Glycerol/organic acid-based ternary deep eutectic solvents as a green approach to recover chitin with different molecular weight from seafood waste.

In this study, we designed a green and efficient approach for the fractionation of high-purity chitin with tunable molecular weights from seafood waste. This was achieved by using ternary deep eutectic solvents (TDESs) composed of choline chloride as a hydrogen bond acceptor, glycerol as the polyol-based hydrogen bond donor, together with lactic acid or malic acid. Two binary DESs and four TDESs were evaluated for their ability to recover chitin. The extracted chitin exhibited not only high yield with excellent protein and mineral removal, but also high purity with similar crystallinity patterns as standard chitin. However, the average molecular weights, viscosity behavior and morphology of chitin extracted by DESs were varied and influenced by organic acid to glycerol molar ratios. The molecular weights of chitin extracted by lactic acid-based TDES ranged from 264 kDa to 541 kDa, but malic acid-based TEDS displayed a stronger depolymerization effect, resulting in chitin with a smaller molecular weight of less than 300 kDa. Lactic acid-based TDES revealed that the purity of chitin remained higher than 92 % after three cycles. This sustainable and environmentally friendly extraction system holds great potential to recover chitin from seafood waste, opening a new era for chitin extraction and applications.

Investigating migration potential of a new rechargeable antimicrobial coating for food processing equipment.

Antimicrobial coatings are designed to inhibit the growth of pathogens and have been used to reduce foodborne illness bacteria on food processing equipment. Novel N-halamine based antimicrobial coatings are highly advantageous due to their unique properties and low cost, and are being investigated for applications in food safety, health care, water and air disinfection, etc. In this study, we evaluated the chemical safety of a novel N-halamine antimicrobial polymer coating (Halofilm) for use on food processing equipment. Migration tests were performed on stainless steel tiles prepared with four different treatment groups: negative control, positive control, Halofilm coating without chlorination, and Halofilm coating with chlorination. An LC-MS/MS method was developed and validated for four formulation components: polyethylenimine (PEI), Trizma® base, hydantoin acrylamide (HA) and dopamine methacrylamide (DMA), followed by stability and recovery tests. Migration tests were conducted at 40 °C with three food simulants (10, 50 and 95% ethanol/water) to mimic various food properties, and aliquots of migration extracts were analyzed at 2, 8, 72, 240 and 720 h. In general, measured concentration levels were consistent among simulant types for the four tested chemicals. Chlorinated tiles had non-detects for three analytes (PEI, HA and DMA), and less than 0.05 mg/kg of HA migration over 30 days. A chlorination step could possibly change the measured mass (m/z) hence leading to non-detects in targeted LC-MS/MS. In non-chlorinated tiles, all four compounds were detected during the migration test. This suggests that addition of the chlorination step may have a stabilizing effect on the polymer. Additionally, full scan high resolution mass spectrometry (HRMS) analysis was employed to screen for migration of other extractable and leachable (E&L) chemicals, which led to the identification of eight common E&L chemicals. To our knowledge, this is the first report evaluating chemical migration from an N-halamine antimicrobial polymer coating product.

Zwitterion and N-halamine functionalized cotton wound dressing with enhanced antifouling, antibacterial, and hemostatic properties.

With emerging needs of wound care management, a multi-functional wound dressing is needed. To prevent infection and reduce patient suffering, antibacterial efficacy against a broad-spectrum of bacteria plus robust antifouling are among the most preferred properties. In this study, a wound dressing was created with antibacterial and anti-fouling capabilities is presented. The approaches used a synthesized tri-functional copolymer comprised of an N-halamine precursor moiety, a marine-inspired surface binding dopamine moiety, and a zwitterionic anti-adhesion moiety bonded onto a commercial cotton gauze. The resulting HaloCare™ wound dressing demonstrated >99.99 % inactivation within 5 min against E. coli and a panel of ESKAPE pathogens plus achieved 98.77 % reduction of non-specific protein binding. HaloCare was also shown to be compatible with hemostatic agents without impacting hemostatic efficacy. HaloCare shows great potential particularly in traumatic injury events as an infection preventing and hemostatic wound management system.

