Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4° and 10°C

The effect of mint ( Mentha piperita ) essential oil (0·5, 1·0, 1·5 and 2·0%, v/w) on Salmonella enteritidis and Listeria monocytogenes in a culture medium and three model foods; tzatziki (pH 4·5), taramosalata (pH 5·0) and pâté (pH 6·8), inoculated at 10⁷ cfu g^-1, at 4° and 10°C for ca 1 week was studied. In the culture medium supplemented with the essential oil, no growth was observed over 2 d at 30°C determined by a conductance method with a Malthus 2000 growth analyser. Salmonella enteritidis died in tzatziki in all treatments and declined in the other foods except for pâté at 10°C as judged with viable counts. Listeria monocytogenes populations showed a declining trend towards the end of the storage period but was increased in pâté. Mint essential oil antibacterial action depended mainly on its concentration, food pH, composition, storage temperature and the nature of the micro-organism.     

Effect of oregano essential oil on microbiological and physico-chemical attributes of minced meat stored in air and modified atmospheres

AIMS: This study aimed to determine the combined effect of packaging (air, modified atmosphere) with or without the addition of essential oil not only on the selection of microbial association of meat but also to determine any significant difference in microbial metabolites produced from the prevailing bacteria. METHODS AND RESULTS: Samples of minced meat were mixed with different concentration of oregano essential oil (0, 0.05, 0.5 and 1% v/w) and packed under aerobic or with modified atmosphere (Mixed Gas Modified Atmosphere--MGMA, 40% CO2/30% N2/30% O2; or CO2 Modified Atmosphere--COMA, 100% CO2) and stored at 5 degrees C. In all packaging conditions, only concentrations of 0.5% and 1% oregano oil were effective. Inhibition was evident in the order air < MGMA < COMA. Oregano essential oil delayed glucose and lactate consumption aerobically as well as under MGMA. pH changes were also evident. Furthermore, proteolysis was significantly inhibited in aerobically stored samples, and so was the production of acetate under MAP. Similar results were obtained for the other organic acids eluted from HPLC column. CONCLUSIONS: Oregano essential oil delayed microbial growth and suppressed the final counts of the spoilage micro-organisms. It also caused a pronounced alteration in the physico-chemical properties of the minced meat. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial analysis alone as spoilage index may misrepresent the effect of a hurdle such as essential oils on spoilage.     

Ecophysiological attributes of a Lactobacillus sp. and a Pseudomonas sp. on sterile beef fillets in relation to storage temperature and film permeability

AIMS: To determine the combined effect of packaging film and temperature on the rate and type of end-products caused by the growth of two main contrasting prevailing organisms in air and 100% CO2, Pseudomonas sp. and Lactobacillus sp., respectively. METHODS AND RESULTS: Pseudomonas sp. and Lactobacillus sp. were inoculated individually on sterile meat fillets. The samples were packed in air or 100% CO2, using a high and a low permeable film, and stored at 0 and 10 degrees C. Pseudomonas sp. grew aerobically and in 100% CO2 using high permeable film at both storage temperatures, while film permeability significantly affected the growth of Lactobacillus sp. only at 10 degrees C. Enzymatic kits and HPLC and GC analysis were used to determine the chemical changes of the samples throughout storage. Pseudomonas sp. presented a greater rate of consumption of glucose and lactate than Lactobacillus sp. in samples stored aerobically or with high permeable film. Propanol-1 and two unidentified organic acids were present only in samples inoculated with Pseudomonas sp., while acetaldehyde, ethanol, diacetyl and acetoin were detected in samples inoculated with Lactobacillus sp. CONCLUSION: Since different microbial species and introduction of new packaging methods affect spoilage reactions of meat either qualitatively or quantitatively, a combination of several chemical indicators should be thoroughly investigated. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides information on how and when such potential indicators can be exploited for the benefit of the industry and consumer.     

