Total and pathogenic Vibrio parahaemolyticus in shrimp: Fast and reliable quantification by real-time PCR

Vibrio parahaemolyticus is found in aquatic environments and is the leading cause of gastroenteritis due to seafood consumption worldwide. We evaluated a quantitative real-time PCR (Q-PCR) assay with hydrolysis probes, to determine whether this method could be used for the efficient counting of total, tdh and trh-positive V. parahaemolyticus in shrimps. We assessed the specificity of this assay, using 62 strains from 12 non target bacterial species of the Vibrio, Photobacterium, Shewanella and Aeromonas genera. Only V. parahaemolyticus with the appropriate target gene generated a fluorescent signal. We assessed the robustness of this assay, by analyzing spiked shrimps by Q-PCR and traditional culture methods, using most probable number (MPN)-PCR. After a 6 h enrichment period, the assay successfully detected total and pathogenic V. parahaemolyticus in shrimps samples spiked with less than 5 V. parahaemolyticus cells/g of shrimp. The Q-PCR results obtained were compared with those obtained by most probable number (MPN)-PCR format. An excellent correlation between the two methods was observed in all cases (R² > 0.9742). The application of this Q-PCR assay to 85 natural shrimp samples also resulted in the successful quantification of V. parahaemolyticus in this matrix, and the counts obtained were correlated with those obtained by (MPN)-PCR (P = 0.2598). Thus, this rapid and sensitive Q-PCR can be used to quantify V. parahaemolyticus in natural shrimp samples. This assay could be proposed, in response to the demands of the European Commission, as a tool for testing for the presence of vibrios in crustaceans, making it possible to legislate in this domain.     

Effect of different fermentation parameters on l-lactic acid production from liquid distillery stillage

Expansion of lactic acid applications, predominantly for the preparation of biodegradable polymers increased the research interest for new, economically favourable production processes. Liquid stillage from bioethanol production can be an inexpensive, valuable source of nutrients for growth of lactic acid bacteria. Utilisation of residual biomass with spent fermentation media as a functional animal feed can greatly influence the process value and its ecological aspect. In this paper, the kinetics of lactic acid and biomass production on liquid stillage by Lactobacillus rhamnosus ATCC 7469 was studied. In addition, the impact of temperature, inoculum concentration, shaking and pH control by addition of CaCO₃ was evaluated. Maximal lactic acid yield of 73.4%, as well as high biomass production (3 × 10⁸ CFU ml⁻¹) were achieved under selected conditions (41 °C, 5% (v/v) of inoculum, 1% (w/v) of CaCO₃, initial pH of 6.5 and shaking rate of 90 rpm). These results were achieved without supplementation of the stillage with nitrogen or mineral sources.     

Molecular typing of Vibrio parahaemolyticus isolates from the middle-east coastline of China

The occurrence of outbreaks of Vibrio parahaemolyticus gastroenteritis in China highlights the need for strain characterization and subtyping of this pathogenic species. A total of 56 epidemiologically-unrelated strains of V. parahaemolyticus were isolated from clinical samples, seafood and various environmental sites in the middle-east coastline of China from 2006 to 2008. The isolates were characterized using four molecular typing methods, including ribotyping, enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), pulsed-field gel electrophoresis (PFGE), and sequence analysis of the gyrB gene. Genetic profiles of cluster analysis from these molecular typing tests clearly showed that there were differences in potential pathogenicity among isolates from seafood and its environments. Genetic characterization of two isolates (F13 and QS2) that originated from seafood demonstrated that they were potentially pathogenic. Discriminatory indices of four typing methods for the 56 V. parahaemolyticus isolates were differentiated by Simpson's Index of Diversity. The discriminatory index of ERIC-PCR typing was maximal (D = 0.942), while that of sequence analysis of the gyrB gene was minimal (D = 0.702). The discriminatory ability was greatly enhanced (D = 0.966) when ERIC-PCR was coupled with sequence analysis of the gyrB gene. These results suggest that ERIC-PCR combined with sequence analysis of gyrB gene may be a reliable, rapid typing strategy for V. parahaemolyticus strains.     

