Elham Shokooh Saremi; Mohammad Bagher Habibi Najafi; Mohammad Hossein Hadad Khodaparast; Masoumeh Bahreini
Abstract
Introduction: It has been well demonstrated that vegetables provide, in addition to other basic nutrients, bioactive substances with beneficial effects on human health. In fact, the consumption of vegetables has been associated with lower incidence and lower mortality rates of cancer and cardiovascular ...
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Introduction: It has been well demonstrated that vegetables provide, in addition to other basic nutrients, bioactive substances with beneficial effects on human health. In fact, the consumption of vegetables has been associated with lower incidence and lower mortality rates of cancer and cardiovascular diseases in humans. Increasing demand for natural additives has shifted the attention from synthetic to natural antimicrobial agents. Leafy vegetables are found to be good source of antimicrobial agents. This study was aimed to examine the antimicrobial activity of leaf extracts of pimpinella affinis. Pimpinella affinis is a member of the family Apiaceae. This biennial herb grows up to 110 cm tall and is native in central and northern parts of Iran. In traditional medicine this herb is being used as carminative agent, appetizer, diuretic, antispasmodic drug, antimicrobial, sedative and lactation medication. It has also been distinguished as an antioxidant and antibacterial agent. There are several methods of obtaining extract from plants including maceration, super critical fluid extraction, subcritical water extraction, microwave and ultrasonic assisted method.
Materials and Methods: After collection from natural habitats of Pimpinella affinis in Mazandaran Province, it was then approved by the Department of Botany of Faculty of Agriculture of University of Sari. The plant was dried in a dry and dark place away from the sun and then was pulverized in the mill and sieved by a mesh of 80 (800 microns). Pimpinella affinis extract obtained by using maceration extraction (ME), ultrasonic assisted method (UAE) and supercritical fluid extraction (SFE). Ethanol: water in 50:50 ratio used as solvent for extraction. Total phenolic content of different extracts was measured by Folin-ciocalteu method. The phenolic compounds fractions were determined using Liquid chromatography–mass spectrometry system. After preparing the mother culture medium, the bacteria were cultured in MHB medium (37 ° C) for 24-18 hours. Stock solutions were prepared from each of the extracts. Serial dilutions of the extracts at concentrations of 0.01 to 10 mg / ml in 2.5% dimethyl sulfoxide were prepared and then sterilized with 0.22 μm pore size syringe filter. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration assay (MBC) were determined by micro dilution method for listeria monocytogenes (ATCC19112), staphylococcus aureus (ATCC25923), pseudomonas aeruginosa (ATCC9027) and Escherichia coli (ATCC25922). Antimicrobial growth was inhibited by measuring the absorbance and ELISA Reader was used to determine the growth rate of microorganisms, and the first house with the lowest absorption read as MIC (mg /ml) was determined. Statistical analysis of MIC, MBC and phenolic compounds of extracts results was done in a completely randomized design and using SPSS software version 20. The comparison of the means was done using Duncan’s test and one-way ANOVA method. In order to reduce the error, all experiments were performed in triplicate.
Results and Discussion: Total phenolic content of extracts ranged between 1502.25 to 1836.69 mg GA/100g E. The results showed that the ultrasonic assisted method have highest total phenolic content and the least phenolic content was observed in extract which obtained by supercritical fluid extraction. Chlorogenic Acid, Cafeic acid and Apigenin-6-C-glucoside were the predominant fractions in Pimpinella affinis which detected by Liquid chromatography–mass spectrometry system. The least and highest amount of MIC and MBC were belonged to ultrasonic assisted and supercritical fluid extracts, respectively. Staphylococcus aureus was most sensitive and Escherichia coli and Pseudomonas aeruginosa were most resistance bacteria. Pimpinella extract due to having phenolic compounds such as Gallic acid, Cafeic acid, Chlorogenic Acid, Kaempferol and Apigenin showed antimicrobial activity and can be used as natural antimicrobial agent.
Safieh Rajabzadeh; Masoumeh Bahreini; Mirza Mohammadreza Sharifmoghadam
Abstract
Introduction: Foodborne pathogens are a major public health problem. Despite the increase in management and consumer interest in food safety, the number of food poisoning cases has been continuously increased in recent years. For example in 2010, human salmonellosis cases were reported in Europe and ...
