Enzyme-catalyzed change of antioxidants content and antioxidant activity of asparagus juice

A pectolytic enzyme preparation from Aspergillus niger (pectinase AN) decreased most rutin content and antioxidant activity of asparagus juice. To investigate the mechanism of such loss, we analyzed several possible related enzyme activities in pectinase AN. We found that the activity of pectinase AN to oxidize guaiacol had no significant difference with or without the presence of H2O2; thus it was laccase activity, not peroxidase (PO) activity, that pectinase AN contained. We did not find any polyphenol oxidase (PPO) activity in pectinase AN. Laccase in pectinase AN could be the major cause of loss of rutin and antioxidant activity of asparagus juice. When most laccase activity of pectinase AN was inactivated after heating at 70 degrees C for 1.5 min and incubated with asparagus juice, the loss rate of rutin was only 9% of that treated with unheated pectinase AN, and the antioxidant activity was even increased. Rhamnosidase activity was detected in pectinase AN and can change rutin in asparagus juice to quercetin-3-glucoside, which has higher antioxidant activity than rutin. This may explain the increase of antioxidant activity of asparagus juice treated with heated pectinase AN that still contained some rhamnosidase activity. The discovery of our research is helpful to produce juice with high antioxidant activity and high health benefits in the juice industry.     

Steam-blanched highbush blueberry (Vaccinium corymbosum L.) juice: phenolic profile and antioxidant capacity in relation to cultivar selection

High-quality standards in blueberry juice can be obtained only taking into account fruit compositional variability and its preservation along the processing chain. In this work, five highbush blueberry cultivars from the same environmental growing conditions were individually processed into juice after an initial blanching step and the influence was studied of the cultivar on juice phenolic content, distribution and relative antioxidant activity, measured as scavenging capacity on the artificial free-radical 2,2-diphenyl-1-picrylhydrazyl (DPPH*). A chromatographic protocol was developed to separate all main phenolic compounds in berries. A total of 15 glycosylated anthocyanins, catechin, galactoside, glucoside, and rhamnoside quercetin 3-derivatives, and main benzoic and cinnamic acids were identified. The total content and relative distribution in anthocyanins, chlorogenic acid, and quercetin of each juice were dependent upon cultivar, and the total content was highly correlated (rxy=0.97) to the antioxidant capacity. A selective protective effect of berry blanching in juice processing can be observed on more labile anthocyanin compounds.     

Activity and concentration of polyphenolic antioxidants in apple juice. 3. Stability during storage

Kinetic data are reported describing the stability of various classes of polyphenolic antioxidants in an apple juice enriched in these compounds as a function of storage temperature and oxygen concentration. The most thermally sensitive compounds were the various quercetin glycosides and epicatechin, whereas phloridzin and chlorogenic acid were more stable. The quercetin glycosides showed differences in their stability: quercetin galactoside approximately quercetin rhamnoside > quercetin glucoside/rutinoside > quercetin arabinoside. The effect of the presence of oxygen on the degradation rates was clear for only quercetin and to a lesser extent for epicatechin. Accelerated shelf-life testing of enriched apple juice during 4 days at 80 degrees C showed decreases in the antioxidant activity of 20-40%. The parameters obtained were used to predict the stability at different storage conditions. Calculations showed that polyphenolic antioxidants and antioxidant activity of enriched apple juice will be quite stable at ambient or refrigerated storage conditions up to half a year.     

Activity and concentration of polyphenolic antioxidants in apple juice. 2. Effect of novel production methods

There is a great interest in food components that possess possible health-protecting properties, as is the case with flavonoids. Previous research showed that conventional apple juice processing resulted in juices poor in flavonoids and with a low antioxidant activity. This paper shows that it is possible to improve flavonoid content in juice and its antioxidant activity by applying an alcoholic extraction either on the pulp or on the pomace. The levels of flavonoids and chlorogenic acid in enriched juice were between 1.4 (chlorogenic acid) and 9 (quercetin glycosides) times higher than in conventional apple juice. In enriched juice the antioxidant activity was 5 times higher than in conventional apple juice, with 52% of the antioxidant activity of the originating fruits present. The novel processing method had similar effects for three apple cultivars tested (Elstar, Golden Delicious, and Jonagold). The taste and color of enriched juice were different from those of conventional juice.     