Lipid components and water soluble metabolites in salted and dried tuna (Thunnus thynnus L.) roes

The salted and dried product of tuna roe (bottarga) is a seafood characteristic of the Mediterranean area and exported all over the world. Samples of bottarga from bluefin tunas (Thunnus thynnus, L.) caught in the southwest Mediterranean sea were analysed. The samples were characterised by high content of marine wax esters (55–67 mol% of lipid classes), of docosahexaenoic (22:6 n-3, 25 w%) and oleic (18:1 n-9, 19 w%) fatty acids. Cholesterol was detected as 7–9 w% of lipids. Free fatty acids, index of lipid hydrolysis, represented 32–39 mol% over total fatty acids. Among metabolites, nutrients as taurine, nicotinamide and β-alanine, were found. The microflora comprised staphylococci, enterococci (2.2 log₁₀ CFU/g) and lactic acid bacteria (3 log₁₀ CFU/g). The food-borne pathogens Staphylococcus aureus, Listeria monocytogenes and Salmonella spp. were not detected. These findings indicate tuna bottarga as valuable source of nutrients.     

Pasteurization of grapefruit juice using a centrifugal ultraviolet light irradiator

Studies are lacking on the nonthermal pasteurization of liquid foods using UV irradiators that centrifugally form very thin films to overcome the problem of limited penetration depth of UV. Grapefruit juice inoculated with Escherichia coli or Saccharomyces cerevisiae was processed at the following conditions: UV dose 4.8–24 mJ/cm²; treatment time 3.2 s, cylinder rotational speed 450–750 rpm, cylinder inclination angle 15–45°, outlet temperature 11 °C, and flow rate 300 ml/min, and was stored for 35 days. Appropriate dilutions of the samples were pour plated with TSA and TSA + 3% NaCl for E. coli and Sabouraud dextrose agar (SDA) and SDA + 5% NaCl for S. cerevisiae. Nonthermal UV processing at 19 mJ/cm², 450 rpm and 15° reduced E. coli in grapefruit juice by 5.1 log₁₀. A dose of 14 mJ/cm² reduced S. cerevisiae by 6.0 log₁₀. Inactivation increased linearly with increasing UV dose. The inactivations at 600 and 750 rpm were similar, and were better than at 450 rpm. The results at 30° and 45° were similar, and were better than at 15°. The occurrence of sublethal injury in either microorganism was not detected. Storing UV processed grapefruit juice at 4 and 10 °C reduced the surviving E. coli to below 1 log₁₀ cfu/ml in 14 days. Processing UV juice reduced the population of S. cerevisiae to less than 1 log₁₀ cfu/ml where it remained for 35 days during refrigerated storage. These results suggest that grapefruit juice may be pasteurized using a nonthermal UV irradiator that centrifugally forms a thin film.     

Preparation of eicosapentaenoic acid concentrates from sardine oil by Bacillus circulans lipase

An extracellular lipase derived from Bacillus circulans, isolated from marine macroalga, Turbinaraia conoides, was used to prepare n-3 polyunsaturated fatty acid (PUFA) concentrates from sardine oil triglycerides. The enzyme was purified 132-fold with specific activity of 386 LU/mg. The purified lipase was able to enrich sardine oil with 37.7 ± 1.98% 20:5n-3 and 5.11 ± 0.14% 18:3n-3 in the triglyceride fraction after 3 h of hydrolysis. Lower hydrophobic constants of n-3 fatty acids (18:3n-3_logP = 5.65; 20:5n-3_logP = 5.85, respectively) than n-6 (20:4n-6_logP = 6.16) resulted in higher hydrolytic resistance of the former toward lipase, leading to their enrichment in the triglyceride fraction. Lipase-catalysed hydrolysis of sardine oil for 3 h, followed by urea complexation, provided free fatty acids containing 51.3 ± 4.65% 20:5n-3. The purified methyl ester of 20:5n-3 (68.29 ± 2.15%) from the urea concentrate was attained by chromatography on argentated neutral alumina.     

