The antimicrobial effect of thyme essential oil, nisin and their combination against Escherichia coli O157:H7 in minced beef during refrigerated storage

The antimicrobial effect of thyme essential oil (EO) at supplementation levels of 0.3%, 0.6% or 0.9%, nisin at 500 or 1000 IU/g, and their combination, on Escherichia coli O157:H7 was examined in both tryptic soy broth (TSB) and minced beef meat. EO at 0.3% possessed a weak antibacterial activity against the pathogen in TSB, whereas at 0.9% showed unacceptable organoleptic properties in minced meat. Thus, only the level of 0.6% of EO was further examined against the pathogens in minced meat. Treatment of minced beef meat with EO at 0.6% showed an inhibitory activity against E. coli O157:H7 during storage at 10 °C, but not at 4 °C. Treatment of minced beef meat or TSB with nisin at 500 or 1000 IU/g did not show any antibacterial activity against E. coli O157:H7. The combination of EO at 0.6% and nisin at 500 or 1000 IU/g showed an additive effect against the pathogen, which was higher during storage at 10 °C than at 4 °C.     

Verocytotoxigenic Escherichia coli O157 in beef and sheep abattoirs in Ireland and characterisation of isolates by Pulsed-Field Gel Electrophoresis and Multi-Locus Variable Number of Tandem Repeat Analysis

This study aimed to investigate verocytotoxigenic Escherichia coli O157 in the largest beef and sheep slaughter plants in Ireland over a one-year period. Samples consisted of pooled rectal swabs (n = 407) and pooled carcass swabs (n = 407) from 5 animals belonging to the same herd or flock and minced meat (n = 91) from the same sampling date. E. coli O157 isolates were characterised using PCR for a range of genes, i.e. 16S, rfbE, fliC, vtx1, vtx2, eaeA and confirmed VTEC O157 isolates were tested for antimicrobial susceptibility and typed using Pulsed-Field Gel Electrophoresis (PFGE) and Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA). VTEC O157 was isolated from 7.6% and 3.9% of bovine rectal and carcass swab samples and from 5.8% and 2.9% of ovine rectal and carcass swab samples respectively. None of the bovine minced meat samples (n = 77) and only one of the 14 ovine minced meat samples was positive for VTEC O157. Following PFGE and MLVA, cross contamination from faeces to carcasses was identified. While PFGE and MLVA identified the same clusters for highly related strains, MLVA discriminated better than PFGE in addition to being more rapid and less labour intensive. Results showed that cattle and sheep presented for slaughter in Ireland harbour VTEC O157, and although the levels entering the food chain are low, this should not be overlooked as possible sources of zoonotic infection; molecular typing was able to demonstrate relationships among strains and could be used to elucidate the sources of human infection.     

A Weibull model to describe antimicrobial kinetics of oregano and lemongrass essential oils against Salmonella Enteritidis in ground beef during refrigerated storage

The antimicrobial effect of oregano (Origanum vulgare L.) and lemongrass (Cymbopogon citratus (DC.) Stapf.) essential oils (EOs) against Salmonella enterica serotype Enteritidis in in vitro experiments, and inoculated in ground bovine meat during refrigerated storage (4 ± 2 °C) for 6 days was evaluated. The Weibull model was tested to fit survival/inactivation bacterial curves (estimating of p and δ parameters). The minimum inhibitory concentration (MIC) value for both EOs on S. Enteritidis was 3.90 μl/ml. The EO concentrations applied in the ground beef were 3.90, 7.80 and 15.60 μl/g, based on MIC levels and possible activity reduction by food constituents. Both evaluated EOs in all tested levels, showed antimicrobial effects, with microbial populations reducing (p ≤ 0.05) along time storage. Evaluating fit-quality parameters (RSS and RSE) Weibull models are able to describe the inactivation curves of EOs against S. Enteritidis. The application of EOs in processed meats can be used to control pathogens during refrigerated shelf-life.     

