I. Viñas

Microbiological quality of fresh, minimally-processed fruit and vegetables, and sprouts from retail establishments

A survey of fresh and minimally-processed fruit and vegetables, and sprouts was conducted in several retail establishments in the Lleida area (Catalonia, Spain) during 2005-2006 to determine whether microbial contamination, and in particular potentially pathogenic bacteria, was present under these commodities. A total of 300 samples--including 21 ready-to-eat fruits, 28 whole fresh vegetables, 15 sprout samples and 237 ready-to-eat salads containing from one to six vegetables--were purchased from 4 supermarkets. They were tested for mesophilic and psychrotrophic aerobic counts, yeasts and moulds, lactic acid bacteria, Enterobacteriaceae, presumptive E. coli and Listeria monocytogenes counts as well as for the presence of Salmonella, E. coli O157:H7, Yersinia enterocolitica and thermotolerant Campylobacter. Results for the fresh-cut vegetables that we analyzed showed that, in general, the highest microorganism counts were associated with grated carrot, arugula and spinach (7.8, 7.5 and 7.4 log cfu g(-1) of aerobic mesophilic microorganisms; 6.1, 5.8 and 5.2 log cfu g(-1) of yeast and moulds; 5.9, 4.0 and 5.1 log cfu g(-1) lactic acid bacteria and 6.2, 5.3 and 6.0 log cfu g(-1) of Enterobacteriaceae). The lowest counts were generally associated with fresh-cut endive and lettuce (6.2 and 6.3 log cfu g(-1) of aerobic mesophilic microorganisms; 4.4 and 4.6 log cfu g(-1) of yeast and moulds; 2.7 and 3.8 log cfu g(-1) lactic acid bacteria and 4.8 and 4.4 log cfu g(-1) of Enterobacteriaceae). Counts of psychrotrophic microorganisms were as high as those of mesophilic microorganisms. Microbiological counts for fresh-cut fruit were very low. Sprouts were highly contaminated with mesophilic (7.9 log cfu g(-1)), psychrotrophic microorganisms (7.3 log cfu g(-1)) and Enterobacteriaceae (7.2 log cfu g(-1)) and showed a high incidence of E. coli (40% of samples). Of the samples analyzed, four (1.3%) were Salmonella positive and two (0.7%) harboured L. monocytogenes. None of the samples was positive for E. coli O157:H7, pathogenic Y. enterocolitica or thermotolerant Campylobacter.

Effect of protective agents, rehydration media and initial cell concentration on viability of Pantoea agglomerans strain CPA-2 subjected to freeze-drying

The effect of initial cell density, protective agents and rehydration media on the viability of biocontrol agent Pantoea agglomerans CPA‐2 when subjected to freeze‐drying was studied. Several additives were tested as protective agents against freeze‐drying injury. Maximum viability of the bacterial cells was obtained with disaccharides (survival levels >60%). Freeze‐dried samples were rehydrated with several media; the highest percentage viability was obtained with 10% non‐fat skim milk (100%+). The effect of initial bacterial load on the final recovery was dependent on protectant but not on rehydration media. Sucrose was an effective protectant when a high initial concentration (1010 cfu ml−1) was used; the opposite occurred with non‐fat skim milk. The use of 1010 cfu ml−1 as an initial concentration, sucrose as a protectant and non‐fat skim milk as a rehydration medium enabled 100% of P. agglomerans viability to be conserved after freeze‐drying. Results suggest the possibility of achieving a good formulation system for the studied biocontrol agent with a high number of viable cells to be used toward pathogens, which is desirable for the industrial development of the product.

Effect of freeze drying and protectants on viability of the biocontrol yeast Candida sake.

The effects of freezing method, freeze drying process, and the use of protective agents on the viability of the biocontrol yeast Candida sake were studied. Freezing at -20 degrees C was the best method to preserve the viability of C. sake cells after freeze drying using 10% skim milk as a protectant (28.9% survival). Liquid nitrogen freezing caused the highest level of damage to the cells with viability < 10%. Different concentrations of exogenous substances including sugars, polyols, polymers and nitrogen compounds were tested either alone or in combination with skim milk. There was little or no effect when additives were used at 1% concentration. Galactose, raffinose and sodium glutamate at 10% were the best protective agents tested alone but the viability of freeze-dried C. sake cells was always < 20%. Survival of yeast cells was increased from 0.2% to 30-40% by using appropriate protective media containing combinations of skim milk and other protectants such as 5% or 10% lactose or glucose, and 10% fructose or sucrose.

