Health risk assessment of some heavy metals detected in edible fishes of Gorgan Bay, Caspian Sea (Iran), for human.

Just recently, heavy metals have been dramatically detected in aquatic animals, especially in fishes; hence, documented method to assess their health risks for humans who eat these contaminated fishes could be helpful. For the sake of this aim, the health risk assessment of four heavy metals including arsenic, cadmium, lead, and copper in three main edible fishes caught from Gorgan bay was studied. Mullet, roach, and common carp have been caught randomly in winter and spring of 2021 from three different fishing sites of Gorgan bay, including Miankaleh wetland, estuary of Qaresu River, and mouth of Chapaghli canal. Heavy metal measurement has been done with MOOPAM method, and risk assessment of fish consumption was assessed by estimated daily (EDI) and weekly intakes (EWI), target hazard quotient (THQ), hazard index (HI), and target cancer risk (TCR) indices. In this study, estimated daily intake (EDI) was much lower than the tolerable daily intake. Although TCR of cadmium within all fishes and arsenic in two fishes were more than acceptable value, THQ and HI values were less than one, and TCR of lead as well as copper was lower than the critical values, which suggests heavy metals of the Gorgan bay have no serious risks to consumer health.

Bioaccumulation of Some Hazardous Heavy Metals in Three Edible Fishes Caught from Gorgan Bay, Caspian Sea.

A survey was conducted to evaluate heavy metal accumulation in edible fishes caught from Gorgan Bay, as a part of the Caspian Sea ecosystem. Fish samples including mullet, roach, and common carp were just randomly prepared from fishing locations in three different areas of Gorgan Bay including estuary, channel, and Miankale wetland twice the fishing seasons. A total of 135 samples were measured using an atomic absorption spectrometer to 4 heavy metals of arsenic (As), lead (Pb), cadmium (Cd), and copper (Cu) (a total of 540 metal samples). The highest accumulation of As was related to the mullet of estuary, and the lowest contamination was related to the mouth of the channel. In relation to Cd, the highest pollution in all three species related to the fishes caught from the estuary and the lowest accumulation related to the Miankaleh wetland. In the case of metal Pb, results showed the most contamination related to the Mullet caught from Miankaleh wetland and the lowest accumulation related to the roach of the mouth of the channel. In all three fish species, the highest contamination of Cu was in the estuary and the lowest was in the fish caught from Miankaleh wetland. In general, the highest accumulation belonged to all three species located in estuaries. The probable reason can be pouring river pollution to the bay more than wetlands and channel. Due to the enclosure of the Caspian Sea and consequently the Gulf of Gorgan, limiting the fishing times and points to the periods of least incoming pollutants is very helpful for public health, especially food hygiene and fishing from this ecologically sensitive and vulnerable basin.

Study of growth kinetic and gastrointestinal stability of acid-bile resistant Lactobacillus plantarum strains isolated from Siahmazgi traditional cheese.

In this study, 22 Lactobacillus plantarum strains isolated from Siahmazgi traditional cheese were evaluated using different tests including resistance to low pH (1.50 and 2.50) and bile salt (0.50 and 1.00%), growth kinetic at low pH values and survival under simulated gastric and intestinal fluids. All the strains retained their viability at pH 2.50. However, the survival of all of the isolates was decreased at pH 1.50. Ten out of 22 strains which were able to tolerate low pH were selected for further investigations. All the selected isolates were able to grow at low pH. Strain F2 showed the highest specific growth rate. Five out of 10 isolates showed a significant decrease in bacterial count varied from 2.00 to 7.00 log CFU mL-1 during 3 hr exposure to 0.50% bile salt, while five isolates represented resistance to 0.50% bile during 3 hr. A significant reduction was observed in survival of all of the isolate at 1.00% bile salt concentration. Furthermore, viability of the selected isolates was lowered during 1 hr incubation under gastric conditions, while it remained unchanged within next 2 hr. Although, no significant changes were seen in bacterial count of the selected isolates during 1 hr of exposure to simulated intestinal condition, the survival of the isolates was relatively reduced after 3 hr. In conclusion, five out of 22 examined L. plantarum isolates showed appropriate resistance properties, therefore, could be good candidates for further examinations including functional and safety evaluation supporting their use as probiotics.

