Fabrication and characterization of kidney bean (Phaseolus vulgaris L.) protein isolate–chitosan composite films at acidic pH

The kidney bean protein isolate-chitosan (KPI/CH) composite films were fabricated at acidic pH to design potential carriers for antimicrobials (e.g., nisin). Tensile and thermo-mechanical properties, surface hydrophobicity and surface free energy (γ) as well as microstructure of the composite films were evaluated. A peelable and self-supported film was obtained for the KPI alone, exhibiting the typical stiff behavior. In contrast, the complex between KPI and CH produced less rigid and much more flexible films, with decreased elastic modulus (EM), storage modulus (E′) and glass transition temperature (T_g). The inclusion of CH enhanced surface hydrophobicity of the films. Concomitantly, the surface free energy (γ) of the films exhibited a reduced tendency. Upon increasing CH-to-KPI ratios, the microstructures of the films changed gradually from protein continuous to bi-continuous, CH continuous pattern. Moreover, the KPI/CH blend films exhibited a visible compositional gradient, reflecting that the CH was rich in near-surface of the films, especially air side. Antimicrobial activity of the films was also analyzed, indicating the test bacteria, Bacillus subtilis, Escherichia coli and Salmonella were sensitive to KPI/CH composite films, in a CH-to-KPI dependent manner. The results presented here supported that the KPI/CH blend films are expected to serve as antimicrobial packaging for food or the carrier of antimicrobials (e.g., nisin).     

Toona sinensis and its major bioactive compound gallic acid inhibit LPS-induced inflammation in nuclear factor-κB transgenic mice as evaluated by in vivo bioluminescence imaging

In the present study, we investigated the anti-inflammatory effects of a nutritious vegetable Toona sinensis (leaf extracts, TS) and its major bioactive compound gallic acid (GA) by analysing LPS-induced NF-κB activation in transgenic mice, using bioluminescence imaging. Mice were challenged intraperitoneally with LPS (1 mg/kg) and treated orally with TS or GA (100 or 5 mg/kg, respectively). In vivo and ex vivo imaging showed that LPS increased NF-κB luminescence in the abdominal region, which was significantly inhibited by TS or GA. Immunohistochemical and ELISA analyses confirmed that TS and GA inhibited LPS-induced NF-κB, interleukin-1β, and tumour necrosis factor-α expression. Microarray analysis revealed that biological pathways associated with metabolism and the immune responses were affected by TS or GA. Particularly, LPS-induced thioredoxin-like 4B (TXNL4B) 2 expression in the small intestine, and TXNL4B, iNOS, and COX-2 expression in RAW 264.7 cells were significantly inhibited by TS or GA. Thus, the anti-inflammatory potential of TS was mediated by the downregulation of NF-κB pathway.     

Attenuation of iNOS and COX2 by blueberry polyphenols is mediated through the suppression of NF-κB activation

Treatment of BV2 microglial cells with blueberry extracts has been shown to be effective in reducing lipopolysaccharide (LPS)-induced proinflammatory mediators such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), inducible NO synthase (iNOS), and cyclooxygenase 2 (COX2). The current study explored the possibility that the down-regulation of iNOS and COX2 by blueberry extracts was mediated through NF-κB signaling pathway. A column-purified fraction of polyphenol-enriched blueberry extract (PC18) was used to treat LPS-activated BV2 cells. The results thus far showed that blueberry polyphenols significantly suppressed iNOS and COX2 promoter activities. In addition, blueberry polyphenols inhibited NF-κB nuclear translocation in LPS-activated BV2 cells. These findings suggested that the beneficial effects of blueberries may involve direct modulation of oxidative stress and/or inflammatory signaling cascades.     

A Porphyra columbina hydrolysate upregulates IL-10 production in rat macrophages and lymphocytes through an NF-κB,and p38 and JNK dependent mechanism

The marine environment represents a relatively untapped source of functional ingredients. Here we characterise a hydrolysate obtained from Phorphyra columbina (PcRH) and its effects on primary splenocytes, macrophages and T lymphocytes in vitro. Our product had a high degree of hydrolysis, due to the use of a mixture of endo-peptidase and exo-peptidase, and was enriched in Asp, Ala and Glu. PcRH had mitogenic effects on rat splenic lymphocytes. IL-10 secretion was enhanced by PcRH in splenocytes (235%), macrophages (150%) and in lymphocytes (472%), while the production of TNFα and other proinflammatory cytokines by macrophages was inhibited (15–75%), especially under lipopolysaccharide stimulation. The effect of the hydrolysate on IL-10 was evoked by JNK, p38 MAPK and NF-κB dependent pathways in T lymphocytes. We conclude that PcRH has immunomodulatory effects on macrophages and lymphocytes, activating NF-κB and MAPK dependent pathways, and predominantly inducing IL-10 production.     

