The recognition of high molecular weight melanoidins as the main components responsible for radical-scavenging capacity of unheated and heat-treated Canadian honeys

As the Maillard reaction is known to occur in heat-treated foods, unheated and heated honey samples were subjected to the activity-guided fractionation and size-exclusion chromatography to compare the degree of browning, radical scavenging activity (ORAC) and molecular size of the fractions obtained. Heat-treatment increased browning in the fractions of light- and medium-coloured honeys (p < 0.002), accelerated the formation of high molecular weight melanoidins (85–232 kDa) and significantly increased ORAC values (p < 0.0001). In contrast, melanoidin content and ORAC decreased in the fractions of heat-treated dark buckwheat honey (p < 0.001). Unheated dark honey contained a significantly higher amount of melanoidins than other honeys (p < 0.007). Together, results showed that at low initial concentration of melanoidins, heat-treatment accelerated formation of new melanoidins and increased ORAC, while at high concentration it caused decrease and a reduction of radical scavenging activity. This study emphasises the importance of non-enzymatic browning in the prediction of the antioxidant pool in thermally processed honeys.     

The influence of protein–flavonoid interactions on protein digestibility in vitro and the antioxidant quality of breads enriched with onion skin

Different types of breads enriched with onion skin were studied. The objectives were twofold: to show and examine protein–phenolic interactions and to discuss results concerning phenolic content, antioxidant activity and protein digestibility in the light of in vitro bioaccessibility. Phenolic contents and antiradical abilities were linked with the level of onion skin supplement however, the amounts determined were significantly lower than expected. Fortification influenced protein digestibility (a reduction from 78.4% for control breads to 55% for breads with a 4% supplement). Electrophoretic and chromatographic studies showed the presence of indigestible protein–flavonoid complexes – with molecular weights about 25 kDa and 14.5 kDa; however, the reduction of free amino group levels and the increase in chromatogram areas suggest that flavonoids also bind to other bread proteins. The interaction of phenolics with proteins affects antioxidant efficacy and protein digestibility; thus, they have multiple effects on food quality and pro-health properties.     

Antioxidant activity of vinegar melanoidins

Melanoidins, the brown polymers formed through Maillard reaction during vinegar process, are one of major high-molecular-weight fractions of vinegar. In this study the antioxidant activity of high-molecular-weight fractions (MW > 3500 Da) separated from ethanol-supernatant extraction of concentrated Zhenjiang aromatic vinegar was evaluated by different in vitro tests: the DPPH radical scavenging activity, the reducing power, total phenolic content, and the inhibitory effect on hydroxyl radical. Each individual fraction was found to have antioxidant activity in all the model systems tested. The high-molecular-weight fractions of vinegar (MW > 3500 Da) were separated into different fractions by DEAE-Sepharose fast flow. The fractions eluted by 0.2 mol/L NaCl and 0.3 mol/L NaCl with higher phenolic content have stronger DPPH radical scavenging activity and reducing power. Antioxidant activity in hydroxyl radical system was not correlated with phenolic content. Two phases which have stronger effects on Maillard reaction products (MRPs) in production process were examined. Decoction, storing and aging may affect vinegar’s antioxidant activity. The present results support the concept that melanoidins formed during vinegar production process may have health promotion activity.     

Storage-induced chemical changes in active components of honey de-regulate its antibacterial activity