Biological characterization of two marine Bdellovibrio-and-like organisms isolated from Daya bay of Shenzhen, China and their application in the elimination of Vibrio parahaemolyticus in oyster

Bdellovibrio-and-like organisms (BALOs) are a group of highly motile delta-proteobacteria that prey on other gram-negative bacteria. However, nothing is known of the application potential of marine BALOs in safeguarding seafood safety. Here, biological characterization of two marine BALOs strains and their application in the elimination of Vibrio parahaemolyticus in oyster (Crassostrea ariakensis) at the laboratory scale were investigated.BALOs strains BDH12 and BDHSH06 were isolated from sediment of Daya bay in Shenzhen of China, with Shewanella putrefaciens strain 12 and V. parahaemolyticus strain SH06 as preys, respectively, when using double layer agar technique. They were identified as BALOs morphologically by transmission electron microscopy, while partial 16S rDNA sequencing analysis revealed that they showed no close relationships with members of the known genera Bdellovibrio, Bacteriolyticum, Bacteriovorax, or Peredibacter.Biological characterizations revealed that both strains had the optimal pH, salinity and temperature at 7.2, 3% and 30 °C, correspondingly. They could not utilize autoclaved, dead cells as hosts. Prey range analysis revealed that individually, BDH12 and BDHSH06 lysed 82.5% (47 strains) and 84.2% (48 strains) of the total 57 preys tested respectively. In combination, they lysed 98.2% (56 of 57) strains. All strains of V. parahaemolyticus, Vibrio cholerae and Vibrio alginolyticus tested could be lysed by both strains.A 7-day laboratory-scale V. parahaemolyticus elimination experiment in oyster showed that in the control, the cell counts of total vibrios and V. parahaemolyticus strain Vp plus in water and in oyster intestines were on the rise, whereas in the BALOs treated groups, their numbers were down from 8.09 ± 0.05 log CFU/ml and 8.02 ± 0.04 log CFU/ml to 2.39 ± 0.01 log CFU/ml and 2.33 ± 0.01 log CFU/ml, respectively. The same patterns could also be observed in oyster intestines. Results of this study indicate the feasibility of using BALOs to biologically control or even eliminate V. parahaemolyticus in seafood oyster.     

Change in attachment of Salmonella Typhimurium, Yersinia enterocolitica, and Listeria monocytogenes to pork skin and muscle after hot water and lactic acid decontamination

The attachment of Salmonella enterica subsp. enterica serovar Typhimurium, Yersinia enterocolitica, and Listeria monocytogenes to pig skin and muscle tissue decontaminated with 80 °C water or 55 °C, 1% lactic acid for 5 and 15 s was investigated. Attachment properties differed between skin and muscle surfaces. A significantly higher number of firmly attached bacteria was found on the decontaminated skin surface compared to the non-treated skin surface, both on hot water (P < 0.0001) and on lactic acid treated skin (P < 0.001). At the muscle surfaces, no such difference in attachment were shown between hot water treated surfaces and non-treated surfaces. In contrast, for lactic acid decontamination, significantly fewer bacteria attached to the treated muscle surfaces (P < 0.0001). The study did not show significant differences in surface attachment, between Salmonella, Yersinia and Listeria, which indicate that surface and environmental factors may influence attachment more than bacterial properties. A more profound location of attached bacteria at muscle compared to skin was indicated. Confocal laser scanning microscopy studies showed that bacteria located in deep tissue structures of non-decontaminated and decontaminated skin and muscle surfaces. In the latter, bacteria tended to “hide” between the muscle fibres and may be entrapped at those sites. The finding of changed attachment properties at skin after decontamination may play a role in cross- and recontamination, during subsequent meat processing.     