Domestic processing of onion bulbs (Allium cepa) and asparagus spears (Asparagus officinalis): effect on flavonol content and antioxidant status.

Two commonly consumed plant foods, onion bulbs and asparagus spears, were subjected to typical domestic processing, including chopping, maceration, and boiling. The impact of these processes on flavonol content was assessed. Further, the consequences of these processes on the antioxidant capacity of the tissues were evaluated with the beta-carotene bleaching method. Chopping significantly affected rutin content in asparagus, yielding an 18.5% decrease in 60 min; but in onions, quercetin 3,4'-diglucoside (Q(DG)) and quercetin 4'-glucoside (Q(MG)) were virtually unaffected by chopping. Boiling for 60 min had more severe effects, as it caused overall flavonol losses of 20.6 and 43.9% in onions and asparagus, respectively. Chopping of tissues did not considerably influence the antioxidant capacity, but boiling did provoke notable changes.     

Structure-antioxidant efficiency relationships of phenolic compounds and their contribution to the antioxidant activity of sea buckthorn juice

The phenolic composition of juice derived from fruits of sea buckthorn (Hippophae rhamnoides) was investigated by high-performance liquid chromatography (HPLC) with diode array and electrochemical detection. Flavonols were found to be the predominating polyphenols while phenolic acids and catechins represent minor components. Of the seven flavonols identified, isorhamnetin 3-O-glycosides were the most important representatives quantitatively. However, because of their structural properties, they were poor radical scavengers as shown by electron spin resonance spectroscopy. Phenolic compounds such as quercetin 3-O-glycosides, catechins, and hydroxybenzoic acids with a catechol structure exhibited good antioxidant capacities, but their concentration in sea buckthorn juice was small. These phenolic compounds, determined by HPLC, accounted for less than 5% of the total antioxidant activity of the filtered juice. Ascorbic acid was shown to be the major antioxidant in sea buckthorn juice. Because of its high concentration of 1.22 g/L, it contributes approximately 75% to total antioxidant activity. The remaining difference can be attributed to higher molecular weight flavan-3-ols (proanthocyanidins), which were determined photometrically after acid depolymerization to colored anthocyanidins.     

Physico-chemical characteristics of nanovesicle-carbohydrate complexes in grape juice concentrate

It is generally assumed that polyphenols, such as anthocyanins in fruit juice, exist in a free soluble state and are readily available for absorption in the gastro-intestinal tract. In the present study, we have investigated the interaction of polyphenols with soluble carbohydrate polymers, such as pectin and lipid nanovesicles, that are generated during homogenization of the fruit tissue during juice extraction. A commercially available grape juice concentrate contained nearly 25% of polyphenol fraction bound to macromolecules that were nondialyzable. Treatment of dialyzed juice with cellulase, pectinase, and beta-galactosidase did not cause the release of bound polyphenols; however, treatment with triton X-100 caused an increased release of bound polyphenols. The dialyzate contained relatively more -3-O glucosides and -3-O-acetoyl glucosides in comparison to the bound fraction which was enriched in -3-O-coumaroyl glucosides, suggesting qualitative differences in the bound and the free anthocyanin composition. Electron microscopic analysis of the juice fractions revealed the presence of electron-dense nanovesicle-fiber complexes ranging from 10 to 200 nm in diameter. Such complexes were absent in the dialyzate fraction. Cellulase treatment did not change the morphology of the complexes; however, treatment with pectinase and beta-galactosidase disrupted the complexes, releasing vesicular structures, suggestive of the pectin nature of the fibrous matrix. The dialyzed and the dialyzate fractions also showed differences in their 1H NMR and fluorescence spectral characteristics. The dialyzed fraction containing polyphenol-pectin complexes showed no superoxide scavenging capacity, reduced hydroxyl radical scavenging activity, and high 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, indicating potential changes in functionality because of the complex formation.     

