Microbiological quality of fresh lettuce from organic and conventional production

Previously there was no available information on the levels of indicator bacteria and the prevalence of pathogens in fresh lettuce grown in organic and conventional farms in Spain. A total of 72 lettuce samples (18 farms for 4 repetitions each) for each type of the agriculture were examined in order to assess the bacteriological quality of the lettuces, in particular the prevalence of selected pathogens. The lettuce samples were analyzed for the presence of aerobic mesophilic, psychrotrophic microorganisms, yeasts and moulds, Enterobacteriaceae, mesophilic lactic acid bacteria, Pseudomonas spp. and presumptive Escherichia coli, Salmonella spp. and Listeria monocytogenes. The mean aerobic mesophilic counts (AM) were 6.35 ± 0.69 log₁₀ cfu g⁻¹ and 5.67 ± 0.80 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. The mean counts of psychrotrophic microorganisms were 5.82 ± 1.01 log₁₀ cfu g⁻¹ and 5.41 ± 0.92 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. Yeasts and moulds (YM) mean counts were 4.74 ± 0.83 log₁₀ cfu g⁻¹ and 4.21 ± 0.96 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. Lactic acid bacteria (LAB) were present in low numbers and the mean counts were 2.41 ± 1.10 log₁₀ cfu g⁻¹ and 1.99 ± 0.91 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. Pseudomonas spp. mean counts were 5.49 ± 1.37 log₁₀ cfu g⁻¹ and 4.98 ± 1.26 log₁₀ cfu g⁻¹ in organic and conventional lettuce, respectively. The mean counts for Enterobacteriaceae were 5.16 ± 1.01 log₁₀ cfu g⁻¹ and 3.80 ± 1.53 log₁₀ cfu g⁻¹ in organic and conventional lettuce, respectively. E. coli was detected in 22.2% (16 samples) of organic lettuce and in 12.5% (9 samples) of conventional lettuce. None of the lettuce samples was positive for E. coli O157:H7, L. monocytogenes and Salmonella spp. From the samples analyzed by principal component analysis (PCA) a pattern with two different groups (conventional and organic) can be observed, being the highest difference between both kinds of samples the Enterobacteriaceae count.     

Combined washing effect of noni extract and oregano essential oil on the decontamination of Listeria monocytogenes on romaine lettuce

Combined washing effect of noni extract (NE) and oregano essential oil (OE) on the decontamination of Listeria monocytogenes on romaine lettuce was examined. The fractional inhibitory concentration index (FICI) between NE and three different essential oils (cinnamon leaf, thyme and OE) was determined. Fractional inhibitory concentration index results indicated that OE only had an additive effect (FICI = 1). Noni extract/oregano essential oil combined washing resulted in the greatest reduction in viable cell numbers (3.42 log CFU g⁻¹) relative to non-washed samples. Furthermore, the combined washing was more effective than sodium hypochlorite washing in terms of reducing the microbial load (>1.44-log reduction). Washing treatment did not change the surface colour or total phenolic content in lettuce samples. These results suggest that NE/OE combined washing is applicable as a novel decontamination treatment to ensure the microbial safety of romaine lettuce without changing the quality.     

Differences in biofilm formation of produce and poultry Salmonella enterica isolates and their persistence on spinach plants

Spinach plants were irrigated biweekly with water containing 2.1 log CFU Salmonella/100 ml water (the maximum Escherichia coli MPN recommended by the Leafy Greens Marketing Agreement; LGMA), or 4.1 CFU Salmonella/100 ml water to determine Salmonella persistence on spinach leaves. Green Fluorescent protein expressing Salmonella were undetectable by most-probable number (MPN) at 24 h and 7 days following each irrigation event. This study indicates that Salmonella are unlikely to persist on spinach leaves when irrigation water is contaminated at a level below the LGMA standards. In a parallel study, persistence of Salmonella isolated from poultry or produce was compared following biweekly irrigation of spinach plants with water containing 6 log CFU Salmonella/100 ml. Produce Salmonella isolates formed greater biofilms on polystyrene, polycarbonate and stainless steel surfaces and persisted at significantly higher numbers on spinach leaves than those Salmonella from poultry origin during 35 days study. Poultry Salmonella isolates were undetectable (<1 log CFU/g) on spinach plants 7 days following each irrigation event when assayed by direct plating. This study indicates that Salmonella persistence on spinach leaves is affected by the source of contamination and the biofilm forming ability of the strain.     