Bacterial synergism or antagonism in a gel cassette system

The growth and the metabolic activity of Shewanella putrfaciens, Brochothrix thermosphacta, and Pseudomonas sp., when cultured individually or in all possible combinations in gel cassettes system supplemented with 0.1% glucose at 5 degrees C, were investigated. The overall outcome was that the coexistence of the above-mentioned microorganisms affected not only each growth rate but also their type of metabolic end products compared to the control cultures. These effects were varied and depended on the selection of the combination of the tested bacteria. For example, the growth of Pseudomonas sp. strains cocultured with either B. thermosphacta or S. putrefaciens strains resulted in different effects: a promoting one for the first and an inhibitory one for the second. Moreover, the production of formic acid and two unidentified organic acids (peaks a and b) was characteristic in all cases in which S. putrefaciens was cultured.     

Survival of Salmonella enteritidis and Listeria monocytogenes on salad vegetables

The influence of initial head-spaces of air – 4.9% CO₂/2.1% O₂/93% N₂ and 5% CO₂/5.2% O₂/89.8% N₂ – on Salmonella enteritidis and Listeria monocytogenes, and on microbial association with shredded carrots and lettuce was studied at 4 °C. Both these pathogens survived but did not grow in any vegetable regardless of the packaging system used. Total viable count, lactic acid bacteria and pseudomonads were also monitored. Lactic acid bacteria were the predominant organisms in all samples. The pH dropped significantly during the storage of vegetables.     

Effect of NaCl and KCl on fate and growth/no growth interfaces of Listeria monocytogenes Scott A at different pH and nisin concentrations

AIMS: The fate of Listeria monocytogenes Scott A, was studied in broth, at different a(w)s (by adding NaCl or KCl from 0.0 to 1.4 mol l(-1)), pHs (from 4.0 to 7.3 by adding lactic acid), and nisin concentrations (from 0 to 100 IU ml(-1)). METHODS AND RESULTS: Increasing salt and nisin concentrations and decreasing pH resulted in lower growth rates and extended lag phases. At pH 4.5 no growth was observed while in presence of nisin and/or 1 mol l(-1) salts of both kinds, L. monocytogenes Scott A was inactivated. Equal-molar concentrations of NaCl or KCl (similar a(w)), exerted similar effects against L. monocytogenes in terms of lag phase duration, growth or death rate. The growth boundaries of L. monocytogenes Scott A at 5 degrees C were also estimated by growth/no growth turbidity data, modeled by logistic polynomial regression. The concordance of logistic models, were 99.6 and 99.8% for NaCl and KCl, respectively. CONCLUSIONS: The growth interfaces derived by both NaCl and KCl models were almost identical. Hence, NaCl can be replaced by KCl without risking the microbiological safety of the product. Increasing nisin concentrations markedly affected the interface resulting in a more inhibitory environment for L. monocytogenes Scott A. Low to medium salt concentrations (0.3-0.7 mol l(-1) of either NaCl or KCl) provided a protective effect against inhibition of L. monocytogenes Scott A by nisin. SIGNIFICANCE AND IMPACT OF THE STUDY: Modelling the growth boundaries not only contributes to the development of safer food by providing useful data, but can also be used to study interactions between factors affecting initiation of growth of pathogenic micro-organisms.     