Combined effects of Ziziphora clinopodioides essential oil and lysozyme to extend shelf life and control Listeria monocytogenes in Balkan-style fresh sausage.

This study was done to evaluate the effects of different concentrations of Ziziphora clinopodioides essential oil (ZCEO) (0, 0.1, and 0.3%) and lysozyme (0 and 400 µg/g) on control of Listeria (L.) monocytogenes and also microbial, chemical, and organoleptic properties of Balkan type fresh sausage under modified atmosphere packaging (MAP) during 13-day storage at refrigerated condition. Results revealed that treated sausages had a slower rate of increase in microbial count than control and sausages containing ZCEO (0.3%) and lysozyme (400 µg/g) possess the lowest microbial count at the end of the storage period. A reduction between 0.90 and 2.05 log CFU/g in L. monocytogenes was recorded for the treated sausage samples in comparison with control samples after 13 days of storage. Based on chemical findings, at the end of the storage, TVB-N value in the control sample gradually increased to 34.30 mg/100 g, whereas TVB-N values of the treated samples with each of the lysozyme and ZCEO alone or in combination were below 25 mg/100 g during the entire storage period. The final TBARS value for the control sample was 0.58 mg malondialdehyde/kg, while the TBARS values for the treated samples remained lower as 0.46 mg malondialdehyde/kg. Regarding sensory attributes, adding ZCEO results in insignificant lower scores in odor and taste than control in the early days of the study (p > .05). It can be argued that ZCEO alone or in combination with lysozyme showed good antimicrobial and antioxidant activities and may have this potential to be used as a preservative in fresh sausage without any significant adverse sensory effects (p > .05).

Phytochemical composition and in vitro safety evaluation of Ziziphora clinopodioides Lam. ethanolic extract: Cytotoxicity, genotoxicity and mutagenicity assessment.

ETHNOPHARMACOLOGICAL RELEVANCE: The application of the herb Ziziphora clinopodioides Lam. in folk medicine and as a food additive has been recommended due to its many claimed bioactivities. Regardless of the plant benefits, its safety considerations are largely unknown. AIM OF THE STUDY: The aim of the present research was to determine the chemical compositions and cytotoxicity, genotoxicity, and mutagenicity potentials of the ethanolic extract of Ziziphora clinopdioides Lam. (EEZC). MATERIALS AND METHODS: GC-MS and LC-MS analysis were used for chemical composition determination. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and trypan blue exclusion dye assays were used for cytotoxicity and the Comet assay was employed for genotoxicity assessment on human blood lymphocytes. Also, the Ames Salmonella/microsome test was carried out for the evaluation of mutagenicity. RESULTS: Pulegone was the main component of the n-hexane fraction. Different phenolic acids and flavonoids were detected by LC-MS. The cytotoxicity study indicated a conspicuous decline in human lymphocyte viability ranging from 52% to 100% as showed by the MTT assay and 67% up to 100% by the trypan blue assay, at 1 and 10 mg/mL, respectively. The Comet assay results revealed a dose dependent genotoxicity, in so much as 90% and 98% of the cells were screened as damaged at concentrations of 5 and 10 mg/mL, respectively. An incidence rate of 8% and 13% of grade 4 damage was observed at 5 and 10 mg/mL, respectively. Additionally, the DNA damage index (DI) was elevated dose-dependently by a rising concentration of the extract, wherein the DI at 10 mg/mL concentration was 2.22, which was 22 times greater than that of negative control, and even more than positive control. The Ames test exhibited no signs of mutagenicity for neither Salmonella typhimurium TA98 nor TA100 strains, accompanied or unaccompanied by S9 metabolic activation. CONCLUSION: Results indicated a dose-dependent cytotoxicity and genotoxicity potential of the EEZC on human lymphocytes, suggesting that this plant should be used with caution by consumers, even in the food and pharmaceutical industries. Since the plant usage in daily life continues to increase due to its ever growing phytotherapical and phytonutritional properties, it may pose a health risk by its high concentration's uptake. Although no mutagenicity of this extract was observed in this study, further research is recommended to clarify the mutagenic risks of this herb.

