Vivian C.H. Wu

A review of microbial injury and recovery methods in food

The existence of injured microorganisms in food and their recovery during culturing procedures is critical. Microbial injury is characterized by the capability of a microorganism to return to normalcy during a resuscitation process in which the damaged essential components are repaired. Injury of microorganisms can be induced by sublethal heat, freezing, freeze-drying, drying, irradiation, high hydrostatic pressure, aerosolization, dyes, sodium azide, salts, heavy metals, antibiotics, essential oils, sanitizing compounds, and other chemicals or natural antimicrobial compounds. Injured microorganisms present a potential threat in food safety since they may repair themselves under suitable conditions. Detection of injured microorganisms can be important to practical interpretations of data in food microbiology. This review provides an overview of microbial injury in food and discusses the development of recovery methods for detecting injured foodborne microorganisms.

Antimicrobial action of the American cranberry constituents; phenolics, anthocyanins, and organic acids, against Escherichia coli O157:H7

We investigated the antimicrobial effect of constituents of the American cranberry (Vaccinium macrocarpon); sugar plus organic acids, phenolics, and anthocyanins, against Escherichia coli O157:H7. Each fractional component was assayed over a 24-h period with 5-log initial inocula to determine the minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and log CFU/ml reductions, at their native pH and neutral pH. Each fraction produced significant reductions (P<0.05) at the native pH: MICs for sugars plus organic, phenolics, and anthocyanins were 5.6/2.6 Brix/acid (citric acid equivalents) 2.70g/L (gallic acid equivalent), and 14.80mg/L (cyanidin-3-glucoside equivalent), respectively. Sugars plus organic acids at native pH (3) produced a reduction below detectable limits (<1 log CFU/ml) compared to the control at 24h for 11.3/5.2 and 5.6/2.6 Brix/acid. Phenolics at native pH (4) produced reductions below detectable limits compared to the control at 24h and initial inocula for treatments of 5.40 and 2.70g/L. Anthocyanins at native pH (2) produced reductions below detectable limits for treatments of 29.15 and 14.80mg/L cyanidin-3-glucoside equivalents. Neutralized phenolics and anthocyanins had the same MIC and MBC as those at their native pH. Neutralized sugars plus organic acids did not inhibit bacterial growth compared to the control. Neutralized phenolics reduced bacteria below detectable limits in treatments of 5.40g/L and 2.70g/L compared to the control. Neutralized anthocyanins reduced bacterial growth below detectable limits at the concentration of 29.15mg/L, but at 14.80mg/L there was no significant reduction. Stationary-phase cells of E. coli O157:H7 were treated with 5% of each fraction in 0.8% NaCl for 20min and viewed under transmission electron microscopy. All fractions caused significant damage compared the control. Sugars plus organic acids caused visible osmotic stress, while phenolics and anthocyanins caused disintegration of the outer membrane.

Application of cranberry concentrate (Vaccinium macrocarpon) to control Escherichia coli O157:H7 in ground beef and its antimicrobial mechanism related to the downregulated slp, hdeA and cfa

The possible use of cranberry concentrate (CC) as a natural food preservative was studied by examining its antimicrobial effect on the growth of Escherichia coli O157:H7 inoculated in ground beef, its organoleptical effect on beef patties, and its antimicrobial mechanism on the gene regulation level. Inoculated ground beef was added with CC and stored at 4 degrees C for 5 days. Bacteria were detected on day 0, 1, 3, and 5. Cranberry concentrate (2.5%, 5%, and 7.5% w/w) reduced total aerobic bacteria 1.5 log, 2.1 log, and 2.7 log CFU/g and E. coli O157:H7 0.4 log, 0.7 log, and 2.4 log CFU/g, respectively, when compared to the control on day 5. Fifty panelists evaluated the burgers supplemented with CC. No differences in appearance, flavor, and taste were found among burgers with 0%, 2.5%, and 5% CC. The expression of E. coli O157:H7 cyclopropane fatty acyl phospholipid synthase (cfa), hypothetical protein (hdeA), outer membrane porin protein C (ompC), hyperosmotically inducible periplasmic protein (osmY), and outer membrane protein induced after carbon starvation (slp) genes with or without CC (2.5% v/v) treatment was investigated by quantitative real-time PCR. Compared to the control, slp, hdeA, and cfa were markedly downregulated, ompC was slightly downregulated, while osmY was slightly affected.