Transfer of Listeria innocua from contaminated compost and irrigation water to lettuce leaves

Many foodborne outbreaks of some pathogens such as Escherichia coli O157:H7, Salmonella or Listeria have been associated with the consumption of contaminated vegetables. Contaminated manure and polluted irrigation water are probable vehicles for the pathogens. The aim of this study was to determine the potential transfer of Listeria innocua from soil fertilized with contaminated compost or irrigated with contaminated water to the edible parts of lettuce grown on these soils together with its survival in lettuce and in soil under field conditions during two different seasons. Moreover, its survival on lettuce sprinkled with contaminated irrigation water was evaluated. L. innocua survived in soil samples for 9 weeks at high concentrations, 10⁵ cfu gdw⁻¹ in fall and 10³ cfu gdw⁻¹ in spring. Pathogen survived better in fall, indicating an important influence of temperature and humidity. L. innocua population in lettuce leaves was very high on lettuce leaves after sprinkling, but decreased to undetectable levels at field conditions. There was also transfer of L. innocua from soil contaminated with compost or irrigated with contaminated water to lettuce leaves, mainly to the outer ones. Survival profiles of L. innocua on lettuce and soil samples contaminated either by application of contaminated compost or surface irrigation water was similar. Our results indicated that contaminated compost and contaminated irrigation water can play an important role in the presence of foodborne pathogens on vegetables.     

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.     

Effects of X-ray radiation on Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri inoculated on shredded iceberg lettuce

The main goal of this investigation was to study the efficacy of X-ray doses (0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 1.5 and 2.0 kGy) on inoculated Escherichia coli O157: H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri on shredded iceberg lettuce. The second goal was to study the effect of X-ray on the inherent microflora counts and visual color of shredded iceberg lettuce during storage at 4 °C for 30 days. Treatment with 1.0 kGy X-ray significantly reduced the population of E. coli O157: H7, L. monocytogenes, Salmonella enterica and S. flexneri on shredded iceberg lettuce by 4.4, 4.1, 4.8 and 4.4-log CFU 5 cm⁻², respectively. Furthermore, more than a 5 log CFU reduction of E. coli O157: H7, L. monocytogenes, S. enterica and S. flexneri was achieved with 2.0 kGy X-ray. Treatment with X-ray reduced the initial microflora on iceberg lettuce and kept them significantly (p < 0.05) lower than the control during storage at 4 °C and 90% RH for 30 days. Treatment with X-ray did not significantly (p > 0.05) change the green color of iceberg lettuce leaves. Treatment with X-ray significantly reduced selected pathogens and inherent microorganisms on shredded iceberg lettuce leaves, which could be a good alternative to other technologies for produce (lettuce) industry.     

Yersinia enterocolitica in slaughter pig tonsils: Enumeration and detection by enrichment versus direct plating culture