To elucidate reasons for the observed variability in the antibacterial activity of honeys, we analysed a causal relationship between (a) honey floral sources and the activity and (b) the effect of honey storage on stability of compounds conferring this activity. Honeys from diverse floral sources were screened against Escherichia coli (ATCC 14948) and Bacillus subtilis (ATCC 6633) using the broth microdilution method. Among “active” honeys, 37% originated from buckwheat, 18% from clover and 12% from blueberry, indicating that these floral sources produced phytochemical(s) that inhibited bacterial growth. The stability of the putative phytochemical(s) was analysed in “active” honeys (MIC₉₀ 6.25% v/v) by measuring the activity every 3–6 months for a period of 1–3 years. A sharp decline in activity against both bacteria was observed in the first 3–6 months of storage. The decline coincided with major changes in chemical composition of honeys which included a significant change in colour (p < 0.0025), extremely significant change in concentration of UV-absorbing compounds (p < 0. 0001) and appearance of melanoidins. While these changes reduced E. coli sensitivity to honey, it rendered B. subtilis completely insensitive. Thus, the data indicates that the presence of phytochemical(s) conferring the antibacterial activity is sensitive to storage. The de-regulation of the antibacterial activity with the concomitant appearance of melanoidins suggests that the active phytochemical components might be sequestered into melanoidin aggregates, losing their function.     

A survey of Irish fruit and vegetable waste and by-products as a source of polyphenolic antioxidants

In this study, fruit and vegetable by-product and waste sources in Ireland were tested for their antioxidant activity and polyphenol content. The highest levels of antioxidants measured by both ferric reducing antioxidant power (FRAP) and diphenyl-picrylhydrazyl (DPPH) assays, were detected in whole kiwifruit. Of the vegetable by-products, broccoli stems showed the best antioxidant potential. The level of polyphenols as assessed by the Folin–Ciocalteu Reagent (FCR) was significantly correlated with the level of polyphenols by HPLC–DAD (r = 0.93). The level of polyphenols assessed by HPLC–DAD was not significantly correlated with the antioxidant assays. Apple pomace and vegetable by-products were found to be good sources of both polyphenols and antioxidants and due to their abundance may be exploitable resources to use as food ingredients.     

Raw Millefiori honey is packed full of antioxidants

Total polyphenols, flavonoids and antioxidant power of raw honey samples from two of the most common Italian varieties, i.e., Millefiori and Acacia, were evaluated. Phenolic content, expressed as caffeic acid equivalents, ranged from 12.5 to 17.5 mg/100 g and from 3 to 11 mg/100 g in Millefiori and Acacia honeys, respectively. All Millefiori samples exhibited the highest flavonoid concentration being between 1.23 and 2.93 mg catechin equivalents (CE)/100 g honey. Total flavonoids in 100 g Acacia honeys were in the range of 0.45–1.01 mg CE. Acacia honeys had lower total antioxidant power, as assessed by ferric reducing/antioxidant power assay, than Millefiori. The relationship between phenolic content and antioxidant power was discussed. Comparative experimental analysis was performed with an artificial honey and processed honeys. Raw Millefiori honey is rich in both amount and variety of antioxidant substances, and its inclusion in the diet may be recommended to complement other polyphenol sources.     

Preparation and anti-influenza A virus activity of κ-carrageenan oligosaccharide and its sulphated derivatives

Carrageenans, the sulphated galactans derived from red algae, are attracting increasing interest in developing potential anti-viral drugs. In this study, low molecular weight κ-carrageenan oligosaccharides (KCO) and their sulphated derivatives (KCO-S) were prepared, and their anti-influenza A virus (IAV) properties were investigated. The results indicated that KCO and KCO-S could effectively inhibit IAV multiplication in MDCK cells in a dose-dependent manner. Furthermore, a structure–activity relationship study showed that the degree of sulphation and molecular weight were the main factors that influenced the anti-IAV activity of κ-carrageenan oligosaccharide. The most active κ-carrageenan oligosaccharide had a sulphate content of 0.8–1.0 mole/mole of disaccharide and a molecular weight of 1–3 kDa. In addition, KCO and KCO-S could significantly improve survival and decrease pulmonary viral titres in IAV-infected mice. Moreover, the antiviral effect of KCO and KCO-S does not seem to be dependent on the interferon system. In conclusion, carrageenan oligosaccharide and its sulphated derivative have good inhibitory actions on IAV replication in vitro and in vivo.     