Minimizing the level of Bacillus cereus spores in farm tank milk

In a year-long survey on 24 Dutch farms, Bacillus cereus spore concentrations were measured in farm tank milk (FTM), feces, bedding material, mixed grass and corn silage, and soil from the pasture. The aim of this study was to determine, in practice, factors affecting the concentration of B. cereus spores in FTM throughout the year. In addition, the results of the survey were used in combination with a previously published modeling study to determine requirements for a strategy to control B. cereus spore concentrations in FTM below the MSL of 3 log₁₀ spores/L. The B. cereus spore concentration in FTM was 1.2 ± 0.05 log₁₀ spores/L and in none of samples was the concentration above the MSL. The spore concentration in soil (4.9 ± 0.04 log₁₀ spores/g) was more than 100-fold higher than the concentration in feces (2.2 ± 0.05 log₁₀ spores/g), bedding material (2.8 ± 0.07 log₁₀ spores/g), and mixed silage (2.4 ± 0.07 log₁₀ spores/g). The spore concentration in FTM increased between July and September compared with the rest of the year (0.5 ± 0.02 log₁₀ spores/L difference). In this period, comparable increases of the concentrations in feces (0.4 ± 0.03 log₁₀ spores/g), bedding material (0.5 ± 0.05 log₁₀ spores/g), and mixed silage (0.4 ± 0.05 log₁₀ spores/g) were found. The increased B. cereus spore concentration in FTM was not related to the grazing of cows. Significant correlations were found between the spore concentrations in FTM and feces (r = 0.51) and in feces and mixed silage (r = 0.43) when the cows grazed. The increased concentrations during summer could be explained by an increased growth of B. cereus due to the higher temperatures. We concluded that year-round B. cereus spores were predominantly transmitted from feeds, via feces, to FTM. Farmers should take measures that minimize the transmission of spores via this route by ensuring low initial contamination levels in the feeds (<3 log₁₀ spores/g) and by preventing growth of B. cereus in the farm environment. In addition, because of the extremely high B. cereus spore concentrations in soil, the contamination of teats with soil needs to be prevented.     

Occurrence and distribution of Vibrio parahaemolyticus in retail oysters in Sao Paulo State, Brazil

Vibrio parahaemolyticus is a potentially pathogenic bacterium that occurs naturally in estuarine environments worldwide, and is often associated with gastroenteritis in humans following consumption of raw bivalve mollusks, especially raw oysters. The occurrence of total and pathogenic V. parahaemolyticus in 74 samples of raw oysters collected in restaurants, supermarkets, groceries and beach huts in Sao Paulo State, was monitored between February 2006 and January 2007. Enumeration of V. parahaemolyticus was performed according to the most probable number (MPN) procedure. Five to ten typical colonies were selected from thiosulfate-citrate-bile salts-sucrose (TCBS) agar plates for confirmation by the presence of the species-specific gene tlh and the virulence genes tdh and trh by multiplex PCR. V. parahaemolyticus was detected in 100% of samples. The densities of total V. parahaemolyticus varied from 1.78 to 6.04 log₁₀ (MPN/g), with higher densities being detected in fall and summer, and lower densities in winter (P < 0.05). There was no statistical difference among densities of V parahaemolyticus regarding the site of collection. None of the 1943 V. parahaemolyticus isolates contained tdh and/or trh. These data provide information for the assessment of exposure to V. parahaemolyticus in oysters consumed in Sao Paulo, State, Brazil.     