Tonsil samples from 139 slaughter pigs were examined for the presence of pathogenic Yersinia enterocolitica by enrichment procedures based on the standard method ISO 10273:2003. In addition, samples were tested by direct plating method to evaluate its efficiency compared to the enrichment culture methods and to quantify the level of contamination in porcine tonsils. In total, 52 samples (37.4%) were positive for pathogenic Y. enterocolitica, all belonging to bioserotype 4/O:3. Fifty out of the 52 positive samples (96.2%) were detected by direct plating. Enumeration showed an average concentration of 4.5 log₁₀ CFU g⁻¹ and 4.4 log₁₀ CFU g⁻¹ tonsil on Salmonella-Shigella-desoxycholate-calcium chloride (SSDC) and cefsulodin-irgasan-novobiocin (CIN) agar plates, respectively. The enrichment procedures recommended by the ISO 10273:2003 method were not optimal for the isolation of pathogenic Y. enterocolitica from pig tonsils: two days enrichment in irgasan-ticarcillin-potassium chlorate (ITC) broth resulted in an isolation rate of 84.6%, while 5 days enrichment in peptone-sorbitol-bile (PSB) broth recovered only 59.6% of positive samples. Reducing the enrichment time in PSB from 5 to 2 days resulted in a significantly higher recovery rate (94.2%) and might serve as an appropriate enrichment protocol for the isolation of pathogenic Y. enterocolitica from pig tonsils. Compared to enrichment culture methods, results based on direct plating can be obtained in a shorter time course and provide quantitative data that might be needed for further risk assessment studies.     

Antimicrobial activity of oregano oil against antibiotic-resistant Salmonella enterica on organic leafy greens at varying exposure times and storage temperatures

The objective of this study was to evaluate the effectiveness of oregano oil on four organic leafy greens (Iceberg and Romaine lettuces and mature and baby spinaches) inoculated with Salmonella Newport as a function of treatment exposure times as well as storage temperatures. Leaf samples were washed, dip inoculated with S. Newport (6-log CFU/ml) and dried. Oregano oil was prepared at 0.1, 0.3, and 0.5% concentrations in sterile phosphate buffered saline (PBS). Inoculated leaves were immersed in the treatment solution for 1 or 2 min, and individually incubated at 4 or 8 °C. Samples were taken at day 0, 1, and 3 for enumeration of survivors. The results showed that oregano oil was effective against S. Newport at all concentrations. S. Newport showed reductions from the PBS control of 0.7–4.8 log CFU/g (Romaine lettuce), 0.8–4.8 log CFU/g (Iceberg lettuce), 0.8–4.9 log CFU/g (mature spinach), and 0.5–4.7 log CFU/g (baby spinach), respectively. The antibacterial activity also increased with exposure time. Leaf samples treated for 2 min generally showed greater reductions (by 1.4–3.2 log CFU/g), than those samples treated for 1 min; however, there was minimal difference in antimicrobial activity among samples stored under refrigeration and abuse temperatures. This study demonstrates the potential of oregano oil to inactivate S. Newport on organic leafy greens.     

Combined effect of enterocin AS-48 and high hydrostatic pressure to control food-borne pathogens inoculated in low acid fermented sausages

The single and combined effects of enterocin AS-48 and high hydrostatic pressure (HHP) on Listeria monocytogenes, Salmonellaenterica, and Staphylococcus aureus was investigated in fuet (a low acid fermented sausage) during ripening and storage at 7 °C or at room temperature. AS-48 (148 AU g⁻¹) caused a drastic 5.5 log cfu g⁻¹ decrease in L. monocytogenes (P < 0.001) and a significant (P < 0.01) inhibition (1.79 logs) for Salmonella at the end of ripening (10 d). After pressurization (400 MPa) and storage Listeria counts remained below 5 cfu g⁻¹ in all fuets containing AS-48 (pressurized or not). HHP alone had no anti-Listeria effect. HHP treatment significantly reduced Salmonella counts, with lowest levels in pressurized fuets with AS-48. S. aureus showed similar growth for all treatments and storage conditions. These results indicate that AS-48 can be applied alone to control L. monocytogenes and combined with HHP treatment to control Salmonella in fuets.     