Dynamic modeling of Listeria monocytogenes growth in pasteurized milk

AIMS: The development and validation of a dynamic model for predicting Listeria monocytogenes growth in pasteurized milk stored at both static and dynamic temperature conditions. METHODS AND RESULTS: Growth of inoculated L. monocytogenes in a commercial pasteurized whole milk product was monitored at various isothermal conditions from 1.5 to 16 degrees C. The kinetic parameters of the pathogen were modelled as a function of temperature using a square root type model, which was further validated using data from 92 published growth curves from eight different milk products. Compared to four published models for L. monocytogenes growth, the model developed in this study performed better, with a per cent discrepancy and bias of 49.1 and -1.01%, respectively. The performance of the model in predicting growth at dynamic temperature conditions was evaluated at four different fluctuating temperature scenarios with periodic temperature changes from -2 to 16 degrees C. The prediction of growth at dynamic storage temperature was based on the square root model in conjunction with the differential equations of the Baranyi and Roberts model, which were numerically integrated with respect to time. The per cent relative errors between the observed and the predicted growth of L. monocytogenes were less than 10% for all temperature scenarios tested. CONCLUSIONS: Available models from experiments conducted in laboratory media may result in significant overestimation of L. monocytogenes growth in pasteurized milk because they do not take into account factors such as milk composition (e.g. natural antimicrobial compounds present in milk) and the interactions of the pathogen with the natural microflora. The product-targeted model developed in the present study showed a high performance in predicting growth of L. monocytogenes in pasteurized milk under both static and dynamic temperature conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Temperature fluctuations often occur during the transportation and storage of pasteurized milk. A high performance, dynamic model for the growth of L. monocytogenes can be a useful tool for effective management and optimization of product safety and can lead to more realistic estimations of pasteurized-milk related safety risks.     

Comparative studies of the growth of Staphylococcus carnosus with or without glucose

Lactic acid and acetic acid are metabolic products of Staphylococcus carnosus irrespective of the media used. Lactate was produced at the expense of glucose (when available) while acetic acid was produced at the expense of lactate under aerobic or semi-aerobic conditions. The production of acetic acid could affect seriously the flavour and aroma of fermented sausages.     

The effect of the olive phenolic compound, oleuropein, on growth and enterotoxin B production by Staphylococcus aureus

The presence of low concentrations (0.1% w/v) of oleuropein, a phenolic compound extracted from olives, delayed the growth of Staphylococcus aureus in NZ amine A and brain heart infusion media modified by the addition of growth factors and glucose (NZA + and BHI +), as indicated by changes in conductance, whilst higher concentrations (0.4–0.6% w/v) inhibited growth completely. Intermediate concentrations of oleuropein (0.2%) prevented growth in BHI + but allowed growth to occur in NZA + despite an extended lag phase (30 h). Concentrations of oleuropein > 0.2% inhibited growth and production of enterotoxin B in both types of media. Lower levels (0.1%) did not affect the final viable count and production of toxin in BHI + but decreased the number of viable organisms and reduced the toxin production in NZA + by eightfold. An increase in the concentration of oleuropein resulted in a decrease in the amount of glucose assimilated and consequently the amount of lactate produced. In addition, oleuropein prevented the secretion of a number of exoproteins. Addition of oleuropein during the exponential phase appeared to have no effect on the growth of Staph. aureus in NZA +.     

Differential Listeria monocytogenes Strain Survival and Growth in Katiki,a Traditional Greek Soft Cheese,at Different Storage Temperatures

