Emulsifying ability of porphyran prepared from dried nori, Porphyra yezoensis, a red alga

A suspension of low-quality dried nori processed from Porphyra yezoensis, a red alga, was autoclaved at 120 degrees C for 30 min, and from the supernatant, five preparations of porphyran of differing molecular masses and chemical compositions were obtained by preprecipitation with ethanol at stepwise-increasing concentrations of 50 and 67% followed by size-exclusion chromatography. The porphyran preparations exhibited a high emulsifying activity index and high emulsion stability over a wide range of pH and temperature and also in the presence of sodium chloride. An adequately high coefficient of correlation between the median diameter of oil droplets and their 3,6-anhydrogalactose content suggests that 3,6-anhydrogalactose could take part in emulsification with porphyran.     

Interaction of porphyran with a hydrophobic surface and stabilization of liposomes

Three porphyran preparations with high emulsifying ability and varying molecular mass, 3,6-anhydrogalactose content, and sulfate content without any protenaceous component were prepared from dried nori processed from Porphyra yezoensis, a red alga. Each of these preparations was applied to demonstrate adsorption or binding to the surface of oil droplets. The decrease in porphyran concentration of the aqueous phase of O/W emulsions prepared with porphyran and with toluidine blue (TB)-porphyran complex formed by adding TB to the O/W emulsions indicated ready adsorption to the surface of oil droplets. The decrease in zeta-potential of the O/W emulsions suggested that the sulfate groups of the adsorbed porphyran were oriented toward the external aqueous phase. A biomolecular interaction analysis exhibited rapid binding of porphyran to C16-alkane, probably through 3,6-anhydrogalactose. Porphyran-coated liposomes were tolerant to digestion with phospholipase D. The increased molecular weight of the porphyran preparations had an increased effect on these characteristics. The results of this study demonstrate that the emulsifying ability of porphyran is derived from the adequate adsorption to the surface of oil droplets and that porphyran could be effectively applied to stabilize liposomes.     

红娘鱼降血压肽的酶解制备工艺优化及酶解产物的抗氧化活性研究

为有效利用红娘鱼制备降血压肽,以红娘鱼鱼糜为原料提取蛋白,并对其进行酶解制备降血压肽。以血管紧张素转换酶ACE抑制率和水解度为指标,通过响应面分析法对酶解红娘鱼鱼糜蛋白制备降血压肽的工艺条件进行优化,并对最优条件下制备的酶解产物进行分子量和抗氧化活性测定。结果表明,碱性蛋白酶是制备降血压肽的最适蛋白酶,响应面法优化制备降血压肽的最佳酶解条件为pH值9、酶与底物的比值(酶底比)1.4%、温度54℃、时间2 h,此条件下酶解制得的降血压多肽ACE抑制率理论值为88%,实际值为89.3%;经高效液相色谱(HPLC)分析可得酶解产物相对分子量<2 000 Da。通过测定酶解产物样品的1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除率、羟自由基(·OH)清除率及还原力判定其体外抗氧化活性,结果表明酶解产物具有较强抗氧化活性。本研究结果为红娘鱼的高值化利用提供了数据支持和理论基础。     

马面鱼皮胶原抗氧化肽的分离制备及稳定性研究

为促进对马面鱼皮资源的综合利用,开发高附加值产品,本试验以DPPH自由基清除率和水解度(DH)为评价指标,探讨马面鱼皮胶原蛋白的最佳酶解工艺,并采用超滤和凝胶柱层析法分离制备抗氧化肽,通过超高效液相色谱-质谱联用(UPLC-MS)法对其进行结构解析。此外,还探讨了pH值、温度及体外模拟消化对多肽抗氧化活性的影响。结果表明,利用双酶分步酶解法可制备高活性抗氧化多肽,即在底物浓度3%,加酶量3 600 U·g^-1以及温度50℃的条件下先用Proteasea A ‘Amano’2G酶解3 h,再用酸性蛋白酶酶解2 h,清除DPPH自由基的IC₅₀值为13.03 mg·mL ^-1。经超滤及柱层析分离后,可得到抗氧化活性较高的A₁组分,其清除DPPH自由基的 IC₅₀值为1.80 mg·mL^-1。稳定性研究结果表明,所制备的胶原蛋白抗氧化肽热稳定性好,在偏酸性条件下能保持较高的活性,经体外模拟胃肠消化后仍能保持较高的抗氧化活性。根据UPLC-MS分析推测A₁的氨基酸序列可能为Gly-Glu-Gly-Ala-Cys-Asn或Asn-Glu-Gly-Ala-Cys-Gly。本研究结果为马面鱼皮的高值化利用及高活性抗氧化肽的筛选提供了一定的理论依据。     