Smearing of soft cheese with Enterococcus faecium WHE 81, a multi-bacteriocin producer,against Listeria monocytogenes

Enterococcus faecium WHE 81, a multi-bacteriocin producer, was tested for its antimicrobial activity on Listeria monocytogenes in Munster cheese, a red smear soft cheese. The naturally delayed and superficial contamination of this type of cheese allowed the use of E. faecium WHE 81 at the beginning of the ripening as a surface culture. A brine solution inoculated at 10⁵ CFU of E. faecium WHE 81 per mL was sprayed on the cheese surface during the first smearing operation. On day 7, smearing of cheese samples with a brine solution at 10² CFU of L. monocytogenes per mL yielded initial cell counts of approximately 50 CFU g⁻¹ of the pathogen on the cheese surface. Although, in some instances, L. monocytogenes could survive (<50 CFU g⁻¹) in the presence of E. faecium WHE 81, it was unable to initiate growth. In control samples however, L. monocytogenes counts often exceeded 10⁴ CFU g⁻¹. In other respects, E. faecium WHE 81, which naturally existed in Munster cheese, did not adversely impact on the ripening process.     

Effects of Pseudomonas chlororaphis and gaseous chlorine dioxide on the survival of Salmonella enterica on tomatoes

Control of Salmonella enterica on tomatoes is important for food safety. The aim of this research was to evaluate the survival of Salmonella enterica serovars Montevideo (SM) and Typhimurium (ST) on tomatoes exposed to gaseous chlorine dioxide and Pseudomonas chlororaphis (Pc). Pc was applied to stem scars of tomatoes prior to inoculations with SM and ST. Tomatoes were treated with gaseous ClO₂ at 0.4 mg L^?1 for 2 and 4 h (90% R.H. 13 °C), respectively. At 4 h of ClO₂ treatment, SM and ST populations were reduced to 0.82 and <0.30 log CFU g^?1, respectively. Tomatoes treated with SM and ST had 5.42 and 5.37 log CFU g^?1 of Salmonella. Tomatoes treated with Pc + Salmonella count was 2.59 (treated) and 5.83 log CFU g^?1 (control). Salmonella survival was similar at 2 and 4 h of ClO₂ treatment. Application of ClO₂ and Pc may reduce contamination of tomatoes by Salmonella serovars.     

The influence of headspace and dissolved oxygen level on growth and haemolytic BL enterotoxin production of a psychrotolerant Bacillus weihenstephanensis isolate on potato based ready-to-eat food products

The major objective of this study was to determine the influence of the initial headspace and dissolved O₂ level and vacuum packaging on growth and diarrhoeal enterotoxin production by Bacillus weihenstephanensis on potato based ready-to-eat food products. In general, the lower the initial headspace or dissolved O₂ level the slower the maximum growth rate (μ_max, log₁₀ CFU g⁻¹ d⁻¹), the longer the lag phase duration (λ, d) and the smaller the maximum population density (N_max, log₁₀ CFU g⁻¹) became. The slowest μ_max, the longest λ and the smallest N_max were generally found for growth under vacuum packaging. This implies shorter shelf-lives will occur at higher initial headspace or dissolved O₂ levels as the growth of B. weihenstephanensis to the infective dose of 10⁵ CFU g⁻¹ in such atmospheres takes a shorter time. Significant consumption of dissolved O₂ only occurred when growth shifted from the lag to the exponential phase and growth generally transitioned from the exponential to the stationary phase when the dissolved O₂ levels fell below ca. 75 ppb. Diarrhoeal enterotoxin production (determined via detection of the L2 component of haemolytic BL) was similar for growth under initial headspace O₂ levels of 1-20.9%, and was only reduced when growth took place under vacuum packaging. The reduction in L2 production when growth took place under vacuum was most probably related to the low final cell densities observed under this condition. Both growth and L2 production were inhibited over a 32-day incubation period at 7 °C by 40% CO₂ irrespective of the headspace or dissolved O₂ levels. The results illustrate the importance of residual O₂ and CO₂ on the shelf-stability and safety of modified atmosphere packaged potato based ready-to-eat food products with regards to B. weihenstephanensis.     