A Broad-Spectrum Antimicrobial and Antiviral Membrane Inactivates SARS-CoV-2 in Minutes.

SARS-CoV-2, the virus that caused the COVID-19 pandemic, can remain viable and infectious on surfaces for days, posing a potential risk for fomite transmission. Liquid-based disinfectants, such as chlorine-based ones, have played an indispensable role in decontaminating surfaces but they do not provide prolonged protection from recontamination. Here a safe, inexpensive, and scalable membrane with covalently immobilized chlorine, large surface area, and fast wetting that exhibits long-lasting, exceptional killing efficacy against a broad spectrum of bacteria and viruses is reported. The membrane achieves a more than 6 log reduction within several minutes against all five bacterial strains tested, including gram-positive, gram-negative, and drug-resistant ones as well as a clinical bacterial cocktail. The membrane also efficiently deactivated nonenveloped and enveloped viruses in minutes. In particular, a 5.17 log reduction is achieved against SARS-CoV-2 after only 10 min of contact with the membrane. This membrane may be used on high-touch surfaces in healthcare and other public facilities or in air filters and personal protective equipment to provide continuous protection and minimize transmission risks.

Assessment of the hepatocyte protective effects of gypenoside and its phosphorylated derivative against DHAV-1 infection on duck embryonic hepatocytes.

BACKGROUND: Duck viral hepatitis (DVH) is an acute disease of young ducklings with no effective veterinary drugs for treatment. Gynostemma pentaphyllum is a well-known traditional Chinese medicine that plays an important role in the treatment of various diseases. Gypenoside (GP), one of the main ingredients of Gynostemma pentaphyllum, was reported with good hepatoprotective effects. However, its low solubility limits its application in the clinics. To improve its solubility and bioactivity, a phosphorylated derivative of gypenoside (pGP) was prepared by the sodium trimetaphosphate-sodium tripolyphosphate (STMP-STPP) method. An infrared spectroscopy method was applied to analyse the structures of GP and pGP. Then, a methyl thiazolyl tetrazolium (MTT) colorimetric assay was applied to study the hepatocyte protective efficacy of these two drugs against duck hepatitis A virus type 1 (DHAV-1) infection, and qPCR, TUNEL labelling and flow cytometry methods were used to study the relevant hepatocyte protective in vitro. RESULTS: The infrared spectroscopy detection results showed that the phosphorylation modification of GP was successful. The MTT colorimetric assay results showed that both GP and pGP possessed good hepatocyte protective efficacy in vitro, and pGP performed better than GP when the drug was added before or after virus inoculation. Furthermore, the qPCR results revealed that both drugs could effectively inhibit the adsorption (when adding GP and pGP pre-virus inoculation), replication and release of DHAV-1, and the viral inhibition rate of pGP was greater than that of GP. The subsequent TUNEL labelling and flow cytometry assays showed that both GP and pGP could significantly inhibit duck embryo hepatocyte apoptosis induced by DHAV-1, and the inhibition effect of pGP was much stronger than that of GP. CONCLUSIONS: GP exerts good hepatocyte protective efficacy not only by inhibiting the proliferation of DHAV-1 but also by inhibiting duck embryonic hepatocyte apoptosis induced by DHAV-1, and phosphorylation modification significantly improves the antiviral and the anti-apoptotic effects of GP. Therefore, pGP has the potential to be developed into a novel drug against DHAV-1 infection.

Evaluation of the Therapeutic Effect of a Flavonoid Prescription against Rabbit Hemorrhagic Disease In Vivo.