超滤对血橙果汁中抗氧化成分及总抗氧化能力的影响

采用管式聚偏氟乙烯(PVDF)超滤膜，探讨了超滤对血橙果汁中抗氧化成分及总抗氧化能力的影响。结果表明，其对维生素C和花青素类成分的保存率约为85%左右，对几种酚酸和类黄酮组分（除槲皮素外）的保存率均在90%以上。同未杀菌的血橙原汁相比，巴氏杀菌汁的总抗氧化能力(TAA)损失率为23%，而超滤澄清汁仅为10%左右，主要与维生素C和花青素含量损失有关。     

Effect of mash maceration on the polyphenolic content and visual quality attributes of cloudy apple juice

The effects of enzymatic mash treatments on yield, turbidity, color, and polyphenolic content of cloudy apple juice were studied. Using HPLC-ESI-MS, cryptochlorogenic acid was identified in cv. Brettacher cloudy apple juice for the first time. Commercial pectolytic enzyme preparations with different levels of secondary protease activity were tested under both oxidative and nonoxidative conditions. Without the addition of ascorbic acid, oxidation substantially decreased chlorogenic acid, epicatechin, and procyanidin B2 contents due to enzymatic browning. The content of chlorogenic acid as the major polyphenolic compound was also influenced by the composition of pectolytic enzyme preparations because the presence of secondary protease activity resulted in a rise of chlorogenic acid. The latter effect was probably due to the inhibited protein-polyphenol interactions, which prevented binding of polyphenolic compounds to the matrix, thus increasing their antioxidative potential. The results obtained clearly demonstrate the advantage of the nonoxidative mash maceration for the production of cloud-stable apple juice with a high polyphenolic content, particularly in a premature processing campaign.     

Comparison of antioxidant potency of commonly consumed polyphenol-rich beverages in the United States

A number of different beverage products claim to have antioxidant potency due to their perceived high content of polyphenols. Basic and applied research indicates that pomegranate juice (PJ), produced from the Wonderful variety of Punica granatum fruits, has strong antioxidant activity and related health benefits. Although consumers are familiar with the concept of free radicals and antioxidants, they are often misled by claims of superior antioxidant activity of different beverages, which are usually based only on testing of a limited spectrum of antioxidant activities. There is no available direct comparison of PJ's antioxidant activity to those of other widely available polyphenol-rich beverage products using a comprehensive variety of antioxidant tests. The present study applied (1) four tests of antioxidant potency [Trolox equivalent antioxidant capacity (TEAC), total oxygen radical absorbance capacity (ORAC), free radical scavenging capacity by 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP)]; (2) a test of antioxidant functionality, that is, inhibition of low-density lipoprotein (LDL) oxidation by peroxides and malondialdehyde methods; and (3) evaluation of the total polyphenol content [by gallic acid equivalents (GAEs)] of polyphenol-rich beverages in the marketplace. The beverages included several different brands as follows: apple juice (3), a?aí juice (3), black cherry juice (3), blueberry juice (3), cranberry juice (3), Concord grape juice (3), orange juice (3), red wines (3), iced tea beverages (10) [black tea (3), green tea (4), white tea (3)], and a major PJ available in the U.S. market. An overall antioxidant potency composite index was calculated by assigning each test equal weight. PJ had the greatest antioxidant potency composite index among the beverages tested and was at least 20% greater than any of the other beverages tested. Antioxidant potency, ability to inhibit LDL oxidation, and total polyphenol content were consistent in classifying the antioxidant capacity of the polyphenol-rich beverages in the following order: PJ>red wine>Concord grape juice>blueberry juice>black cherry juice, a?aí juice, cranberry juice>orange juice, iced tea beverages, apple juice. Although in vitro antioxidant potency does not prove in vivo biological activity, there is also consistent clinical evidence of antioxidant potency for the most potent beverages including both PJ and red wine.     

