A novel antifungal nanoemulsion based on reuterin-assisted synergistic essential oils: Preparation and in vitro/in vivo characterization.

This research aimed to develop, optimize, and evaluate a new antifungal nanoemulsion system based on the crude reuterin-synergistic essential oils (EOs) hybrid to overcome the EOs application limits. At first, the antifungal effects of the Lactobacillus plantarum and Lactobacillus reuteri cell-free extracts (CFE) were tested against the Botrytis cinerea, Penicillium expansum, and Alternaria alternata as indicator fungus using broth microdilution method. The L. reuteri CFE with the MIC of 125 µL/mL for B. cinerea and 250 µL/mL for P. expansum and A. alternata showed more inhibitory effects than L. plantarum. Next, reuterin as a significant antibacterial compound in the L. reuteri CFE was induced in glycerol-containing culture media. To reach a nanoemulsion with maximum antifungal activity and stability, the reuterin concentration, Tween 80 %, and ultrasound time were optimized using response surface methodology (RSM) with a volumetric constant ratio of 5 % v/v oil phase including triple synergistic EOs (thyme, cinnamon, and rosemary) at MIC concentrations. Based on the Box-Behnken Design, the maximum antifungal effect was observed in the treatment with 40 mM reuterin, 1 % Tween 80, and 3 min of ultrasound. The growth inhibitory diameter zones of B. cinerea, P. expansum, and A. alternata were estimated 6.15, 4.25, and 4.35 cm in optimum nanoemulsion, respectively. Also, the minimum average particle size diameter (16.3 nm) was observed in nanoemulsion with reuterin 40 mM, Tween 80 5 %, and 3 min of ultrasound treatment. Zeta potential was relatively high within -30 mV range in all designed nanoemulsions which indicates the nanoemulsion's stability. Also, the prepared nanoemulsions, despite initial particle size showed good stability in a 90-d storage period at 25 °C. In vivo assay, showed a significant improvement in the protection of apple fruit treated with reuterin-EOs nanoemulsions against fungal spoilage compared to free reuterin nanoemulsion. Treatment of apples with nanoemulsion containing 40 mM reuterin showed a maximum inhibitory effect on B. cinerea (5.1 mm lesion diameter compared to 29.2 mm for control fruit) within 7 d at 25 °C. In summary, the present study demonstrated that reuterin-synergistic EOs hybrid with boosted antifungal activities can be considered as a biopreservative for food applications.

Lactic acid bacteria isolated from traditional Iranian butter with probiotic and cholesterol-lowering properties: In vitro and in situ activity.

Producing butter from yogurt is known as a traditional way practiced in Iran and elsewhere, particularly in rural areas. Lactic acid bacteria (LAB) with probiotic and cholesterol-lowering properties were isolated from traditional butter collected in different regions of Iran. Then, isolates were identified and applied as adjunct culture in industrial butter production. Ten samples of traditional Iranian butter were collected from local farms. Fifty-four isolates were considered LAB due to biochemical examinations. Molecular techniques then identified 10 strains showing high cholesterol reduction ability and tolerated bile and acid. The sequence analysis revealed that four isolates belonged to Enterococcus durans, four isolates to Lactobacillus, one isolate to Pediococcus, and one isolate to Neoscardovia. Lactobacillus brevis IBRC-M 11044, Pediococcus pentosaceus IBRC-M 11045, Neoscardovia arbecensis IBRC-M 4391 4378, and Lactobacillus pentosus IBRC-M 11043 were selected and applied as adjunct culture in producing four treatments of industrial butter. All examined strain treatments showed significant changes in cholesterol level of butter samples. Furthermore in all samples, the cholesterol content was significantly lower than control (p < .5). The highest level of cholesterol reduction was achieved in the butter sample prepared by Lactobacillus pentosus IBRC-M 11045. Sensory analysis showed that the butter sample with Neoscardovia arbecensis IBRC-M 4391 4378 was more acceptable than other butter samples. Due to our finding, it is valuable to incorporate these strains in butter production and introduce novel functional butter to market.

The Emulsifier Carboxymethylcellulose Induces More Aggressive Colitis in Humanized Mice with Inflammatory Bowel Disease Microbiota Than Polysorbate-80.