Evaluation of the phenolic content,antioxidant activity and colour of Slovenian honey

Honey samples from the seven most common honey types in Slovenia were screened for total phenolic content by the modified Folin–Ciocalteu method, for potential antioxidant activity using the ferric reducing antioxidant power (FRAP) assay and by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method for antiradical activity. In addition the colour characteristics of honey samples were analysed. The results of the study showed that total phenolic content, antioxidant activity and colour parameters differ widely among different honey types. Phenolic content expressed as gallic acid equivalent ranged from 44.8 mg/kg in acacia honey to 241.4 mg/kg in fir honey. Antioxidant activity was the lowest in the brightest acacia and lime honeys and the highest in darker honeys, namely fir, spruce and forest. The colour of the Slovenian honeys, analysed in this study was very variable and ranged from pale yellow to dark brown. Correlations between the parameters analysed were found to be statistically significant (p < 0.05).     

Structural characterisation and antioxidant activity of melanoidins from high-temperature fermented apple

High-temperature fermented apple is a new fermented product with characteristic black appearance, which is prepared through natural fermentation of whole fresh apple at controlled high temperature (60–90 °C) and humidity (relative humidity of 50–90%). In this work, melanoidins from high-temperature fermented apple were obtained by ultrafiltration and subsequent diafiltration. The structural characterisation of melanoidins was investigated in the present study. Results showed that the molecular weight of the obtained melanoidins was about 179 kDa and the melanoidins were comprised primarily of furans by pyrolysis–gas chromatography–mass spectrometry. Antioxidant activities of the high-temperature fermented apple and its melanoidins were evaluated by ABTS, DPPH, hydroxyl radicals, ferric reducing antioxidant power and Fe²⁺-chelating assays. Both high-temperature fermented apple and its melanoidins showed extremely high antioxidant activities in scavenging hydroxyl radicals. High correlation between the antioxidant activity assayed methods and the physicochemical characteristics of melanoidins was obtained. The contribution of melanoidins to the total antioxidant capacity of high-temperature fermented apple was determined.     

Characteristics and antioxidant activity of ultrafiltrated Maillard reaction products from a casein–glucose model system

Maillard reaction products (MRPs) were prepared from casein–glucose by refluxing for 130 min at 102 °C and initial pH 12.0 without pH control to investigate the characteristics of casein–glucose Maillard reaction and the antioxidant activity difference among different fractions of MRPs. Browning and intermediate products increased, however, the pH of the system decreased with increase in the heating time. Free amino group content decreased 78% during first 10 min and did not change nearly thereafter. Amino acid analysis indicated that lysine and arginine decreased significantly, and casein was partially hydrolysed to peptides or free amino acid. High molecular weight compounds were dominant in the MRPs, determined by high performance gel-filtration chromatography. After ultrafiltration, antioxidant activity of each MRPs fraction was investigated by DPPH radical-scavenging activity, reducing power, Fe²⁺ chelating activity and lecithin oxidation assay. MRPs of different molecular weight exhibited distinctly different antioxidant activities.     

The relationship between the content of Maillard reaction-like products and bioactivity of Canadian honeys

Honey possesses antioxidant and antibacterial activities; however the compounds that are responsible for these activities are still under investigation. To determine whether there are common features/compounds that underlie the antioxidant and antibacterial activities, 20 Canadian honeys were analysed using the oxygen radical absorbance capacity (ORAC) and the broth micro-dilutions assay, respectively. Principal component analysis revealed the highest correlation between ORAC and Maillard reaction-like products (MRLP) (R = 0.938, p < 0.0001). In addition, the extremely significant correlations between ORAC, MRLPs, phenolic content and honey colour may suggest that these compounds represent the same chemical entity and exert their antioxidant activity while being part of a higher molecular weight structure. Moreover, the antioxidant activity and MRLPs were also the dominant variables contributing to the antibacterial activity against Escherichia coli (R = 0.737, p < 0.0001 and R = 0.65, p < 0.002, respectively) however no such correlation against Bacillus subtilis. In conclusion, MRLPs in honey appear to underlie the antioxidant and partially the antibacterial activity against E. coli.     