Phytochemical stability and color retention of copigmented and processed muscadine grape juice

Muscadine (Vitis rotundifolia) grape juice was assessed for color and phytochemical stability as influenced by anthocyanin copigmentation with a water-soluble rosemary extract, fortification with ascorbic acid, and processing by heat or high hydrostatic pressure (HHP). The roles of polyphenolic cofactors in the presence and in the absence of ascorbic acid were assessed as a means to improve the overall processing stability of the juice. Addition of rosemary extract from 0 to 0.4% (v/v) readily formed copigment complexes with anthocyanins and resulted in concentration-dependent hyperchromic shifts from 10 to 27% that corresponded to increased antioxidant activity. The presence of ascorbic acid was generally detrimental to juice quality, especially in the presence of rosemary extract, and resulted in overall anthocyanin, ascorbic acid, and antioxidant activity losses. Although thermal and high-pressure processing methods were detrimental to juice quality, HHP resulted in greater losses after processing, likely due to action from residual oxidase enzymes. Although physicochemical attributes were enhanced by copigmentation with rosemary extract, methods to inactivate residual enzymes should be addressed prior to copigmentation to prevent degradation of anthocyanins in the presence of ascorbic acid.     

Effect of buckwheat extract on the antioxidant activity of lipid in mouse brain and its structural change during in vitro human digestion

This study was conducted to investigate the effects of buckwheat ( Fagopyrum esculentum Moench cv. Yangjul No. 2) extract on the antioxidant activity of lipids in mouse brain and the structural change during in vitro human digestion. Buckwheat was collected from a wild farm and extracted with water. The buckwheat extracts were then passed through an in vitro human digestion model that simulated the composition of the mouth, stomach, and small intestine juice. The results confirmed that the main phenolics of buckwheat extract were rutin, quercitrin, and quercetin. The rutin content increased with digestion of the buckwheat (from 48.82 to 96.34 μg/g) and rutin standard samples (from 92.76 to 556.56 μg/g). Antioxidant activity was more strongly influenced by in vitro human digestion of both buckwheat and rutin standard. After digestion by the small intestine, the antioxidant activity values were dramatically increased (from 5.06 to 87.82%), whereas the antioxidant activity was not influenced by digestion in the stomach for both buckwheat extract and rutin standard. Inhibition of lipid oxidation of buckwheat in mouse brain lipids increased after digestion in the stomach for both buckwheat extract and the rutin standard. The major finding of this study was that in vitro human digestion may be an important modulator of the antioxidant capacity of buckwheat and that this may be because in vitro human digestion increased the antioxidative activity via an increase in antioxidants such as rutin and quercetin.     

Effects of extraction methods on phenolic contents and antioxidant activity in aerial parts of Potentilla atrosanguinea Lodd. and quantification of its phenolic constituents by RP-HPLC

The effects of different solvent systems (methanol, ethanol, acetone, and their 50% aqueous concentrations) and extraction procedures (microwave, ultrasound, Soxhlet and maceration) on the antioxidant activity of aerial parts of Potentilla atrosanguinea were investigated by three different bioassays: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays and ferric reducing antioxidant potential (FRAP). The 50% aqueous ethanol extracts exhibited strong antioxidant activity measured in terms of Trolox equivalent antioxidant capacity (TEAC) [(54.34 to 122.96, 29.82 to 101.22 and 13.64 to 41.43) mg of Trolox/g] with ABTS (*+), DPPH (*) and FRAP assays, respectively. In general, TEAC of Soxhlet extracts was found to be 1.8 and 3 times higher than ultrasound and maceration but slightly (1.2 times) higher than microwave. A positive correlation (r(2) = 0.931 to 0.982) was observed between total polyphenol (TPC) and total flavonoid (TFC) contents which ranged between 26.7 to 30.7 mg/g gallic acid equivalent and 16.8 to 20.8 mg/g quercetin equivalent respectively, with antioxidant activity. In addition, some of its bioactive phenolic constituents which contribute largely toward antioxidant potential such as chlorogenic acid, catechin, caffeic acid, p-coumaric acid and quercetin were also quantified in different extracts by RP-HPLC.     