Biological control of major postharvest pathogens on apple with Candida sake

Epiphytic microorganisms isolated from apples, pears and the surfaces of apple leaves were screened for antagonistic activity against Penicillium expansum (blue-mold), Botrytis cinerea (gray-mold) and Rhizopus nigricans (Rhizopus rot) on apple (Malus domestica). A total of 933 bacteria and yeasts were tested in primary screening against P. expansum. Ninety-two strains reduced the lesion size on apples by more than 50%, 72 of which were isolated from the surface of apples. For secondary screening against P. expansum, B. cinerea and R. nigricans, 31 strains were selected. The most promising isolate, CPA-1, was identified as Candida sake. This yeast, isolated from apples in storage season was very effective against all three diseases. Wounded Golden Delicious apples protected with the yeast suspension at a concentration of 2.6 x 10(6) CFU/ml and inoculated with conidia of B. cinerea and R. nigricans of 10(5) and 10(4) conidia/ml, respectively, did not develop rot. Complete control of P. expansum was obtained at the same concentration of the antagonist with a pathogen inoculum concentration of 10(3) conidia/ml. This strain, also provided excellent control of rot development under cold storage conditions. The strain of Candida sake can grow actively in aerobic conditions. In drop-inoculated wounds of apples, the populations of C. sake increased by more than 50-fold during the first 24 h at 20 degrees C. The maximum population of C. sake on apple wounds was the same at 20 as at 1 degrees C and was recovered after three and twenty days, respectively.

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 log(10) cfu g(-1) and 5.67 +/- 0.80 log(10) cfu g(-1) from organic and conventional lettuce, respectively. The mean counts of psychrotrophic microorganisms were 5.82 +/- 1.01 log(10) cfu g(-1) and 5.41 +/- 0.92 log(10) cfu g(-1) from organic and conventional lettuce, respectively. Yeasts and moulds (YM) mean counts were 4.74 +/- 0.83 log(10) cfu g(-1) and 4.21 +/- 0.96 log(10) 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 log(10) cfu g(-1) and 1.99 +/- 0.91 log(10) cfu g(-1) from organic and conventional lettuce, respectively. Pseudomonas spp. mean counts were 5.49 +/- 1.37 log(10) cfu g(-1) and 4.98 +/- 1.26 log(10) cfu g(-1) in organic and conventional lettuce, respectively. The mean counts for Enterobacteriaceae were 5.16 +/- 1.01 log(10) cfu g(-1) and 3.80 +/- 1.53 log(10) 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.

Biological control of postharvest pear diseases using a bacterium, Pantoea agglomerans CPA-2

Epiphytic microorganisms isolated from the fruits and leaf surfaces of apples and pears were screened for antagonistic activity against Penicillium expansum on pears. From 247 microorganisms tested for antagonistic properties against P. expansum, a bacterium strain identified as Pantoea agglomerans (CPA-2) was selected. This bacterium was very effective against Botrytis cinerea, P. expansum and Rhizopus stolonifer. Complete control at the three tested concentrations (2 x 10(7), 8 x 10(7) and 1 x 10(8) CFU ml(-1)) was obtained on wounded pears inoculated with 10(3), 10(4) and 10(5) conidia ml(-1) of P. expansum and R. stolonifer. At 8 x 10(7) CFU ml(-1), Pan. agglomerans reduced B. cinerea decay by more than 80% at the three concentrations of the pathogen. In over 3 years of experiments in semicommercial trials, Pan. agglomerans provided excellent control against B. cinerea and P. expansum under cold storage, either in air or in low oxygen atmospheres. Equal control was obtained with Pan. agglomerans at 8 x 10(7) CFU ml(-1), as with the fungicide imazalil at commercial doses, against both pathogens. Pan. agglomerans grew well inside wounds on pears at both room and cold temperatures and under modified atmospheres. In contrast, it grew poorly on the surface of intact fruit.