Combined antimicrobial effect of oregano essential oil and caprylic acid in minced beef

Oregano essential oil (OEO) and caprylic acid (CA) are highly aromatic natural antimicrobials with limited individual application in food. We proved their combined additive effect when used in meat. Application of 0.5% CA and 0.2% OEO (v/w) with 0.1% of citric acid in vacuum packed minced beef inoculated with Listeria monocytogenes at a concentration of 5 log cells/g reduced counts of lactic acid bacteria by 1.5 log CFU/g and counts of psychrotrophic bacteria and L. monocytogenes by more than 2.5 log CFU/g at the end of storage at 3 °C for 10 days. In sensory evaluation the samples with OEO showed during the whole experiment statistically better scores than control, whereas the samples treated with CA showed worse colour attributes.     

Biological activity and chemical composition of different berry juices

Total phenolics, total anthocyanins, mineral content, radical scavenging activity and antiproliferative activity against human cancer cell lines were evaluated in fresh pressed juices of five different berries. Total phenolic content ranged from 133.0 to 260.3 mg of gallic acid equivalents/100 g of fresh weight, for red currant and black currant, respectively. Bilberry juice contained the highest amount of total anthocyanins (0.18%). Significant correlation between total phenolics content and radical scavenging activity was observed (r = −0.980; p < 0.01). All examined juices showed antiproliferative activity in dose-dependent manner with IC₅₀ ranging from 10.2 to 70.5 μl/ml. Black currant juice was the most effective inhibitor of proliferation in all cell lines tested (HeLa, Fem X, LS 174, MCF-7 and PC-3). Significant correlations of acidity and total anthocyanin content with antiproliferative activity of berry juices on HeLa cells, Fem X cells and MCF-7 cells were noticed. Berry juices are good sources of some minerals and contribute significantly to daily intake of these micronutrients.     

Salmonella surveillance and control at post-harvest in the Belgian pork meat chain

Salmonella remains the primary cause of reported bacterial food borne disease outbreaks in Belgium. Pork and pork products are recognized as one of the major sources of human salmonellosis. In contrast with the primary production and slaughterhouse phases of the pork meat production chain, only a few studies have focussed on the post-harvest stages. The goal of this study was to evaluate Salmonella and Escherichia coli contamination at the Belgian post-harvest stages. E. coli counts were estimated in order to evaluate the levels of faecal contamination. The results of bacteriological analysis from seven cutting plants, four meat-mincing plants and the four largest Belgian retailers were collected from official and self-monitoring controls. The prevalence of Salmonella in the cutting plants and meat-mincing plants ranged from 0% to 50%. The most frequently isolated serotype was Salmonella typhimurium. The prevalence in minced meat at retail level ranged from 0.3% to 4.3%. The levels of Salmonella contamination estimated from semi-quantitative analysis of data relating to carcasses, cuts of meat and minced meat were equal to −3.40 ± 2.04 log CFU/cm², −2.64 ± 1.76 log CFU/g and −2.35 ± 1.09 log CFU/g, respectively. The E. coli results in meat cuts and minced meat ranged from 0.21 ± 0.50 to 1.23 ± 0.89 log CFU/g and from 1.33 ± 0.58 to 2.78 ± 0.43 log CFU/g, respectively. The results showed that faecal contamination still needs to be reduced, especially in specific individual plants.     

The spoilage characteristics of Brochothrix thermosphacta and two psychrotolerant Enterobacteriacae in vacuum packed lamb and the comparison between high and low pH cuts

The spoilage potential of Brochothrix thermosphacta, Serratia proteamaculans and Rahnella aquatilis was investigated in vacuum packaged high (5.9 to 6.4) and low (5.4 to 5.8) pH lamb. Vacuum packaged fore shank (m. extensor carpi radialis) and striploins (m. longissimus dorsi) (n = 306) inoculated with ~ 100 CFU of individual bacteria were stored for twelve weeks at temperatures − 1.5, 0, 2 and 7 °C. Spoilage characteristics and bacterial numbers were recorded and analysed in comparison to un-inoculated control samples. All three bacterial species were shown to grow in vacuum packaged lamb of pH values between 5.4 and 6.4, when stored at chilled temperatures (− 1.5 to 7 °C) for up to 84 days. B. thermosphacta and S. proteamaculans caused spoilage to the meat under these conditions whilst R. aquatilis spoiled high pH meat at 7 °C. These results go against previous beef models stipulating that Brochothrix and Enterobacteriacae species cannot grow on or cause spoilage of low pH meat in the absence of oxygen.     