HPLC–MS identification of phenols in hazelnut (Corylus avellana L.) kernels

In whole hazelnut kernels, as the main product of hazelnut (Corylus avellana L.), phenols were analysed in 20 hazelnut cultivars by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS). Twenty-three compounds from different phenolic groups were detected, and 15 of them were identified. In hazelnut kernels, these substances were detected: nine flavan-3-ols, two benzoic acids (gallic and protocatechuic acid), three flavonols and phloretin glycoside. Total phenol concentrations ranged from 70 to 478 mg gallic acid equivalents per kg hazelnut kernels. A high content level of total phenols was observed in the ‘Tonda Gentile delle Langhe’ and ‘Lewis’ cultivars, which was followed by the ‘Corabel’, ‘Fertile de Coutard’, ‘Daria’ and ‘Tonda Gentile Romana’ cultivars. Similarly, the highest antioxidative activity, measured by employing DPPH-antiradical assay, was also found in the ‘Tonda Gentile delle Langhe’ cultivar, followed by the ‘Fertile de Coutard’.     

Determination of minor carbohydrates in carrot (Daucus carota L.) by GC–MS

Minor carbohydrates present in carrot (Daucus carota L.) have been studied by GC–MS analysis of their trimethylsilyl derivatives because of their remarkable role in a variety of biological functions. Scyllo-inositol and sedoheptulose (d-altro-2-heptulose), identified for the first time in this paper were present in all the carrots analysed in concentrations ranging 1.5–5.8 and 1.4–24.6 mg g⁻¹ dried weight, respectively. Other minor carbohydrates detected in carrot were myo- inositol (2.2–9.8 mg g⁻¹) and mannitol (traces-1.3 mg g⁻¹). Whereas small amounts (close to 2 mg g⁻¹) of scyllo-inositol were experimentally determined in other vegetables from the Apiaceae family (parsley, coriander and fennel), sedoheptulose was only detected at trace levels.     

Structure–physicochemical function relationships of 7S globulins (vicilins) from red bean (Phaseolus angularis) with different polypeptide constituents

Red bean 7S globulins (vicilins) with different polypeptide constituents or heterogeneity were fractionated using acidic extraction (at 0.5 M NaCl) and anion-exchange column chromatography. The physicochemical and conformational properties, including amino acid composition, net charge and/or surface hydrophobicity (H₀), protein solubility (PS), thermal and emulsifying properties, as well as secondary, tertiary and/or quaternary conformations, were evaluated. There were distinct differences in zeta potential, H₀, DSC characteristics, emulsifying activities and tertiary and/or quaternary conformations among the vicilins with different polypeptide constituents, but the PS and secondary conformation were slightly different. The PS as a function of pH and thermal stability were closely related to their surface charge and/or hydrophobicity. The emulsifying ability and the emulsion stability were closely dependent on the PS and H₀, and even the flexibility in tertiary and/or quaternary conformations. These results suggested good relationships between the physicochemical functions and conformational features of red bean vicilins, which could be of great help for further utilization of legume proteins as a potential functional ingredient.     

GABA shunt and polyamine degradation pathway on γ-aminobutyric acid accumulation in germinating fava bean (Vicia faba L.) under hypoxia

GABA shunt and polyamine degradation pathway on γ-aminobutyric acid (GABA) accumulation in germinating fava bean under hypoxia was investigated. GABA content, GAD and DAO activity were significantly increased under hypoxia treatment. Glu and polyamine contents enhanced largely and thus supplied as sufficient substrates for GABA formation. In contrast, GABA content decreased, mainly in the embryo, after removing the hypoxia stress. DAO activity, Glu and polyamines contents decreased, while an increment of GAD activity was observed. This indicated that GAD activity can be not only regulated by hypoxia, but by the rapid growth of embryo after the recovery from hypoxia stress. When treated with AG, DAO activity was almost inhibited completely, and the GABA content decreased by 32.96% and 32.07% after treated for 3 and 5 days, respectively. Hence, it can be inferred that about 30% of GABA formed in germinating fava bean under hypoxia was supplied by polyamine degradation pathway.     