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Introduction: Foodborne pathogens are a major public health problem. Despite the increase in management and consumer interest in food safety, the number of food poisoning cases has been continuously increased in recent years. For example in 2010, human salmonellosis cases were reported in Europe and in 2011, a multistate foodborne E.coli O157:H7 outbreak affected 8 people and also a larger multistate Listeria monocytogenes outbreak caused 146 infections and 29 deaths. Traditional culture methods for detection of microorganisms in food are laborious and time consuming. Due to Economic impact of foodborne contamination and diseases, there are great efforts to develop more sensitive methods for rapid and accurate pathogen detection and identification in foodstuffs. Recently, molecular techniques such as PCR based DNA amplification have been employed that offer several advantages over the classical microbiological methods, such as shortening time of analysis, lowering detection limits, increasing specificity and having potential for automation. In this study we used multiplex PCR system, for the simultaneous detection of the three pathogens (salmonella spp., L. monocytogenes and E. coli O157:H7) in artificially inoculated lettuce. Materials and methods: Bacterial strains used in this study, Salmonella entrica serovar Typhimurium PTCC1709, Listeria monocytogenes PTCC1298 and E. coli O157:H7 NCTC12900, were grown in Trypticase Soy Broth at 37°C and then serial dilutions of strains from 100 to 106 cfu/ml (1 to 1000000 cfu/ml) were prepared using normal saline (0.85 g/l). Lettuce samples were cut into pieces of 5 ± 0.2 grams. To sterilize, the pieces overwhelm in 3% sodium hypochlorite solution for 15 min and in order to eliminate extra chlorine ions, the samples moved to sterile distilled water containing some drops of 1% sodium thiosulfate for 2 min. Bacterial strains were inoculated on lettuce as pure and mixed cultures separately. For the mixed cultures 100 µl of each bacterial strain inoculated on lettuce (in total 300 µl). The eluted bacteria were re-suspended in 100 µl of sterile normal saline and used for DNA extraction. DNA extraction was performed on each bacterial strain before and after inoculation to lettuce. The target genes were the rfb gene (antigen O157 producer) for E. coli O157:H7, the invA gene (invasion protein A) for salmonella spp., and the prfA gene (transcriptional activator of the virulence factor) for L. monocytogenes. The genes described here have been used as the most specific and reliable genetic targets for the above microorganisms. As an internal control gene, the 16S rRNA gene was targeted in the presence of bacterial DNA. Monoplex PCR reactions were performed in a final volume of 25 µl. Master mix composition was as follows: PCR buffer 10X, 2.5 µl; MgCl2 25mM, 2.5 µl; Taq DNA Polymerase 5 U/µl, 0.2 µl; dNTPs 10 mM, 0.4 µl; F/R primers 10 pmol, 1µl; extracted DNA as template, 2 µl and distilled water, 15.4 µl. Thermal cycler conditions were as follows: predenaturation at 94°C for 5min; 35 cycles consisting of denaturation at 94°C for 30s, annealing at 55°C for 30s and extension at 72°C for 60s; and final elongation at 72°C for 7 min. Multiplex PCR reactions were performed in a final volume of 25µl using 4 µl of total extracted DNA from three pathogens as template (mixture of three bacteria). Master mix composition was as follows: PCR buffer 10X, 2.5 µl; MgCl2 25 mM, 2.5 µl; Taq DNA Polymerase 5U/µl, 0.5 µl; dNTPs 10mM, 1 µl; EC-F/R, 1 µl; SAL-F/R, 0.8 µl and LIS-F/R, 1 µl (concentration of each primer was 10 pmol) and distilled water 8.9 µl. Thermal cycler conditions were as follows: pre-denaturation at 94°C for 3 min; 35 cycles consisting of denaturation at 94°C for 30s, annealing at 57°C for 30s and extension at 72°C for 90s; and final elongation at 72°C for 10 min. PCR products were visualized via gel electrophoresis, with 1% agarose gels. Results and Discussion: The main focus of this work was on the use of rapid method instead of culturing stages for detection of foodborne pathogens from ready-to-eat vegetables. Lettuce was used as a model for ready-to-eat vegetables and inoculated with E. coli O157:H7, salmonella spp. and L. monocytogenes. Multiplex PCR was performed after elution of bacteria from lettuce and the test provided a detection limit of 1 cfu/1g for three foodborne pathogenic bacteria The primer selection and multiplex PCR optimization allowed the setting of a robust method with performances comparable to a duplex PCR system, when tested on a complex food system. The sensitivity and robustness of the method proposed, together with its ability to perform well on a complex food matrix, make it a suitable method to be implemented in control laboratories for the detection of the target pathogens in food samples.
Neda Nayyeri; Mohammad Reza Edalatian Dovom; Mohammad Bagher Habibi Najafi; Masoumeh Bahreini
Abstract
Introduction : Nowadays, attention to foods free from chemical preservatives is on the rise. Recently, consumers have concerned about foods containing these preservatives in their formulation .Therefore,the use of antimicrobial peptides produced by Lactic acid bacteria (LAB) are strongly highlighted. ...