Commonly used synthetic dietary emulsifiers, including carboxymethylcellulose (CMC) and polysorbate-80 (P80), promote intestinal inflammation. We compared abilities of CMC vs. P80 to potentiate colitis and impact human microbiota in an inflammatory environment using a novel colitis model of ex-germ-free (GF) IL10-/- mice colonized by pooled fecal transplant from three patients with active inflammatory bowel diseases. After three days, mice received 1% CMC or P80 in drinking water or water alone for four weeks. Inflammation was quantified by serial fecal lipocalin 2 (Lcn-2) and after four weeks by blinded colonic histologic scores and colonic inflammatory cytokine gene expression. Microbiota profiles in cecal contents were determined by shotgun metagenomic sequencing. CMC treatment significantly increased fecal Lcn-2 levels compared to P80 and water treatment by one week and throughout the experiment. Likewise, CMC treatment increased histologic inflammatory scores and colonic inflammatory cytokine gene expression compared with P80 and water controls. The two emulsifiers differentially affected specific intestinal microbiota. CMC did not impact bacterial composition but significantly decreased Caudoviricetes (bacteriophages), while P80 exposure non-significantly increased the abundance of both Actinobacteria and Proteobacteria. Commonly used dietary emulsifiers have different abilities to induce colitis in humanized mice. CMC promotes more aggressive inflammation without changing bacterial composition.

In vitro degradation of low-density polyethylene by new bacteria from larvae of the greater wax moth, Galleria mellonella.

Three bacterial species isolated from whole body extracts of the greater wax moth larvae, Galleria mellonella, were evaluated for their ability to utilize low-density polyethylene (LDPE) as a sole carbon source in vitro. These bacteria were identified as Lysinibacillus fusiformis, Bacillus aryabhattai, and Microbacterium oxydans. Their ability to biodegrade LDPE was assessed by growth curves, cell biomass production, polyethylene (PE) weight loss, and the presence of LDPE hydrolysis products in the growth media. Consortia of these bacteria with three other bacteria previously shown to degrade LDPE (Cupriavidus necator H16, Pseudomonas putida LS46, and Pseudomonas putida IRN22) were also tested. Growth curves of the bacteria utilizing LDPE as a sole carbon source revealed a peak in cell density after 24 h. Cell densities declined by 48 h but slowly increased again to different extents, depending on the bacteria. Incubation of LDPE with bacteria isolated from greater wax moth larvae had significant effects on bacterial cell mass production and weight loss of LDPE in PE-containing media. The bacterial consortia were better able to degrade LDPE than were the individual species alone. Gas chromatographic analyses revealed the presence of linear alkanes and other unknown putative LDPE hydrolysis products in some of bacterial culture media.

Effect of medium and aggregation on antibacterial activity of nanodiamonds.

Fluorescent nanodiamonds are widely used as abrasives, optical or magnetic labels, in drug delivery or nanoscale sensing. They are considered very biocompatible in mammalian cells. However, in bacteria the situation looks different and results are highly controversial. This article presents a short review of the published literature and a systematic experimental study of different strains, nanoparticle sizes and surface chemistries. Most notably, particle aggregation behaviour and bacterial clumping are taken into consideration to explain reduced colony counts, which can be wrongly interpreted as a bactericidal effect. The experiments show no mechanism can be linked to a specific material property, but prove that aggregation and bacteriostatic effect of nanodiamond attachment play a significant role in the reported results.

Challenges with Verifying Microbial Degradation of Polyethylene.

Polyethylene (PE) is the most abundant synthetic, petroleum-based plastic materials produced globally, and one of the most resistant to biodegradation, resulting in massive accumulation in the environment. Although the microbial degradation of polyethylene has been reported, complete biodegradation of polyethylene has not been achieved, and rapid degradation of polyethylene under ambient conditions in the environment is still not feasible. Experiments reported in the literature suffer from a number of limitations, and conclusive evidence for the complete biodegradation of polyethylene by microorganisms has been elusive. These limitations include the lack of a working definition for the biodegradation of polyethylene that can lead to testable hypotheses, a non-uniform description of experimental conditions used, and variations in the type(s) of polyethylene used, leading to a profound limitation in our understanding of the processes and mechanisms involved in the microbial degradation of polyethylene. The objective of this review is to outline the challenges in polyethylene degradation experiments and clarify the parameters required to achieve polyethylene biodegradation. This review emphasizes the necessity of developing a biochemically-based definition for the biodegradation of polyethylene (and other synthetic plastics) to simplify the comparison of results of experiments focused for the microbial degradation of polyethylene.