Inhibition of Bacillus cereus spore outgrowth and multiplication by chitosan

Bacillus cereus is an endospore-forming bacterium able to cause food-associated illness. Different treatment processes are used in the food industry to reduce the number of spores and thereby the potential of foodborne disease. Chitosan is a polysaccharide with well-documented antibacterial activity towards vegetative cells. The activity against bacterial spores, spore germination and subsequent outgrowth and growth (the latter two events hereafter denoted (out)growth), however, is poorly documented. By using six different chitosans with defined macromolecular properties, we evaluated the effect of chitosan on Bacillus cereus spore germination and (out)growth using optical density assays and a dipicolinic acid release assay. (Out)growth was inhibited by chitosan, but germination was not. The action of chitosan was found to be concentration-dependent and also closely related to weight average molecular weight (M_w) and fraction of acetylation (F_A) of the biopolymer. Chitosans of low acetylation (F_A = 0.01 or 0.16) inhibited (out)growth more effectively than higher acetylated chitosans (F_A = 0.48). For the F_A = 0.16 chitosans with medium (56.8 kDa) and higher M_w (98.3 kDa), a better (out)growth inhibition was observed compared to low M_w (10.6 kDa) chitosan. The same trend was not evident with chitosans of 0.48 acetylation, where the difference in activity between the low (19.6 kDa) and high M_w (163.0 kDa) chitosans was only minor. In a spore test concentration corresponding to 10²-10³ CFU/ml (spore numbers relevant to food), less chitosan was needed to suppress (out)growth compared to higher spore numbers (equivalent to 10⁸ CFU/ml), as expected. No major differences in chitosan susceptibility between three different strains of B. cereus were detected. Our results contribute to a better understanding of chitosan activity towards bacterial spore germination and (out)growth.     

Isomaltulose production by free cells of Serratia plymuthica in a batch process

Free cells of Serratia plymuthica were used to convert sucrose into isomaltulose and trehalulose reducing sugars. The effects of substrate concentration and temperature were observed in a batch process with flasks shaken at 150 rpm. The experimental design and response surface methodology analysis indicated that the conversion parameters had significant influence (p < 0.05) on sucrose conversion, and that a valid model was obtained after an analysis of variance (F_model = 10.48 > F_charted = 5.79) to obtain a response surface and isocurve. Conversion was favoured when a 30% sucrose solution and temperature of 25 °C were used, which resulted in a high conversion into isomaltulose – over 70% – and 7–8% trehalulose. Small amounts of glucose (5–7%) and fructose (5–8%) were formed in the reaction medium.     

Aerobic bacterial, coliform, Escherichia coli and Staphylococcus aureus counts of raw and processed milk from selected smallholder dairy farms of Zimbabwe

A cross sectional study was conducted to enumerate total viable bacteria (TBC), coliforms, Escherichia coli and Staphylococcus aureus in raw (n = 120) and processed (n = 20) milk from individual farms from three smallholder dairy schemes of Zimbabwe between October, 2009 and February, 2010. Data on management factors were collected using a structured questionnaire. A standard pour plate technique was used to enumerate total viable bacteria, while for coliforms, E. coli and S. aureus, counts were assessed by the spread plate technique. The association of total viable bacterial counts and management factors was assessed using univariable and a linear regression model. The log₁₀ TBC for raw milk differed significantly (P < 0.05) amongst the schemes with the lowest (5.6 ± 4.7 log₁₀ cfu/ml) and highest (6.7 ± 5.8 log₁₀ cfu/ml) recorded from Marirangwe and Nharira respectively. The mean log₁₀ of TBC of processed milk (6.6 ± 6.0 log₁₀ cfu/ml) were marginally higher than those of raw milk (6.4 ± 5.6 log₁₀ cfu/ml) but not significant (P > 0.05). The coliform, E. coli and S. aureus counts for raw milk significantly differed (P < 0.05) amongst the study areas. The variation in TBC, coliforms, E. coli and S. aureus counts amongst the schemes could be attributed to differences in milking hygiene where farms with more access to training and monitoring of microbiological quality of milk had lower counts. Linear regression analysis revealed dairy scheme, delivery time and season of milking as independently associated with increased TBC of raw milk. The high TBC of raw and processed milk generally indicated low levels of milking hygienic practices, and high level of post-processing contamination, respectively. The high TBC, coliform, E. coli and S. aureus counts of both raw and processed milk may present a public health hazard. Thus, educating the farmers on general hygienic practices, quickening the delivery of milk to collection centres, or availing cooling facilities on-farm will improve the microbiological quality and safety of milk.     

