UV-C treatment. Furthermore, the non-enveloped human noroviruses (HuNoVs), had predicted D 90 values of 69.1, 89, and 77.6 J/m2 for genogroups GI, GII, and GIV, respectively. Murine norovirus (MNV-1) of GV with a D 90 = 100 J/m2 was identified as a potential conservative surrogate for UV-C inactivation of these HuNoVs. This study provides useful insights for the identification of potential non-pathogenic (to humans) surrogates to understand inactivation kinetics and their use in experimental validation of UV-C disinfection systems. This approach can be used to narrow the number of surrogates used in testing UV-C inactivation of other human and animal ssRNA viral pathogens for experimental validation that can save cost, labor and time.

Propidium monoazide for viable Salmonella enterica detection by PCR and LAMP assays in comparison to RNA-based RT-PCR, RT-LAMP, and culture-based assays.

Rapid and sensitive detection of live/infectious foodborne pathogens is urgently needed in order to prevent outbreaks and food recalls. This study aimed to (1) evaluate the incorporation of propidium monoazide (PMA) into PCR or LAMP assays to selectively detect viable Salmonella Enteritidis following sublethal heat or UV treatment, and autoclave sterilization; and (2) compare the detection of PMA-PCR and PMA-LAMP to DNA-based PCR and LAMP (without PMA), RNA-based RT-PCR and RT-LAMP, and culture-based methods. Nucleic acids (DNA or RNA) from 1-mL S. Enteritidis samples were used for PCR, RT-PCR, LAMP, and RT-LAMP assays. Serially diluted samples were plated on Xylose Lysine Tergitol-4 agar for cultural enumeration. Comparable detection of overnight cultured S. Enteritidis was obtained by PMA-PCR, PCR, and RT-PCR, though 1 to 2 log less sensitive than cultural assays. PMA-LAMP and RT-LAMP showed similar detection of overnight cultures, being 1 to 2 log less sensitive than the LAMP assay, and ∼4 log less than culture-based detection. Autoclaved S. Enteritidis did not test positive by RNA-based methods or PMA-PCR, but PMA-LAMP showed detection of 1 log CFU/mL. PMA-PCR and RT-PCR showed comparable detection of sublethal heat-treated cells to cultural assays, while PMA-LAMP showed 1 to 2 log less detection. Our results suggest that PMA-PCR and PMA-LAMP assays are not suitable for selective viable cell detection after UV treatment. While PMA-LAMP assay needs optimization, PMA-PCR shows promise for live/viable S. Enteritidis detection. PMA-PCR shows potential for routine testing in the food industry with results within 1-day, albeit depending on the inactivation method employed.

Encapsulation of vitamin D3 in gum arabic to enhance bioavailability and stability for beverage applications.

Delivery of vitamin D3 (VD3 ) in foods should exhibit desirable physicochemical characteristics and improves absorption. In this study, gum arabic (GA) was investigated as a VD3 carrier to encapsulate VD3 . VD3 dissolved in 5 mL ethanol corresponding to 0.3 to 6.0% mass of GA, was blended in 5.0% w/v GA solution, followed by freeze drying. The encapsulation efficiency decreased while loading capacity increased with an increased amount of VD3 . At the highest VD3 level, the loading capacity (3.47%) was the highest, and the encapsulation efficiency (61.24%) was satisfactory, and the treatment was further studied. The magnitude of negative zeta-potential increased from 3.1 to 31.0 mV at pH 2.0 to 7.4. During the 100-day storage at 3 °C of capsules reconstituted at pH 2.0 to 7.4, the hydrodynamic diameter decreased at all pH conditions, most evident for reduction to 81.3 nm at pH 7.4, and no precipitation was observed, indicating the significance of steric repulsion on capsule stability. Bioaccessibility of VD3 in capsules (95.76%) was significantly higher than the nonencapsulated VD3 (68.98%). The in vivo pharmacokinetic study in Sprague-Dawley rats after a single-dose of 300 µg VD3 showed the area-under-curve of serum 25(OHD) level in 48 hr of the encapsulation treatment was 4.32-fold of the nonencapsulated VD3 and more than twice higher than the VD3 -GA physical mixture. During 2-week supplementation of 60 µg VD3 /d, rats receiving capsules or physical mixture had 25(OH)D levels of at least 81 ng/mL higher than that of the nonencapsulated VD3 group. The studied encapsulation system holds great potential as a value-added ingredient to supplement VD3 in beverages with a wide pH range. PRACTICAL APPLICATION: The findings of this study demonstrated the improved dispersion stability and absorption of vitamin D3 after encapsulation in gum arabic. The capsules exhibited good dispersion stability across a pH range between 2.0 and 7.4, showing potential application in beverages. Furthermore, the enhanced absorption of VD3 after encapsulation highlights the nutritional benefits of the studied encapsulation system.

