Heterologous expression of Thrombocidin-1 in Pichia pastoris: Evaluation of its antibacterial and antioxidant activity.

The antimicrobial peptide Thrombocidin-1 (TC-1) isolated from human blood that derived from NAP-2 by deleting of two amino acids from C-terminal region. In this study, a C-terminal 6 _ His tagged recombinant TC-1 was expressed as a secreted peptide in Pichia pastoris, for the first time. The recombinant P. pastoris was inoculated in to BMMY culture medium, incubation with 5 µl/ml absolute methanol for 72 h at 30 °C. The TC-1 peptide was concentrated with nickel affinity chromatography and electrophoresis on 16% acrylamide gels. The molecular weight of recombinant TC-1 is approximately 8 kDa and under these conditions, the concentration of TC-1 considered 190 µg/ml that determined by the Bradford method. The antimicrobial activity test (Minimum Inhibitory Concentration and Minimum Bactericidal Concentration) was done against: Listeria monocytogenes, Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa. The growth of these pathogenic bacteria was limited when we used peptide at a concentration of as low as 19.56 µg/ml. Based on DPPH radical scavenging (DPPH-RS) activity and reducing power assays, this peptide showed relatively good antioxidant potential in comparison with standard antioxidant used in this study (BHT). Due to the existence of TC-1 in blood, which makes it safe for human consumption, and the good results of its antimicrobial and antioxidant activity, it can be introduced as a good alternative and a novel effective peptide to food industry for bio-preservation.

Development of shelf life kinetic model for fresh rainbow trout (Oncorhynchus mykiss) fillets stored under modified atmosphere packaging.

The sensory, chemical (based on the thiobarbituric acid, total volatile basic nitrogen and trimethylamine), and microbial quality (based on the total viable count and lactic acid bacteria count) of the rainbow trout stored under modified atmosphere packaging (MAP) conditions was evaluated. Four different gas combinations, including P1 (80% CO2, 10% N2, 10% O2), P2 (60% CO2, 20% N2, 20% O2), P3 (60% CO2, 40% N2, 0% O2), and P4 (40% CO2, 30% N2, 30% O2), were used. Also, the fish packages were stored at four constant temperatures (including 0, 5, 10, and 15 °C) for 12 days. The absence of oxygen in P3 and high concentration of carbon dioxide in P1 extended the shelf life by delaying the chemical, microbial, and sensory spoilage. Over the storage time of trout fillets in MAP, the rate of chemical reactions significantly increased while the sensory scores decreased. Based on the Arrhenius kinetic modeling for the spoilage reactions of the sensory (total acceptance) and chemical (total volatile basic nitrogen) indices, the shelf life was extended for P3 and succeedingly, for P1 packaging.

Optimization of the production conditions of the lipase produced by Bacillus cereus from rice flour through Plackett-Burman Design (PBD) and response surface methodology (RSM).

In this study, the screening of lipase positive bacteria from rice flour was carried out by Rhodamin B agar plate method. Bacillus cereus was identified by 16S rDNA method. Screening of the appropriate variables and optimization of the lipase production was performed using Plackett-Burman design (PBD) and response surface methodology (RSM). Among the isolated bacteria, an aerobic Bacillus cereus strain was recognized as the best lipase-producing bacteria (177.3 ± 20 U/ml). Given the results, the optimal enzyme production conditions were achieved with coriander seed extract (CSE)/yeast extract ratio of 16.9 w/w, olive oil (OO) and MgCl2 concentration of 2.37 g/L and 24.23 mM, respectively. In these conditions, the lipase activity (LA) was predicted 343 U/mL that was approximately close to the predicted value (324 U/mL), which was increased 1.83 fold LA compared with the non-optimized lipase. The kinetic parameters of Vmax and Km for the lipase were measured 0.367 µM/min.mL and 5.3 mM, respectively. The lipase producing Bacillus cereus was isolated and RSM was used for the optimization of enzyme production. The CSE/yeast extract ratio of 16.9 w/w, OO concentration of 2.37 g/L and MgCl2 concentration of 24.23 mM, were found to be the optimal conditions of the enzyme production process. LA at optimal enzyme production conditions was observed 1.83 times more than the non-optimal conditions. Ultimately, it can be concluded that the isolated B. cereus from rice flour is a proper source of lipase.

Application of intelligent modeling to predict the population dynamics of Pseudomonas aeruginosa in Frankfurter sausage containing Satureja bachtiarica extracts.