Production of wara, a West African soft cheese using lemon juice as a coagulant

As an important protein source for West African consumers, wara cheese made from the leave extract of Calotropis procera has extremely short shelf life of only 2-3 days [Adegoke, G. O., Nse, E. N., & Akanni, A. O. (1992). Effects of heat, processing time, and pH on the microflora, aflatoxin content, and storability of wara, a soft white cheese. Die Nahrung, 36(3), 259-264; Umoh, V. J., & Solomon, O. (2001). Safety assessment and critical control point of milk product and some cereal beverages in Northern Nigeria. In: Proceedings of USDA/USAID/NIGERIA international conference on food safety and security, August 1-3 (pp. 122-127). Ibadan, Nigeria: IITA; Belewu, M. A., Belewu, K. Y., & Nkwunonwo, C.C. (2005). Effect of biological and chemical preservatives on the shelflife of West African soft cheese. African Journal of Biotechnology, 4, 1076-1079; Adetunji, A. O., Alonge, D. O., & Chen, J. (Unpublished). Microbial quality of wara, a southwestern Nigerian soft cheese]. Lemon juice was used in this study as a substitute coagulant during wara manufacture in order to improve the microbial quality of wara. The cheese was manufactured from pasteurized milk inoculated with 10¹ or 10² CFU ml⁻¹ of Listeria monocytogenes. Samples of the milk or cheese were taken along the manufacturing steps and during a 5 d storage period at 15 and 28 °C in order to determine the populations of L. monocytogenes, total aerobes, Enterobacteriaceae, and psychrotrophs, as well as mold and yeast. On the 4th day of storage, portions of the un-inoculated control cheese from 28 °C were deep fried in vegetable oil, mimicking the practice of West African local cheese processors. The results showed that L. monocytogenes, at both inoculation levels, did not survive the manufacture of wara. In samples initially inoculated with 10¹ CFU ml⁻¹ of L. monocytogenes, the Enterobacteriaceae counts decreased from the initial 1.78 to 1.00 Log₁₀ CFU g⁻¹ with the addition of lemon juice, and became undetectable (<1.00 Log₁₀ CFU g⁻¹) at the curdling point as well as during the 5 d storage period at both temperatures. The total aerobic counts increased from the undetectable level on the 1st day of storage to 7.65 and 3.39 Log₁₀ CFU g⁻¹, respectively at 28 or 15 °C on the 5th day of storage. The psychrotrophic, as well as the yeast and mold counts increased from the undetectable levels on the 1st day of storage to 7.11 and 5.03 Log₁₀ CFU g⁻¹, respectively at 28 °C. At 15 °C however, the population of pyschrotrophs remained undetectable throughout the 5 d storage period whereas, the yeast and molds count increased to 3.08 Log₁₀ CFU g⁻¹ on day 3 before quickly decreasing to the undetectable levels on the 5th day of storage. A similar trend was observed in cheese made from the milk with an initial Listeria inoculation level of 10² CFU ml⁻¹. The results of this study showed that lemon juice significantly reduced the populations of the sampled microorganisms, especially the populations of Enterobacteriaceae.     

Nutrient contents of kale (Brassica oleraceae L. var. acephala DC.)