Phenolic extraction from apple peel by cellulases from Thermobifida fusca

With the optimization of the pretreatment conditions for the crude Thermobifida fusca cellulase activity and phenolic release from apple peel, we focused on the activity of individual purified cellulase related to the antioxidant activity. The overall phenolic release was significantly increased in a synergistic manner with combined pretreatment, not with individual pretreatment such as boiling, acid, and pectinase treatment. Approximately 60 mg of reducing sugar equivalent were produced per g of apple peel by treatment with T. fusca crude extract, and up to 3 times more reducing sugars were released when the apple peel was boiled and then treated with acid and pectinase. There was good correlation between the release of phenolics and reducing sugar by cellulase treatment and also between the amount of total phenolics and antioxidant capacity by each enzyme treatment (r2> 0.95). Among the tested enzymes purified from T. fusca cell extract, cellulase activity on apple peel was the highest with cellulase 6A (Cel 6A; 43% digestion), and the highest antioxidant capacity was obtained by incubation with Cel 6B (16 mg vitamin C equiv/g). Synergism in the activity was found from the combined treatment with Cel 6A and 6B in both cellulase activity and antioxidant capacity after 20 h of incubation. Cel 9A (progressive endocellulase) exhibited greater cellulase activity and antioxidant capacity than Cel 9A cd which lacks in cellulose-binding module, indicating that the cellulose-binding domain might play important roles in cellulolysis of apple peel. This study could provide some insights into the action mechanism of various cellulases on the digestion of cellulose-containing byproducts and expand the opportunity for cellulase utilization in the extraction of functional ingredients from the plant-derived byproducts.     

Enzyme-assisted processing increases antimicrobial and antioxidant activity of bilberry

The effects of nine cell wall-degrading enzymes on the antimicrobial and antioxidant activities of bilberry were studied. Antimicrobial activity was measured using the human pathogens Salmonella enterica sv. Typhimurium and Staphylococcus aureus as test strains. Enzyme treatments liberated phenolics from the cell wall matrix, which clearly increased the antimicrobial activity of berry juices, press cakes, and berry mashes on the basis of plate counts. Antibacterial effects were stronger against Salmonella than against Staphylococcus bacteria. In general, the increase in activity measured as colony-forming units per milliliter was 3-5 logarithmic units against Salmonella and 1-2 units against Staphylococcus bacteria. Increase in antimicrobial activity was observed only in acidic conditions, which is also the natural environment in various berry products, such as juices. The activity profile of the pectinase preparation affected the chemistry of the phenolics due to the presence of deglycosylating activities in some preparations. The difference in phenolic profiles was reflected in the antimicrobial effects. Bilberry mashes treated with Pectinex Ultra SP-L, Pectinex 3 XL, and Pectinex BE XXL were most efficient against Salmonella bacteria, whereas mashes treated with Pectinex Smash, Pectinex BE 3-L, and Biopectinase CCM showed the strongest antimicrobial activity against Staphylococcus bacteria. Due to the liberation of phenolics from the cell wall matrix the antioxidant activity measured as radical scavenging activity was also increased on average about 30% by the enzymatic treatments. The highest increase in phenolic compounds was about 40%. Highest increases in anthocyanins and in antioxidant activity were observed in berry mash treated with Pectinex Smash XXL enzyme, and the lowest increase was observed after treatment with Pectinex BE 3-L. Enzyme-assisted processing is traditionally used to improve berry and fruit juice yields. However, enzymatic treatments also have an impact on the functional properties of the products. The increased liberation of phenolics from the cell wall matrix can prolong the shelf life of berry products by limiting the growth of contaminants during processing or storage. The increased amount of phenolic compounds may also have a positive effect on gut well-being.     

Antioxidant activity of protein-bound quercetin

Bovine serum albumin (BSA) was derivatized by covalent attachment of different amounts of quercetin [ratios of BSA to quercetin were 20:1, 10:1, 7:1, 5:1, and 2:1 (w/w)]. The antioxidant activity of the protein-phenol derivatives was investigated using a modified TEAC assay. The results show that the covalent attachment of quercetin to BSA decreases the total antioxidant activity in comparison to an equivalent amount of free quercetin depending on the degree of derivatization. The derivative with the highest amount of covalently bound quercetin (the 2:1 derivative) showed an antioxidant activity of only 79% compared to an equivalent amount of free quercetin. After the enzymatic proteolysis of the BSA-quercetin derivatives with trypsin, the total antioxidant activity of the degradation products increases in comparison to the respective undigested derivatives but does not reach the activity of an equivalent amount of free quercetin. Even after 240 min of tryptic degradation, there is still a lack in antioxidant activity (for the 7:1 derivative nearly 33%) as compared to free quercetin.     

Inhibitory effect of a quercetin metabolite, quercetin 3-O-beta-D-glucuronide, on lipid peroxidation in liposomal membranes.