Katiki Domokou is a traditional Greek cheese, which has received the Protected Designation of Origin recognition since 1994. Its microfloras have not been studied although its structure and composition may enable (or even favor) the survival and growth of several pathogens, including Listeria monocytogenes. The persistence of L. monocytogenes during storage at different temperatures has been the subject of many studies since temperature abuse of food products is often encountered. In the present study, five strains of L. monocytogenes were aseptically inoculated individually and as a cocktail in Katiki Domokou cheese, which was then stored at 5, 10, 15, and 20°C. Pulsed-field gel electrophoresis was used to monitor strain evolution or persistence during storage at different temperatures in the case of the cocktail inoculum. The results suggested that strain survival of L. monocytogenes was temperature dependent since different strains predominated at different temperatures. Such information is of great importance in risk assessment studies, which typically consider only the presence or absence of the pathogen.Listeria monocytogenes is a ubiquitous food-borne pathogen associated with outbreaks of listeriosis from consumption of various food commodities, especially dairy products, seafood, and meat (2, 26). The pathogen is of great health concern for the food industry because it is characterized by high mortality rates, amounting to 20 to 30% (14). Due to the severity of illness, especially for pregnant women, neonates, the elderly, and immunodeficient people, the level of the pathogen in food should remain low to ensure safe food products.The new regulation of the European Union (EU) for microbiological criteria for L. monocytogenes in foods has set maximum levels of 100 CFU g⁻¹ at the time of consumption for soft cheeses (8). In fact, the new EC 2073/2005 regulation in annex I lists the microbiological criteria for foodstuffs, which are classified into food safety criteria and process hygiene criteria. According to the new EU regulation, food safety criteria are those which “define the acceptability of a product or a batch of foodstuff applicable to products placed on the market” (8).Legislative amendments regarding the presence of L. monocytogenes in ready-to-eat (RTE) foods are of great importance. Indeed, for the first time RTE foods are legislatively distinguished according to the target population for which they are intended, i.e., whether they are intended for consumption (i) by infants, (ii) by people with special medical conditions (immunocompromised), or (iii) by other target human subpopulations. In the most recent amendment the RTE foods other than those intended for infants or for those with special medical needs are further subdivided into foods that are able to support the growth of L. monocytogenes and those that are not. Products with pH ≤ 5.0 and water activity of ≤0.94 and products with a shelf life of less than 5 days are automatically considered to belong to the category of RTE foods that are unable to support the growth of L. monocytogenes (8). The regulation also states that “other categories of products can also belong to this category, subject to scientific justification.” Last but not least, the food safety criteria for L. monocytogenes are adjusted according to the bacteria''s temporal stage in the food chain. Thus, for RTE foods that are able to support the growth of L. monocytogenes, the new regulation demands the absence of the pathogen (in 25 g) “before the food has left the immediate control of the food business operator, who has produced it” but allows up to 100 CFU g⁻¹ in “products placed on the market during their shelf life.” The 100-CFU g⁻¹ limit also applies throughout the shelf life of marketed RTE foods unable to support L. monocytogenes growth (8).The pH and the water activity of Katiki Domokou (Katiki), a spreadable RTE traditional Greek cheese, are within the limits mentioned in the regulation. This product, a white cheese with a creamy structure, was traditionally produced from goat milk or from a mixture of goat and sheep milk. It has been recognized as a Protected Designation of Origin product since 1994 (www.greekcheese.gr), and its consumption has readily increased in the last few years. The milk is initially pasteurized and cooled at 27 to 28°C. Coagulation is then conducted with or without the addition of rennet, and the mixture is left to stand at 20 to 22°C. The curd is pulped and placed in cloth sacks for draining, with high final moisture (ca. 75%) and low salt content (ca. 1%) and pH (4.3 to 4.5) while it is stored at 4 to 5°C.The quantitative estimation of kinetic parameters related to growth, survival, and death of L. monocytogenes has been described previously (2, 14, 20). The kinetic parameters of L. monocytogenes during storage at different temperatures have been the subject of many studies since temperature abuse of food products is often encountered (25, 28). However, strain characteristics or viability have not been taken into account (or have not been considered) as yet (20). This may explain the variability of findings in regard to different storage conditions (7, 17). Pulsed-field gel electrophoresis (PFGE) is a powerful subtyping tool, a gold standard for epidemiology, which provides repeatable results. It has the ability to generate profiles of a wide range of microorganisms and to discriminate strains with high fidelity (11, 19). PFGE has been used in several studies to type strains of epidemiological interest as well as to trace contaminants in the food chain (12, 13, 18).The purpose of the present study was to assess the survival of five strains of L. monocytogenes inoculated either individually or as a cocktail in Katiki cheese. The cheese was stored at 5, 10, 15, and 20°C over a period of 1 month. PFGE was used to monitor the strain(s) that might survive and/or grow at different temperatures in a complex ecosystem like Katiki. The strains used in the study to form the inoculum consisted of two type strains of serotype 4b and three isolates belonging to our laboratory collection that were isolated from soft cheese and the conveyor belt of RTE foods. The strains were chosen on the basis of their source of isolation since this could be crucial to the interpretation of the data. The population was monitored throughout storage with respect to its quantitative as well as its qualitative evolution.