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.

Shelf life extending of probiotic beef patties with polylactic acid-ajwain essential oil films and stress effects on Bacillus coagulans.

Meat and meat products are prone to the microbial and chemical spoilage, due to the high nutritional content. This study investigated the effect of polylactic acid (PLA) films incorporated with ajwain essential oil (AEO) on microbial (total viable count [TVC], psychrotrophic bacterial count [PTC], Enterobacteriaceae, Pseudomonas spp., yeast and mold (Y&M), and also Bacillus coagulans [BCG]), chemical (pH, peroxide value [PV], thiobarbituric acid-reactive substance [TBARS], and TVN values), and sensorial properties of beef patties, as well as survivability of BCG during refrigerated storage. Results showed that all microbial counts of samples were significantly increased, except BCG, during storage but the lowest TVC of samples was achieved in samples wrapped with PLA-1% AEO (8 log colony forming units per gram [CFU/g]) at 12th of storage, which is significantly lower than control treatments (10.66 log CFU/g). The best results in all treatments are those wrapped by PLA-1% AEO in all evaluated characteristics. At the final day of storage, PTC (8.82 log CFU/g), Enterobacteriaceae (5.05 log CFU/g), Pseudomonas spp. (9.08 log CFU/g), Y&M (4.69 log CFU/g), and also pH (4.5), PV (5.12 meq/kg), TBARS (2.92 MDA/kg), and TVN (14.43 mgN/100 g) values of PLA-1% AEO treatments were significantly lower than control samples. AEO-PLA films reduce the survival of BCG in raw patties, which reached 6.19 log CFU/g in PLA-1% AEO treatments, although increasing the concentration of AEO in packaging PLA films led to the maintenance of BCG viability during the cooking process by increasing the AEO in PLA films. Overall, results showed shelf life of beef patties is extended 3 days more (150%) by wrapping with PLA films incorporated with 1% AEO.

Shelf life extension of minced squab using poly-lactic acid films containing Cinnamomum verum essential oil.

The effects of poly-lactic acid films containing different concentrations of Cinnamomum verum essential oil (CVEO) on microbial, chemical and sensory criteria of minced squab were assessed during the storage at 4 °C for 12 days. The counts of total aerobic bacteria, psychrotrophic bacteria, lactic acid bacteria, pseudomonads and Enterobacteriacea of wrapped samples with PLA films containing 0.6 and 0.9 % CVEO never reached 7 log CFU/g during the storage. Counts of E. coli and L. monocytogenes spiked to the PLA-wrapped with 0.6 or 0.9 CVEO squab samples were an order and 2 orders of magnitude lower, respectively, after the storage comparing to control. Also, samples wrapped with PLA films containing different concentrations of CVEO had lower total volatile base nitrogen, and thiobarbituric acid reactive substances values vs. control. Finally, applying of PLA films containing different concentration of CVEO, led to minor changes in sensory characteristics (P > 0.05).

Development of high-resolution melting (HRM) assay to differentiate the species of Shigella isolates from stool and food samples.

Shigella species, a group of intracellular foodborne pathogens, are the main causes of bacillary dysentery and shigellosis in humans worldwide. It is essential to determine the species of Shigella in outbreaks and food safety surveillance systems. The available immunological and molecular methods for identifying Shigella species are relatively complicated, expensive and time-consuming. High resolution melting (HRM) assay is a rapid, cost-effective, and easy to perform PCR-based method that has recently been used for the differentiation of bacterial species. In this study, we designed and developed a PCR-HRM assay targeting rrsA gene to distinguish four species of 49 Shigella isolates from clinical and food samples and evaluated the sensitivity and specificity of the assay. The assay demonstrated a good analytical sensitivity with 0.01-0.1 ng of input DNA template and an analytical specificity of 100% to differentiate the Shigella species. The PCR-HRM assay also was able to identify the species of all 49 Shigella isolates from clinical and food samples correctly. Consequently, this rapid and user-friendly method demonstrated good sensitivity and specificity to differentiate species of the Shigella isolates from naturally contaminated samples and has the potential to be implemented in public health and food safety surveillance systems.

Development of Antibacterial Biocomposites Based on Poly(lactic acid) with Spice Essential Oil (Pimpinella anisum) for Food Applications.