Probing the interactions between hardness and sensory of pistachio nuts during storage using principal component analysis.

Instrumental hardness and sensory characteristics of pistachio nuts such as flavor, texture, and overall acceptability were measured in three different packages (atmosphere, vacuum, and oxygen scavenger), at three different conditions temperatures (20, 35, and 50˚C) during 12 weeks' storage. Results showed that the effect of temperature, storage time, and interaction effects of packaging and temperature, and packaging and storage time, and packaging and temperature and stoarge time on instrumental hardness were significant (p < 0.01). In sensory evaluation, the effect of treatments on sensory attributes was not significant. Instrumental hardness and sensory texture had a high correlation with each other. Principal component analysis (PCA) of the data obtained for the samples permitted the reduction of the variables to two principal components, which together explained 60.7% of the total variability.

Effect of the milk fat content and starter culture selection on proteolysis and antioxidant activity of probiotic yogurt.

In this study, the effects of milk fat content (0%, 2% and 3.5%) and starter culture (autochthonous or commercial) on physicochemical properties, degree of proteolysis, antioxidant activity and viability of Lactobacillus acidophilus, within 21 days storage of probiotic yogurt at 5 ± 1 °C were investigated. Statistical analysis showed that the type of starter culture had a significant effect (P < 0.05) on proteolysis and antioxidant activity, in such a way that both of them were increased until the 14th day of storage but they decreased after this period. Similarly, the pH value of all samples decreased during storage time. It ranged from 3.84-4.34 and 4.18-4.43 for yogurt samples made by autochthonous and commercial starter culture, respectively. According to the results, the survival of Lactobacillus acidophilus decreased during storage time (P < 0.05), although it stood at recommended levels for health effects (at least 106 cfu/ml in traditional yogurt). Milk fat content did not have significant effect on the survival of probiotic organisms (P < 0/05).

Microbial degradation of low-density polyethylene and synthesis of polyhydroxyalkanoate polymers.

We have characterized the ability of eight bacterial strains to utilize powdered low-density polyethylene (LDPE) plastic (untreated and without any additives) as a sole carbon source. Cell mass production on LDPE-containing medium after 21 days of incubation varied between 0.083 ± 0.015 g/L cell dry mass (cdm) for Micrococcus luteus IRN20 and 0.39 ± 0.036 g/L for Cupriavidus necator H16. The percent decrease in LDPE mass ranged from 18.9% ± 0.72% for M. luteus IRN20 to 33.7% ± 1.2% for C. necator H16. Linear alkane hydrolysis products from LDPE degradation were detected in the culture media, and the carbon chain lengths of the hydrolysis products detected varied, depending on the species of bacteria. We also determined that C. necator H16 produced short-chain-length polyhydroxyalkanoate biopolymers, while Pseudomonas putida LS46 and Acinetobacter pittii IRN19 produced medium-chain-length biopolymers while growing on polyethylene powder. Cupriavidus necator H16 accumulated poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-V) polymers to 3.18% ± 0.4% of cdm. The monomer composition of the PHB-V was 94.9% ± 0.61% 3-hydroxybutyrate and 5.03% ± 0.56% 3-hydroxyvalerate. This is the first report that provides direct evidence for simultaneous bioconversion of LDPE plastic to biodegradable polyhydroxyalkanoate polymers.

Interactions between polyols and wheat biopolymers in a bread model system fortified with inulin: A Fourier transform infrared study.