Rabbit hemorrhagic disease (RHD) is an acute, high fatal contagious disease induced by rabbit hemorrhagic disease virus (RHDV) with acute severe hepatic injury and causes huge economic loss worldwide. In order to develop an effective and reliable drug to treat this disease in clinic, a prescription formulated with baicalin, linarin, icariin, and notoginsenoside R1 (BLIN) according to the theory of syndrome differentiation and treatment in traditional Chinese veterinary medicine was applied to investigate its curative effects against RHD in vivo. The preliminary study results showed that BLIN prescription exerted good curative effect on RHD therapy. To further validate the curative effect and to investigate the possible related curative mechanisms of this drug, the survival rates, the plasma biochemical indexes of hepatic function, the plasma evaluation indexes of oxidative injury, and the RHDV gene expression levels were detected and then the correlation among these indexes was also analyzed. These results showed that BLIN prescription could significantly increase the survival rate, reduce the hepatic injury severity, alleviate the oxidative injury, and decrease the RHDV gene expression level in rabbits infected with RHDV. All these results indicate that BLIN prescription possesses outstanding curative effect against RHD, and the curative mechanism may be related to its antioxidant and anti-RHDV activities. Therefore, this prescription can be expected to be exploited into a new candidate for RHD therapy in clinic.

The reduction of Salmonella on chicken skin by the combination of sodium dodecyl sulfate with antimicrobial chemicals and coating wax microemulsions.

Chickens with high populations of various microorganisms arrive at processing facilities. Salmonella species are one of the important foodborne pathogens commonly found in poultry products. Various intervention strategies are implemented during poultry processing to reduce microorganisms in the products, including pre-scald bird brushes, multi-stage scalding, antimicrobial applications, etc. In this study, the effects of adding sodium dodecyl sulfate (SDS) to chlorine (Cl) and peracetic acid (PAA) against Salmonella were investigated. In addition, the efficacy of wax coating the skin to reduce Salmonella attachment was studied. Skin samples were collected following the 4 different methods of (1) euthanized-dry hand-de-feathered carcasses, (2) carcasses rinsed in tap water and mechanically de-feathered, (3) carcasses soft scalded and mechanically de-feathered, and (4) from carcasses hard scalded and mechanically de-feathered. It was shown that 0.5% SDS was able to reduce Salmonella both loosely (34, 28, 42, and 13%, respectively) and firmly (29, 39, 32, and 53%, respectively) attached in the 0.005% Cl-treated samples, but did not increase antimicrobial efficacy of 0.2% PAA. Moreover, carnauba wax coating significantly (P ≤ 0.05) reduced Salmonella attachment on all 4 types of chicken skins, 1.57, 0.71, 0.74, and 0.84 log cfu/sample on dry hand de-feathered, tap water rinsed, soft-scalded and hard-scalded chicken skins, respectively. Beeswax coating did not affect Salmonella attachment regardless of types of chicken skins. Overall, the addition of SDS improved antimicrobial activity of Cl, but not for PAA. Moreover, carnauba wax coating was an effective intervention to reduce Salmonella on chicken skin.

Baicalin Inhibits Biofilm Formation and the Quorum-Sensing System by Regulating the MsrA Drug Efflux Pump in Staphylococcus saprophyticus.

Staphylococcus saprophyticus (S. saprophyticus) is one of the main pathogens that cause serious infection due to its acquisition of antibiotic resistance. The efflux pump decreases antibiotic abundance, and biofilm compromises the penetration of antibiotics. It has been reported that baicalin is a potential agent to inhibit efflux pumps, biofilm formation, and quorum-sensing systems. The purpose of this study was to investigate whether baicalin can inhibit S. saprophyticus biofilm formation and the quorum-sensing system by inhibiting the MsrA efflux pump. First, the mechanism of baicalin inhibiting efflux was investigated by the ethidium bromide (EtBr) efflux assay, measurement of ATP content, and pyruvate kinase (PK) activities. These results revealed that baicalin significantly reduced the efflux of EtBr, the ATP content, and the activity of PK. Moreover, its role in biofilm formation and the agr system was studied by crystal violet staining, confocal laser scanning microscopy, scanning electron microscopy, and real-time polymerase chain reaction. These results showed that baicalin decreased biofilm formation, inhibited bacterial aggregation, and downregulated mRNA transcription levels of the quorum-sensing system regulators agrA, agrC, RNAIII, and sarA. Correlation analysis indicated that there was a strong positive correlation between the efflux pump and biofilm formation and the agr system. We demonstrate for the first time that baicalin inhibits biofilm formation and the agr quorum-sensing system by inhibiting the efflux pump in S. saprophyticus. Therefore, baicalin is a potential therapeutic agent for S. saprophyticus biofilm-associated infections.