Anti-inflammatory effect of flavonoids isolated from Korea Citrus aurantium L. on lipopolysaccharide-induced mouse macrophage RAW 264.7 cells by blocking of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signalling pathways

Citrus fruits are an abundant source of various flavonoids, which have been used as a traditional herbal medicine in Korea and China. Most flavonoids are known to have anti-oxidant, anti-bacterial and analgesic properties. In this study, it was examined whether flavonoids (nobiletin, naringin and hesperidin) isolated from Korea Citrus aurantium L. inhibited the pro-inflammatory mediators including cytokines by blocking nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signalling in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The flavonoids suppressed mRNA and protein expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in LPS-induced macrophages. The molecular mechanism was associated with inhibition of the degradation/phosphorylation of I-κB-α and nuclear translocation of the NF-κB p-65 as well as phosphorylation of MAPK by flavonoids. These results suggest that flavonoids have anti-inflammatory effects by suppressing expression of COX-2, iNOS and cytokines by blocking the NF-κB and MAPK signalling in RAW 264.7 macrophages.     

Effect of γ-irradiation on antioxidant activity of black pepper (Piper nigrum L.)

Antioxidant activity and EPR investigations of irradiated ground black pepper (Piper nigrum L.) were evaluated. The black pepper was exposed to γ-irradiation at doses from 5 to 30 kGy. The effect of irradiation on antioxidant properties of black pepper extracts was investigated by radical-scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, by determination of reducing power and content of thiobarbituric acid-reactive substances. Some significant changes were observed in creation of thiobarbituric acid-reactive substances (TBARS). Difference between non-irradiated and irradiated samples at the legal European limit dose of 10 kGy reached, on average, 23% and, at the Food and Drug Administration (FDA) 30 kGy limit, 33%. Irradiation affected significantly the DPPH radical-scavenging activity and reducing power of ground black pepper extracts. The γ-radiation treatment of ground black pepper samples observed by EPR, resulted in the production of three paramagnetic species (GI–GIII) characterized by different origin, thermal behaviour and stability. The axially symmetric EPR resonances, GI and GII, were assigned to the carbohydrate radical structures. The spin Hamiltonian parameters of GIII possessed the characteristic features of “cellulosic” radical species. The EPR measurements, performed 20 weeks after the radiation process, confirmed that temperature increase from 298 to 353 K, caused significant decrease of integral EPR signal intensity for γ-irradiated samples (∼40%), compared to the reference (non-irradiated) ground black pepper, where only 13% drop was found. Significant correlation between EPR and thiobarbituric acid methods was assessed by study of antioxidant activity changes in relation to irradiation doses and also in the case of spice storage, between EPR and reducing power methods.     

Identification of calpastatin, μ-calpain and m-calpain in Atlantic salmon (Salmo salar L.) muscle

A soft fish muscle is generally considered as a poor quality trait among consumers and producers. This degradation and softening of post mortem muscle is thought to be partly caused by proteolytic enzymes such as the calpain system. Separation and identification of μ-calpain and m-calpain and their inhibitor – calpastatin, from Atlantic salmon (Salmo salar) muscle were for the first time assessed in this study. A two-step chromatography approach was used, starting with a hydrophobic interaction column and followed by an anion exchange column. Calpastatin was successfully separated from calpain by hydrophobic interaction chromatography, and following the anion exchange chromatography, two forms of calpastatin (I and II) and two forms of calpain (micro (μ) and milli (m)) were revealed. The proteolytic activity of μ-calpain was detectable with column chromatography, but not consistently detected with casein zymography, and m-calpain was detected with both chromatography and casein zymogram. The proteolytic activity of m-calpain per g muscle was 15 times higher than that of μ-calpain. μ-Calpain had a temperature optimum of 15 °C and a maximum calcium requirement at 0.2 mM, while m-calpain had temperature optimum at 25 °C and a maximum calcium requirement of 0.6 mM. The two forms of calpastatin differed in inhibitory activity with calpastatin II having the highest activity. Both calpastatins tolerated heat treatment, as previously seen for mammals, and they kept their activity when stored at −80 °C, but not at −20 °C. The calpain to calpastatin ratio was 1:4.5 as observed for beef muscle. This study provides evidence that two calpain isoforms, likely to be μ- and m-calpain, in addition to two forms of calpastatin exist in Atlantic salmon muscle.     