Effect of Lactobacillus plantarum and chitosan in the reduction of browning of pericarp Rambutan (Nephelium lappaceum)

The effects of Lactobacillus plantarum alone or in combination with chitosan were evaluated on quality and color retention in rambutan fruits (Nephelium lappaceum) stored at 25 °C and 10 °C with 75 ± 2.5% of relative humidity for 10 and 15 days, respectively. The development of the microorganisms was evidenced by viability analyses and lactic acid production. The application of L. plantarum significantly improved color retention (a* and L*), and reduced weight losses. The lactobacilli, alone or in combination with chitosan, preserved fruit quality characteristics such as firmness, total soluble solids and titratable acidity. The lactobacilli application on rambutan pericarp produced acidification of pericarp and avoided the browning; thereby desiccation was prevented due to biofilm formation.     

Efficacy of neutral electrolyzed water for sanitization of cutting boards used in the preparation of foods

The effectiveness of neutral electrolyzed water (NEW) to sanitize cutting boards used for food preparation was investigated. Cutting boards made of hardwood and bamboo were inoculated with Escherichia coli K12 and Listeria innocua, dried for 1 h, washed, rinsed and sanitized with NEW, sodium hypochlorite (NaClO) solution, or tap water (control). After each washing protocol, surviving bacterial populations were determined. Results showed that both NEW and NaClO sanitizing solutions produced similar levels of bacterial reductions. In manual washing, the population reductions by NEW and NaClO were 3.4 and 3.6 log₁₀ CFU/100 cm² for E. coli, and 4.1 and 3.9 log₁₀ CFU/100 cm² for L. innocua, respectively. In the automatic washing, the reductions by NEW and NaClO were 4.0 and 4.0 log₁₀ CFU/100 cm² for E. coli, and 4.2 and 3.6 log₁₀ CFU/100 cm² for L. innocua, respectively. No significant differences (P > 0.05) were observed in surviving bacteria counts when comparing board material types.     

Thermal inactivation of Salmonella spp. during conching

Although chocolate is a microbiologically stable product it has been described as a vehicle for Salmonella spp. Because of the low water activity (a_w) and the high fat content of chocolate Salmonella spp. shows an increased heat resistance, even during the thermal process of chocolate making. The aim of this study was to evaluate the thermal inactivation of Salmonella spp. during conching in various masses of chocolate and cocoa butter at different temperatures (50-90 °C). The effect of thermal treatment on Salmonella spp. was determined with the MPN (Most-Probable-Number) method. Results of thermal treatment showed approximate D-values for cocoa butter from D_50°C = 245 min to D_60°C = 306 min, for cocoa liquor from D_50°C = 999 min to D_90°C = 26 min and for dark chocolate of D_50°C = 1574 min. z-values were found to be z = 20 °C in cocoa liquor and z = 14 °C in dark chocolate. This study demonstrates that the conching process alone does not ensure the inactivation of Salmonella spp. in different chocolate masses and that an additional decontamination step at the beginning of the process as well as an HACCP concept is necessary during chocolate production to guarantee the absence of Salmonella spp. in chocolates and related products.     

Fat content increases the lethality of ultra-high-pressure homogenization on Listeria monocytogenes in milk