In vitro synergistic effect of baicalin with azithromycin against Staphylococcus saprophyticus isolated from francolins with ophthalmia.

Francolins ophthalmia is often caused by resistant conditional pathogenic bacteria. Conditional pathogenic Staphylococcus saprophyticus is a potential reservoir of macrolides antibiotics resistance gene. Baicalin has been reported as a potential agent to synergistically inhibit the replication of Staphylococcus. The objective of this study was to isolate the pathogen of the francolins ophthalmia, identify the antibiotic resistance profile of isolated S. saprophyticus, and investigate the effect of baicalin combined with azithromycin (Azm) against azithromycin resistant S. saprophyticus (ARSS). The ARSS was isolated and identified from francolins suffered from ophthalmia by phenotypic and molecular biology methods. The antibiotic resistance profile was identified by Kirby-Bauer method. Then the minimal inhibitory concentration (MIC) of Azm in absence and presence of a sub-inhibitory concentration baicalin/verapamil was determined to assess the effect that baicalin combined with Azm against ARSS. ARSS was isolated and identified from francolins experienced ophthalmia. The isolated ARSS was resistant to 11 among the 13 antibiotics that were tested. The synergistic effect of baicalin and Azm was noticed with a reduction rate varied from 2 to 128-fold. It appears from this study that S. saprophyticus can cause francolins ophthalmia and baicalin may be used as a natural agent resistance inhibitor for ARSS.

Conformal Hydrogel Coatings on Catheters To Reduce Biofouling.

Reducing biofouling while increasing lubricity of inserted medical catheters is highly desirable to improve their comfort, safety, and long-term use. We report here a simple method to create thin (∼30 µm) conformal lubricating hydrogel coatings on catheters. The key to this method is a three-step process including shape-forming, gradient cross-linking, and swell-peeling (we label this method as SGS). First, we took advantage of the fast gelation of agar to form a hydrogel layer conformal to catheters; then, we performed a surface-bound UV cross-linking of acrylamide mixed in agar in open air, purposely allowing gradual oxygen inhibition of free radicals to generate a gradient of cross-linking density across the hydrogel layer; and finally, we caused the hydrogel to swell to let the non-cross-linked/loosely attached hydrogel fall off, leaving behind a surface-bound, thin, and mostly uniform hydrogel coating. This method also allowed easy incorporation of different polymerizable monomers to obtain multifunctionality. For example, incorporating an antifouling, zwitterionic moiety sulfobetaine in the hydrogel reduced both in vitro protein adsorption and in vivo foreign-body response in mice. The addition of a biocidal N-halamine monomer to the hydrogel coating deactivated both Staphylococcus aureus ( S. aureus) and Escherichia coli ( E. coli) O157:H7 within 30 min of contact and reduced biofilm formation by 90% compared to those of uncoated commercial catheters when challenged with S. aureus for 3 days. The lubricating, antibiofouling hydrogel coating may bring clinical benefits in the use of urinary and venous catheters as well as other types of medical devices.

Scalable and Rechargeable Antimicrobial Coating for Food Safety Applications.