A piezoelectric immunosensor for specific capture and enrichment of viable pathogens by quartz crystal microbalance sensor, followed by detection with antibody-functionalized gold nanoparticles

A sensitive bacteria enrichment and detection system for viable Escherichia coli O157:H7 was developed using a piezoelectric biosensor-quartz crystal microbalance (QCM) with antibody-functionalized gold nanoparticles (AuNPs) used as detection verifiers and amplifiers. In the circulating-flow QCM system, capture antibodies for E. coli O157:H7 were first immobilized onto the QCM chip. The sample containing E. coli O157:H7 was circulated through the system in the presence of 10 ml of brain heart infusion (BHI) broth for 18 h. The cells of E. coli O157:H7 specifically captured and enriched on the chip surface of the QCM were identified by QCM frequency changes. Listeria monocytogenes and Salmonella Typhimurium were used as negative controls. After bacterial enrichment, detection antibody-functionalized AuNPs were added to enhance the changes in detection signal. The use of BHI enrichment further enhanced the sensitivity of the developed system, achieving a detection limit of 0-1 log CFU/ml or g. The real-time monitoring method for viable E. coli O157:H7 developed in this study can be used to enrich and detect viable cells simultaneously within 24h. The unique advantages of the system developed offer great potential in the microbial analysis of food samples in routine settings.

Evaluation of thin agar layer method for recovery of acid-injured foodborne pathogens.

The thin agar layer (TAL) method of Kang and Fung was used to enumerate acid-injured foodborne pathogens. This method involves overlaying 14 ml of nonselective medium (tryptic soy agar [TSA]) onto a prepoured and solidified pathogen-specific, selective medium in a petri dish. After surface plating, injured cells resuscitated and grew on TSA during the first few hours of incubation; then, the selective agents from the selective medium diffused to the top layer, interacted with the recovered microorganisms, and started to produce typical reactions. Foodborne pathogens were exposed to 2% acetic acid for 1, 2, or 4 min, and the recovery rate with the TAL method was compared with the rate of TSA and pathogen-specific, selective media. No significant difference occurred between TSA and TAL (P > 0.05) for enumeration of acid-injured Escherichia coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus, and Yersinia enterocolitica, and both recovered significantly higher numbers than the selective medium for each respective pathogen (P < 0.05). For recovery of acid-injured Listeria monocytogenes, no difference (P > 0.05) occurred among TSA, TAL, and selective media. However, fewer cells were recovered in the selective media. The TAL method is a one-step, convenient procedure for recovery of acid-injured cells.

A simple instrument-free gaseous chlorine dioxide method for microbial decontamination of potatoes during storage

An instrument-free gaseous chlorine dioxide (ClO(2)) method to control microorganisms on potatoes during storage was developed. Gaseous ClO(2) was generated by combining an equal amount of impregnated sodium chlorite and activating acids in a sachet without using any solution or equipment. After activation by mixing, the sachet was placed in the application area. The decontamination efficiency of ClO(2) on natural microbiota including total microorganisms, yeasts and molds, and inoculated Pseudomonas aeruginosa on potatoes was investigated. Different treatments using 2, 3, and 4 g of materials and various time intervals (2.5 and 5 h) to generate 16, 20, 24, 30, 32, and 40 mg/L of ClO(2) were evaluated. The results were effective for natural microbiota, showing over a 5 log CFU/potato reduction with a 4 g treatment after 5 h. For P. aeruginosa, there was almost a 6 log CFU/potato reduction after 5 h of the 4 g treatment. The lowest treatment tested (2 g at 2.5 h) showed reductions of 1.7, 1.9, and 2.3 log CFU/potato for total microorganisms, yeasts and molds, and P. aeruginosa, respectively. Gaseous ClO(2) did not affect the overall visual quality of the potato. The residue of ClO(2) decreased to <1 mg/L after 14 days for each treatment, indicating ClO(2) dissipates naturally over time.

Phytochemicals in Lowbush Wild Blueberry Inactivate Escherichia coli O157:H7 by Damaging Its Cell Membrane

The antimicrobial activity and model of action of polyphenolic compounds extracted from lowbush wild blueberries (LWB) were studied against Escherichia coli O157:H7. Polyphenols in LWB were extracted using 80% vol/vol methanol and designated as total blueberry phenolics (TBP). The fraction was further separated by a C-18 Sep-Pak cartridge into monomeric phenolics acids (MPA) and anthocyanins plus proanthocyanidins (A&P). The A&P fraction was further separated into anthocyanins and proanthocyanidins using a LH-20 Sephadex column. Each fraction was diluted in 0.85% wt/vol NaCl, inoculated with E. coli O157:H7 to achieve 8 log colony-forming units (CFU)/mL, and incubated at 25 °C for 1 h. The survival populations of E. coli O157:H7 in the phenolic fractions were determined by a viable cell counts method. The permeability of the cell membrane of E. coli O157:H7 was determined using LIVE/DEAD viability assay, and the damage was visualized by using transmission electron microscopy (TEM). Significant (p<0.05) reductions of 5 log CFU/mL of E. coli O157:H7 were observed for MPA at 0.4 g/L gallic acid equivalents (GAE), A&P at 0.9 g/L GAE, and anthocyanins at 0.65 g/L GAE. Reductions of 6-7 CFU/mL were observed for MPA at 0.8 g/L GAE, A&P at 1.8 g/L GAE, and anthocyanins at 1.3 g/L GAE compared to the control. The cell membrane of E. coli O157:H7 exhibited a significantly increased permeability when treated with proanthocyanidins (0.15 g/L GAE), A&P (0.45 g/L GAE), anthocyanins (0.65 g/L GAE), and TBP (0.14 g/L GAE). TEM confirmed the inactivation and increased membrane permeability of E. coli O157:H7. This study demonstrated the antimicrobial effect of polyphenols from LWB against E. coli O157:H7 and the probable mode of action.