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Introduction : Nowadays, attention to foods free from chemical preservatives is on the rise. Recently, consumers have concerned about foods containing these preservatives in their formulation .Therefore,the use of antimicrobial peptides produced by Lactic acid bacteria (LAB) are strongly highlighted. Ribosomally-synthesized peptides, which possess antimicrobial properties, are produced by a vast range of organisms including prokaryotes and Eukaryotes.Yeasts are the most important microorganisms which are responsible for fruit juice and soft drink spoilage. In case of growth and production of by-products like CO2, acid and other contaminants, the spoilage will be visible.Lactic acid bacteria isolated from natural, local sources such as dairy products have presented potentially antifungal features against food spoilage fungi. Among these, Rodotorula and Penicillium are regarded as the most critical genera in fruit juice spoilage.The main objective ofthis study was the evaluation of anti-yeast activity of Lactobacillus plantarum isolates from different stages of Lighvan production against Rodotorulamucilaginosa as fruit juice spoilage indicator. In the next step, technological parameters effects were analyzed on antimicrobial potential of cell-free supernatant of these isolates. Finally, we aimed at finding the most effective isolate on aforementioned yeast.Materials and methods: NineteenLb.plantarumisolates, which were identified previously, were subjected to antifungal assay. For this purpose, RodotorulamucilaginosaPTCC 5257 was selected. Agar spot and Well Diffusion Assay (WDA) were applied for antifungal assay in solid and liquid media, respectively.Determination of yeast colony: Following the cultivation of yeasts in Potato Dextrose Broth, it was determined using Spectrophotometer.Preparation of Lb.plantarum cell-free supernatant (CFS) was carried out. In agar spot method, clear zones of inhibition around the spotted colonies were evaluated after 24-48h incubation. In WDA, CFS of Lb.plantarum isolates were poured in wells and clear zones were evaluated around each well after 24-48 h incubation.Technological properties: The influence of different levels of pH (2, 3, 4, 5, 6, and 7) was analyzed on CFS of Lb.plantarum isolates. This assay was done according to Wang et al., 2011. Finally, results were reported using the measurement of clear zone diameter in mm. All experiments were performed in duplicates.The effects of various temperatures were applied on CFS and remaining the antifungal activity was evaluated according to Rouse et al., 2008. Finally, all CFS of Lb.plantarum isolates were subjected to Proteniase K and antifungal properties of CFS were assayed by WDA according to Ghrairi et al., 2005.Results and discussion:Agar spot results showed the highest and lowest clear zone of inhibition related to C28 and LF49 with 16 and 6 mm diameter, respectively. In this method, 11 out of 19 isolates (60%) presented anti-yeast activity with clear zone formation. In comparison between two incubation temperatures (25 and 30◦C), all isolates stronger clear zone in 30◦C than in 25◦C. This was due to enhancement of yeast growth in 25C rather than in 30C. Also, with respect to the mesophilic nature of Lb.plantarum isolates, the possibility of metabolite production are more likely in 30C. It was reported that antifungal activity of LAB is mostly due to synergistic effect of lactic acid and acetic acid. In agar spot, some colony-associated antimicrobial compounds are responsible for antifungal activity. In WDA, 8 out of 19 isolates (42%) were positive for their inhibitory effects. The highest anti-yeast activity was seen at pH=2. It seems that antimicrobial compounds are likely more stable at acidic conditions than at alkaline ones. Among isolates, C28 presented the highest stability at alkaline conditions. With pH increase, the antifungal activity decreased so that no anti-yeast activity was seen at pH=7. Regarding the different temperatures, we should mention that thermal resistance of isolates' CFSwitnessed declining trend with increasing of temperature. This fact implies the presence of low- molecular weight compounds in CFS. Finally, all isolates' CFS was subjected to proteinase K. All isolates have lost their anti-yeast activity after enzyme treatment showing their proteinaceous nature.Conclusion: In WDA, the number of positive isolates showing anti-yeast activity declined in comparison with agar spot. Since some isolates retain their inhibitory activity toward food spoilage yeast at low pH, their CFS can be applied in acidic foods like fruit juice. Also, some isolates showed their antifungal activity at high temperatures (80C and 100C) which are applied for fruit juice pasteurization, so they can be applied in fruit juices as a bio-preservative.
Masoumeh Bahreini; Mohammad Bagher Habibi Najafi; Mohammad Reza Bassami; Eisa Jahed
Abstract
Ozone is a strong oxidant and a potent disinfecting agent. There are numerous application areas of ozone in food industry. While there are many investigations on the application of ozone in food industry, relatively little information is available on the potential of ozone to reduce microbial populations ...