Stepwise regression, Genetic Algorithm-Artificial Neural Network (GA-ANN) and Co-Active Neuro Fuzzy Inference System (CANFIS) were used to predict the effect of Satureja extracts (water and ethanol) on the population dynamics of Pseudomonas aeruginosa in a complex food system (Frankfurter sausage). The stepwise regression, GA-ANN and CANFIS were fed with four inputs: concentration (at five levels 0, 2000, 4000, 6000 and 8000 ppm), type of extract (water and ethanol), temperature (at three levels 5, 15 and 25°Ð¡) and time (1-20 days). The results showed that the stepwise regression was good for modeling the population dynamics of P. aeruginosa (R(2) = 0.92). It was found that ANN with one hidden layer comprising 14 neurons gave the best fitting with the experimental data, so that made it possible to predict with a high determination coefficient (R(2) = 0.98). Also, an excellent agreement between CANFIS predictions and experimental data was observed (R(2) = 0.96). In this research, GA-ANN was the best approach to simulate the population dynamics of P. aeruginosa. Furthermore, Satureja bachtiarica ethanol extract was able to reduce P. aeruginosa population, showing stronger effect at 5 °C and the concentration of 8000 ppm.

Bio-synthesis, purification and structural analysis of Cyclosporine-A produced by Tolypocladium inflatum with valorization of agro-industrial wastes.

Cyclosporine A (CyA) holds significant importance as a strategic immunosuppressive drug for organ transplant patients. In this study, we aimed to produce pure and cost-effective Cyclosporine A (CyA) by fermenting a culture medium containing dairy sludge, using Tolypocladium inflatum PTCC 5253. Following the fermentation stage, ethyl acetate extraction and fast protein liquid chromatography were employed for sample purification. The initial evaluation of the effectiveness of CyA obtained from these processes was performed through bioassay, wherein the antimicrobial clear zone diameter was found to be larger compared to the sample obtained from the fermentation culture. The concentration of CyA was determined using high-performance liquid chromatography, yielding values of 334 mg/L, 456 mg/L, and 578 mg/L for the fermented, extracted, and purified samples, respectively. Further analysis utilizing liquid chromatography tandem mass spectrometry (LC/MS/MS) confirmed a purity of 91.9% and proper agreement with the standard sample based on the ion intensity of Z/m 1205. To validate the structure of CyA, nuclear magnetic resonance spectroscopy, Fourier-transform infrared (FT-IR), and Raman spectroscopy were employed. X-ray diffraction and differential scanning calorimetry analyses demonstrated that the purified CyA exhibited a crystal structure similar to the standard sample, characterized by two broad peaks at 2θ = 9° and 20°, and comparable glass transition temperatures (57-68 °C for the purified sample; 53-64 °C for the standard sample). Dynamic light scattering analysis confirmed a uniform particle size distribution in both the purified and standard samples. The zeta potentials of the purified and standard samples were determined to be - 25.8 ± 0.16 and - 23.63 ± 0.12 mV, respectively. Our results demonstrate that dairy sludge can serve as a suitable culture medium for the production of (CyA).

Structural characterization of exopolysaccharide from Leuconostoc mesenteroides P35 and its binding interaction with bovine serum albumin using surface plasmon resonance biosensor.

This paper describes the structural elucidation of Leuconostoc mesenteroides P35 exopolysaccharide (EPS-LM). Ln. mesenteroides P35 strain was isolated from a French goat cheese for its capacity to produce EPS increasing the viscosity of a whey-based fermentation medium. The chemical structure of EPS-LM analysis was elucidated by determination of optical rotation degree, macromolecular characterization, sugar units and methylation analyses, FT-IR, 1D NMR spectroscopy (1H and 13C NMR), 2D NMR spectroscopy (1H1H COSY, HSQC and HMBC). EPS-LM was a high molecular weight (ranging from 6.7 × 106 Da to 9.9 × 106 Da) dextran that is composed of only d-glucose units containing α (1 â†’ 6) linkages and paltry α (1 â†’ 3) branches. Since polysaccharide-protein interactions can be exploited to control and design food matrices, EPS-LM interactions with bovine serum albumin (the main constituent of bovine plasma) were investigated by surface plasmon resonance (SPR). Kinetic properties of EPS-LM binding with immobilized BSA via showed an increase of EPS-LM affinity (equilibrium constant (Kd)) for BSA from (2.50 ± 0.01) × 10-5 M-1 at 298 K to (9.21 ± 0.05) × 10-6 M-1 at to 310 K. The thermodynamic parameters revealed that van der Waals and hydrogen binding forces play a major role in the interaction of EPS-LM with BSA. However, EPS-LM-BSA interaction was non-spontaneous, entropy driven and an EPS-LM - BSA binding process was endothermic (ΔG > 0). The structural findings suggested that Ln. mesenteroides P35 α-D-glucan might find widespread technological applications in the biopolymer, medical and food industries.