One of the ways to improve food safety and reduce community health risks is fortification of these products with inulin. Inulin, in spite of the effects and nutritional benefits, will also have undesirable effects on the quality and shelf life of bread. In this study, the interactions between polyols as improvers (i.e. glycerol, sorbitol and propylene glycol) and major biopolymers of wheat flour (i.e. starch and gluten) were examined in model systems fortified with Serish inulin by Fourier transform infrared (FTIR) spectroscopy. The changes in starch structure were estimated focusing on the ratios of the heights of the bands at 1047 and 1022 cm-1 which expresses the quantity of ordered starch to amorphous starch. At first and 5th days of storage, this ratio of control sample was higher than polyol treated samples. It was proved from Gaussian-Lorenzian curve fitting that the relative contribution of characteristic peaks of ß-turns and intramolecular ß-sheets was consecutively increased when polyol proportion of models increased. Whereas, content of intermolecular ß-sheets and α-helix was slightly decreased with increasing of polyols in the models. Briefly, polyols especially 5% propylene glycol, could be used to reduce the undesirable effects of inulin on the quality parameters of dough and bread.

In Vitro Evaluation of Antimold Activity of Annatto Natural Dye and Its Effects on Microbial, Physicochemical, and Sensory Properties of Bread.

In vitro antimold activity of annatto natural dye against Aspergillus niger, Neurospora sitophila, and Rhizopus stolonifer was evaluated with agar well diffusion and agar dilution assays. The effects of adding annatto dye (0, 0.5, 0.75, and 1%) to bread formulations were then examined. Total microbial and fungal counts, moisture, color, radical scavenging activity, and sensory analysis of bread were monitored. A. niger was the fungus most sensitive to annatto dye in vitro. Increased dye concentrations resulted in significant decreases ( P < 0.05) in moisture, total microbial and fungal counts, and L*, a*, and b* values of bread. The highest antioxidant activity was achieved in the bread with 1% of annatto dye. All bread containing annatto dye had acceptable sensory attributes as determined by panelists. The addition of annatto dye resulted in bread with a longer shelf life and acceptable sensory qualities.

The biodiversity of Lactobacillus spp. from Iranian raw milk Motal cheese and antibacterial evaluation based on bacteriocin-encoding genes.

Lactobacilli, as the largest group of lactic acid bacteria, produce large amounts of antimicrobial metabolites such as organic acids, fatty acids, ammonia, hydrogen peroxide, diacetyl and bacteriocin, which inhibit the growth of pathogenic bacteria and increase shelf life of food. The aim of this study was to identify the Lactobacillus spp. isolated from Iranian raw milk Motal cheese and to detect the presence of bacteriocin genes in the isolated Lactobacillus strains exhibiting antimicrobial activity. For this purpose, 6 Motal cheese samples from Dasht-e-Moghan region, Iran, were subjected to microbial characterization. Nineteen Lactobacillus spp. were isolated and subsequently identified based on biochemical and molecular methods. According to the sequencing of isolates, Lactobacillus spp. consisted primarily of Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus buchneri. The identified isolates were then evaluated for antimicrobial activity against Escherichia coli ATCC 25922, Listeria innocua ATCC 33090 and Staphylococcus aureus ATCC 25923. The results of PCR analysis using specific primers of genes encoding Bacteriocin, revealed the presence of Plantaricin A and Plantaricin EF in all Lactobacillus plantarum isolates and Brevicin 174A in 5 of Lactobacillus brevis isolates, whereas the gene encoding Pediocin PA-1 was not observed in any of examined isolates. It is therefore concluded that bacteriocinogenic isolates could be recommended as suitable candidates to be used as starter, adjunct-starter or antimicrobial agents for production of fermented and non-fermented products.

Synergistic effects of some essential oils against fungal spoilage on pear fruit.