Bovine Milk Exosomes Affect Proliferation and Protect Macrophages against Cisplatin-Induced Cytotoxicity.

BACKGROUND: Exosomes are extracellular vesicles involved in intercellular communication. The objectives were to characterize bovine milk exosomes (BME) and determine its effect on RAW 264.7 macrophages. METHODS: BME were isolated using differential centrifugation and characterized by particle size and the presence of exosomal markers Alix, TSG101, and CD81. The effect of in vitro digestion and different pH on the stability of BME was investigated. The biological activity of BME in RAW 264.7 macrophages was conducted by assessing proliferation and cell cycle. Moreover, the protective effect of exosomes on cisplatin-induced cytotoxicity was evaluated. RESULTS: BME have an average particle size of 106.8 ± 3.4 nm and expressed Alix, TSG101, and CD81. TSG101 was detected after digestion and exposure to different pH values. Cell-cycle analysis showed that BME reduced the percentage of apoptotic cells while arresting the cells in G2/M phase accompanied by differential expression of proliferation markers p53, p21, cyclin D1, and ß-catenin. Exosomes protected macrophages against cisplatin-induced cytotoxicity. CONCLUSION: Our results showed for the first time the effect of BME on the proliferation of RAW 264.7 macrophages and its protective effect against chemotherapeutic drug-induced cytotoxicity. Potential effect of BME on immune system must be studied.

Heat sensitization of hepatitis A virus and Tulane virus using grape seed extract, gingerol and curcumin.

Human noroviruses (HNoV) and hepatitis A virus (HAV) are predominantly linked to foodborne outbreaks worldwide. As cell-culture systems to propagate HNoV in laboratories are not easily available, Tulane virus (TV) is used as a cultivable HNoV surrogate to determine inactivation. Heat-sensitization of HAV and TV by "generally recognized as safe'' (GRAS) substances can potentially reduce their time-temperature inactivation parameters during processing to ensure food safety. Curcumin, gingerol (from ginger), and grape seed extract (GSE) reportedly have anti-inflammatory, immune-modulating and antiviral properties. The objective of this study was to determine and compare the D-values and z-values of HAV and TV at 52-68 °C with or without curcumin (0.015 mg/ml), gingerol (0.1 mg/ml), or GSE (1 mg/ml) in 2-ml glass vials. HAV at ~7 log PFU/ml and TV at ~6 log PFU/ml were diluted in phosphate buffered saline (PBS) and added to two sets of six 2-mL sterile glass vials. One set served as the control and the second set had the three extracts individually added for thermal treatments in a circulating water bath for 0-10 min. The D-values for TV in PBS ranged from 4.55 ± 0.28 to 1.08 ± 0.16 min, and for HAV in PBS ranged from to 9.21 ± 0.24 to 0.67 ± 0.19 min at 52-68 °C. Decreased D-values (52-58 °C) for TV with curcumin ranging from 4.32 ± 0.25 to 0.62 ± 0.17 min, gingerol from 4.09 ± 0.18 to 0.72 ± 0.09 min and GSE from 3.82 ± 0.18 to 0.80 ± 0.07 min, with similar trends for HAV were observed. The linear model showed significant differences (p < 0.05) between the D-values of HAV and TV with and without plant extracts for most tested temperatures. This suggests that GRAS substances can potentially lower temperature and time regimens needed to inactivate HAV and TV.

Antiviral effects of blueberry proanthocyanidins against Aichi virus.

Blueberry polyphenols are known for their high antioxidant and antimicrobial potential. Aichi virus (AiV) is an emerging human enteric virus that causes gastroenteritis outbreaks worldwide. This study aimed to (1) determine the time- and dose-dependent effects of blueberry proanthocyanidins (B-PAC) against AiV over 24 h at 37 °C; (2) gain insights on their mode of action using pre- and post-treatment of host cells and Transmission Electron Microscopy; and (3) determine their anti-AiV effects in model foods and under simulated gastric conditions. AiV at ∼5 log PFU/ml was incubated with equal volumes of commercial blueberry juice (BJ, pH 2.8), neutralized BJ (pH 7.0), B-PAC (2, 4, and 10 mg/ml) prepared either in 10% ethanol, apple juice (AJ), 2% milk, simulated gastric fluid (SGF, pH 1.5) or simulated intestinal fluid (SIF, pH 7.5), and controls (malic acid (pH 3.0), phosphate buffered saline (pH 7.2), apple juice (pH 3.6) and 2% milk) over 24 h at 37 °C, followed by standard plaque assays. Each experiment was replicated thrice and data were statistically analyzed. Differences in AiV titers with 1 mg/ml B-PAC were 2.13 ±â€¯0.06 log PFU/ml lower after 24 h and ≥3 log PFU/ml (undetectable levels) lower with 2 and 5 mg/ml B-PAC compared to AiV titers in PBS after 24 h and 3 h, respectively. BJ at 37 °C resulted in titer differences (lower titers compared to PBS) of 0.17 ±â€¯0.06, 1.27 ±â€¯0.01, and 1.73 ±â€¯0.23 log PFU/ml after 1, 3, and 6 h and ≥3 log PFU/ml after 24 h. Pre- and post-treatment of host cells with 0.5 mg/ml B-PAC caused titer decreases of 0.62 ±â€¯0.33 and 0.30 ±â€¯0.06 log PFU/ml, respectively suggesting a moderate effect on viral-host cell binding. B-PAC at 2 mg/ml in AJ caused titer differences of ≥3 log PFU/ml after 0.5 h, while differences of 0.84 ±â€¯0.03 log PFU/ml with 5 mg/ml B-PAC in milk, and ≥3 log PFU/ml with B-PAC at 5 mg/ml in SIF after 30 min were obtained. This study shows the ability of BJ and B-PAC to decrease AiV titers to potentially prevent AiV-related illness and outbreaks.

