AFM study of the differential inhibitory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negative bacteria

(−)-Epigallocatechin-3-gallate (EGCG), a main constituent of tea catechins, affects Gram-positive and Gram-negative bacteria differently; however, the underlying mechanisms are not clearly understood. Atomic force microscopy (AFM) was used to compare morphological alterations in Gram-positive and Gram-negative bacteria induced by EGCG and by H₂O₂ at sub-minimum inhibitory concentrations (MICs). EGCG initially induced aggregates in the cell envelopes of Staphylococcus aureus and eventually caused cell lysis, which was not observed in cells treated with H₂O₂. It initially induced nanoscale perforations or microscale grooves in the cell envelopes of Escherichia coli O157:H7 which eventually disappeared, similar to E. coli cells treated with H₂O₂. An E. coli O157:H7 tpx mutant, with a defect in thioredoxin-dependent thiol peroxidase (Tpx), was more severely damaged by EGCG when compared with its wild type. Similar differing effects were observed in other Gram-positive and Gram-negative bacteria when exposed to EGCG; it caused aggregated in Streptococcus mutans, while it caused grooves in Pseudomonas aeruginosa. AFM results suggest that the major morphological changes of Gram-negative bacterial cell walls induced by EGCG depend on H₂O₂ release. This is not the case for Gram-positive bacteria. Oxidative stress in Gram-negative bacteria induced by EGCG was confirmed by flow cytometry.     

The antioxidant activity and transcellular pathway of Asp‐Leu‐Glu‐Glu in a Caco‑2 cell monolayer

Asp‐Leu‐Glu‐Glu (DLEE) is one of the antioxidant peptides purified from Chinese dry‐cured Xuanwei ham in our previous study. In the current work, the stability in a simulated digestion system, the transportation pathway and the antioxidant ability of DLEE were further investigated in a Caco‐2 cell monolayer. In the simulated gastrointestinal digestion system, no oligopeptides were generated. In the transport trial, the inhibitors cytochalasin D increased the transport of DLEE across the Caco‐2 cell monolayer, with P_app values of 3.22 × 10^?6 cm s^?1. A decreased expression occludin was observed with the DLEE incubation in the cell monolayer, and the antioxidant activity showed to be increased gradually in basolateral side. This study indicates that the absorption of DLEE could mainly occur via paracellular transport and provides information about its antioxidant activity after being absorbed across a cell monolayer.     

The influence of milk with different compositions on the bioavailability of blackberry polyphenols in model sports nutrition beverages

This study aimed to determine whether the fat and protein content in dairy-fruit blends prepared using full-fat, semi-skimmed, skimmed or high-protein milk in combination with freeze-dried blackberry puree influenced the bioaccessibility and bioavailability of blackberry polyphenols and organic acids. Samples were subjected to in vitro digestion, after which bioavailability was analysed with a Caco-2 cell model. The addition of puree to milk resulted in the formation of protein aggregates, which partially protected anthocyanins during digestion. Milk fat may have increased the bioaccessibility of anthocyanins while reducing permeability through the Caco-2 cells, leading to a lower bioavailability in tested samples.     

Antioxidant properties of 1,2,3,4,6-penta-O-galloyl-β-d-glucose from Elaeocarpus sylvestris var. ellipticus

The antioxidant potential of 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG), isolated from Elaeocarpus sylvestris var. ellipticus, was investigated by various established systems based on cell-free and cell system experiments, such as radical detection, antioxidant enzyme assay, lipid peroxidation detection, and cell viability assay. PGG was found to quench the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and intracellular reactive oxygen species. PGG recovered the cellular antioxidant enzyme activities of superoxide dismutase, catalase, and glutathione peroxidase, which were reduced by H₂O₂ treatment, thereby resulting in the inhibition of lipid peroxidation. Cytoprotective effects of PGG were based on the results of DNA fragmentation, mitochondrial membrane potential (Δψ), apoptotic body formation, and caspase-3 activity. The results suggest that PGG protects cells against H₂O₂-induced cell damage via antioxidant properties.     