The development of natural protective agents as alternatives to chemical fungicides is currently in the spotlight. In the present investigation, chemical composition and antifungal activities of thyme, cinnamon, rosemary and marjoram essential oils (EO), as well as synergism of their possible double and triple combinations were investigated. The compositions of the oils were determined by GC/MS. For determination of antifungal activity against Penicillium expansum and Botrytis cinerea, a broth microdilution method was used. The possible interactions of some essential oil combinations were performed by the two and three-dimensional checkerboard assay and isobologram construction. An in vivo antifungal assay was performed by artificial wounding of pear fruits. The maximum antifungal activity was demonstrated by thyme and cinnamon oils which displayed lower MIC values whereas rosemary and marjoram oils with MIC range between 2500 and 10,000µg/mL exhibited weak antifungal activities against tested fungi. In synergy testing, some double combinations (thyme/cinnamon, thyme/rosemary, cinnamon/rosemary) were found to be synergistic (FICi≤0.5). The triple combination of thyme, cinnamon and rosemary was synergistic for B. cinerea and P. expansum (FICi values of 0.5 and 0.375, respectively); while combination of cinnamon, marjoram and thyme exhibited additive and synergistic effect against P. expansum (FIC=0.625) and B. cinerea (FIC=0.375) respectively. The usage of a mathematical Gompertz model in relation to fungal kinetics, showed that the model could be used to predict growth curves (R2=0.993±0.05). For B. cinerea, Gompertz parameters for double and triple combination treatments showed significant increase in lag phase (1.92 and 2.92days, respectively) compared to single treatments. Increase lag time up to 2.82days (P<0.05) also observed in P. expansum treated by triple combination of EOs. Base on the results, the lowest maximum growth rate (0.37mm/day) was observed in B. cinerea treated by triple combination of thyme, cinnamon and rosemary. The in vivo test also demonstrated considerable inhibitory effects of EO combination treatments. Average lesion diameter of pears treated with triple combination of cinnamon/rosemary/thyme (78, 1250, 39µg/mL) was 6mm and 8mm against B. cinerea and P. expansum respectively, in 10days at 25°C. Results also showed that double combination of thyme/cinnamon (78, 156µg/mL) has more inhibitory effect than single EO treatments.

Development of sourdough fermented date seed for improving the quality and shelf life of flat bread: study with univariate and multivariate analyses.

The aim of this work was to study the effects of a novel sourdough system prepared by wheat flour supplemented by combination of pulverized date seed, Lactobacillus plantarum, and/or Lactobacillus brevis as well as Saccharomyces cerevisiae on the sourdough characteristics, quality, sensory, texture, shelf life and image properties of Barbari flat bread. The highest sourdough acidity and bread specific volume was obtained with co-culture of Lb. plantarum + Lb. brevis + S. cerevisiae. The results suggest that fermentation is a potential bioprocessing technology for improving sensory aspects of bread supplemented with pulverized date seed, as a dietary fiber resource. Texture analysis of bread samples during 7 days of storage indicated that the presence of pulverized date seed in sourdough was able to diminish bread staling. The interaction of baker's yeast and lactic acid bacteria (LAB) has led to increase the particle average size of bread crumb and decrease the area fraction than the LAB samples. It was observed that all treatments of sourdough Barbari breads had higher cell wall thickness than the control Barbari bread. Avrami non-linear regression equation was chosen as useful mathematical model to properly study bread hardening kinetics. In addition, principal component analysis (PCA) allowed discriminating among sourdough and bread specialties. Partial least squares regression (PLSR) models were applied to determine the relationships between sensory and instrumental data.

Serish inulin and wheat biopolymers interactions in model systems as a basis for understanding the impact of inulin on bread properties: a FTIR investigation.

In this study the interactions between Serish root inulin and the main biopolymer types of wheat flour namely gluten, starch and phospholipid, were investigated in different model systems using Fourier transform infrared (FTIR) spectroscopy to unravel the underlying physical mechanism by which inulin impacts dough and bread properties. Interactions of inulin with starch and phospholipid were not considerable compared to gluten, but it was also clear that the modes of these interactions varied with the type and the amount of additives used in model formulation. This study revealed that when inulin is added to gluten, water redistribution promotes partial dehydration of gluten and collapse of ß-spirals into intermolecular ß-sheet structures; this trans-conformations might be due to physical reasons are believed to further impact the poor quality of bread containing added inulin. Upon performing Gaussian-Lorenzian curve fitting, it was observed that by adding of inulin to model systems, the relative contribution of characteristic peaks of ß-turn and intramolecular ß-sheet was progressively decreased whereas intermolecular ß-sheet and α-helix contents were increased.

Characterization of fructan extracted from Eremurus spectabilis tubers: a comparative study on different technical conditions.

