Food Protection Trends - July 2010 - 405

preservatives (4, 7) for controlling or eliminating pathogenic bacteria while maintaining overall product quality (7) represents an interesting approach to improving the microbial safety of fresh produce. A wide range of natural products from plants, animals or microorganisms can be used to extend food shelf life. The antimicrobial properties of some types of bacteria are of interest to researchers involved in finding new biocontrol strategies to be used in foods. The native microorganisms present on the surface of fresh produce are assumed to play an important role in maintaining the safety of ready-to-eat vegetables (12) by out-competing pathogens for physical space and nutrients and/or by producing antagonistic compounds that negatively affect the viability of pathogens (10, 13). Another alternative for biopreservation is the use of plant extracts. Spice oleoresins constitute the true essence of spices in their most concentrated form, containing volatile as well as non-volatile components and differing from essential spice oils in that they have all the flavoring ingredients of a particular spice. In addition, they are the most convenient substitutes for raw spices in the food processing industry, since they are free of bacteria and may be standardized to a desired flavor strength (15). It is well known that the antimicrobial potency of essential oils and oleoresins is generally lower in food systems than in vitro, depending on the food composition, processing steps and storage temperature, all of which could strongly influence the effectiveness of these antimicrobial agents (4). Accordingly, larger amounts of oleoresins are required in food systems, which could seriously interfere with the food’s final sensory properties. Among several oleoresins that may be useful as antimicrobial agents, oregano and rosemary may have the greatest potential for use in industrial applications (5). The incidence of foodborne infections caused by bacterial pathogens, especially Escherichia coli O157:H7, continues to be a problem in the industry. This pathogen attaches preferentially to cut edges of vegetables and penetrates into the plant tissue, where it remains unaffected by various sanitizing treatments (19). These facts have led investigators to search for novel methods of

controlling E. coli O157:H7 contamination in vegetables (6, 18). Raw materials, such as carrots, have been subjected to processing operations that commonly increase the risk of contamination by this pathogen (11). In this context, the aims of the present work were to evaluate the effectiveness of the endogenous microflora of sliced and grated carrots in controlling E. coli O157:H7 and to determine the effectiveness of oregano and rosemary oleoresins to control pathogen growth. In addition, the ability of the oleoresins to control growth of the native microflora were evaluated at two storage temperatures, 10 and 20°C.

BHI at two consecutive 24-h intervals immediately before each experiment.

Effectiveness of endogenous microflora on E. coli O157:H7 control
Grated or sliced carrots (100 g) were inoculated with 100 µl of E. coli O157:H7 active culture in the form of several drops. Carrot samples were vigorously shaken to distribute the inoculum and immediately covered with film (Resinite AF 50). The pathogen was inoculated at three levels (low: 2–3 log10, medium: 4–5 log10 and high: 6–7 log10, approximately). To account for possible growth of native E. coli strains, a control sample without added E. coli O157:H7 was used. Carrot samples were stored for 72 h at saturated relative humidity and two temperatures: 10°C (representing inadequate refrigeration storage) or 20°C (representing an abusive, room temperature). Each assay was performed in duplicate in four independent experimental runs.

MATERIALS AND METHODS
Sample preparation
Carrots (Daucus carota L.) were grown and harvested at optimal maturity in Sierra de los Padres, Mar del Plata, Argentina, and were immediately transported to the laboratory. Carrots of about 140–180 mm in length and 40 mm in diameter (upper end) were precleaned with tap water, knife-peeled, topped and then grated or sliced by use of a multi-purpose belt cutting machine manufactured by Braun (Kronberg, Germany). Finally, carrots were washed, centrifuged and aseptically transferred to packages of PET materials (10 × 10 cm).

Effect of oregano and rosemary on E. coli O157:H7 inoculated in carrot samples
Rosemary (Rosmarinus officinalis) and oregano (Origanum vulgare) oleoresins used in this work were provided by Pionherb, Buenos Aires, Argentina. Food grade oleoresins were obtained from fresh herbs by alcohol steam distillation. One hundred grams of grated or sliced carrots were thoroughly mixed with oleoresins at two concentrations: 0.5 and 1% w/w. Samples with oleoresins were immediately inoculated with E. coli O157:H7 by placing 100 µl bacterial suspension on grated or sliced carrots in the form of several drops, reaching a pathogen concentration of approximately 104–105 CFU/g, representing a medium contamination level. Inoculated carrot samples were shaken vigorously to evenly distribute the inoculum and immediately covered with film (Resinite AF 50). To compare the effectiveness of oleoresins on E. coli O157:H7 growth, a sample without oleoresin was used as control. Carrot samples were stored for 96 h at 10°C or 20°C. Each assay was performed in duplicate in four independent experimental runs.

Culture maintenance and inoculum preparation
E. coli O157:H7, ATCC 25158 provided by CIDCA (Centro de Investigación y Desarrollo en Criotecnología de Alimentos, La Plata, Argentina) was used. In previous work, Moreira et al. (11) reported no significant differences in sensitivity to essential oils among different E. coli strains (E. coli O157:H7, ATCC 25158, ATCC 32922 and CI and CII, isolated from foods). For this reason, only one strain of E. coli O157:H7 (ATCC 25158) was used in the present work. A stock culture was maintained in tryptic soy broth (Britania, Buenos Aires, Argentina) at 4°C. Before use, E. coli O157:H7 was cultured in brain heart infusion broth (BHI, Britania, Buenos Aires, Argentina) for 24 h at 37°C. The culture was transferred to 9.0 ml of

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Food Protection Trends - July 2010

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