The effect of Zataria multiflora Boiss Essential oil on the growth and citrinin production of Penicillium citrinum in culture media and cheese.

The effect of Zataria multiflora Boiss Essential oil (EO) on the growth, spore production, and citrinin production of Penicellium citrinum PTCC 5304 in the culture media as well as Iranian ultra-filtered white cheese in brine was investigated. Radial growth and spore production on the potato dextrose agar (PDA) were effectively inhibited by EO in a dose-dependent manner. At 200 ppm, the radial growth and sporulation declined by 92% and 100%, respectively. The growth was completely prevented at 400 ppm of EO on PDA and the minimum fungicidal concentration (MFC) of the oil was estimated at 400 ppm. Furthermore, the Zataria multiflora also significantly suppressed the mycelial growth and citrinin production in broth medium at all investigated concentrations (P < 0.05). At 150 ppm of EO, the citrinin accumulation and mycelial growth reduced by 88.6% and 89.6%, respectively. The EO was tested at all concentrations and the findings show an inhibitory effect of P. citrinum against the radial fungal growth and citrinin production in cheese. However, no concentration of EO could completely inhibit the growth and production of citrinin in cheese. We therefore concluded that Zataria multiflora has the potential to substitute the antifungal chemicals as a natural inhibitor to control the growth of molds in foods such as cheese.

Nanocomposite films based on CMC, okra mucilage and ZnO nanoparticles: Physico mechanical and antibacterial properties.

This work examined the physico mechanical parameters and antibacterial activity of CMC/okra mucilage (OM) blend films containing ZnO nanoparticles (NPs). Different proportions of CMC and okra mucilage (100/0; 70/30; 60/40 and 50/50 respectively), were mixed and casted to posterior analysis of formed films. The more colored films were obtained by higher contents of okra mucilage and adding ZnO nanoparticles. The incorporation of ZnO NPs into CMC film decreased the elongation at the break (EB) value of the films and increased the tensile strength (TS) value of the film. With increase in CMC concentration in the films, higher water vapor permeability and higher solubility in water were achieved. Microstructure analysis using SEM showed a smooth and compact surface morphology, homogeneous structure, and a rough surface for CMC, CMC+ZnO, and CMC/OM30%+ZnO, respectively. Nanocomposite films presented antibacterial activity against tested bacteria. Films contained okra mucilage showed more antibacterial activity. The inhibitory activities of resultant films were stronger against S. aureus than E. coli.

Phenotypic and genotypic characterization of antibiotic resistance of methicillin-resistant Staphylococcus aureus isolated from hospital food.

BACKGROUND: Pathogenic biotypes of the Methicillin-resistant Staphylococcus aureus (MRSA) strains are considered to be one of the major cause of food-borne diseases in hospitals. The present investigation was done to study the pattern of antibiotic resistance and prevalence of antibiotic resistance genes of different biotypes of the MRSA strains isolated from various types of hospital food samples. METHODS: Four-hundred and eighty-five raw and cooked hospital food samples were cultured and MRSA strains were identified using the oxacillin and cefoxitin disk diffusion tests and mecA-based PCR amplification. Isolated strains were subjected to biotyping and their antibiotic resistance patterns were analyzed using the disk diffusion and PCR methods. RESULTS: Prevalence of S. aureus and MRSA were 9.69 and 7.62%, respectively. Meat and chicken barbecues had the highest prevalence of MRSA. Prevalence of bovine, ovine, poultry and human-based biotypes in the MRSA strains were 8.10, 8.10, 32.43 and 48.64%, respectively. All of the MRSA strains recovered from soup, salad and rice samples were related to human-based biotypes. MRSA strains harbored the highest prevalence of resistance against penicillin (100%), ceftaroline (100%), tetracycline (100%), erythromycin (89.18%) and trimethoprim-sulfamethoxazole (83.78%). TetK (72.97%), ermA (72.97%), msrA (64.86%) and aacA-D (62.16%) were the most commonly detected antibiotic resistance genes. CONCLUSIONS: Pattern of antibiotic resistance and also distribution of antibiotic resistance genes were related to the biotype of MRSA strains. Presence of multi-drug resistance and also simultaneous presence of several antibiotic resistance genes in some MRSA isolates showed an important public health issue Further researches are required to found additional epidemiological aspects of the MRSA strains in hospital food samples.