Improving control of green and blue molds of oranges by combining Pantoea agglomerans (CPA-2) and sodium bicarbonate

The potential of using Pantoea agglomerans (strain CPA-2) alone, or in combination with sodium bicarbonate or sodium carbonate solutions, for control of Penicillium digitatum (green mold) and Penicillium italicum (blue mold) on oranges was investigated under ambient (20 °C) and cold storage (3 °C) conditions. P. agglomerans controlled both pathogens on oranges at 2 × 108 cfu ml-1. The biocontrol agent was found to be completely tolerant to 2% sodium bicarbonate at room temperature, although its culturability was reduced by > 1000-fold after 30 min in 2% sodium carbonate. The efficacy of P. agglomerans for control of green mold was improved when combined with sodium bicarbonate, resulting in complete and 97.6% reduction of decay incidence at 3 °C and 20 °C, when compared to untreated controls. Satisfactory results were also obtained with the combined treatment for control of blue mold. P. agglomerans grew well inside wounds on oranges at both 20 °C and 3 °C. In contrast, it showed a reduced growth on the surface of intact fruit. Sodium bicarbonate at 2% concentration did not noticeably affect antagonist population development. Thus, use of bicarbonate treatment at 2% followed by the antagonist P. agglomerans CPA-2 could be an alternative to chemicals for control of postharvest diseases on oranges.

Possible involvement of hydrogen peroxide in the development of resistance mechanisms in ‘Golden Delicious’ apple fruit

Apples (Malusdomestica L. cv Golden Delicious) were picked 7 days before (harvest 1), or after (harvest 2) the commercial harvest time. Changes in H2O2 levels and in the activity of the H2O2-generating enzyme superoxide dismutase (SOD) and H2O2-scavenging enzymes catalase (CAT) and unspecific peroxidase (POX) were estimated immediately after harvest and during the first 24 h following wounding and inoculation with Penicillium expansum. Fruit from harvest 1 had lower decay incidence and severity of infection than those from harvest 2. Immediately after wounding or inoculation, the less susceptible fruit (harvest 1) showed a significant increase in H2O2 levels concomitantly with higher activity of SOD. No significant changes in CAT and POX were found. In susceptible fruit (harvest 2), both H2O2 levels and SOD activity in wounded/inoculated fruit remained similar to those in control fruit. In contrast, CAT and POX activity significantly increased as a consequence of wounding and inoculation. Collectively, these results provide evidence that harvest date is important in determining the susceptibility of ‘Golden Delicious’ fruit to P. expansum, and that H2O2 and associated metabolism induced by wounding could play a role in defence mechanisms in this fruit.

Post‐harvest biological control by Pantoea agglomerans (CPA‐2) on Golden Delicious apples

Aims: To investigate the potential of Pantoea agglomerans to control the major post‐harvest diseases on Golden Delicious apples.

Viability, efficacy, and storage stability of freeze-dried biocontrol agent Candida sake using different protective and rehydration media.

Viability, efficacy against Penicillium expansum on Golden Delicious apples, and storage stability of freeze-dried Candida sake strain CPA-1 were studied. The effect of several protective agents and rehydration media was investigated in the freeze drying of C. sake. Skimmed milk at 10% concentration was a good rehydration medium for all protectants tested. In general, good viability results were obtained when the same solution was used as a protectant and as a rehydration medium. The best survival was obtained when C. sake cells were protected with 10% lactose + 10% skimmed milk and rehydrated with skimmed milk (85% viability). The potential for biocontrol of the best freeze-dried treatments against P. expansum on apples was compared with that of fresh cells. Freeze-dried treatments at 1 x 10(7) CFU/ml reduced the incidence of decay by 45 to 66%. The best biocontrol effect was obtained with cells that had been freeze dried using 10% lactose + 10% skimmed milk as a protectant and 1% peptone as a rehydration medium, with a 66% reduction in rot incidence. However, in all treatments, the efficacy of freeze-dried cells was significantly lower than fresh cells. The stability of freeze-dried samples decreased during storage and was influenced by storage temperature. In the best treatment, storage of C. sake cells for 60 days at 4 degrees C resulte in final concentrations of 2.5 x 10(8) CFU/ml, which was a 10-fold reduction in relation to the initial starting concentration of cells prior to freeze drying.