Investigation on the extractability of melanoidins in portioned espresso coffee

Coffee melanoidins have attracted interest as a result of its potential health benefits. This investigation aims to elucidate the extraction behavior of melanoidins and their populations during the preparation of portioned espresso coffee and its relationship with the antioxidant activity of the coffee brew. Filter-paper pods, FAP capsule, and clone capsule containing light roasted coffee have been investigated. An accumulative fractionation approach has applied to model the extraction kinetics of melanoidins, melanoidin populations, browning, chlorogenic acids (CGA), and antioxidant activity. Melanoidins were very efficiently extracted in clone capsules since less than 9 s was necessary to extract the 50% of the melanoidin content as compared with pods and FAP capsules, and the kinetic of extraction is slower than CGA. The extraction profile of melanoidins and browning fitted better with the antioxidant capacity than CGA and total solids profile. Melanoidin populations were obtained according to ethanol solubility. Total melanoidin content and the ratio between melanoidin populations did not change during extraction volume for espresso coffee. Melanoidin populations soluble at 75% ethanol showed the highest antioxidant activity. However, melanoidins with higher antioxidant activity are extracted at higher volumes. This investigation could make possible the adjustment of the technological requirements of espresso coffeemakers to produce an espresso coffee with high levels of beneficial compounds.     

Chemical properties and antioxidant activity of exopolysaccharides from mycelial culture of Cordyceps sinensis fungus Cs-HK1

Exopolysaccharide (EPS) was produced in a mycelial liquid culture of the Cordyceps sinensis fungus, Cs-HK1, at a rate proportional to mycelial growth. The crude EPS isolated from the culture medium by ethanol precipitation contained 65–70% sugar and about 25% protein, suggesting that the EPS was composed of polysaccharide–protein complexes. It exhibited a wide molecular weight (MW) range from about 5 kDa to more than 200 kDa. The intrinsic viscosity as an index of average MW of EPS, increased during the period of rapid mycelial growth to 11.0 dL/g on day 5, and remained relatively stable between day 5 to day 8 (stationary growth period). Both infrared spectrometry of EPS and gas chromatography of acetylated EPS suggested that the EPS had a β-d-glucan backbone. The EPS showed moderate antioxidant activities with a Trolox equivalent antioxidant capacity of 35–40 μmol Trolox/g and a ferric reducing ability of plasma of 50–52 μmol Fe(II)/g.     

Relative antioxidant and cytoprotective activities of common herbs

Many studies have been carried out on bioactivities of individual herbs, however, no collective study on their comparative antioxidant and cytoprotective activities against oxidative damage has been reported. We selected 17 common commercial herbs and studied their relative phenolic contents, antioxidant activities, and cytoprotective activities on gap–junction intercellular communication and antioxidative enzymes in vitro under the same conditions. Total polyphenol content ranged from 464 to 870 gallic acid equivalents (GAE) mg/100 g and total flavonoid content from 212 to 494 catechin equivalents (CE) mg/100 g. Among the samples, chamomile, rosehip, hawthorn, lemon verbena, and green tea contained relatively high total phenolics (769–844 mg GAE/100 g) and flavonoids (400–4 mg CE/100 g). Chamomile also showed the highest antioxidant activity with 960 mg/100 g of vitamin C equivalent (VCE), followed by hawthorn (929 mg VCE/100 g) and black tea (916 mg VCE/100 g). Total phenolic and total flavonoids showed a higher correlation with antioxidant activity. Most of herbs enhanced cell viability and showed protective effects against oxidative stress induced by hydrogen peroxide in Chinese hamster lung fibroblast (V79-4) cells. Furthermore, herbs used in this study showed higher protective effect on gap–junction intercellular communication (GJIC) as compared to gallic acid and catechin, and also enhanced activity of the antioxidative enzymes such as superoxide dismutase (SOD) and catalase (CAT) in a dose-dependent manner.     