Listeria monocytogenes CCUG 15526 was inoculated at a concentration of approximately 7.0 log₁₀ cfu/mL in milk samples with 0.3, 3.6, 10, and 15% fat contents. Milk samples with 0.3 and 3.6% fat content were also inoculated with a lower load of approximately 3.0 log₁₀ cfu/mL. Inoculated milk samples were subjected to a single cycle of ultra-high-pressure homogenization (UHPH) treatment at 200, 300, and 400 MPa. Microbiological analyses were performed 2 h after the UHPH treatments and after 5, 8, and 15 d of storage at 4°C. Maximum lethality values were observed in samples treated at 400 MPa with 15 and 10% fat (7.95 and 7.46 log₁₀ cfu/mL), respectively. However, in skimmed and 3.6% fat milk samples, complete inactivation was not achieved and, during the subsequent 15 d of storage at 4°C, L. monocytogenes was able to recover and replicate until achieving initial counts. In milk samples with 10 and 15% fat, L. monocytogenes recovered to the level of initial counts only in the milk samples treated at 200 MPa but not in the milk samples treated at 300 and 400 MPa. When the load of L. monocytogenes was approximately 3.0 log₁₀ cfu/mL in milk samples with 0.3 and 3.6% fat, complete inactivation was not achieved and L. monocytogenes was able to recover and grow during the subsequent cold storage. Fat content increased the maximum temperature reached during UHPH treatment; this could have contributed to the lethal effect achieved, but the amount of fat of the milk had a stronger effect than the temperature on obtaining a higher death rate of L. monocytogenes.     

Hollow fibre-based liquid phase microextraction combined with high-performance liquid chromatography for the analysis of flavonoids in Echinophora platyloba DC. and Mentha piperita

A simple, inexpensive and efficient three phase hollow fibre liquid phase microextraction (HF-LPME) technique combined with HPLC was used for the simultaneous determination of flavonoids in Echinophora platyloba DC. and Mentha piperita. Different factors affecting the HF-LPME procedure were investigated and optimised. The optimised extraction conditions were as follows: 1-octanol as an organic solvent, pH_donor = 2, pH_acceptor = 9.75, stirring rate of 1000 rpm, extraction time of 80 min, without addition of salt. Under these conditions, the enrichment factors ranged between 146 and 311. The values of intra and inter-day relative standard deviations (RSD) were in the range of 3.18–6.00% and 7.25–11.00%, respectively. The limits of detection (LODs) ranged between 0.5 and 7.0 ng mL⁻¹. Among the investigated flavonoids quercetin was found in E. platyloba DC. and luteolin was found in M. piperita. Concentration of quercetin and luteolin was 0.015 and 0.025 mg g⁻¹ respectively.     

Greek dry-salted olives: Monitoring the dry-salting process and subsequent physico-chemical and microbiological profile during storage under different packing conditions at 4 and 20 °C

Naturally black olives cv. Thassos were processed in dry salt according to industrial procedure by uniformly dispersing 40 kg of coarse salt in 100 kg freshly harvested olives. Dry-salting process was monitored by measuring selected physico-chemical (weight loss, NaCl content, pH, a_w, reducing sugars) and microbiological parameters (total viable counts, lactic acid bacteria, enterobacteria, yeasts, pseudomonads). Total weight loss amounted to 21 g/100 g after 80 days of dry-salting. Salt content in the fruits increased to 7.4 g/100 g followed by a decrease in water activity from 0.98 to 0.76. The pH did not change significantly presenting a slight decrease from 5.25 to 4.92. The initial microflora of the fruits comprised of lactic acid bacteria (4.1 log₁₀ cfu/g), yeasts (5.7 log₁₀ cfu/g), enterobacteria (3.7 log₁₀ cfu/g ) and pseudomonads (4.0 log₁₀ cfu/g). In the end of dry-salting, no microbial groups were enumerated apart from yeasts, due to the low water activity of the product. After dry-salting, olives were packed in HDPE bags under air (control samples) and 100 ml/100 ml CO₂. Another lot of fruits was dipped in 1 g/100 ml solution of potassium sorbate for 10 min prior to packing in the same bags under aerobic conditions. All packages were stored at 4 and 20 °C for a period of 180 days. During storage, the microbial flora comprised of yeasts, while no lactic acid bacteria, enterobacteria, pseudomonads or Staphylococcus aureus were detected as the low water activity/high salt content does not favour their growth. At 4 °C, the population of yeasts declined steadily throughout storage, but to a different extend depending on the packing treatment. At 20 °C, only potassium sorbate was effective in suppressing yeast growth. All packing treatments prevented fungal growth at both storage temperatures, apart from the samples stored in air. The pH, a_w and salt content did not change significantly throughout storage.