Fructose, glucose and sucrose, as the major soluble sugars and citric and malic acids, as the major organic acids, were identified and determined in kale (Brassica oleraceae L. var. acephala DC., black cabbage) leaves. Fructose was the predominant sugar (2011 mg 100 g⁻¹ dry wt) identified, followed by glucose (1056 mg 100 g⁻¹ dry wt) and sucrose (894 mg 100 g⁻¹ dry wt). The contents of citric and malic acids were at 2213 and 151 mg 100 g⁻¹ dry wt in the leaves. The 16:0, 18:2n − 6 and 18:3n − 3 fatty acids were the most abundant fatty acids in the leaves. Considering the level of these fatty acids, 18:3n − 3 was found to be the highest (85.3 μg g⁻¹ dry wt), contributing 54.0% of the total fatty acid content. Linoleic acid (18:2n − 6), being the second most abundant fatty acid was present at 18.6 μg g⁻¹ dry wt, contributing 11.8% of the total fatty acid content. In the seed oil of kale, 22:1n − 9 was the most abundant fatty acid (4198 μg g⁻¹ dry wt, 45.7%), with 18:2n − 6 (1199 μg g⁻¹ dry wt, 12.3%) and 18:1n − 9 (1408 μg g⁻¹ dry wt, 14.8%) being the second next most abundant fatty acids. The most abundant amino acid was glutamic acid (Glu) which was present at 33.2 mg g⁻¹ dry wt. Aspartic acid, which was the second most abundant amino acid, was present at 27.6 mg g⁻¹ dry wt and accounted for 10.2% of the total amino acid content of kale leaf. The amino acid content was assessed by comparing the percentages of the essential amino acids in kale leaf versus those of a World Health Organization (WHO) standard protein. The protein of kale leaf compares well with that of the WHO standard. Only one amino acid, lysine, had a score that fell below 100%; the lysine score of kale leaf was 95%. This study attempts to contribute to knowledge of the nutritional properties of the plant. These results may be useful for the evaluation of dietary information.     

Evaluation of a novel antimicrobial (lauric arginate ester) substance against biofilm of Escherichia coli O157:H7, Listeria monocytogenes,and Salmonella spp.

The purpose of this study was to evaluate the activity of a novel antimicrobial substance lauric arginate ester (LAE) against selected foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp.) in biofilm. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined and showed that LAE exhibits a strong antimicrobial activity. Biofilms were grown on abiotic stainless steel, rubber, MBEC biofilm device) and biotic (lettuce) surfaces. The efficacy of LAE (50, 100 and 200 ppm) at reducing the biofilm cells on these surfaces was examined by applying LAE for 2 h. Results revealed that LAE exhibited the reduction in biofilm bacteria up to 7 log CFU cm^?2, 3.5 log CFU cm^?2, 4.0 log CFU peg^?1 and 1.5 log CFU cm^?2 on stainless steel, rubber, MBEC and lettuce surfaces, respectively. Overall, these results suggest that LAE has been shown to be a potential alternative to control bacteria in biofilm mode in food industry.     

A probiotic soy-based innovative product as an alternative to petit-suisse cheese

The present study aimed to develop a probiotic soy-based product similar to petit-suisse cheese and to evaluate its perspectives regarding potential for consumer health benefits, sensory acceptability, and instrumental texture during storage. Three different trials were studied: MP (milk-based petit-suisse – control); MSP (mixed product with milk cream and soy); SP (soymilk-based product). The formulations were produced with an ABT culture, containing Lactobacillus acidophilus La-5, Bifidobacterium animalis Bb-12, and the starter Streptococcus thermophilus and stored at 4 °C for up to 28 days. Bb-12 viability remained always above 8 log cfu g⁻¹ for all trials, whereas viability of La-5 was satisfactory at the end of storage for MP (7.56 log cfu g⁻¹) and MSP (6.49 log cfu g⁻¹), but only up to 21 days (6.84 log cfu g⁻¹) for SP. The pH remained stable and was lower for MSP (p < 0.05), whereas instrumental hardness and gumminess increased in soy-based products (MSP and SP) and decreased in the control (MP). SP had the highest sensory score means (6.4) on day 21, being sensorially attractive to provide consumers with a functional food without dairy ingredients and with high viability of the probiotic microorganisms.     

Reduction of Escherichia coli O157:H7 viability on leafy green vegetables by treatment with a bacteriophage mixture and trans-cinnamaldehyde