Occurrence of β-casein fragments in cold-stored and curdled river buffalo (Bubalus bubalis L.) milk

The safeguard of river buffalo Mozzarella cheese, a Protected Designation of Origin dairy product, has prompted an analytical study to trace the milk and curd used as raw material in cheesemaking. This is to prevent the illegal use of milk or curd from different geographical areas outside of those indicated in the official production protocol. For this purpose, we studied primary proteolysis occurring in fresh and frozen milk and curd to identify a molecular marker that could indicate the raw material used. Whole casein from frozen river buffalo milk was separated using cation-exchange chromatography and sodium dodecyl sulfate-PAGE, and a protein component with an estimated molecular weight of 15.3 kDa was detected. This protein component was revealed in fresh river buffalo milk as a faint electrophoresis band, which drastically increased in intensity in refrigerated and frozen milk as well as in curd and was found to be associated with β-CN through hydrophobic interaction. By using matrix-assisted laser desorption/ionization-time of flight peptide mass mapping, this component was identified as the C-terminal fragment f(69-209) of β-CN (expected molecular weight of 15,748.8 Da). β-Casein f(69-209), originating from the early hydrolysis of Lys₆₈-Ser₆₉ by plasmin, has no counterpart in bovine milk. The increased rate of hydrolysis by plasmin toward the cleavage site Lys₆₈-Ser₆₉ has to be ascribed to the elevated proline content of the peptide 61-73. The favored production of β-CN f(69-209) has also drawn attention to the complementary proteose peptone β-CN f(1-68) that is presumed to play a physiological role in inducing milk secretion similar to that of β-CN f(1-29). The higher in vivo and in vitro production rate, compared with γ₁-CN formation, indicates that β-CN f(69-209) and its complementary fragment are candidate molecular markers to evaluate milk and curd freshness. We used indirect ELISA analysis based on the determination of remaining nonhydrolyzed β-CN to perform a quantitative evaluation of proteolysis.     

Use of sodium nitrite in salt-curing of Atlantic salmon (Salmo salar L.) – Impact on product quality

Quality changes during processing and quality differences in smoked fillets of Atlantic salmon (4–5 kg) salted with nitrite salt compared to table salted fillets were measured. Quality parameters from right-side fillets dry salted with nitrite salt were compared with the respective left-side fillets treated the same way with table salt. Ten raw right-side fillets were analysed and used as raw material reference. Use of nitrite salt in salt-curing of smoked salmon affected colour to a more reddish hue, tended to increase carotenoid stability and displayed positive effects on NaCl diffusivity. Only slight weight changes and change in texture properties were revealed. The use of nitrite salt displayed no adverse effects like increased content of N-nitrosoamines in smoked products. In fact, significant lower contents of N-nitrosoamines were found in nitrite salted smoked fillets compared to smoked fillets salted with table salt. Relatively high amounts of residual nitrite in nitrite-treated fillets seem to be the most prominent adverse effect caused by the use of nitrite salt in salt-curing of smoked Atlantic salmon.     

Determination of flavonoid level variation in onion (Allium cepa L.) infected by Fusarium oxysporum using liquid chromatography–tandem mass spectrometry

In order to evaluate the flavonoid level variation in an onion (Allium cepa L.) infected by Fusarium oxysporum, the bulbs of a healthy onion and of an infected one were analysed for flavonoids via high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS). Among eleven flavonoids characterised, isorhamnetin 4′-O-galactoside (8) was identified in an onion for the first time. When the healthy bulb was inoculated with the fungus, the two quercetin derivatives (4 and 7) and the two isorhamnetin derivatives (5 and 9) underwent concentration changes typical for the defense materials against pathogens. The yellow granules that were accumulated on the abaxial epidermal cell layers after 8 days of inoculation were confirmed as quercetin (10) and isorhamnetin (11). It was deduced that they were produced from flavonoids 4, 5, 7 and 9 by hydrolysis enzyme of the fungus.     