Compositions of anthocyanins in blackberry juice and their thermal degradation in relation to antioxidant activity

This study investigated the compositions of anthocyanins in blackberry juice and the effect of heating (70, 80, and 90?°C) on individual anthocyanins, as well as its impact on antioxidant activity. The results showed that five anthocyanins were identified, including cyanidin (Cy)-3-O-glucoside (1), Cy-3-O-arabinoside (2), Cy-3-O-malonyl-glucoside (3), Cy-3-O-dioxalyl-glucoside (4), and the Cy derivative (5). The degradation rates of individual anthocyanins (1–4) followed the first-order reaction kinetics. Unexpectedly, the amount of the Cy derivative (5) was observed to slightly increase in temperature. At the same temperature, the half-lives (T _1/2) values of each anthocyanin (1–4) significantly differed (P?<?0.05). Maximum of the T _1/2 values were observed with Cy-3-O-dioxalyl-glucoside (T _1/2 values ranged 9.48–3.54?h) and exhibited relatively resistant stability. Moreover, the T _1/2 values of Cy-3-O-glucoside were higher than Cy-3-O-arabinoside and Cy-3-O-malonyl-glucoside. The antioxidant activity of blackberry juice reduced with the decreasing of total anthocyanins. These results demonstrated that the time dependency of anthocyanins degradation and antioxidant capacity were strongly correlated in blackberry juice.     

Impact of high pressure processing on total antioxidant activity,phenolic, ascorbic acid,anthocyanin content and colour of strawberry and blackberry purées

The present study was undertaken to assess the effect of high pressure treatments and conventional thermal processing on antioxidant activity, levels of key antioxidant groups (polyphenols, ascorbic acid and anthocyanins) and the colour of strawberry and blackberry purées. Bioactive compounds (cyanidin-3-glycoside, pelargonidin-3-glucoside, ascorbic acid) and antioxidant activity were measured in strawberry and blackberry purées subjected to high pressure treatment (400, 500, 600 MPa/15 min/10–30 °C) and thermal treatments (70 °C/2 min). Samples were assessed immediately after processing. Different pressure treatments did not cause any significant change in ascorbic acid (p > 0.05). In contrast, following thermal processing (P₇₀ ≥ 2 min) ascorbic acid degradation was 21% (p < 0.05) as compared to unprocessed purée. However, no significant changes in anthocyanins were observed between pressure treated and unprocessed purées (p > 0.05), whereas conventional thermal treatments significantly reduced the levels (p < 0.05). In general, antioxidant activities of pressure treated strawberry and blackberry purées were significantly higher (p < 0.05) than in thermally processed samples. Colour changes were minor (ΔE) for pressurised purées but the differences were slightly higher for thermally treated samples. Redness of purées was well retained in high pressure treated samples. Therefore processing strawberry and blackberry by high pressure processing could be an efficient method to preserve these products quality. Hence high pressure processing (HPP) at moderate temperatures may be appropriate to produce nutritious and fresh like purées.Industrial relevanceThis research paper provides scientific evidence of the potential benefits of high pressure processing in comparison to thermal treatments in retaining important bioactive compounds. Antioxidant activity (ARP), ascorbic acid, and anthocyanins after exposure to high pressure treatments (400–600 MPa) were well retained. Our results also show that redness and colour intensity of strawberry and blackberry purées were better preserved by high pressure processing than conventional thermal treatment. From a nutritional perspective, high pressure processing is an attractive food preservation technology and offers opportunities for horticultural and food processing industries to meet the growing demand from consumers for healthier food products. Therefore high pressure processed foods could be sold at a premium than their thermally processed counterparts as they will have retained their fresh-like properties.     

