Richard A. Holley

Antibacterial activity of selected fatty acids and essential oils against six meat spoilage organisms

The antibacterial activity of selected fatty acids and essential oils was examined against two gram-negative (Pseudomonas fluorescens and Serratia liquefaciens) and four gram-positive (Brochothrix thermosphacta, Carnobacterium piscicola, Lactobacillus curvatus, and Lactobacillus sake) bacteria involved in meat spoilage. Various amounts of each preservative were added to brain heart infusion or MRS (deMan, Rogosa and Sharpe) agars, and the minimum inhibitory concentration was determined for each organism. Essential oils were analysed by gas-liquid chromatography to determine the concentration of selected components commonly found in spices. B. thermosphacta, P. fluorescens and S. liquefaciens were not affected by fatty acids, and generally overcame the inhibitory effect of essential oils after 24 h of exposure. Among the fatty acids, lauric and palmitoleic acids exhibited the greatest inhibitory effect with minimum inhibitory concentrations of 250 to 500 micrograms/ml, while myristic, palmitic, stearic and oleic acids were completely ineffective. For essential oils, clove, cinnamon, pimento, and rosemary were found to be the most active. The 1/100 dilution of those oils inhibited at least five of the six tested organisms. A relationship was found between the inhibitory effect of essential oils and the presence of eugenol and cinnamaldehyde.

Inhibition of surface spoilage bacteria in processed meats by application of antimicrobial films prepared with chitosan

A study was undertaken to evaluate the feasibility of using antimicrobial films, designed to slowly release bacterial inhibitors, to improve the preservation of vacuum-packaged processed meats during refrigerated storage. The antimicrobial films were prepared by incorporating acetic or propionic acid into a chitosan matrix, with or without addition of lauric acid or cinnamaldehyde, and were applied onto bologna, regular cooked ham, or pastrami. At various times during storage, packages were opened and the amounts of antimicrobial agents remaining in the chitosan matrix were measured. Regardless of film composition or meat product type, propionic acid was nearly completely released from the chitosan matrix within 48 h of application, whereas release of acetic acid was more limited, with 2-22% of the acid remaining in chitosan after 168 h of storage. Addition of lauric acid, but not cinnamaldehyde, to the chitosan matrix generally reduced the release of acetic acid significantly (P < or = 0.05) and the release was more limited onto bologna than onto ham or pastrami. In addition, the efficacies of the various films for inhibiting bacterial growth were tested against indigenous lactic acid bacteria and Enterobacteriaceae, and against Lactobacillus sakei or Serratia liqueficiens, surface-inoculated onto the meat products. Whereas lactic acid bacteria were not affected by the antimicrobial films under study, the growth of Enterobacteriaceae and S. liquefaciens was delayed or completely inhibited as a result of film application. Strongest inhibition was observed on drier surfaces (bologna), onto which acid release was slower, and with films containing cinnamaldehyde, as a result of its greater antimicrobial activity under these conditions.

Mechanisms of bactericidal action of cinnamaldehyde against Listeria monocytogenes and of Eugenol against L. monocytogenes and Lactobacillus sakei

ABSTRACT The spice oil components eugenol and cinnamaldehyde possess activity against both gram-positive and gram-negative bacteria, but the mechanisms of action remain obscure. In broth media at 20°C, 5 mM eugenol or 30 mM cinnamaldehyde was bactericidal (>1-log reduction in the number of CFU per milliliter in 1 h) to Listeria monocytogenes. At a concentration of 6 mM eugenol was bactericidal to Lactobacillus sakei, but treatment with 0.5 M cinnamaldehyde had no significant effect. To investigate the role of interference with energy generation in the mechanism of action, the cellular and extracellular ATP levels of cells in HEPES buffer at 20°C were measured. Treatment of nonenergized L. monocytogenes with 5 mM eugenol, 40 mM cinnamaldehyde, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 5 min prevented an increase in the cellular ATP concentration upon addition of glucose. Treatment of energized L. monocytogenes with 40 mM cinnamaldehyde or 10 μM CCCP caused a rapid decline in cellular ATP levels, but 5 mM eugenol had no effect on cellular ATP. Treatment of L. sakei with 10 mM eugenol prevented ATP generation by nonenergized cells and had no effect on the cellular ATP of energized cells. CCCP at a concentration of 100 μM had no significant effect on the cellular ATP of L. sakei. No significant changes in extracellular ATP were observed. Due to their rapidity, effects on energy generation clearly play a major role in the activity of eugenol and cinnamaldehyde at bactericidal concentrations. The possible mechanisms of inhibition of energy generation are inhibition of glucose uptake or utilization of glucose and effects on membrane permeability.

Interaction of monolaurin, eugenol and sodium citrate on growth of common meat spoilage and pathogenic organisms.