Ultrasensitive detection of micrococcal nuclease activity and Staphylococcus aureus contamination using optical biosensor technology-A review.

One of the most common and important pathogenic bacteria is Staphylococcus aureus (S. aureus) which is known as a foodborne illness all over the world. The detection of micrococcal nuclease (MNase) can act as a unique diagnostic biomarker for the identification of S. aureus. So far, various complex methods have been introduced for the evaluation of S. aureus bacterium. However, they have different limitations such as labor-intensive, inaccurate results and time-consuming procedures. Thus, it is of particular attention to develop fast, easy, simple and more approachable detection methods based on nanotechnology and MNase detection. In this review, recent advances and modern techniques of ultrasensitive biosensors based on quantum dots (QDs), noble metal and magnetic nanoparticles (NPs), graphene oxide (GO) nanosheets, and also transfer energy strategy have been discussed for the identification of MNase activity and S. aureus contamination. Besides, advantages and disadvantages of different types of fluorescent, phosphorescent and colorimetric biosensors have been discussed.

Microwave-assisted hydrodistillation extraction of essential oil from coriander seeds and evaluation of their composition, antioxidant and antimicrobial activity.

Coriander seeds essential oil is used in food preparation, perfume, cosmetics and pharmaceuticals. In this study, extraction of essential oil from coriander seeds was done by hydrodistillation (HD) and microwave assisted hydrodistillation (MAHD) methods. Chemical composition, total phenol contents, antimicrobial and antioxidant properties of essential oils were measured and the results were compared between HD and MAHD methods. Scanning electron microscopy (SEM) results showed that the essential oils had inhibitory effect on the bacterial membrane and cell wall. Results showed that total phenol contents and antioxidant activity increased under heat and microwave conditions. Coriander seeds essential oil had a very strong effect on Candida albicans. Gram-positive bacteria were more sensitive to the essential oil of coriander seeds than Gram-negative bacteria. The essential oil extracted by MAHD showed better antimicrobial activity, higher phenols yield and antioxidant activity. According to the results of GC-MS, linalool was the most common constituent of both essential oils.

Use of Plantago major seed mucilage as a novel edible coating incorporated with Anethum graveolens essential oil on shelf life extension of beef in refrigerated storage.

In this study, Plantago major seed mucilage (PMSM) was extracted from whole seeds using hot-water extraction (HWE). The dill (D) essential oil components were identified through gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) and its antioxidant properties were examined through the methods of 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant potential (FRAP) and ß-carotene-linoleic acid assay (B-CL). Total phenolic content (TPC) was characterized through the Folin-Ciocalteu method and the antimicrobial effect was evaluated on 10 pathogenic microorganisms. PMSM edible coating incorporated were prepared in four different concentrations of essential oils, including 0, 0.5, 1 and 1.5% (w/w). The control and the coated beef samples were analyzed periodically for microbiological (total viable count, psychrotrophic count, Escherichia coli, Staphylococcus aureus and fungi), chemical (thiobarbituric acid, peroxide value and pH), and sensory characteristics. The IC50, FRAP, B-CL and TPC of the dill essential oil were equal to 11.44µg/ml, 9.45mmol/g, 82.86 and 162.65µg/ml GAE, respectively. PMSM extended the microbial shelf life of beef by 3days, whereas the PMSM+0.5%D, PMSM+1%D and PMSM+1.5%D resulted in a significant shelf life extension of the beef by 6, 9 and 9days, respectively, as compared to the control samples.

Characterization of antioxidant-antibacterial quince seed mucilage films containing thyme essential oil.

In this study thyme essential oil (TEO) concentrations ranging from 0% to 2.0%, incorporated in quince seed mucilage (QSM) film were used. Antibacterial activity, physical, mechanical, barrier and antioxidant properties of QSM films were evaluated. The antimicrobial activity of the QSM films incorporated with thyme essential oil was screened against 11 important food-related bacterial strains by agar disc-diffusion assay. Films containing 1% of thyme essential oil were effective against all test microorganisms and exhibited a strong inhibitory effect on the growth of Shewanella putrefaciens, Listeria monocytogenes and Staphylococcus aureus. QSM films exhibited some antioxidant activity, which was significantly improved by the addition of the essential oil. A reduction of the glass transition temperature, as determined by differential scanning calorimetry (DSC), was caused by addition of thyme essential oil into the QSM films. Scanning electron microscopy was carried out to explain structure-property relationships. Incorporating thyme essential oil into edible QSM films provides a novel way to improve the safety and shelf life of ready-to-eat foods.