Phenolic Content and Antioxidant Activity of Raspberry and Blackberry Cultivars

ABSTRACT: Raspberry (Aksu Kırmızısı, Rubin, Newburgh, Hollanda Boduru, Heritage) and blackberry (Bursa 1, Bursa 2, Jumbo, Chester) cultivars were assayed for antioxidant activity (determined as 2,2-azino-di-[3-ethylbenzothialozine-sulphonic acid][ABTS], 2,2-diphenyl-1-picrylhydrazyl radical [DPPH], and cupric ion reducing antioxidant capacity [CUPRAC]), total phenol, total flavonoid, and total anthocyanin contents. In addition, 10 anthocyanins and anthocyanidins were determined in raspberry and blackberry by liquid chromatography-mass spectrometry (LC-MS/MS). Raspberry and blackberry had the highest ABTS, DPPH, CUPRAC, total phenol, and total flavonoid contents in methanol extracts, whereas total anthocyanin contents were the highest in water extracts. The antioxidant activity of the raspberry and blackberry was directly related to the total amount of phenolic compounds detected in the raspberry and blackberry. All antioxidant activity values were highly correlated with anthocyanin content in blackberry (0.93 ≤ r ≤ 0.99, P = 0.05). On the other hand, high correlation between total flavonoid content and antioxidant activity was recorded in water extract of blackberry (0.91 ≤ r ≤ 0.93, P = 0.05). ABTS value was highly correlated with total flavonoid content in methanol extract (r = 0.90), whereas total flavonoid content was relatively less correlated with DPPH (r = 0.85) and CUPRAC (r = 0.89).     

Oryzadine,a new alkaloid of Oryza sativa cv. Heugjinjubyeo, attenuates oxidative stress-induced cell damage via a radical scavenging effect

The antioxidant properties of oryzadine, a new alkaloid, obtained from Oryza sativa cv. Heugjinjubyeo was investigated by applying various methods based on cell-free and cell experiments. Oryzadine showed scavenging effects on the hydroxyl radical, superoxide radical, and intracellular reactive oxygen species (ROS). Oryzadine inhibited H₂O₂-induced DNA damage, which was demonstrated by DNA tail formation, lipid peroxidation which was demonstrated by the formation of thiobarbituric acid reactive substance (TBARS), and protein oxidation which was demonstrated by protein carbonyl formation. Therefore, oryzadine protected H₂O₂-induced cell damage. Our results show that the cytoprotective effects of oryzadine stem from its ability to inhibit H₂O₂-induced apoptosis, as demonstrated by a decrease in apoptotic body formation and the inhibition of mitochondrial membrane potential (ΔΨ) loss. Taken together, these findings suggest that oryzadine protected cells against H₂O₂-induced cell damage via ROS scavenging effect. Therefore, oryzadine could be considered a significant natural source of antioxidant.     

Inhibitory effect of the ethyl acetate fraction from astringent persimmon on H2O2-induced oxidative stress in HepG2 cells

Persimmon cv. Sangjudungsi (Diospyros kaki Thunb.) is a major astringent cultivar in Korea. A phenolic extract of cv. Sangjudungsi was obtained using acetone with homogenization, then sequentially fractionated into n-hexane, chloroform, ethyl acetate, n-butanol, and water fractions. These 5 fractions were used to evaluate levels of total phenolics and total flavonoids, and the antioxidant capacities, and to investigate whether the fractions protected human hepatoma HepG2 cells from deleterious oxidative stress. The ethyl acetate fraction had the highest levels of total phenolics and total flavonoids, and the greatest antioxidant capacity. Under oxidative stress caused by H₂O₂, the ethyl acetate fraction at non-toxic concentrations significantly (p<0.05) restored the viability of HepG2 cells in a dose-dependent manner, compared with a control. The ethyl acetate fraction also alleviated intracellular oxidative stress, possibly due to effective antioxidant activities in cells. Astringent persimmons are a good source of antioxidants for reduction of oxidative stress.     