Microbiological and organoleptic characteristics of beef trim and ground beef treated with acetic acid, lactic acid, acidified sodium chlorite, or sterile water in a simulated commercial processing environment to reduce Escherichia coli O157:H7 and Salmonella

The objective of this study was to validate the effectiveness of acetic and lactic acids (2% and 5%), acidified sodium chlorite (1000 ppm), and sterile water in reducing Escherichia coli O157:H7 and Salmonella Typhimurium in inoculated beef trim in a simulated processing environment. Samples were collected to assess microbial characteristics at three processing points. Results from this study indicate that all treatments, including sterile water, reduced pathogen concentrations (P < 0.05) of both E. coli O157:H7 and Salmonella Typhimurium in ground beef up to 0.5 and 0.6 log by 24 h, respectively. In some cases, there were no significant differences between the antimicrobial treatments and the sterile water using this application method. Triangle sensory test results of non-inoculated beef indicated there were no differences (P < 0.05) in the means of correct responses between controls or antimicrobial treatments at 6 or 24 h. While interventions are important for beef trim, use of the interventions must be validated under industry conditions to ensure proper effectiveness.     

Protective action of Lactobacillus curvatus CRL705 on vacuum-packaged raw beef. Effect on sensory and structural characteristics

Lactobacillus curvatus CRL705 was examined for its effectiveness as protective culture in the biopreservation of vacuum-packaged fresh beef stored during 60 days at 2 °C. For this purpose, L. curvatus CRL705, producer of lactocin 705 and lactocin AL705, was inoculated on the meat surface (10⁶ cfu g⁻¹). This microorganism became the dominating population throughout the storage period controlling the growth of Brochothrix thermosphacta and spoilage lactic acid bacteria naturally present on the meat. When the microstructural characteristics of the meat were evaluated using light microscopy, beef samples inoculated with the bioprotective culture showed a 10 days delay for the appearance of tissue degradation signs. Sensory analysis demonstrated that beef samples treated with L. curvatus CRL705 only developed an “acid” off-flavor after 60 days of refrigerated storage, and no undesirable off-odors were found. Therefore, inoculation with this bacteriocinogenic strain would provide an additional hurdle to improve storage life of refrigerated vacuum-packaged beef without affecting its sensory and structural characteristics.     

Characterization of the Enterobacteriaceae community that developed during storage of minced beef under aerobic or modified atmosphere packaging conditions

The whole cell protein and macrorestriction analysis of DNA of Enterobacteriaceae isolates recovered from minced beef stored at 0, 5, 10 and 15 °C aerobically and under modified atmosphere packaging consisting of 40% CO₂-30% O₂-30% N₂ in the presence (MAP+) and absence (MAP−) of oregano essential oil were studied. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) profiles obtained from whole cell protein analysis of the Enterobacteriaceae isolates revealed seven groups. Moreover, application of a modified PFGE protocol with XbaI restriction, resulted into 19 different fingerprints. The Enterobacteriaceae community of fresh meat consisted of Serratia liquefaciens and Serratia proteamaculans. S. liquefaciens strain VK23 was the dominant isolate of Enterobacteriaceae for the most conditions adopted, except 10 °C and 15 °C under MAP + and 10 °C under MAP−. In the latter cases, Hafnia alvei represented the dominant fingerprint. Citrobacter freundii was recovered from minced beef stored aerobically, while H. alvei and Proteus vulgaris were recovered under MAP. Storage conditions affected the Enterobacteriaceae community; modified atmosphere packaging increased both species and strain diversity.     

The effect of phytic acid on oxidative stability of raw and cooked meat

The effects of phytic acid addition (0.1, 1 and 5 mM) to pork and beef homogenates on TBARS and metmyoglobin levels in raw meat, and TBARS and heme iron contents in cooked meat during 3 days of storage at 4 °C were investigated. Also, the role of inositol as a potential synergist of IP₆ (phytic acid) was examined. IP₆ effectively decreased the TBARS accumulation in raw and cooked meat homogenates. The metmyoglobin formation was inhibited in raw beef by phytic acid in a dose-dependent manner. The effect of IP₆ was more pronounced in cooked meat than in raw and in cooked beef homogenates more than pork. Inositol did not enhance antioxidant action of phytic acid in minced meat.     