Optimization of extraction, antioxidant activity and functional properties of quince seed mucilage by RSM.

The effect of extraction temperature (25-65°C), extraction time (5-25 min) and water to seed ratio (10:1-50:1) on yield, protein content, turbidity, viscosity, antioxidant activity, emulsion stability and foam stability of quince seed mucilage was investigated using response surface methodology (RSM). Experiments were designed according to Central Composite Face Center Design with these seven factors, including central and axial points. Numerical optimization determined the optimum extraction conditions based on the highest yield, viscosity, antioxidant activity, emulsion stability and foam stability and the lowest protein content and turbidity as being time 5 min, temperature 65°C and water to seed ratio 25.1:1. At this optimum point, extraction yield, protein content, turbidity, viscosity, antioxidant activity, emulsion stability and foam stability were found to be 11.58 (%), 2.71 (%), 068 (Å), 1473.96 (mPas), 29.88 (%), 94.89 (%) and 21.36 (%), respectively. The results demonstrated that quince seed mucilage had desirable antioxidant activity, viscosity, emulsifying and foaming properties.

Effect of quince seed mucilage edible films incorporated with oregano or thyme essential oil on shelf life extension of refrigerated rainbow trout fillets.

The effects of quince seed mucilage film (QSMF) containing oregano (O) or thyme (T) essential oil on shelf life extension of rainbow trout (Oncorhynchus mykiss) fillets during refrigerated storage (4°C) were evaluated over a period of 18days. Films were prepared in four different concentrations of essential oils, including 0, 1, 1.5 and 2%. The control and the wrapped fillet samples were analyzed periodically for microbiological (aerobic and psychrotrophic count, Pseudomonas spp., H2S-producing bacteria, lactic acid bacteria, and Enterobacteriaceae), chemical (TBA, TVB-N, TMA-N), and sensory characteristics. Bacteria grew most quickly in trout fillets stored in air, followed by those wrapped with QSMF and the lowest counts were in wrapped samples with QSMF+2%T. Pseudomonas spp., Enterobacteriaceae and LAB counts were significantly lower in samples wrapped with QSMF+2%T. The lowest TBA value was obtained in fillets wrapped QSMF containing 2% oregano essential oil. The strong antioxidant activity of QSMF+2%O was related to the composition of oregano essential oil. The GC analysis of essential oil components revealed that carvacrol (81.85%) was the major component of oregano essential oil. TBA value varied for all treatments and remained lower than 2mg MDA/kg throughout storage. The formation of TVB-N, TMA-N increased with time of storage. TVB-N and TMA-N correlated well with the microbiological data, indicating that along with TVB-N, TMA-N may serve as a useful index for fillets spoilage. QSMF extended the microbial shelf life of rainbow trout fillets by 2days, whereas the QSMF+1%O, QSMF+1.5%O, QSMF+2%O, QSMF+1%T, QSMF+1.5%T and QSMF+2%T resulted in a significant shelf life extension of the trout fillets by 3, 5, 9, 6, 10 and 11days, respectively, as compared to the control samples.

Physical, barrier and antioxidant properties of a novel plasticized edible film from quince seed mucilage.

In this study, we investigated the potential of quince seed mucilage (QSM) as a new source for preparation of edible films and determined the physical, mechanical, barrier, antioxidant, microstructural and thermal properties. QSM films were prepared by incorporation of three levels of glycerol (25-50%, w/w). As glycerol concentration increased, water vapor permeability (WVP), oxygen permeability (O2P), elongation at break (EB), water solubility and moisture content of QSM films increased while, tensile strength (TS), density and surface hydrophobicity decreased significantly. The measurement of color values showed that by the increasing of the glycerol concentration in polymer matrix, the b and L values increased while ΔE value decreased. Microscopic views indicated smooth and uniform surface morphology without obvious cracks, breaks, or openings on the surfaces after the incorporation of glycerol as a plasticizer. The results of the present study suggest that QSM as a new antioxidant edible film with interesting specifications can potentially be used for packaging of a wide range of food products.