Effect of processing on the texture and structure of raspberry (cv. Heritage) and blackberry (cv. Thornfree)

This paper reports separate studies of the effect of pre-treatments (CaCl₂, low methoxyl pectin (LMP), and combined solutions) and the effect of freezing method (at four different rates) and thawing mode (at two different rates) on objective parameters, structure and sensory characteristics of fresh raspberries and blackberries. After that, the effect of a complete freezing process combining the best pre-treatments with the best freezing/thawing conditions found for each fruit was investigated. Kramer Shear Cell (KSC), back extrusion, compression and multiple penetration tests were used to measure fruit texture objectively. For calcium and LMP pre-treatments, which were applied separately, texture parameters were significantly higher in samples treated at the highest concentrations (100 mM of CaCl₂ for both fruits and 0.3 and 3% of LMP for raspberry and blackberry, respectively) compared to fresh controls. Blackberry structure was more susceptible than raspberry structure to the effect of pre-treatments. For the combined pre-treatments, the highest texture parameters were found in the samples treated with CaCl₂ (100 mM) and LMP (0.1%) in the case of raspberries and CaCl₂ (100 mM) and LMP (3%) in the case of blackberries. Combined pre-treatment did not increase firmness with respect to that of samples treated only with calcium, which indicates that CaCl₂ preserved the raspberry structure more efficiently during processing. Fruits frozen by forced convection with liquid nitrogen vapour at –40 °C were significantly firmer. Raspberries should be thawed at 5 °C, whereas blackberries may be thawed at room temperature. Sensory analysis showed that the blackberry structure was more resistant to freezing. In both fruits, over the complete process parameter values were again highest in the samples treated with 100 mM CaCl₂, applied either separately or in combination with LMP. In raspberry, panellists detected no significant differences between sensory texture parameters of the different samples, and in blackberry, panellists found no significant differences between any of the sensory characteristics. Multiple penetration maximum force (F _MP) was the parameter that best expressed product firmness for both fresh and frozen raspberries, whereas compression slope (S _C) best reflected changes in blackberries. SEM mainly corroborated results from objective texture parameters.     

Effects of nutrition strategy on the levels of nutrients and bioactive compounds in blackberries

The effects of nutrition strategy on levels of nutrients and bioactive compounds in fruit and leaves of blackberries were studied in greenhouse-grown blackberry plants fertilised with combinations of two levels (low, high) of nitrogen (60 and 100 kg ha⁻¹, respectively) and potassium (66.4 and 104 kg ha⁻¹, respectively). Plant concentrations of organic phytochemicals were quantitatively analysed by high-performance liquid chromatography. High amounts of both fertilisers produced high amounts of all nutrients and bioactive compounds analysed in fruit except total acidity and ellagic acid. There were major differences in compounds affecting taste in fruit, e.g., sugars (fructose and glucose), total soluble solids and pH, and also in anthocyanin content. The concentrations of secondary metabolites, vitamin C and ellagic acid in fruit also varied significantly between treatments, although the differences were smaller. Storage of blackberries showed variable effects in the different levels of compounds, and the changes found were small. Nutrient regime did not affect blackberry leaves to the same extent, and only minor changes were found. The findings show that by optimising plant nutrition, phytonutrient levels can be maximised and maintained in fresh and stored berry crops, especially those grown in greenhouses, where conditions can easily be regulated.     

Antioxidative effects of whey protein on peroxide-induced cytotoxicity

Myoblastic toxicity is a major adverse effect caused by reactive oxygen species (ROS) when exercising heavily. Although protection or alleviation of ROS toxicity can be achieved by administration of antioxidant vitamins such as vitamin E and vitamin C, their protective effect remains controversial. Thus, alternative natural antioxidants may be potential candidates for foods for athletes. In this research, we investigated the antioxidative effect of whey protein against hydrogen peroxide (H₂O₂) toxicity using C₂C₁₂ myoblasts. Whey protein pre-incubation prevented the decrease in cell viability after H₂O₂ treatment. The production of 8-hydroxydeoxyguanosine associated with DNA oxidative damage was also inhibited by the whey protein pre-incubation. Endogenous antioxidant defense, such as glutathione, catalase, and superoxide dismutase activity, was also modulated by the antioxidant. At the same time, enhanced mRNA expression levels of heme oxygenase-1 and NADPH quinone oxidoreductase-1 were observed in cells pre-incubated with whey protein before H₂O₂ abuse. These findings suggest that whey protein improved the antioxidant capacity against acute oxidative stress through multiple pathways and this protein may serve as an alternative source of antioxidants for prevention of athletic injuries caused by ROS.     