To study the antioxidant activity of quercetin 3-O-beta-D-glucuronide (Q3GA), which is one of the quercetin metabolites in the blood after intake of quercetin-rich food, the inhibitory effect of Q3GA on lipid peroxidation was estimated using phosphatidylcholine large unilamellar vesicles (PC LUV) as a biomembrane model. Iron ion, an aqueous peroxyl radical generator, a peroxynitrite generator, or lipoxygenase was used as the inducer of lipid peroxidation. In all cases, Q3GA inhibited lipid peroxidation significantly, although its inhibitory effect was lower than that of quercetin aglycon. The ultrafiltration of PC LUV containing Q3GA revealed that Q3GA has low but significant affinity with the membranes of phospholipid bilayers. It is therefore likely that Q3GA acts as an efficient antioxidant in membranous lipid peroxidation through its localization in the phospholipid bilayer. This conjugated quercetin metabolite seems to retain the ability to protect cellular and subcellular membranes from peroxidative attack by reactive oxygen species and peroxidative enzymes.     

Cellulolytic and pectolytic activity of streptomycetes isolated from root-free soil,rhizosphere and mycorrhizosphere of pine (Pinus sylvestris L.)

Summary Our studies have revealed that streptomycetes inhabiting root-free soil and the root zone of pine trees differ in their capacity to produce cellulolytic and pectolytic enzymes. Most of the root-zone organisms but only a few of the root-free soil isolates exhibited cellulolytic activity. A few of the root-zone organisms but no soil isolate showed pectolytic activity. In general the cellulolytic activity was higher in cellulase producers from the root zone than in those derived from the root-free soil. The streptomycetes studied produced only endopolymethylgalacturonase. The mean total activity of this enzyme was higher in the rhizosphere isolates but the mean specific activity was higher in the mycorrhizosphere organisms.     

Enzyme-assisted extraction of antioxidative phenols from black currant juice press residues (Ribes nigrum).

Enzymatic release of phenolic compounds from pomace remaining from black currant (Ribes nigrum) juice production was examined. Treatment with each of the commercial pectinolytic enzyme preparations Grindamyl pectinase, Macer8 FJ, Macer8 R, and Pectinex BE, as well as treatment with Novozym 89 protease, significantly increased plant cell wall breakdown of the pomace. Each of the tested enzyme preparations except Grindamyl pectinase also significantly enhanced the amount of phenols extracted from the pomace. Macer8 FJ and Macer8 R decreased the extraction yields of anthocyanins, whereas Pectinex BE and Novozym 89 protease showed no effect. A decrease in pomace particle sizes from 500-1000 microm to <125 microm increased the phenol yields 1.6-5 times. Black currant pomace devoid of seeds gave significantly higher yields of phenols than pomace with seeds and seedless wine pomace. Four selected black currant pomace extracts all exerted a pronounced antioxidant activity against human LDL oxidation in vitro when tested at equimolar phenol concentrations of 7.5-10 microM.     

Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements

A cellular antioxidant activity (CAA) assay for quantifying the antioxidant activity of phytochemicals, food extracts, and dietary supplements has been developed. Dichlorofluorescin is a probe that is trapped within cells and is easily oxidized to fluorescent dichlorofluorescein (DCF). The method measures the ability of compounds to prevent the formation of DCF by 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-generated peroxyl radicals in human hepatocarcinoma HepG2 cells. The decrease in cellular fluorescence when compared to the control cells indicates the antioxidant capacity of the compounds. The antioxidant activities of selected phytochemicals and fruit extracts were evaluated using the CAA assay, and the results were expressed in micromoles of quercetin equivalents per 100 micromol of phytochemical or micromoles of quercetin equivalents per 100 g of fresh fruit. Quercetin had the highest CAA value, followed by kaempferol, epigallocatechin gallate (EGCG), myricetin, and luteolin among the pure compounds tested. Among the selected fruits tested, blueberry had the highest CAA value, followed by cranberry > apple = red grape > green grape. The CAA assay is a more biologically relevant method than the popular chemistry antioxidant activity assays because it accounts for some aspects of uptake, metabolism, and location of antioxidant compounds within cells.