Antimicrobial evaluation of novel poly-lactic acid based nanocomposites incorporated with bioactive compounds in-vitro and in refrigerated vacuum-packed cooked sausages.

Biodegradability and antimicrobial activity of food packaging materials are among the most attractive parameters in modern food industries. In order to develop biodegradable poly-lactic acid (PLA) film to antibacterial nanocomposites, different concentration of Zataria multiflora Bioss. essential oil (ZME), propolis ethanolic extract (PEE) and cellulose nanofiber (CNF) were incorporated to the polymer by solvent casting method. The resulting films were characterized by mechanical and physical tests and their antimicrobial application was evaluated in-vitro against four common foodborne pathogens and in vacuum-packed cooked sausages during refrigerated storage. Mechanical examination revealed that addition of ZME and PEE made films more flexible and incorporation of CNF improved almost all mechanical parameters tested. Moreover, according to physical analysis, incorporation of 0.5% v/v ZME to the composite primary solutions improved water vapor permeability of the resulting films. Almost all of the active films were effective against the tested bacteria except for PLA/PEE films, and maximum antibacterial effects recorded for the films containing both ZME and PEE. Based on the microbiological and sensory evaluation of the sausages, all of the PLA/1%ZME/PEE composites increased the shelf life to >40days. The results indicate that incorporation of natural antimicrobial substances such as ZME and PEE to packaging material could be an interesting approach in development of active packaging material without significant negative effect on polymer technical properties.

Effect of PLA films containing propolis ethanolic extract, cellulose nanoparticle and Ziziphora clinopodioides essential oil on chemical, microbial and sensory properties of minced beef.

This study was conducted to examine the effects of polylactic acid (PLA) film containing propolis ethanolic extract (PE), cellulose nanoparticle (CN) and Ziziphora clinopodioides essential oil (ZEO) on chemical, microbial and sensory properties of minced beef during storage at refrigerated temperature for 11days. The initial total volatile base nitrogen (TVB-N) was 8.2mg/100g and after 7days reached to 29.1mg/100g in control, while it was lower than 25mg/100g for treated samples. At the end of storage time in control samples peroxide value (PV) reached to 2.01meqperoxide/1000g lipid, while the values for the treated samples remained lower than 2meqperoxide/1000g lipid. Final microbial population decreased approximately 1-3logCFU/g in treated samples compared to control (P<0.05). Films containing 2% ZEO alone and in combination with different concentrations of PE and CN extended the shelf life of minced beef during storage in refrigerated condition for at least 11days without any unfavorable organoleptic properties.

The effect of alginate and chitosan concentrations on some properties of chitosan-coated alginate beads and survivability of encapsulated Lactobacillus rhamnosus in simulated gastrointestinal conditions and during heat processing.