Methylglyoxal-derived advanced glycation end products induce matrix metalloproteinases through activation of ERK/JNK/NF-κB pathway in kidney proximal epithelial cells

Food Science and Biotechnology - The accumulation of reactive α-dicarbonyl leading to advanced glycation end products (AGEs) have been linked to pathophysiological diseases in many studies,...     

Dicaffeoylquinic acids in Yerba mate (Ilex paraguariensis St. Hilaire) inhibit NF-κB nucleus translocation in macrophages and induce apoptosis by activating caspases-8 and -3 in human colon cancer cells

Scope : The biological functions of caffeoylquinic acid (CQA) derivatives from various plant sources have been partially elucidated. The objectives were to isolate and purify diCQAs from Yerba mate tea leaves and assess their anti‐inflammatory and anti‐cancer capabilities in vitro and explore their mechanism of action. Methods and results : Methanol extracts of dried mate leaves were resolved by flash chromatography and further purified resulting in two fractions one containing 3,4‐ and 3,5‐diCQAs and the other 4,5‐diCQA with NMR‐confirmed structures. Both fractions inhibited LPS‐induced RAW 264.7 macrophage inflammation by suppressing nitric oxide/inducible nitric oxide and prostaglandin E₂/cyclooxygenase‐2 pathways through inhibiting nucleus translocation of Nuclear factor κB subunits, p50 and p65. The diCQA fractions inhibited Human colon cancer cells CRL‐2577 (RKO) and HT‐29 cell proliferation by inducing apoptosis in a time‐ and concentration‐dependent manner, but did not affect the protein levels of p21, p27, p53, and Bax:Bcl‐2 ratio in RKO cells. In HT‐29 cells, however, the diCQA fractions increased Bax:Bcl‐2 ratio. The diCQA fractions increased the activation of caspase‐8 leading to cleavage of caspase‐3 in both RKO and HT‐29 colon cancer cells. Conclusion : The results suggest that diCQAs in Yerba mate could be potential anti‐cancer agents and could mitigate other diseases also associated with inflammation.     

Effect of γ-irradiation on the physicochemical and sensory properties of cashew nuts (Anacardium occidentale L.)

The present study evaluated cashew nuts' quality as a function of γ-irradiation dose in order to determine dose levels causing minimal undesirable changes to the product. Physicochemical (colour, peroxide value, hexanal content, fatty acid composition, volatile compounds) and sensory (colour, texture, odor, taste) properties of cashew nuts were determined after irradiation at doses up to 7 kGy. Results showed a five-fold increase in peroxide value (PV) and a two-fold increase in hexanal content after irradiation at a dose of 7 kGy. Of the fatty acids determined, stearic acid concentration increased while oleic acid decreased with irradiation dose. PUFA were unaffected by irradiation. Volatile compounds such as aldehydes, ketones and alcohols increased after irradiation indicating enhanced lipid oxidation. Colour parameter a∗ increased (p < 0.05) at doses >3 kGy while colour parameters L∗ and b∗ remained unaffected by irradiation. Sensory analysis showed that cashew nuts remained organoleptically acceptable at doses <3 kGy.     

Anti-inflammatory effect of the hexane fraction from Orostachys japonicus in RAW 264.7 cells by suppression of NF-κB and PI3K-Akt signaling

We investigated the anti-inflammatory effect of the hexane fraction from Orostachys japonicus (OJH) in LPS (lipopolysaccharide)-stimulated RAW 264.7 cells. Pretreatment with OJH dose-dependently reduced the cellular NO (nitric oxide) concentration and also inhibited expression of iNOS (inducible nitric oxide synthase) protein and mRNA. By the prevention of IκBα (inhibitory factor kappa B alpha) phosphorylation and degradation, OJH inhibited LPS-induced NF-κB (nuclear factor-kappa B) activation. OJH had no effect on the LPS-induced phosphorylation of ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), or p38, whereas it attenuated the phosphorylation of Akt in a dose-dependent manner. In addition, OJH suppressed the LPS-induced expression of LPS receptors CD14 and TLR4 (toll like receptor 4). These results suggest that OJH may interrupt LPS-induced pro-inflammatory cascades through inhibition of NF-κB and Akt activation.