The antimicrobial effect of thyme essential oil, nisin, and their combination against Listeria monocytogenes in minced beef during refrigerated storage

The antimicrobial effect of thyme essential oil (EO) at 0.3%, 0.6%, or 0.9%, nisin at 500 or 1000IU/g, and their combination against Listeria monocytogenes was examined in both tryptic soy broth (TSB) and minced beef meat. Thyme EO at 0.3% possessed a weak antibacterial activity against the pathogen in TSB, whereas at 0.9% showed unacceptable organoleptic properties in minced meat. Thus, only the level of 0.6% of EO was further examined against the pathogen in minced meat. Treatment of minced beef meat with nisin at 500 or 1000IU/g showed antibacterial activity against L. monocytogenes, which was dependent on the concentration level of nisin and the strains used. Treatment of minced beef meat with EO at 0.6% showed stronger inhibitory activity against L. monocytogenes than treatment with nisin at 500 or 1000IU/g. All treatments showed stronger inhibitory activity against the pathogens at 10 degrees C than at 4 degrees C. The combined addition of EO at 0.6% and nisin at 500 or 1000IU/g showed a synergistic activity against the pathogen. Most efficient among treatments was the combination of EO at 0.6% with nisin at 1000IU/g, which decreased the population of L. monocytogenes below the official limit of the European Union recently set at 2logcfu/g, during storage at 4 degrees C.     

Effect of adding essential oils of coriander (Coriandrum sativum L.) and hyssop (Hyssopus officinalis L.) on the shelf life of ground beef

This study examined the effect of adding essential oils of hyssop and coriander at the highest concentration (0.02% v/w) sensorially acceptable to a panel of assessors on the microbiological and biochemical characteristics of stored ground beef. Vacuum-packed meat was stored at 0.5 ± 0.5 °C and 6 ± 1 °C for 15 days. The greatest beneficial effect of both additives was in inhibiting the development of undesirable sensory changes (extending acceptability by up to 3 days) and the growth of Enterobacteriaceae (by up to approximately 1-2 log cycles compared with the controls). The effect on lactic acid bacteria, total viable bacterial count and other groups of microorganisms investigated was minor (up to 1 log cycle) and similar for both oils. Neither did these additives significantly affect amino nitrogen levels, protease activity, the proportions of meat pigments, protein electropherograms and pH levels. This indicates the limited effect of these essential oils in the concentrations applied on preserving vacuum-packed minced beef.     

Enhancing the thermal destruction of Escherichia coli O157:H7 in ground beef patties by trans-cinnamaldehyde

The effect of trans-cinnamaldehyde (TC) on the inactivation of Escherichia coli O157:H7 in undercooked ground beef patties was investigated. A five-strain mixture of E. coli O157:H7 was inoculated into ground beef (7.0 log CFU/g), followed by addition of TC (0, 0.15, and 0.3%). The meat was formed into patties and stored at 4 °C for 5 days or at −18 °C for 7 days. The patties were cooked to an internal temperature of 60 or 65 °C, and E. coli O157:H7 was enumerated. The numbers of E. coli O157:H7 did not decline during storage of patties. However, cooking of patties containing TC significantly reduced (P < 0.05) E. coli O157:H7 counts, by >5.0 log CFU/g, relative to the reduction in controls cooked to the same temperatures. The D-values at 60 and 65 °C of E. coli O157:H7 in TC-treated patties (1.85 and 0.08 min, respectively) were significantly lower (P < 0.05) than the corresponding D-values for the organism in control patties (2.70 and 0.29 min, respectively). TC-treated patties were more color stable and showed significantly lower lipid oxidation (P < 0.05) than control samples. TC enhanced the heat sensitivity of E. coli O157:H7 and could potentially be used as an antimicrobial for ensuring pathogen inactivation in undercooked patties. However detailed sensory studies will be necessary to determine the acceptability to consumers of TC in ground beef patties.     