BACKGROUND: In this study, chitosan-coated alginate beads were produced with different concentrations of chitosan and alginate to evaluate the survival of encapsulated Lactobacillus rhamnosus GG during exposure to adverse conditions in gastrointestinal simulated juice and heat processing. RESULTS: The encapsulation yield of different encapsulation treatments was between 25 and 53.2%. Although there was a drastic decrease in pH within 48 h of incubation in MRS medium inoculated with free and encapsulated bacteria, no significant changes (P > 0.05) in bacterial count were observed among different encapsulation treatments. Moreover, the survival rate after gastrointestinal juice exposure of all prepared beads was 10-87 times greater than that of free cells and was significantly enhanced by increasing chitosan and alginate concentrations. The encapsulated bacteria survived significantly (P < 0.05) better than the free cells during heat exposure at 55, 60 and 65 °C: free cells experienced about 5 log cycles reduction after heat treatment at 65 °C for 30 min, whereas 40 g L(-1) alginate/10 g L(-1) chitosan-encapsulated L. rhamnosus was reduced by only 2.55 log cycles. CONCLUSION: Encapsulation effectively protected L. rhamnosus against heat treatment and gastrointestinal conditions, and this effect is important in delivering sufficient numbers of viable probiotic bacteria to the gastrointestinal tract.

Effects of Zataria multiflora Boiss. essential oil on growth and gene expression of enterotoxins A, C and E in Staphylococcus aureus ATCC 29213.

Staphylococcal food poisoning results from the consumption of food in which enterotoxigenic staphylococci have grown and produced toxins. The present study was conducted with three principal aims: i) to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Zataria multiflora Boiss. essential oil (EO) against Staphylococcus aureus ATCC 29213, ii) to evaluate the effect of subinhibitory concentrations (subMIC) of EO on the growth of bacteria over 72 h (at 25 and 35 °C), and iii) to investigate the expression of genes involved in the production of staphylococcal enterotoxins SEA, SEC and SEE over 72 h at 35 °C. The MIC and MBC of Z. multiflora Boiss. EO were 0.03 and 0.04%, respectively. Colony counting at 24, 48 and 72 h of 3 day cultures grown in the presence of 75%MIC of the EO showed that the growth rate was reduced 2.16, 2.78 and 2.91 log 10 cfu/ml at 25 °C, and 1.34, 2.35 and 2.57 log 10 cfu/ml at 35 °C, respectively, compared to control cultures. SubMIC levels of EO also significantly decreased the expression of staphylococcal enterotoxin (SE)-related genes and therefore the production of SEs in a dose dependent manner. For example, when cultured with 75% MIC, the transcriptional levels of sea, sec, see and agrA were decreased 11.7, 9.3, 10.45 and 10.3 fold after 18 h and 13.9, 11.21, 12.44 and 12.52 fold after 72 h in comparison to control, respectively.

Generation of a Uracil Auxotroph Strain of the Probiotic Yeast Saccharomyces boulardii as a Host for the Recombinant Protein Production.

BACKGROUND: Saccharomyces boulardii (S. boulardii) is the best known probiotic yeast. The genetic engineering of this probiotic strain requires the availability of appropriate mutants to accept various gene constructs carrying different selection markers. As the auxotrophy selection markers are under focus, we have generated a ura3 auxotroph mutant of S. boulardii for use in further genetic manipulations. METHODS: Classical UV mutagenesis was used for the generation of auxotroph mutants. The mutants were selected in the presence of 5-FOA (5-Fluoroorotic acid), uracil and uridine. Uracil auxotrophy phenotype was confirmed by the ability of mutants to grow in the presence of uracil and the lack of growth in the absence of this compound. To test whether the uracil auxotrophy phenotype is due to the inactivation of URA3, the mutants were transformed with a plasmid carrying the gene. An in vitro assay was used for the analysis of acid and bile resistance capacity of these mutants. RESULTS: Three mutants were found to be ura3 auxotroph as they were able to grow only in the presence of uracil. When the URA3 gene was added, these mutants were able to grow normally in the absence of uracil. Further in vitro analysis showed that the acid and bile resistance capacity of one of these mutants is intact and similar to the wild type. CONCLUSION: A uracil auxotroph mutant of the probiotic yeast, S. boulardii, was generated and characterized. This auxotroph strain may have potential applications in the production and delivery of the recombinant pharmacuetics into the intestinal lumen.