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Ozone is a strong oxidant and a potent disinfecting agent. There are numerous application areas of ozone in food industry. While there are many investigations on the application of ozone in food industry, relatively little information is available on the potential of ozone to reduce microbial populations in fresh-cut fruits and vegetables and the visual quality of lettuce. In this study, ozone water was applied at five concentrations (0.6, 0.8, 1.2, 1.6 and 2 ppm) for four exposure times (1, 3, 5 and 10 min) on natural microflora and E. coli O157:H7 and Salmonella inoculated of lettuce. The reduction in the total bacterial count, yeast and mold, total coliform, as well as lactic acid bacteria counts were examined. The promising results indicated the efficacy of ozone water to reduce the microbial populations on lettuce. In the best condition, the inoculated samples of E. coli O157:H7 (ATCC 35150, NCTC 12900), Salmonella typhi morium ( ATCC 14028, NCTC 12023) and Salmonella enterica subsp, enterica (PTCC 1709) on lettuce were decreased further than 2 log10 cfu/g. The results showed the population of aerobic mesophilic bacteria, yeast/ moulds, total coliforms and lactic acid bacteria were decreased to 1.54, 0.94, 1.94 and 1.35 log10 cfu/g respectively. The method of ozone generation, type of application, as well as the optimal exposure time and concentration of ozone as an antimicrobial agent on lettuce is mentioned in detail.
Masoumeh Bahreini; Mohammad Bagher Habibi Najafi; Mohammad Reza Bassami; Morteza Abbaszadegan
Abstract
We investigated the microbiological quality of raw vegetables in Mashhad, Iran. In order to document the incidence of indicator and pathogenic microorganisms in this area. ninety eghit raw vegetable samples collected during 7 months were analyzed. All samples were examined for the presence of aerobic ...
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We investigated the microbiological quality of raw vegetables in Mashhad, Iran. In order to document the incidence of indicator and pathogenic microorganisms in this area. ninety eghit raw vegetable samples collected during 7 months were analyzed. All samples were examined for the presence of aerobic mesophilic bacteria, coliforms, Enterobacteriaceae, Escherichia coli, Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus, yeast and mould. Our data showed that the incidence levels of aerobic mesophilic bacteria was less than 107 cfu/g on 83% raw vegetables and 100% of raw vegetables were contained Entrobacteriace and total coliforms, and their range varied from 1 log to 6 log cfu/g. The mean counts of Lactic acid bacteria and yeasts and moulds were 4.1 log cfu/g and 4.5 log cfu/g, respectively, and the incidence levels of yeasts and moulds in 83% of raw vegetables was less than 5 log cfu/g. Our results were similar with other studies that determined microbial levels on raw vegetables. 12.7% of samples raw vegetables contained E.coli, but only 4.1% were higher than ≥ 2 log cfu/g. The incidence levels for E.coli O157:H7 and Salmonella on raw vegetables were 6% and 12.7% respectively. 94.9% of raw vegetables were contaminated with S. aureus and 7.8% of them were coagulase positive.
Masoumeh Bahreini; Mohammad Bagher Habibi Najafi; Mohammad Reza Bassami; Morteza Abbaszadegan; Ahmad Reza Bahrami; Hamidreza Ejtehadi
Abstract
The number of vegetable processing plants has been increased during recent years in Iran as many other countries. On the other hand, fresh vegetable products are susceptible to microbial contamination after harvest, processing, handling, packing and distribution. The aim of this study was to determine ...
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The number of vegetable processing plants has been increased during recent years in Iran as many other countries. On the other hand, fresh vegetable products are susceptible to microbial contamination after harvest, processing, handling, packing and distribution. The aim of this study was to determine and evaluate the level of microbial load of vegetables during different cleaning steps in a fresh-cut vegetable processing plant and to identify the critical points in the process lines and operating areas. Samples were taken from the plant before and after washing, disinfection, cutter, drying and packaging and kept in ice pack and delivered to the laboratory. The samples were analyzed for mesophilic aerobic count, yeasts and moulds, lactic acid bacteria, total coliforms, Enterobacteriaceae, E. coli and Staphylococcus aureous as well as for the presence of Salmonella, according to the standard guideline. The amounts of total aerobic bacteria, Enterobacteriaceae, E. coli, yeasts and moulds on surfaces and air were also determined. Results showed that bacteria as well as yeasts and moulds levels were decreased after washing and disinfectation up to 1 and 1.5 log10, respectively, but after that, the microbial load was increased due to secondary contamination. During all steps, salmonella was not detected, but E. coli detected in some of the steps and S. aureous was detected at all steps. The highest levels of total aerobic bacteria, Enterobacteriaceae, E. coli, yeasts and moulds were detected on the equipments (cutters, peeling, centrifuge machines, etc.). Different Hygienic areas should be separated enough to allow maintenance of good hygiene in cleaner areas in primary washing steps. Despite this, the results showed that there is a vital need to improve cleaning and hygienic practices in vegetable processing plants. Several practical recommendations were given for cleaning, design of production lines, training of employees and surface hygiene.
Keywords: Ready to eat vegetables, Microbial evaluation, Microbial quality, Minimal processing, Hygienic level