Environmental surfaces are common avenues for microbial contamination and transmission in food-processing establishments. We recently synthesized a polymer that combines both N-halamine and dopamine functional groups to form a novel antimicrobial coating material. A series of chemical (titration) and biological ("sandwich" and anti-inhabitation) tests were designed to prove the stability and functionality of as-developed coating material. Halamine-dopamine polymer-coated stainless-steel surface inactivated 6 log10 CFU of both Staphylococcus aureus and Escherichia coli O157:H7 under experimental detection limit within 10 min of contact time. After three "discharge-recharge" cycles, the surface maintained the same antimicrobial effectiveness; 60% of the surface chlorine remained after 10 "discharge-recharge" cycles. In addition, the coating thickness and chlorine content could be further tuned through adjusting the formulation of the coating. We also demonstrated that this coating material could be easily scaled up to apply on real food equipment parts through a spray-coating method. Thus our polymer material has great potential to produce a high-performance, low-cost, and easy-to-apply coating on food-associated environmental surfaces for food safety preventive-control applications.

N-halamine incorporated antimicrobial nonwoven fabrics for use against avian influenza virus.

Airborne pathogens are one of the most common avenues leading to poultry diseases. Preventing the avian influenza (AI) virus from entering the chicken hatchery house is critical for reducing the spread and transmission of AI disease. Many studies have investigated the incorporation of antimicrobials into air filters to prevent viruses from entering the indoor environment. N-halamines are one of the most effective antimicrobial agents against a broad spectrum of microorganisms. In this study, 1-chloro-2,2,5,5-tetramethyl-4-imidazolidinone (MC, a variety of N-halamine) was coated on nonwoven fabrics to give the fabric antimicrobial activity against the AI virus. Results showed that MC exhibited potent antiviral activity either in suspension or in the air. Higher concentrations of MC completely inactivated AI viruses and disrupted their RNA, preventing them from being detected by the real time reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Coating the fabrics with MC resulted in remarkably reduced presence of AI virus on the MC-treated fabric in a short period of time. Furthermore, aerosolized AI viruses were completely inactivated when they passed through filters coated with the MC compound. In addition, MC is not volatile and does not release any gaseous chlorine. The active chlorine in the MC compound is stable, and the coating procedure is straightforward and inexpensive. Therefore, this study validates a novel approach to reducing airborne pathogens in the poultry production environment.

Absorbent Pads Containing N-Halamine Compound for Potential Antimicrobial Use for Chicken Breast and Ground Chicken.

N-Halamines are a group of compounds containing one or more nitrogen-halogen covalent bond(s), and the high-energy halide bond provides a strong oxidative state so that it is able to inactivate microorganisms effectively. In this study, the shelf life of chicken breast and ground chicken packed with 1-chloro-2,2,5,5-tetramethyl-4-imidazolidinone (MC, a member oft the N-halamines) treated absorbent pads was investigated during refrigerated storage. Fresh, processed chicken meat in packaging trays loaded with or without MC treated absorbent pads were stored at 4 °C for 11 days. The microbial counts in chicken meat as well as in the food pads were analyzed on days 1, 4, 7, and 11. MC treated pads reduced the levels of the main spoilage-related microorganisms (aerobic plate counts, lactic acid bacteria, Enterobacteriaceae, psychrotrophs, and Pseudomonas spp.) present in the absorbent pads by an average of 3.5 log CFU/g compared to the control. Microbial loads in chicken breast packed with MC coated absorbent pads were 0.3 log CFU/g lower than those in the control, and an approximate 0.2 log CFU/g reduction was observed for ground chicken. Neither the color nor the pH of the meat was negatively impacted by the presence of MC. The populations of inoculated Salmonella and Campylobacter in meat loaded with MC treated absorbent pads were on average lower than those in the controls. Pathogens in the control pads increased to 3.7 and 4.9 log CFU/g, while the MC treated absorbent pads lowered these two bacteria to under the detection limit (l.7 log CFU/g) throughout 11 days of storage.

Antimicrobial Activity of N-Halamine-Coated Materials in Broiler Chicken Houses.