Effect of Zataria multiflora Boiss. essential oil on colony morphology and ultrastructure of Aspergillus flavus.

The mode of inhibitory action of Zataria multiflora Boiss. essential oil (EO) on the fungus, Aspergillus flavus, was studied by colony morphology examination, light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The EO at concentrations used in this study suppressed the size of the colony as well as sporulation. SEM of mycelia treated with given concentrations of EO showed morphological alterations ranging from loss of turgidity and uniformity of mycelia at low concentrations of EO to evident destruction of the hyphae at higher concentration of EO. Semi-thin sections of mycelia exposed to different concentrations of EO were analysed by light microscopy and revealed that the major change at level as low as 50 ppm of EO was limited to vacuolisation of cytoplasm resulting in cell swelling, while at higher concentrations, detachment of the cell membrane from the cell wall, deformation of mycelia and shedding the cytoplasm from the cell were the main alterations. These damages were well documented by TEM, which showed that the main sites of action of EO were the plasma membrane and cell wall. In conclusion, morphological and structural changes observed in this study may be one of the mechanisms involved in growth inhibition of the fungi and reducing aflatoxin production.

Effect of Zataria multiflora Boiss. essential oil, nisin, and their combination on the production of enterotoxin C and alpha-hemolysin by Staphylococcus aureus.

The effect of different concentrations of Zataria multiflora Boiss. essential oil (EO; 0%, 0.005%, and 0.015%), nisin (0, 0.125, and 0.25 microL/mL), and their combinations on the production of staphylococcal enterotoxin C (SEC) and alpha-hemolysin (alpha-toxin) by Staphylococcus aureus at different inoculation levels (10(3), 10(4), and 10(5) cfu/mL) in brain heart infusion broth during storage at 35 degrees C for up to 43 days was evaluated. The SEC production was significantly (p < 0.05) inhibited and the hemolysis due to alpha-toxin was significantly (p < 0.05) reduced by EO concentration at levels 0.015% and 0.005%, respectively. Significant (p < 0.05) inhibitory effect of EO on SEC production at level 0.005% was observed when it was used in combination with nisin = 0.125 microL/mL. The significant (p < 0.05) synergistic effect of EO = 0.005% and nisin = 0.125 microL/mL was also observed as more reduction of hemolysis due to alpha-toxin than EO = 0.005% alone. Further, EO significantly (p < 0.05) prevented SEC production by S. aureus during the manufacturing process of a traditional Iranian white brined cheese (as a food model) even at its lowest concentration (5 microL/100 mL), in this study.

Effect of Zataria multiflora Boiss. essential oil on growth and aflatoxin formation by Aspergillus flavus in culture media and cheese.

The effect of Zataria multiflora Boiss. essential oil (EO) against growth, spore production and aflatoxin formation by Aspergillus flavus ATCC 15546 was investigated in synthetic media as well as Iranian ultra-filtered white cheese in brine. EO effectively inhibited radial growth and spore production on potato dextrose agar (PDA) in a dose-dependent manner. At 200 ppm, the radial growth and sporulation reduced by 79.4% and 92.5%, respectively. The growth was completely prevented at EO400 ppm on PDA, and minimum fungicidal concentration (MFC) of the oil was estimated at 1000 ppm. The oil also significantly suppressed mycelial growth and aflatoxin synthesis in broth medium at all concentrations tested (P<0.05). At 150 ppm of EO, the mycelial growth and aflatoxin accumulation reduced by 90% and 99.4%, respectively. The EO at all concentrations tested, had an inhibitory effect against radial fungal growth and aflatoxin production by A. flavus in cheese. However, no concentration of EO examined was able to completely inhibit the growth and aflatoxin production in cheese. The results suggested the potential substitution of the antifungal chemicals by this EO as a natural inhibitor to control the growth of molds in foods such as cheese.