Antibacterial effects of natural tenderizing enzymes on different strains of Escherichia coli O157:H7 and Listeria monocytogenes on beef

This study determined the efficacy of actinidin and papain on reducing Listeria monocytogenes and three mixed strains of Escherichia coli O157:H7 populations on beef. The average reduction of E. coli O157:H7 was greater than that of L. monocytogenes and higher concentrations of either protease yielded greater reduction in bacterial populations. For instance, actinidin at 700 mg/ml significantly (p ≤ 0.05) reduced the population of L. monocytogenes by 1.49 log cfu/ml meat rinse after 3 h at 25 & 35 °C, and by 1.45 log cfu/ml rinse after 24 h at 5 °C, while the same actinidin concentration significantly reduced the populations of three mixed strains of E. coli O157:H7 by 1.81 log cfu/ml rinse after 3 h at 25 & 35 °C, and 1.94 log cfu/ml rinse after 24 h at 5 °C. These findings suggest that, in addition to improving the sensory attributes of beef, proteolytic enzymes can enhance meat safety when stored at suitable temperatures.     

Antimicrobial and human cancer cell cytotoxic effect of synthetic angiotensin-converting enzyme (ACE) inhibitory peptides

Four peptides with high angiotensin-converting enzyme (ACE) inhibitory effect were separated from beef sarcoplasmic protein hydrolysates using commercial enzymes. They were identified as GFHI, DFHING, FHG, and GLSDGEWQ and their 50% inhibition concentration (IC₅₀) values against ACE were 117, 64.3, 52.9, and 50.5 μg/ml, respectively. These peptides were synthesised and further biological activities of these four peptides were measured, including antimicrobial, cytotoxic effect against cancer cells, and macrophage-stimulating effect. Peptide GLSDGEWQ showed growth inhibition on Salmonella Typhimurium, Bacillus cereus, Escherichia coli, and Listeria monocytogenes at a 100 ppm level but not on Staphylococcus aureus and Pseudomonas aeruginosa. Peptide GFHI showed higher inhibition activity on the growth of E. coli and P. aeruginosa at concentrations of 200 and 400 μg/ml. However, peptide FHG inhibited only P. aeruginosa at 200 and 400 μg/ml. The effect of separated peptides on breast cancer (MCF-7), lung cancer (A549), and stomach cancer (AGS) cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Peptide GFHI showed a slight decrease of MCF-7 cell viability in a dose dependent manner. When 400 μg/ml of peptide GFHI was applied to the AGS cell, its viability was decreased by 75%. However, peptide DFHINQ seemed to act as a nutrient to AGS cell because it increased its viability. None of the four peptides had a cytotoxic effect on A549 cells. Nitric oxide (NO) production of peptide GFHI by stimulation of macrophage was investigated at 100, 300, and 1000 μg/ml concentration. NO was not produced in all treatments. From these results it is expected that the ACE inhibitory peptides identified from beef sarcoplasmic protein hydrolysates have both antimicrobial and cancer cell cytotoxic effects.     

Control of foodborne pathogens on ready-to-eat roast beef slurry by ε-polylysine

This study evaluates the antimicrobial effectiveness of ε-polylysine against Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes in laboratory media and roast beef slurry. ε-Polylysine supplemented laboratory media and roast beef slurry were inoculated with three-strain cocktails of each pathogen and survival was periodically monitored using conventional spread plating. Inoculated laboratory media was stored at room temperature (22 °C) for 48 h, and inoculated roast beef slurry was stored at 4 °C for up to 7 days. Maximum log reductions in laboratory media/roast beef slurry were 6.01 ± 1.43/3.81 ± 0.37, > 7.82 ± 0.05/5.23 ± 0.08, and 4.58 ± 0.86/5.83 ± 0.48 for E. coli O157:H7, S. tyhimurium, and L. monocytogenes, respectively. Injured cells were produced as a result of exposure to polylysine. This study confirms the effectiveness of polylysine against pathogens in laboratory media, and demonstrates its potential as a novel antimicrobial agent in complex food matrix such as roast beef.     