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been recognized as a major foodborne pathogen responsible for frequent gastroenteritis outbreaks. Phages and essential oils can be used as a natural antimicrobial method to reduce bacterial pathogens from the food supply. The objective of this study was to determine the effect of a bacteriophage cocktail, BEC8, alone and in combination with the essential oil trans-cinnameldehyde (TC) on the viability of a mixture of EHEC O157:H7 strains applied on whole baby romaine lettuce and baby spinach leaves. The EHEC O157:H7 strains used were Nal^R mutants of EK27, ATCC 43895, and 472. Exponentially growing cells from tryptic soy (TS) broth cultures were spot inoculated on leaves and dried. EHEC cells were placed at low, medium, and high inoculum levels (10⁴, 10⁵, and 10⁶ CFU/mL, respectively). Appropriate controls, BEC8 (approx. 10⁶ PFU/leaf), and TC (0.5% v/v) were applied on treated leaves. The leaves were incubated at 4, 8, 23, and 37 °C in Petri dishes with moistened filter papers. EHEC survival was determined using standard plate count on nalidixic acid (50 μg/mL) Sorbitol MacConkey agar. No survivors were detected when both leaves were treated with BEC8 or TC individually at low inoculum levels after 24 h at 23 and 37 °C. When the EHEC inoculum size increased and/or incubation temperature decreased, the efficacy of BEC8 and TC decreased. However, when the two treatments were combined, no survivors were detected after 10 min at all temperatures and inoculum levels on both leafy greens. These results indicated that the BEC8/TC combination was highly effective against EHEC on both leafy greens. This combination could potentially be used as an antimicrobial to inactivate EHEC O157:H7 and reduce their incidence in the food chain.     

Molecular ecology and polyphasic characterization of the microbiota associated with semi-dry processed coffee (Coffea arabica L.)

This work was aimed at isolating and identifying the microbiota present during the semi-dry method of coffee processing using polyphasic methods and to evaluate microbial diversity with PCR-DGGE. Samples of Coffea arabica L. were collected during different processing stages in southern Minas Gerais, Brazil. The bacterial and fungal isolates were phenotypically characterised and grouped according to the ARDRA technique, in which the 16–23S and ITS1-5.8S regions of the rDNA were sequenced for species identification. The bacterial counts varied from 3.7 to 7 log CFU g⁻¹. The yeast counts ranged from 3.4 to 6.9 log CFU g⁻¹, and the filamentous fungal population varied from 2 to 3.7 log CFU g⁻¹. Bacillus subtilis, Escherichia coli, Enterobacter agglomerans, Bacillus cereus and Klebsiella pneumoniae were the predominant bacteria detected during the processing of the coffee, and Pichia anomala, Torulaspora delbrueckii and Rhodotorula mucilaginosa were the dominant yeasts. All of the yeast and bacterial species detected by PCR-DGGE were isolated using culture-dependent methods, with the exception of one uncultivable bacterial species. Aspergillus was the most common genus among the filamentous fungal isolates. The use of polyphasic methods allowed a better characterization of the microbiota that is naturally present in semi-dry processed coffee.     

Viability of Lactobacillus acidophilus La-5 added solely or in co-culture with a yoghurt starter culture and implications on physico-chemical and related properties of Minas fresh cheese during storage

The effect of a probiotic culture of Lactobacillus acidophilus (La-5), added solely or in co-culture with a starter culture of Streptococcus thermophilus, on texture, proteolysis and related properties of Minas fresh cheese during storage at 5 °C was investigated. Three cheese-making trials were prepared and produced with no addition of cultures (T1 - control), supplemented with La-5 (T2), and with La-5 + S. thermophilus (T3). Viable counts of La-5 remained above 6.00 log cfu g⁻¹ during the whole storage for T2, reaching 7.00 log cfu g⁻¹ on the 14th day. For T3, the counts of La-5 remained above 6.00 log cfu g⁻¹ after 7 days of storage. Due to the presence of S. thermophilus, T3 presented the highest proteolytic index increase and titratable acidity values. Nevertheless, these results and S. thermophilus addition had no influence on viability of La-5 which presented satisfactory populations for a probiotic food. Moreover, the use of a yoghurt culture for the production of Minas fresh cheese T3 supplemented with La-5 resulted in a good quality product, with a small rate of post-acidification, indicating that traditional yoghurt culture could be employed in co-culture with La-5 to improve the quality of this cheese.