Optimization of Ziziphora clinopodioides L. essential oil nanoencapsulation in chitosan nanocomplex by response surface methodology.

Nano-encapsulation of essential oils, a specific area of interest, can help overcome challenges associated with their commercial use. This study aimed to evaluate the effect of different concentrations of chitosan, Ziziphora clinopodioides L. essential oil (ZcEO), and Sodium-Tri Polyphosphate (TPP), both individually and in interaction, on several properties of EO-loaded chitosan nanoparticles. These properties include particle size (PS), zeta potential (ZP), and encapsulation efficiency (EE) using a two-stage emulsion-ionic gelation approach. The optimization of the parameters was done by response surface methodology using Box-Behnken design. The chemical composition of ZcEO was analyzed as well. The primary compounds in ZcEO were found to be pulegone (29.24 %), 1,3-dimethyl-2-(2-methylpropylidene) imidazolidine (9.05 %), piperitenone (6.65 %), thymol (5.38 %), and carvacrol (5.27 %). The PS ranged from 117.33 to 4934.1 nm, the ZP varied from -1.1 to -30.83 mV, and the EE spanned from 31.74 to 87.04 %. The results showed that an increase in the initial EO content led to a decrease in PS and ZP, but an increase in EE. Moreover, increasing the TPP concentration resulted in an enhancement in PS, ZP, and EE, whereas increasing the Chs concentration led to a slight increase in PS, ZP, and EE. Furthermore, the results of this study proved the interaction effect of different parameters on the responses investigated. Under optimized conditions, the optimal concentrations of chitosan, ZcEO and TPP were attained at 6.768, 6.078, and 7.595 mg/mL respectively. This resulted in a PS of 117.331 nm, a ZP of -20.949 mV, and an EE of 75.385 %. In conclusion, the results suggest that adjusting the concentrations of Chs, EO, and TPP is an effective approach to controlling the properties of NPs and optimizing their performance.

Nutritional, functional, and sensorial properties of oat milk produced by single and combined acid, alkaline, α-amylase, and sprouting treatments.

In this study, the effects of different treatments of the oat slurry on the nutritional, functional, and sensorial properties of oat milk were evaluated. The sprouting and sprouting-acidic treatments have the highest oat milk yield (91.70%) and protein extraction yield (82.74%), respectively. The protein concentrations of alkali, sprouting-acidic, and α-amylase-alkali treatments were significantly (p < .05) higher than other treatments. The alkali treatments showed higher fat content (0.66%). In addition, acidic and alkali treatments in single or combined with other treatments showed the highest dry matter and energy value. The carbohydrate content of α-amylase-alkali treatment (4.35%) was higher than other treatments and also, all acidic treatments showed higher ash content (>1) compared to the other treatments. Furthermore, the sprouting-α-amylase and acidic-α-amylase showed the lowest starch (0.28%) and the highest reducing sugar content (3.15%) compared to the other treatments, respectively. Moreover, the α-amylase-alkali treatment showed the highest total phenolic content and antioxidant activity (342.67 mg GAE/L and 183.08 mg BHT eq/L, respectively). Furthermore, sensory evaluation of most treatments showed acceptable scores (≥7) for consumers, especially in the case of α-amylase, sprouting, and α-amylase-sprouting treatments. Results show that the different treatments had different effects on the nutritional, functional, and sensorial properties of oat milk. In conclusion, from the nutritional and functional point of view, the two-stage treatments were more effective than singular treatments on investigated factors proposing their application in functional plant milk preparation.

Microbial, physiochemical and functional properties of probiotic yogurt containing Lactobacillus acidophilus and Bifidobacterium bifidum enriched by green tea aqueous extract.

In this study, the effect of aqueous extract of green tea on the viability of probiotic bacteria including Lactobacillus acidophilus and Bifidobacterium bifidum and the sensory and physicochemical and functional properties of synbiotic yogurt was investigated during 4 weeks of storage. L. acidophilus and B. bifidum counts did not significantly change in yogurt containing 0.5% and 1% of the extract during storage. Also, the addition of the extract to yogurt highly increased the phenolic compounds, since the amount of phenolic compounds in yogurt containing 0.5% and 1% extract was 660 and 1,123 mg gallic acid/kg, respectively. In addition, a significant increase in the antioxidant activity of yogurt containing green tea extract was observed in comparison with the control. The amount of antioxidant activity increased during 4 weeks of storage, which reached to 4,193 and 7,337 mg BHT eq./kg in probiotic yogurt containing 0.5% and 1% extract, respectively. The acidity increased during 4 weeks of storage, while the pH decreased. Addition of the extract significantly increased the acidity of probiotic yogurt compared with the control (p < .05). In addition, in all studied groups, an increase in syneresis was observed during the study, and the syneresis was greater in yogurt containing aqueous extract of green tea, compared with the control group. Although adding the green tea extract to probiotic yogurt impaired taste, texture, and appearance compared with the plain yogurt, the overall acceptability of these samples was yet above the acceptable level. In conclusion, the results of the study showed that the addition of aqueous extract of green tea increased the antioxidant properties and the amount of phenolic compounds in yogurt, while the viability of probiotic bacteria was not changed. Therefore, the simultaneous use of green tea extract and probiotics in yogurt is recommended as an effective functional food formulation.