The antimicrobial activity of 1-chloro-2,2,5,5-tetramethyl-4-imidazoidinone (MC), a nonbleaching N-halamine compound, was investigated on materials commonly used in broiler production, including stainless steel, galvanized metal, aluminum, plastic, and pressure-treated wood. MC aqueous solutions at 0.02, 0.04, and 0.06% were challenged with Salmonella Typhimurium and Campylobacter jejuni at 6 log CFU/mL, resulting in complete inactivation of both bacteria in 30 min with 0.06% MC. Follow-up experiments were performed using test materials treated with 0.1 and 1% MC and challenged with Salmonella Typhimurium and C. jejuni at 6 log CFU per coupon. Stability of MC on the various surfaces of testing materials was assessed, and the chlorine content of the materials was measured using iodometric thiosulfate titration over a 4-week period. Antimicrobial activities were evaluated by a sandwich test on each sampling day during 4 weeks of storage. On the samples treated with 1% MC, bacteria at 6 log CFU per coupon were completely inactivated within 2 h of contact time. The antimicrobial activity extended to 4 weeks, and the active chlorine atoms in the treated materials decreased from the initial 1016 to 1015 atoms per cm2. Overall, MC had high stability and long-lasting antimicrobial activity, which suggests that MC has high potential for use as a novel antimicrobial agent to lower the microbial load on broiler house materials.

N-Halamine Biocidal Materials with Superior Antimicrobial Efficacies for Wound Dressings.

This work demonstrated the successful application of N-halamine technology for wound dressings rendered antimicrobial by facile and inexpensive processes. Four N-halamine compounds, which possess different functional groups and chemistry, were synthesized. The N-halamine compounds, which contained oxidative chlorine, the source of antimicrobial activity, were impregnated into or coated onto standard non-antimicrobial wound dressings. N-halamine-employed wound dressings inactivated about 6 to 7 logs of Staphylococcus aureus and Pseudomonas aeruginosa bacteria in brief periods of contact time. Moreover, the N-halamine-modified wound dressings showed superior antimicrobial efficacies when compared to commercially available silver wound dressings. Zone of inhibition tests revealed that there was no significant leaching of the oxidative chlorine from the materials, and inactivation of bacteria occurred by direct contact. Shelf life stability tests showed that the dressings were stable to loss of oxidative chlorine when they were stored for 6 months in dark environmental conditions. They also remained stable under florescent lighting for up to 2 months of storage. They could be stored in opaque packaging to improve their shelf life stabilities. In vitro skin irritation testing was performed using a three-dimensional human reconstructed tissue model (EpiDerm™). No potential skin irritation was observed. In vitro cytocompatibility was also evaluated. These results indicate that N-halamine wound dressings potentially can be employed to prevent infections, while at the same time improving the healing process by eliminating undesired bacterial growth.

Effect of subinhibitory concentrations of chlorogenic acid on reducing the virulence factor production by Staphylococcus aureus.

Chlorogenic acid (CA) has been reported to inhibit several pathogens, but the influence of subinhibitory concentrations of CA on virulence expression of pathogens has not been fully elucidated. The aim of this study was to explore the effect of CA on the virulence factor production of Staphylococcus aureus. The minimum inhibitory concentration (MIC) of CA against S. aureus was determined using a broth microdilution method. Hemolysin assays, coagulase titer assays, adherence to solid-phase fibrinogen assays, Western blot, and real-time reverse transcriptase-polymerase chain reaction were performed to evaluate the effect of subinhibitory concentrations of CA on the virulence factors of S. aureus. MIC of CA against S. aureus ATCC29213 was found to be 2.56 mg/mL. At subinhibitory concentrations, CA significantly inhibited the hemolysis and dose-dependently decreased coagulase titer. Reduced binding to fibrinogen and decreased production of SEA were observed with treatment of CA at concentrations ranging from 1/16MIC to 1/2MIC. CA markedly inhibited the expression of hla, sea, and agr genes in S. aureus. These data demonstrate that the virulence expression of S. aureus could be reduced by CA and suggest that CA could be potentially developed as a supplemental strategy to control S. aureus infection and to prevent staphylococcal food poisoning.