Inhibition of verocytotoxigenic Escherichia coli by antimicrobial peptides caseicin A and B and the factors affecting their antimicrobial activities

The antimic robial activities of caseicin A and B antimicrobial peptides (AMPs) were assessed against a selection of verocytotoxigenic Escherichia coli (VTEC) strains (n = 11), other bacterial pathogenic and spoilage bacteria (n = 7), using a model broth system. The ability of the AMPs to retain their antimicrobial activities against a strain of E. coli O157:H7 380-94 under various test conditions (pH, temperature, water activity, sodium chloride concentrations, inoculum size and the presence of competitive microflora) was assessed and the minimum inhibitory concentrations (MIC) and number of surviving E. coli O157:H7 calculated. The mean number of VTEC surviving after exposure to 2 mg/ml caseicin A and B was reduced by 4.96 and 4.19 log₁₀ cfu/ml compared to the respective controls. The susceptibility of E. coli O157:H7 to the caseicin AMPs decreased as temperature, pH, water activity and inoculum size were reduced. The presence of sodium chloride (0.5-2.5%) did not affect the activity of caseicin A (p > 0.05), however it did inhibit the activity of caseicin B. The presence of a competitive microflora cocktail did not significantly (p > 0.05) affect the activities of the AMPs for the majority of the concentrations tested. Using a quantitative PCR assay, the levels of verotoxins (vt1 and vt2) expressed by E. coli O157:H7 following exposure to a sub-inhibitory concentration (0.5 mg/ml) of caseicin A showed that the verotoxin levels did not differ from the levels produced by the control cultures. The antimicrobial activity of caseicin A against E. coli O157:H7 was also tested in a model rumen system, however concentrations of ≥ 2 mg/ml did not significantly (p > 0.05) reduce E. coli O157:H7 numbers in the model system over a 24 h period. The application of caseicin AMPs in food and/or animal production may be valuable in combination with other antimicrobials although further research is required.     

Tunisian Salvia officinalis L. and Schinus molle L. essential oils: their chemical compositions and their preservative effects against Salmonella inoculated in minced beef meat

The essential oils (EOs) extracted from the aerial parts of cultivated Salvia officinalis L. and the berries of Schinus molle L. were analysed by gas chromatography–mass spectrometry (GC–MS) and 68 and 67 constituents were identified, respectively. The major constituents were 1,8-cineole (33.27%), β-thujone (18.40%), α-thujone (13.45%), borneol (7.39%) in S. officinalis oil and α-phellandrene (35.86%), β-phellandrene (29.3%), β-pinene (15.68%), p-cymene (5.43%) and α-pinene (5.22%) in S. molle oil.In its second part, the present study was conducted to evaluate the in vitro antimicrobial activity of both studied EOs. For this purpose, paper disc-diffusion method and broth microdilution test were used. The disc-diffusion method showed significant zone of lysis against all the pathogens studied (gram-negative and gram-positive bacteria, yeast). These activities remained stable after six months, and decreased approximately by 20% after one year of storage of the EOs at 4 to 7 °C. On comparing the efficiency of both EOs, S. officinalis EO exhibited higher antibacterial activity against the majority of strains and especially against Candida albicans (two fold more active according to the inhibition zones values). The minimal inhibitory concentrations (MICs) were reported between 4.5 mg/ml and 72 mg/ml on nutrient broth. The particular chemotype of each EO may be involved in its specific antimicrobial behaviour.Furthermore, the inhibitory effect of these EOs were evaluated against two foodborne pathogens belonging to Salmonella genus, experimentally inoculated (10³ CFU/g) in minced beef meat, which was mixed with different concentrations of the EO and stored at 4 to 7 °C for 15 days. Although the antibacterial activities of both EOs in minced beef meat were clearly evident, their addition had notable effects on the flavour and taste of the meat at concentrations more than 2% for S. molle and 1.5% for S. officinalis. One solution to the above-mentioned problem may be the use of combinations of different food preservation systems. In this context, each of the EOs has been used along with low water activity (addition of NaCl) in addition to low refrigeration temperatures. Results on the Salmonella growth showed that some combinations could be recommended to eliminate germs from minced raw beef. By using this method, a stable and, from a microbiological point of view, safe meat can be produced without substantial loss in sensory quality.Results obtained herein, may suggest that the EOs of S. officinalis and S. molle possess antimicrobial activity, and therefore, they can be used in biotechnological fields as natural preservative ingredients in food and/or pharmaceutical industry.