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.

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.

Effects of Lactobacillus rhamnosus ATCC 7469 on Different Parameters Related to Health Status of Rainbow Trout (Oncorhynchus mykiss) and the Protection Against Yersinia ruckeri.

In the current study, we investigated the effect of a probiotic bacterium (Lactobacillus rhamnosus ATCC 7469) microencapsulated with alginate and hi-maize starch and coated with chitosan on improving growth factors, body composition, blood chemistry, and the immune response of rainbow trout (initial weight: 18.41 ± 0.32 g). Four experimental diets were formulated to feed fish for 60 days. They were control diet without any additive (C), diet added with beads without probiotic (E), a probiotic sprayed to the diet (L.r), and encapsulated probiotic supplemented diet (E-L.r). The results indicated that feeding with E-Lr significantly improved weight gain (84.98 g) and feed conversion ratio (0.95) compared to the other groups (P < 0.05). Also, fish fed E-Lr diet had a significantly higher value of whole-body protein (17.51%), total protein in the blood (4.98 g/dL), lysozyme (30.66 U/mL), alternative complement pathway hemolytic activity (134 U/mL), superoxide dismutase (203 U/mg protein), and catalase (528.33 U/mg protein) (P < 0.05) as compared to those fed the control diet. Similarly, a higher relative expression of immune-related genes such as interleukin-1 (Il-1) and tumor necrosis factor-alpha (TNF-1α) were reported in those fed E-L.r and L.r diets respectively. Interestingly, the fish fed dietary E-L.r had a significantly lower value of lipid in the whole body (4.82%) and cholesterol in the blood (160.67%) in comparison with those fed the control diet (P < 0.05). At the end of the experiment, all groups were challenged by Yersinia ruckeri where the survival rate of rainbow trout fed dietary E-L.r (70.36%) was statistically higher than that of the others (P < 0.05). Overall, the results suggested that encapsulated probiotic Lact. rhamnosus ATCC 7469 acted better than unencapsulated probiotic and has a potential to improve growth performance, flesh quality, and the immune response of rainbow trout.

Effect of Auricularia auricula aqueous extract on survival of Lactobacillus acidophilus La-5 and Bifidobacterium bifidum Bb-12 and on sensorial and functional properties of synbiotic yogurt.

The effect of Auricularia auricula aqueous extract (AAE) on the survival of Lactobacillus acidophilus La-5 and Bifidobacterium bifidum Bb-12, and on chemical and sensory properties of yogurt was investigated during 28 days of storage at 4°C. The use of 0.05% of AAE improved the survival of L. acidophilus La-5 and B. bifidum Bb-12 about 0.35 and 0.58 log CFU/g, respectively. However, AAE in 0.1% concentration enhanced the survival of L. acidophilus La-5 and B. bifidum Bb-12 about 0.43 and 0.51 log CFU/g, respectively. Moreover, 0.1% concentration of AAE drastically increased antioxidant activity and total phenolic content to 115.30 mg BHT eq./kg and 1,057.6 mg Gallic acid/kg after 28 days, respectively. Addition of AAE to the yogurt significantly decreased sensorial acceptance while increased syneresis compared to the control group (p < .05). In conclusion, the results of this study showed that addition of AAE improved probiotic protection and functional properties of the yogurt recommending its application in symbiotic yogurt.

Effect of autochthonous starter cultures isolated from Siahmazgi cheese on physicochemical, microbiological and volatile compound profiles and sensorial attributes of sucuk, a Turkish dry-fermented sausage.

The effect of adding autochthonous starter cultures isolated from Siahmazgi cheese, on the physicochemical parameters and microbial counts of sucuk was investigated during the ripening period. SPME-GC/MS was used in volatile compound analysis and a trained group of panelists carried out sensory analysis of the final product. After preliminary screening, three strains of Lactobacillus plantarum, which possess desirable technological properties, were used to prepare three starter cultures: LBP7, LBP10 and LBP14. The addition of LBP7 and LBP14 starter cultures had a significant effect (P<0.05) on lightness, leading to higher L values compared to control sausages during the ripening period. Both LBP7 and LBP14 sausages showed higher counts of lactic acid bacteria, lower growth of Enterobacteriaceae and Gram-positive catalase-positive cocci and greatly lowered the pH value compared to control sausages throughout the ripening process. At the end of the ripening process, lactic acid bacteria counts were affected (P<0.05) by the addition of starter culture since higher counts were observed in sausages prepared with LBP7 (9.14logCFU/g) and LBP14 (8.96logCFU/g) batches. The decrease of water activity during the ripening of sausages was not affected by the various starters. The texture profiles of all sausages were similar except for LBP10, which showed lower hardness and gumminess during ripening. Under the conditions of the study, volatile compounds were mainly from spices, and no marked differences were found among inoculated sausages. However, sensory evaluation revealed that most of the sensory attributes were scored higher for inoculated sausages than for the control ones. Therefore, LBP7 and LBP14 could be promising candidates for inclusion as starter cultures for the manufacture of sucuk.

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.

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.