Thermal Inactivation Kinetics of Tulane Virus in Cell-Culture Medium and Spinach.

Human noroviruses (HNoVs) cause significant gastrointestinal disease outbreaks worldwide. Tulane virus (TV) is a cultivable HNoV surrogate widely used to determine control measures against HNoVs. The objective of this study was to determine the heat inactivation kinetics (D- and z-values) of TV in cell-culture media and on spiked homogenized spinach using the first-order and Weibull models. TV in cell-culture media at approximately 7 log PFU/mL (PFU-plaque forming unit) in 2-mL glass vials was heated at 52, 54, and 56 °C for up to 10 min in a circulating water bath. Survivors were enumerated using confluent host LLC-MK2 cells in six-well plates by plaque assay. Data from three replicate treatments assayed in duplicate were analyzed statistically. D-values by the first-order model for TV in cell-culture media at 52, 54, and 56 °C were 4.59 ± 0.05, 2.91 ± 0.05, and 1.74 ± 0.07 min, respectively, with a z-value of 9.09 ± 0.01 °C (R2  = 0.997). The Weibull model showed td  = 1 values of 2.53 ± 0.08, 1.99 ± 0.10, and 0.57 ± 0.64 min, respectively, at the same temperatures. The D-values for TV in spinach were 7.94 ± 0.21, 4.09 ± 0.04, and 1.43 ± 0.02 min and the z-value was 10.74 ± 0.01 °C (R2  = 0.98) by the first-order model and 4.89 ± 0.02, 3.21 ± 0.45, and 0.25 ± 0.38 min for the Weibull model at 50, 54, and 58 °C, respectively. In comparison to previously reported results for the cultivable HNoV surrogate, murine norovirus -1, TV in cell-culture media and spiked on spinach homogenates showed lower D- and z-values. TV may not be an ideal HNoV surrogate for heat inactivation studies in cell-culture media or homogenized spinach in vacuum bags.

Monomeric catechin and dimeric procyanidin B2 against human norovirus surrogates and their physicochemical interactions.

Plant polyphenols have shown antiviral activity against several human pathogens, but their physicochemical interactions are not well-understood. The objectives of this study were to compare the antiviral activity between monomeric catechin and dimeric procyanidin B2 (PB2) using cultivable human norovirus surrogates (feline calicivirus (FCV-F9) and murine norovirus (MNV-1)) and to understand their potential antiviral mechanism using virus-like particles (VLPs) and the P domain of human norovirus GII (HNoV GII.4). Surrogate viruses at 5 log PFU/mL were treated with 0.5-5 mg/mL monomeric catechin monohydrate, PB2 or phosphate buffered saline (PBS, pH 7.2; control) at 37 °C over 24 h. Infectivity was determined using plaque assays and data from triplicate experiments were statistically analyzed. PB2 at 0.5 mg/mL and 1 mg/mL reduced FCV-F9 to undetectable levels after 3 h and MNV-1 by 0.21 and 1.23 log PFU after 24 h, respectively. Monomeric catechins at 1 mg/mL reduced FCV-F9 to undetectable levels after 6 h and MNV-1 titers to undetectable levels after 24 h. In addition, PB2 was shown to directly bind the P domain, the main capsid structure of HNoVs in the ratio of 1:1 through spontaneous interactions. Electrostatic interactions played a dominant role between PB2 and the P domain. PB2 significantly altered tertiary but not secondary structures of VLPs. Transmission electron microscopy demonstrated that PB2 aggregated VLPs, further indicating interactions between them. These findings indicate that PB2 causes structural changes of the P domain of VLPs, mainly through direct interaction leading to HNoV inactivation.