Among the main biodegradable food packaging materials, poly-lactic acid (PLA) is a commercially successful polymer used notably in the food packaging industry. In this study, active PLA films containing different percentage of anise essential oil (AE) (0, 0.5, 1 and 1.5% v/v) were developed, and characterized by physical, mechanical and antibacterial analysis. Based on physical examinations, thermal stability of PLA/AE films was greater than the neat PLA film, and the minimum water vapor permeability (WVP) was recorded for PLA/0.5AE film (1.29 × 10 11 g/m s), while maximum WVP was observed for PLA/1.5AE (2.09 × 1011 g/m s). Moreover, the lightness and yellowness of the composites were decreased by the addition of AE. For the PLA composites with 1.5% AE, the tensile strength decreased by 35% and the elongation break increased by 28.09%, comparing to the pure PLA. According to the antibacterial analysis, the minimum inhibitory concentrations of PLA/AE film were 5 to 100 mg/mL and the active composite could create visible inhibition zones of 14.2 to 19.2 mm. Furthermore, the films containing AE inhibited L. monocytogenes and V. parahaemolyticus in a concentration-dependent manner. The confirmation of the success of the incorporation of EOs into the PLA films was further evaluated using principal component analysis, where positive results were obtained. In this context, our findings suggest the significant potency of AE to be used as an antibacterial agent in active food packaging.

RAPD and ERIC-PCR coupled with HRM for species identification of non-dysenteriae Shigella species; as a potential alternative method.

OBJECTIVE: Species identification of Shigella isolates are so prominent for epidemiological studies and infection prevention strategies. We developed and evaluated RAPD and ERIC-PCR coupled with HRM for differentiation of non-dysenteriae Shigella species as potential alternative methods. After isolation of eighteen Shigella strains from faecal specimens collected from children under 2 years of age with diarrhea (n = 143), the species of the isolates were identified by slide agglutination assay. Also, species were identified using developed RAPD-PCR-HRM and ERIC-PCR-HRM techniques. Differentiation of the data sets was measured by principal component analysis as a dimension reduction method. Then, sensitivity and specificity of the methods were evaluated. RESULTS: We found RAPD-PCR-HRM method with high sensitivity and specificity (100 and 85% respectively) to identify non-dysenteriae Shigella species in clinical specimens. However, sensitivity and specificity of ERIC-PCR-HRM were evaluated 33 and 46% respectively and significantly lower than that of RAPD-PCR-HRM assay. Regardless of inherent poor reproducibility of DNA fingerprinting-based methods, RAPD-PCR-HRM assay can be considered as a potential alternative method to identify non-dysenteriae species of Shigella in clinical specimens. As we observed in the current study, HRM technique is more rapid, inexpensive, and sensitive than gel electrophoresis method to characterize PCR amplicons.

Investigating the properties of PLA-nanochitosan composite films containing Ziziphora Clinopodioides essential oil and their impacts on oxidative spoilage of Oncorhynchus mykiss fillets.

The present study aimed to investigate the impact of polylactic acid (PLA) integrated with nanochitosan (NC) composite film and Ziziphora Clinopodioides essential oil (ZCEO) on oxidative spoilage of rainbow trout fillets during a 9-day refrigeration period. For this purpose, first, six degradable films including T1: PLA, T2: PLA + NC, T3: PLA + NC + 1% ZCEO, T4: PLA + NC + 1.5% ZCEO, T5: PLA + 1% ZCEO, and T6: PLA + 1.5% ZCEO were prepared. Next, the properties of the films were investigated. The results of the mechanical tests showed that ZCEO decreased the tensile strength and increased the elongation at the break of the PLA films. However, adding NC improved the mechanical characteristics of the PLA film. The outcomes of physical tests including moisture content (14.02%), solubility (17.41%), water vapor permeability (1.14 × 10-7 g s-1 m-1 Pa-1), and oxygen transmission rate (21.54 cm3.mm/m2.24 hr) showed that adding 1% ZCEO improved the film physical characteristics. Nevertheless, by adding 1.5% concentration EO to the PLA film, the values of water vapor permeability, porosity (according to the scanning electron microscopy), and turbidity increased and cross-sectional pores were observed. Moreover, the films had high antioxidant properties (84.33%). In the next step, the effects of the mentioned films on the oxidative spoilage of rainbow trout fillets were investigated. The results of the chemical analysis in PLA film with the EO compared with the control treatment revealed the increasing trend of oxidation indices, in general. Moreover, increasing the concentration yielded better results such that all treatments containing EO showed satisfactory results up until the storage period ends. In most cases, adding NC affected the mentioned characteristics positively, and the best results were observed in T4 and then in T3. However, based on economics and the better mechanical and physical properties of T3, the film can be applied as an active packaging in fishery products.