Interactions of monolaurin, eugenol (phenolic compound) and sodium citrate (chelator) on the growth of six organisms including common meat spoilage (Lactobacillus curvatus, Lactobacillus sake, Leuconostoc mesenteroides, Brochothrix thermosphacta) and pathogenic (Escherichia coli O157:H7 and Listeria monocytogenes) organisms were investigated. The combinations of 100 to 250 ppm monolaurin with 500 and 1000 ppm eugenol, and 0.2 and 0.4% sodium citrate were more effective than each component separately. More than one combination prevented detectable growth of each organism. Lactic acid bacteria (LAB) and E. coli O157:H7 were most resistant and L. monocytogenes and B. thermosphacta most sensitive to control by the chosen combinations. The presence of sodium citrate was necessary to yield potent inhibition of Lb. curvatus and Lb. sake growth by the monolaurin and eugenol combinations.

Use of natural antimicrobials to increase antibiotic susceptibility of drug resistant bacteria

Plant-derived antibacterial compounds may be of value as a novel means for controlling antibiotic resistant zoonotic pathogens which contaminate food animals and their products. Individual activity of natural antimicrobials (eugenol, thymol, carvacrol, cinnamaldehyde, allyl isothiocyanate (AIT)) and activity when paired with an antibiotic was studied using broth microdilution and checkerboard methods. In the latter assays, fractional inhibitory concentration (FIC) values were calculated to characterize interactions between the inhibitors. Bacteria tested were chosen because of their resistance to at least one antibiotic which had a known genetic basis. Substantial susceptibility of these bacteria toward the natural antimicrobials and a considerable reduction in the minimum inhibitory concentrations (MICs) of the antibiotics were noted when paired combinations of antimicrobial and antibiotic were used. In the interaction study, thymol and carvacrol were found to be highly effective in reducing the resistance of Salmonella Typhimurium SGI 1 (tet A) to ampicillin, tetracycline, penicillin, bacitracin, erythromycin and novobiocin (FIC<0.4) and resistance of Streptococcus pyogenes ermB to erythromycin (FIC<0.5). With Escherichia coli N00 666, thymol and cinnamaldehyde were found to have a similar effect (FIC<0.4) in reducing the MICs of ampicillin, tetracycline, penicillin, erythromycin and novobiocin. Carvacrol, thymol (FIC<0.3) and cinnamaldehyde (FIC<0.4) were effective against Staphylococcus aureus blaZ and in reducing the MICs of ampicillin, penicillin and bacitracin. Allyl isothiocyanate (AIT) was effective in reducing the MIC of erythromycin (FIC<0.3) when tested against S. pyogenes. Fewer combinations were found to be synergistic when the decrease in viable population (log DP) was calculated. Together, fractional inhibitory concentrations < or = 0.5 and log DP<-1 indicated synergistic action between four natural antimicrobials and as many as three antibiotics to which these bacteria were normally resistant.

Interactive inhibition of meat spoilage and pathogenic bacteria by lysozyme, nisin and EDTA in the presence of nitrite and sodium chloride at 24 °C

To develop a nisin- and lysozyme-based antimicrobial treatment for use with processed ham and bologna, in vitro experiments were conducted to determine whether inhibition enhancing interactions occur between the antimicrobials lysozyme, chrisin (a commercial nisin preparation), EDTA, NaCl and NaNO(2). Inhibitory interactions were observed between a number of agents when used against specific pathogenic and spoilage bacteria. The observed interactions included lysozyme with EDTA (Enterococcus faecalis and Weissella viridescens), chrisin with EDTA (all Gram-positive organisms), EDTA with NaCl (Escherichia coli, Salmonella enterica serovar Typhimurium, Serratia grimesii), EDTA with nitrite (E. coli, Lactobacillus curvatus, Leuconostoc mesenteroides, Listeria monocytogenes, S. Typhimurium), chrisin with nitrite (Lc. mesenteroides, L. monocytogenes), and NaCl with nitrite (S. Typhimurium, Shewanella putrefaciens). Previous reports have described interactions between nisin with EDTA that resulted in enhanced antimicrobial effect against Gram-negative bacteria, or lysozyme with nisin against Gram-positive bacteria. These interactions were not observed in these experiments. We observed that unlike previous studies, these experiments were conducted on growing cells in nutrient broth, rather than under conditions of nutrient limitation. We propose that screening of antimicrobials for use in food systems in nutrient-deficient systems is inappropriate and that new protocols should be developed.