Antimicrobial activity of Satureja montana L. essential oil against Clostridium perfringens type A inoculated in mortadella-type sausages formulated with different levels of sodium nitrite

This research evaluated the antimicrobial effect of the winter savory (Satureja montana L.) essential oil (EO) against Clostridium perfringens type A (ATCC 3624) inoculated in mortadella-type sausages formulated with different levels of sodium nitrite (NaNO₂: 0 ppm, 100 ppm and 200 ppm) in addition to EO at concentrations of 0.0%, 0.78%, 1.56% and 3.125% stored at 25 °C for 30 days. The EO extracted by hydrodistillation and analyzed by gas chromatography-mass spectrometry (CG-MS) was tested in vitro using an agar well diffusion method for determination of minimum inhibitory concentration (MIC) on C. perfringens. According to compositional analysis of the winter savory EO, 26 chemical compounds were identified, and the major constituents were thymol (28.99%), p-cymene (12.00%), linalool (11.00%) and carvacrol (10.71%). The results obtained showed that EO applied at a concentration of 1.56%, which was defined as the MIC, exhibited antimicrobial activity against C. perfringens in the in vitro assays, and the transmission electron microscopy (TEM) revealed structural damage and cell lysis of C. perfringens caused by EO treatment. A synergistic effect between NaNO₂ and EO was observed. In mortadella-type sausages formulated with 100 ppm of NaNO₂ and EO at all concentrations tested, the population of target microorganisms was reduced (p ≤ 0.05) compared to control samples during all storage period. This data suggests the potential combined use of savory EO and minimal amounts of the synthetic additive, NaNO₂ to control C. perfringens in mortadella, which goes according to current market trends, where consumers are requesting natural products.     

Caseinophosphopeptides exert partial and site-specific cytoprotection against H2O2-induced oxidative stress in Caco-2 cells

Caseinophosphopeptides can sequester prooxidant metals and scavenge free radicals, and may thus be used as functional food ingredients. The total antioxidant capacity (TEAC and ORAC) of two pools of caseinophosphopeptides (1–3 mg/ml), obtained from casein subjected to simulated gastrointestinal digestion (at two different pH values) and selective precipitation, was evaluated to determine dose–response activity. Pool B (which showed the highest antioxidant capacity due to the presence of more antioxidant amino acids) was used to test its cytoprotective effect against H₂O₂-induced oxidative stress in Caco-2 cells. Caseinophosphopeptides protected the cells against oxidative damage by preserving cell viability, increasing GSH content, inducing catalase enzyme activity, diminishing lipid peroxidation and maintaining a correct cell cycle progression. However, they failed to exert protection at a mitochondrial level (ROS and mitochondrial membrane potential), implying a partial and site-specific effect. Thus, their mechanism of action is not only related to free radical scavenging activity, but also to metal chelation and the modulation of intracellular signaling cascades.     

Polyphenols bioaccessibility and bioavailability assessment in ipecac infusion using a combined assay of simulated in vitro digestion and Caco-2 cell model

In this report, we investigated for the first time the total polyphenols content (TPC) and antioxidant activity before and after digestion of Carapichea ipecacuanha root infusion, better known as ipecac, prepared at different concentrations. An in vitro digestion system coupled to a Caco-2 cell model was applied to study the bioavailability of antioxidant compounds. The ability of ipecac bioaccessible fractions to inhibit reactive oxygen species (ROS) generation at cellular level was also evaluated. The findings revealed that water volume of 50 mL g⁻¹ of sample provided the maximum yield of extraction of TPC and antioxidant activity. Polyphenols increased in content and activity after digestion and they were highly bioavailable (75% of intestinal absorption). Polyphenols were also present in the residual parts which indicate a possible local activity. Results also suggest that ipecac infusion could represent a promising source of effective bioavailable antioxidants to be exploited in functional foods field.     