Lowbush wild blueberries have the potential to modify gut microbiota and xenobiotic metabolism in the rat colon.

The gastrointestinal tract is populated by an array of microbial species that play an important role in metabolic and immune functions. The composition of microorganisms is influenced by the components of the host’s diet and can impact health. In the present study, dietary enrichment of lowbush wild blueberries (LWB) was examined to determine their effect on colon microbial composition and their potential in promoting gut health. The microbial composition and functional potential of the colon microbiota from Sprague Dawley rats fed control diets (AIN93) and LWB-enriched diets (AIN93+8% LWB powder substituting for dextrose) for 6 weeks were assessed using Illumina shotgun sequencing and bioinformatics tools. Our analysis revealed an alteration in the relative abundance of 3 phyla and 22 genera as representing approximately 14 and 8% of all phyla and genera identified, respectively. The LWB-enriched diet resulted in a significant reduction in the relative abundance of the genera Lactobacillus and Enterococcus. In addition, hierarchal analysis revealed a significant increase in the relative abundance of the phylum Actinobacteria, the order Actinomycetales, and several novel genera under the family Bifidobacteriaceae and Coriobacteriaceae, in the LWB group. Functional annotation of the shotgun sequences suggested that approximately 9% of the 4709 Kyoto Encyclopaedia of Gene and Genome (KEGG) hits identified were impacted by the LWB-diet. Open Reading Frames (ORFs) assigned to KEGG category xenobiotics biodegradation and metabolism were significantly greater in the LWB-enriched diet compared to the control and included the pathway for benzoate degradation [PATH:ko00362] and glycosaminoglycan degradation [PATH:ko00531]. Moreover, the number of ORFs assigned to the bacterial invasion of epithelial cells [PATH:ko05100] pathway was approximately 8 fold lower in the LWB group compared to controls. This study demonstrated that LWBs have the potential to promote gut health and can aid in the development of optimal diets.

The effect of American cranberry (Vaccinium macrocarpon) constituents on the growth inhibition, membrane integrity, and injury of Escherichia coli O157:H7 and Listeria monocytogenes in comparison to Lactobacillus rhamnosus.

The antimicrobial properties of the American cranberry were studied against Escherichia coli O157:H7, Listeria monocytogenes, and Lactobacillus rhamnosus to determine the effects on growth inhibition, membrane permeability, and injury. Cranberry powder was separated using a C-18 Sep-Pak cartridge into sugars plus organic acids (F1), monomeric phenolics (F2), and anthocyanins plus proanthocyanidins (F3). Fraction 3 was further separated into anthocyanins (F4) and proanthocyanidins (F5) using an LH-20 Sephadex column. Each fraction was diluted in the brain heart infusion (BHI) broth to determine the minimum inhibitory/bactericidal concentrations (MIC/MBC). L. monocytogenes was the most susceptible to cranberry fraction treatment with the lowest MIC/MBC for each treatment, followed by E. coli O157:H7 and L. rhamnosus. Membrane permeability and potential was studied using LIVE/DEAD viability assay and using Bis (1, 3-dibutylbarbituric acid) trimethine oxonol (DiBAC4), respectively. L. rhamnosus demonstrated the highest permeability followed by E. coli O157:H7, and L. monocytogenes. L. rhamnosus demonstrated the highest recovery followed by E. coli O157:H7, and L. monocytogenes. Each cranberry fraction demonstrated membrane hyperpolarization at their native pH, while F2, F3, and F5 demonstrated membrane depolarization at neutral pH. With this knowledge cranberry compounds may be used to prevent maladies and potentially substitute for synthetic preservatives and antibiotics.

Quantitative structure-activity relationships of antimicrobial fatty acids and derivatives against Staphylococcus aureus

Fatty acids and derivatives (FADs) are resources for natural antimicrobials. In order to screen for additional potent antimicrobial agents, the antimicrobial activities of FADs against Staphylococcus aureus were examined using a microplate assay. Monoglycerides of fatty acids were the most potent class of fatty acids, among which monotridecanoin possessed the most potent antimicrobial activity. The conventional quantitative structure-activity relationship (QSAR) and comparative molecular field analysis (CoMFA) were performed to establish two statistically reliable models (conventional QSAR: R2=0.942, Q2LOO=0.910; CoMFA: R2=0.979, Q2=0.588, respectively). Improved forecasting can be achieved by the combination of these two models that provide a good insight into the structure-activity relationships of the FADs and that may be useful to design new FADs as antimicrobial agents.