Physicochemical characteristics and anticoagulant activities of low molecular weight fractions by free-radical depolymerization of a fucosylated chondroitin sulphate from sea cucumber Thelenata ananas

A process of depolymerization of a new fucosylated chondroitin sulphate from Thelenata ananas by free-radicals was developed. The fractions with different molecular weights and a narrow molecular weight distribution was obtained. The products were characterised by physicochemical properties, FT-IR spectra and NMR spectra. The results confirm the retention of primary structure of the fractions after the depolymerization. Furthermore, the anticoagulant activity of the fractions was evaluated by the activated partial thromboplastin time (APTT). The APTT activity decreases in proportion to the molecular weight following a logarithmic-like function, and the potentiating of prolongation of APTT activity requires at least an oligosaccharide chain of about 6–8 units (6000–7000 Da). Compared to high doses of low molecular weight heparin (LMWH), a more desirable molecular weight fraction (13,950 Da) demonstrates significantly lower anticoagulant activity. Thus, the fraction has more potential as an antithrombotic agent with reduced bleeding risk relative to LMWH.     

Formation of biopolymer particles by thermal treatment of β-lactoglobulin–pectin complexes

The purpose of this study was to prepare and characterize biopolymer particles based on thermal treatment of protein–polysaccharide electrostatic complexes formed from a globular protein (β-lactoglobulin) and an anionic polysaccharide (beet pectin). Initially, the optimum pH and pectin concentration for forming protein–polysaccharide complexes were established by mixing 0.5 wt% β-lactoglobulin solutions with beet pectin (0–0.5 wt%) at different pH values (3–7). Biopolymer complexes in the sub-micron size range (d = 100–300 nm) were formed at pH 5.0 and 0.1 wt% pectin. These particles were then subjected to a thermal treatment (30–90 °C at 0.8 °C min⁻¹). The presence of pectin increased the thermal aggregation temperature of the protein, although aggregate formation was still observed when the protein–polysaccharide systems were heated above about 70 °C. The impact of pH (3–7) on the properties of heat-treated biopolymer particles (83 °C, 15 min, pH 5) was then established. The biopolymer particles were stable to aggregation over a range of pH values, which increased as the amount of pectin was increased. The biopolymer particles prepared in this study may be useful for encapsulation and delivery of bioactive food components, or as substitutes for lipid droplets.     

Application of ultrafiltration in the study of phenolic isolates and melanoidins from pale and black brewers' spent grain

Two types of brewers' spent grain (BSG), pale and black, were studied employing ultrafiltration (UF cut‐off, 10 kDa) to obtain high‐molecular‐weight (HMW) and low‐molecular‐weight (LMW) phenolic fractions from one pale (P2) and one black (B2) BSG extracts. Of the four UF fractions obtained, the HMW B2 fraction had highest level of polyphenols (5.73 ± 0.25 mg GAE g^?1 BSG dw) and protein (18.82 ± 0.41 mg protein g^?1 BSG dw). Metal‐chelating ability and antioxidative properties were also identified. Antioxidant activity evaluated using the DPPH and FRAP assays, indicated that activity in B2 extracts was associated mainly with the HMW fraction (3.10 ± 0.10 and 2.49 ± 0.09 TE g^?1 BSG dw, respectively), whereas in P2 samples, antioxidant activity was highest in LMW fractions (0.58 ± 0.01 and 0.92 ± 0.03 TE g^?1 BSG dw, respectively). The high antioxidant activity and metal‐chelating ability observed for black BSG extracts may be attributed to the presence of coloured melanoidinic structures.     