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).

Nanocomposite active packaging based on chitosan biopolymer loaded with nano-liposomal essential oil: Its characterizations and effects on microbial, and chemical properties of refrigerated chicken breast fillet.

Biodegradable films reinforced with bio-nanomaterials are a solution for developing active packaging systems, shelf-life extension and protection of environment against conventional packaging. This study aimed to characterize the biocompatible chitosan (CS) films formulated with nano-liposomal garlic essential oil (NLGEO) and assess the physicho-mechanical, morphology properties and also microbial and chemical changes in chicken fillets during storage time at 4 °C. NLGEO was obtained by thin-layer hydration-sonication method using glycerol and tween 80 as plasticizer and emulsifier, respectively. Different levels (0, 0.5, 1 and 2%) of NLGEO with average size of ~101 nm were added into the chitosan matrix and films fabricated by casting method. The average size, polydispersity index and zeta potential were ~101 nm, 0.127 and -7.23, respectively. Control samples showed higher values for pH, total volatile nitrogen (TVN), peroxide value (PV), thiobarbituric acid-reactive substances (TBARS), and microbial count including total viable count (TVC), coliforms, Staphylococcus aureus and psychrotroph bacteria than treated samples. The films with higher NLGEO content represented stronger inhibitory effects. The incorporation of NLGEO improved the mechanical properties and water resistance of active films. Microstructure analysis also showed a nearly smooth surface morphology and homogenous structure with a good dispersion for NLGEO films. Significant synergistic effects in chemical and bacterial preservation of chicken fillet samples were observed by NLGEO films. The optimal mechanical and barrier properties of chitosan-NLGEO films introduced it a potential active packaging to extend the shelf life of chicken fillet.

Evaluation of microbiological, chemical, and sensory properties of cooked probiotic sausages containing different concentrations of astaxanthin, thymol, and nitrite.

In this study, the effects of different concentrations of thymol and astaxanthin on control of Clostridium perfringenes and also microbial, chemical, and organoleptic properties of common and probiotic beef cooked sausages containing two levels of nitrite during storage at refrigerated condition during 45 days were evaluated. Based on findings, control group had significantly higher total volatile base nitrogen (TVB-N) than nitrite-, thymol-, and astaxanthin-treated samples. At the end of the storage time in control, thiobarbituric acid reactive substances (TBARS) value reached 1.96 mg/kg, while the values for treated samples remained lower than 1.63 mg/kg. Final count of lactic acid bacteria decreased approximately 1.67-3.79 log CFU/g in treated samples compared with the control group (p < .05). A reduction between 1.46 and 2.46 log CFU/g in C. perfringenes count was recorded for the treated samples in comparison with control group after 45 days of storage.

The effects of storage and hop extract on aroma and flavour compounds in Balkan-style sausages packed under a CO2-containing anaerobic atmosphere.

Changes in the levels of pH, lactic acid, acetic acid, headspace volatile compounds, and spoilage odour intensity during a 35 day refrigerated storage period in two sets of a Balkan-style fresh sausage, a control sausage (CON) and a sausage containing an aqueous hop extract (HOP), packaged under a 20% CO2 and 80% N2 atmosphere were evaluated. Storage resulted in progressive sausage acidification and increased the levels of acetic acid, 1-methylbutanol, ethyl acetate, ethyl hexanoate, and 2-ethylhexanol; all of which are associated with anaerobic microbial metabolism under sugar restricted conditions. Storage decreased the levels of hexanal, heptanal, pentanol, and garlic-derived organosulfur compounds. Hop extract showed oxygen scavenging abilities, and decreased the levels of the volatile compounds derived from lipid auto-oxidation while contributing to the presence of specific terpene compounds. The use of hop extract did not improve the shelf life of sausages packed under anaerobic atmosphere. The spoilage odour appeared in both types of sausages on the 14th day, and it was considered strong from day 21 onwards.