The fructans, inulin and oligofructose, were known to possess many physiologic properties. In the present study, the Box-Behnken design was used to determine the optimum extraction conditions of fructan from Eremurus spectabilis root powder (Serish) with water extraction, direct and indirect ultrasound assisted extraction methods that gave the maximum yield. Sonication amplitude (20-100 %), sonication temperature (30-70 °C) and sonication time (5-40 min) were considered variables of direct and indirect ultrasound extractions while for conventional extraction the following variables were water to solid ratio (30-50 v/w), temperature (40-90 °C) and time (5-40 min). A second-order polynomial model was fitted to each response and the regression coefficients were determined using least square method. There was a good agreement between the experimental data and their predicted counterparts. In addition to establishing the difference of these extraction methods, the scanning electron microscopy, Fourier-transform infrared spectroscopy, zeta potential and particle size analysis have been shown to be useful tools to investigate, approximate and predict characteristics of extracted fructan. Moreover, comparison of conventional extraction, direct sonication extraction, indirect sonication extraction showed the indirect sonication extraction is a suitable method for fructan extraction.

Fractionation of Eremurus spectabilis fructans by ethanol: Box-Behnken design and principal component analysis.

The fructans, inulin and oligofructose, are known to exert many food and pharmaceutical applications and are widely used in functional foods throughout the world for their nutritional and techno-functional properties. In the present study, the Box-Behnken design was used to determine the optimal conditions for fructan precipitation from Eremurus spectabilis root powder (Serish) by adding ethanol that gave the maximum yield. Ethanol-to-syrup (E/S) ratio (2:1-15:1), precipitation temperature (30-60°C) and syrup concentration (10-40°B) were considered variables of fructan precipitation. The most compatible model among mean, linear and quadratic expressions was fitted to each response and the regression coefficients were determined using least square method. There was a good agreement between the experimental data and their predicted counterparts. The optimum conditions for fractionating fructan composition of Serish by ethanol were estimated to be E/S ratio of 8.56, temperature of 23.51°C and initial syrup concentration of 40°B. Precipitation under these optimized conditions achieved the best yield (85.81%), average chain length (12.92) and purity (80.18%). In addition, principal component analysis (PCA) allowed discriminating among precipitated fructan specialties.

Optimisation of ultrasound-assisted extraction of natural pigment from annatto seeds by response surface methodology (RSM).

The present study reports on the extraction of natural pigment from annatto seeds. Response surface methodology (RSM) was used to investigate the effect of process variables on the ultrasound-assisted extraction (UAE). Four independent variables including temperature (20-80°C), sonication time (2-10 min), duty cycle (0.2-0.8s) and the ratio of seeds to the solvent (5-20%) were studied. According to the results, the optimal UAE condition was obtained with a temperature of 72.7°C, extraction time of 7.25 min, the ratio of seed to solvent of 14% and duty cycle of 0.8s. At these conditions, extraction yield determined as 6.35% and the absorbance value as 0.865%. The experimental values under optimal condition were in good consistent with the predicted values, which suggested UAE is more efficient process as compared to conventional extraction.

Genetic algorithm-artificial neural network and adaptive neuro-fuzzy inference system modeling of antibacterial activity of annatto dye on Salmonella enteritidis.

Annatto is commonly used as a coloring agent in the food industry and has antimicrobial and antioxidant properties. In this study, genetic algorithm-artificial neural network (GA-ANN) and adaptive neuro-fuzzy inference system (ANFIS) models were used to predict the effect of annatto dye on Salmonella enteritidis in mayonnaise. The GA-ANN and ANFIS were fed with 3 inputs of annatto dye concentration (0, 0.1, 0.2 and 0.4%), storage temperature (4 and 25°C) and storage time (1-20 days) for prediction of S. enteritidis population. Both models were trained with experimental data. The results showed that the annatto dye was able to reduce of S. enteritidis and its effect was stronger at 25°C than 4°C. The developed GA-ANN, which included 8 hidden neurons, could predict S. enteritidis population with correlation coefficient of 0.999. The overall agreement between ANFIS predictions and experimental data was also very good (r=0.998). Sensitivity analysis results showed that storage temperature was the most sensitive factor for prediction of S. enteritidis population.