Enzymatic inhibition by allyl isothiocyanate and factors affecting its antimicrobial action against Escherichia coli O157:H7

Allyl isothiocyanate (AIT) is derived from the glucosinolate sinigrin found in plants of the family Brassicaceae. It is a well-recognized antimicrobial agent against a variety of organisms, including foodborne pathogens such as Escherichia coli O157:H7. The efficiency of this natural agent in reducing E. coli O157:H7 numbers in food products have been reported. However, few have examined the mechanism by which AIT, and perhaps most of the isothiocyanates, kill E. coli O157:H7. In the present report, AIT showed greater antimicrobial activity at low pH values. For example, at pH 4.5 and 5.5 the MIC was 25 microL/L, while at pH 8.5, 500 microL/L was required to inhibit bacterial growth. This mustard-derived compound exhibited a high decomposition rate in water at 37 degrees C. Its degradation profile contained 3 major products and of these, diallylthiourea represented the largest ( approximately 80%) component. The decomposition products did not show antimicrobial activity towards E. coli O157:H7, even when combined with a sub-lethal dose of AIT (10 microL/L). AIT may only be antimicrobial in its original form and any further degradation in water is undesirable. AIT interactions with thioredoxin reductase and acetate kinase were also subjects of this study. AIT at 10 to 100 microL/L was able to significantly inhibit both enzymes, but only 1 microL/L was needed to decrease the activity of thioredoxin reductase. From these results, it can be postulated that: 1) AIT is a more effective antimicrobial at low pH values and its degradation reduces this activity; 2) decomposition products in water might not participate in the antimicrobial action of AIT; and 3) AIT seems to have a multi-targeted mechanism of action, perhaps inhibiting several metabolic pathways and damaging cellular structures.

Inhibition of bacterial growth on ham and bologna by lysozyme, nisin and EDTA

Abstract Ham and bologna sausages were prepared with or without addition of 500 mg kg −1 lysozyme:nisin, 1:3, and 500 mg. kg −1 EDTA. Sausages were inoculated with one of; Brochothrix thermosphacta , Escherichia coli O157:H7, Lactobacillus sakei , Lactobacillus curvatus , Leuconostoc mesenteroides , Listeria monocytogenes , Salmonella typhimurium , Serratia grimesii or Shewanella putrefaciens , vacuum packed and stored for 4 weeks at 8°C. Plate counts were made on selective and nonselective media. Inhibitor treatment reduced initial populations of B. thermosphacta and Lc. mesenteroides on both meats . Treatment of ham and bologna prevented growth of B. thermosphacta , to week 4. Treatment reduced growth of Lb. curvatus on ham and bologna, to week 3. Treatment of bologna reduced growth of Lc. mesenteroides and L. monocytogenes for 2 weeks. Treatment of ham reduced growth of E. coli O157:H7 for 4 weeks. On treated ham the growth of S. typhimurium was increased from week 3. No difference was observed between control and treatment samples with other organisms.

High Hydrostatic Pressure Effects on the Texture of Meat and Meat Products

High hydrostatic pressure (HHP) treatment can influence meat protein conformation and induce protein denaturation, aggregation, or gelation. The means whereby HHP treatment exerts effects on meat protein structure change are due to the rupture of noncovalent interactions within protein molecules, and to the subsequent re-formation of intra- and inter-molecular bonds within or among protein molecules. Depending upon the meat protein system, the pressure, the temperature, and the duration of the pressure treatment, meat can be either tenderized or toughened. Muscle texture variation induced by heat treatment is due to breakage of hydrogen bonds, whereas changes from high pressure treatment are due to the rupture of hydrophobic and electrostatic interactions. Pressure treatment has little effect on the toughness of connective tissue. Juiciness, springiness, and chewiness are increased upon HHP treatment. Prerigor HHP treatment tenderizes meat, whereas tenderizing effects of postrigor HHP treatment are only measureable if pressure and heat treatment are combined. The limitations and future applications of high pressure technology are also discussed.

Surface Application of Lysozyme, Nisin, and EDTA to Inhibit Spoilage and Pathogenic Bacteria on Ham and Bologna

A study was conducted to determine if the effectiveness of an antimicrobial treatment for cooked ham and bologna would be increased or maintained when applied in a surface coating. Cooked 10-g disks of ham and bologna sausage received one of three treatments: no coating (control), coating with 0.2 g of 7% (wt/vol) gelatin gel (gel-control), or coating with 0.2 g of 7% gelatin gel containing 25.5 g/liter of lysozyme-nisin (1:3) plus 25.5 g/liter of EDTA (gel-treated). The samples were then inoculated with one of six test organisms: Brochothrix thermosphacta, Escherichia coli O157:H7, Lactobacillus sakei, Leuconostoc mesenteroides, Listeria monocytogenes, or Salmonella Typhimurium. Inoculated samples were vacuum packed and stored at 8 degrees C for 4 weeks. The antimicrobial gel treatment had an immediate bactericidal effect up to 4 log CFU/cm2 on the four gram-positive organisms tested (B. thermosphacta, Lactobacillus sakei, Leuconostoc mesenteroides, and Listeria monocytogenes) and inhibited the growth of these organisms during the 4 weeks of storage. The antimicrobial gel treatment also had a bactericidal effect on the growth of Salmonella Typhimurium during storage. The numbers of E. coli O157:H7 on ham were reduced by 2 log CFU/cm2 following treatment with both antimicrobial-containing and non-antimicrobial-containing gels during the 4-week storage period. No effect was observed on the growth of E. coli O157:H7 on bologna.