Differentiation of stx1A gene for detection of Escherichia coli serotype O157: H7 and Shigella dysenteriae type 1 in food samples using high resolution melting curve analysis.

Escherichia coli serotype O157: H7 and Shigella dysenteriae type 1 as the Shiga toxin-producing bacteria cause some acute gastrointestinal and extraintestinal diseases such as hemorrhagic uremic syndrome and bloody diarrhea in human. Stx genes are the key virulence factors in these pathogens. The aim of this study was to develop HRMA assay to differentiate stx1A gene for detection of E. coli serotype O157: H7 and Sh. dysenteriae type 1 and determine the prevalence of these pathogens in food samples using this method. PCR-HRMA assay and gold standard methods have been carried out for identification of pathogens among 135 different food samples. We found HRMA method a sensitive and specific assay (100 and 100%, respectively) for differentiation of stx1A gene, consequently, detection of these pathogens in food samples. Also, the highest prevalence of E. coli serotype O157: H7 and Sh. dysenteriae type 1 harboring stx1A gene was observed in raw milk and vegetable salad samples, respectively. HRMA as a rapid, inexpensive, sensitive and specific method is suggested to be used for differentiation of stx1A gene to detect E. coli serotype O157: H7 and Sh. dysenteriae type 1 as the key pathogens for safety evaluation of food samples.

Incorporation of nanoencapsulated garlic essential oil into edible films: A novel approach for extending shelf life of vacuum-packed sausages.

The efficacy of chitosan (CH) and whey protein (WP) films impregnated with garlic essential oil (GEO, 2% v/v) or nanoencapsulated GEO (NGEO, 2% v/v) to extend the shelf life of refrigerated vacuum-packed sausages were assessed and compared during 50 days. The primary evaluation of GEO and NGEO showed that GEO had a considerable amount of active compounds diallyl sulfide derivatives (~67%) and the mean size and zeta potential of NGEO were 101 nm and -7.27 mV, respectively. Based on the microbiological and lipid stability analysis of the sausages, all active films retarded lipid oxidation and the growth of main spoilage bacterial groups compared to the control, and CH film containing NGEO exhibited the best result with the peroxide value, thiobarbituric acid reactive substances and aerobic plate count of 0.37 (meq/kg lipid), 0.47 (mg malondialdehyde/kg) and 3.69 (log CFU/g), respectively, on day 50. The nanoencapsulation of GEO made no significant differences in the sensory properties comparing to free-GEO samples (P < .05).

Microbial Growth and Biogenic Amine Production in a Balkan-Style Fresh Sausage during Refrigerated Storage under a CO2-Containing Anaerobic Atmosphere: Effect of the Addition of Zataria multiflora Essential Oil and Hops Extract.

Fresh sausages are highly perishable, and the preservatives allowed in these types of meat preparations are limited. Balkan-style fresh sausages were prepared in triplicate without antimicrobials (Control), with an aqueous hops extract (30 mL/kg), with Zataria multiflora Boiss essential oil (1 mL/kg), or a combination of both (15 and 0.5 mL/kg, respectively), and refrigerator-stored under a 20% CO2 and 80% N2 atmosphere. The spoilage microbial growth, i.e., lactic acid bacteria (LAB), Brochothrix thermosphacta, Enterobacteriaceae, Micrococcaceae, molds and yeasts, the pH value, and the production of biogenic amines in the sausages were monitored weekly and compared with a control sausage during a 35-day storage period. Furthermore, 349 colonies of presumptive LAB (isolated from the De Mann, Rogose-Sharpe agar plates) were identified using a MALDI-TOF-based method. Growth levels to ≈ 9 Log colony forming units (CFU) per g were reached by LAB, with a predominance of Lactobacillus sakei. Enterobacteriaceae and B. thermosphacta also showed significant growth (up to 6 Log CFU/g). Biogenic amine levels increased, and tyramine values overcame 250 mg/kg. The study could not demonstrate a significant effect of antimicrobial source treatments in any of the characteristics studied, and thus, the shelf-life of sausages.

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.

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.