Effects induced by the nodulation with Bradyrhizobium japonicum on Glycine max (soybean) metabolism and antioxidant potential

This study was conducted in order to assess the effect of the inoculation of Glycine max (soybean) with Bradyrhizobium japonicum on the metabolite profile and antioxidant potential of its aerial parts. Extracts containing phenolic compounds and organic acids were analysed by HPLC–DAD and HPLC–UV, respectively. Volatiles profile was determined by HS-SPME/GC–IT-MS. The antioxidant activity of acidic and methanolic extracts was assessed against DPPH. Nodulation caused by B. japonicum led to an increase in the contents of phenolic compounds and organic acids. The same was observed for volatile compounds, with compounds like linalyl acetate, menthyl acetate and α-farnesene being detected only in inoculated plants. The phenolic extracts showed stronger antioxidant capacity than the organic acid extracts. In addition, extracts from plants nodulated with B. japonicum exhibited significantly higher antioxidant activity than control samples. These findings suggest that the inoculation with nodulating B. japonicum strains may be employed to manipulate the content of interesting metabolites in G. max aerial parts.     

Protective effect of two edible mushrooms against oxidative cell damage and their phenolic composition

The aim of the study was to investigate phenolic composition, antioxidative, protective and cytotoxic effects of Pleurotus eryngii and Auricularia auricula-judae. Analysis of phenolic compounds in these edible mushrooms species has been carried out by high-performance liquid chromatography (HPLC). Protective effect of these mushrooms on H₂O₂ induced oxidative cell damage was determined by using MTT (3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) assay. Antioxidant activities of the mushrooms extracts were evaluated by using complementary in vitro assays. In addition, the measurement of total antioxidant compounds in the extracts was carried out. All the extracts exhibited protective effect against H₂O₂ induced oxidative cell damage but the highest activity was observed for A. auricula-judae aqueous extract (89.5 ± 1.8% cell viability at 0.1 mg/ml). P. eryngii methanolic extract showed the highest ferrous iron chelating ability (IC₅₀ = 0.42 ± 0.03 mg/ml). A. auricula-judae extracts (at concentration of 0.025–0.100 mg/ml) were not toxic to baby hamster kidney fibroblast cell line (BHK 21). These results suggest that these mushrooms may be used as a potential source of natural antioxidants for food supplementation or in the development of nutraceuticals.     

Multifactorial analysis of acetaldehyde kinetics during alcoholic fermentation by Saccharomyces cerevisiae

Acetaldehyde is the terminal electron acceptor in the alcoholic fermentation by Saccharomyces cerevisiae. Quantitatively the most important carbonyl by-product, it has relevance for ethanol production yields as well as product stabilization and toxicology. The aim of this study was to investigate the effect of various enological parameters on acetaldehyde kinetics during alcoholic fermentations. Two commercial yeast strains were tested in two grape musts and the pH, temperature, SO₂ and nutrient addition were varied. All incubations had uniform kinetics where acetaldehyde reached an initial peak value followed by partial reutilization. Peak acetaldehyde concentrations and residual concentrations after 15 days of fermentations ranged from 62 to 119 mg l^− 1 and 22 to 49 mg l^− 1, respectively. A positive linear relationship was found between peak and final acetaldehyde levels in Gewürztraminer, but not Sauvignon Blanc fermentations, where sluggish fermentations were observed. Several factors had a significant effect on peak and/or final acetaldehyde levels. SO₂ addition, grape cultivar and fermentation nutrition were important regulators of peak acetaldehyde production, while final acetaldehyde concentrations were correlated with SO₂ addition, grape cultivar and temperature. The results allowed to estimate the acetaldehyde increase caused by SO₂ addition to 366 ??g of acetaldehyde per mg of SO₂ added to the must. The course of the final fermentation phase was shown to determine acetaldehyde residues. Comparison of acetaldehyde and hexose kinetics revealed a possible relationship between the time of occurrence of peak acetaldehyde concentrations and the divergence of glucose and fructose degradation rates.