Chemical composition,antioxidant activity and antimicrobial properties of propolis extracts from Greece and Cyprus

Chemical composition, antioxidant activity and in vitro antimicrobial activity of twelve propolis ethanolic extracts (PEE) from mainland Greece, Greek islands, and east Cyprus were determined. The PEE studied contained significant amounts of terpenes and/or flavonoids, anthraquinones – mainly emodin and chrysophanol – and low amounts of phenolic acids and their esters, presenting differences from typical European propolis, and similarities to East Mediterranean propolis. Simple polyphenols and terpenic acids content ranged between 11.9–373.5 and 7.23–286.5 mg/g of PEE, respectively, with anthraquinones representing the 1.3–28.9% of simple polyphenols. Despite differences in composition, the PEE samples exhibited significant antioxidant, antibacterial, and antifungal activities, affecting a wider spectrum of microorganisms than the food grade antibacterial nisin, and presenting lower or no activity against several Lactobacillus strains. The presence of significant amounts of terpenoids seemed to enhance the antimicrobial activity of PEE. The conclusion, given the non-toxic and natural origin of PEE, is that, besides their potential pharmaceutical and nutraceutical value, propolis balsams from Greece and Cyprus are attractive candidates for use as natural antioxidant and microbicidal additives in food systems, especially those containing lactic acid bacteria.     

Milk β-lactoglobulin complexes with tea polyphenols

The effect of milk on the antioxidant capacity of tea polyphenols is not fully understood. The complexation of tea polyphenols with milk proteins can alter the antioxidant activity of tea compounds and the protein secondary structure. This study was designed to examine the interaction of β-lactogolobulin (β-LG) with tea polyphenols (+)-catechin (C), (−)-epicatechin (EC), (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG) at molecular level, using FTIR, CD and fluorescence spectroscopic methods as well as molecular modelling. The polyphenol binding mode, the binding constant and the effects of polyphenol complexation on β-LG stability and secondary structure were determined. Structural analysis showed that polyphenols bind β-LG via both hydrophilic and hydrophobic interactions with overall binding constants of K_C–β-LG = 2.2 (±0.8) × 10³ M⁻¹, K_EC–β-LG = 3.2 (±1) × 10³ M⁻¹, K_ECG–β-LG = 1.1 (±0.6) × 10⁴ M⁻¹ and K_EGCG–β-LG = 1.3 (±0.8) × 10⁴ M⁻¹. The number of polyphenols bound per protein molecule (n) was 1.1 (C), 0.9 (EC), 0.9 (ECG) and 1.3 (EGCG). Molecular modelling showed the participation of several amino acid residues in polyphenol–protein complexation with extended H-bonding network. The β-LG conformation was altered in the presence of polyphenols with an increase in β-sheet and α-helix suggesting protein structural stabilisation. These data can be used to explain the mechanism by which the antioxidant activity of tea compounds is affected by the addition of milk.     

Antioxidant activity and phenolics of an endophytic Xylaria sp. from Ginkgo biloba

The objective of this study was to evaluate the antioxidant activity of cultivated fruiting bodies of an endophytic Xylaria sp. (strain number YX-28), from Ginkgo biloba. The results indicated that the methanol extract exhibited strong antioxidant capacity in both 2,2-diphenyl-1-picrylhydrazyl (DPPH) analysis and β-carotene–linoleic acid model system. Total phenolic and flavonoid contents extracted by different solvents were determined using Folin–Ciocalteu procedure and the flavonoid–aluminium method. The results showed that total phenolic and flavonoid contents were the highest in methanol extract (54.51 ± 1.05 mg gallic acid equivalent/g dry weight and 86.76 ± 0.58 mg rutin equivalent/g dry weight), while the hexane extract was the lowest (9.71 ± 0.57 mg GAE/g dw and 10.14 ± 0.76 mg RE/g dw, respectively). The correlation coefficients from regression analysis showed a positive relationship between total phenolic content in the extracts and DPPH activity (R² = 0.7336), as well as between total flavonoid content and DPPH activity (R² = 0.9392). Furthermore, GC/MS method was used to confirm the presence of phenolics with antioxidant activity in the methanol extract and resulted in the identification of 41 compounds, esters, phenolics, alkanes, carboxylates and alcohols being the main components. In conclusion, cultivated fruiting bodies of Xylaria sp.YX-28 may have potential as natural antioxidant.