Inhibition of lipid oxidation in cooked beef patties by hydrolyzed potato protein is related to its reducing and radical scavenging ability

Protein hydrolysates were prepared by limited alcalase hydrolysis (0.5, 1, and 6 h, corresponding to degrees of hydrolysis of 0.72, 1.9, and 2.3, respectively) of heat-coagulated potato protein. The hydrolysates were characterized for peptide composition, ferric reducing/antioxidant power (FRAP), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging activity, and Fe2+- and Cu2+-chelation capacity. Hydrolyzed and intact proteins were formulated (4%, w/w) into beef patties to determine in situ antioxidant efficacy. Thiobarbituric acid-reactive substances (TBARS) and peroxide value (PV) formed in cooked and PVC-packaged patties during storage (4 degrees C, 0-7 days) were analyzed. Hydrolysis increased the protein solubility by 14-19-fold and produced numerous short peptides (< 6 kDa). The FRAP values of the protein sample (23 micromol/g) increased markedly after hydrolysis but were similar between the three hydrolysates (597-643 micromol/g). Similarly, the ABTS radical-scavenging activity also was increased by hydrolysis and was the greatest for the 1-h hydrolysate. Hydrolysis increased the Cu2+-chelation activity but decreased the Fe2+-chelation ability of the protein. The production of PV in patties after 7 days of storage was lowered 44.9% and 74.5% (P < 0.05), and that of TBARS was reduced 40.9% and 50.3% (P < 0.05), by intact and hydrolyzed proteins, respectively.     

A new procedure to measure the antioxidant activity of insoluble food components

The measurement of antioxidant activity was limited to soluble components to date. Functional groups, which are bound to insoluble matters, may exert antioxidant activity by a surface reaction phenomenon. This hypothesis was tested on the insoluble matters of foods, food ingredients, and Maillard reaction products (MRPs). Insoluble matters were prepared by consecutive washes with water and methanol followed by a lyophilization of the insoluble residue. The measurement was performed by a new procedure using 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazil (DPPH) colored radicals. These insoluble matters always showed antioxidant activity. Alkali hydrolysis reduced up to 90% the antioxidant activity of cereal-based insoluble matters, thus confirming that fiber-bound compounds have a major role in their antioxidant activity. The antioxidant activity of the insoluble MRPs was not significantly affected by processing conditions, but severe treatments increased the ratio between insoluble and soluble matters. The contribution of insoluble matter to total antioxidant activity was limited for fruits and vegetables, but it was relevant for cereal-based foods and increased over 50% for dietary-fiber-rich ingredients.     

Hydrolysis influence on phytochemical composition, antioxidant activity, plasma concentration, and tissue distribution of hydroethanolic Ilex paraguariensis extract components

The infusion of aerial parts of Ilex paraguariensis is widely consumed. Its antioxidant activity suggests an important role of this plant in the treatment/prevention of oxidative stress related diseases. Plant extract active compounds are frequently found in esterified form that may be poorly absorbed. Hydrolysis of the extract is a possible approach to increase its bioavailability. The aim of this study was to perform a phytochemical analysis and evaluate in rats the plasma concentration and tissue distribution of antioxidant compounds in the hydroethanolic extract of Ilex paraguariensis, before and after enzymatic hydrolysis. Both extracts presented high antioxidant activity and phenolic content. Rats given single or repeated doses of the hydrolyzed extract showed increased plasma antioxidant activity and higher plasma levels of caffeic acid. However, no changes of endogenous antioxidants were observed. In conclusion, hydrolysis of the extract of Ilex paraguariensis is a strategy to improve its bioavailability and in vivo antioxidant activity.     

Hydrolysis kinetics of secoisolariciresinol diglucoside oligomers from flaxseed

Flaxseed is the richest dietary source of the lignan secoisolariciresinol diglucoside (SDG) and contains the largest amount of SDG oligomers, which are often hydrolyzed to break the ester linkages for the release of SDG and the glycosidic bonds for the release of secoisolariciresinol (SECO). The alkaline hydrolysis reaction kinetics of SDG oligomers from flaxseed and the acid hydrolysis process of SDG and other glucosides were investigated. For the kinetic modeling, a pseudo-first-order reaction was assumed. The results showed that the alkaline hydrolysis of SDG oligomers followed first-order reaction kinetics under mild alkaline hydrolytic conditions and that the concentration of sodium hydroxide had a strong influence on the activation energy of the alkaline hydrolysis of SDG oligomers. The results also indicated that the main acid hydrolysates of SDG included secoisolariciresinol monoglucoside (SMG), SECO, and anhydrosecoisolariciresinol (anhydro-SECO) and that the extent and the main hydrolysates of the acid hydrolysis reaction depended on the acid concentration, hydrolysis temperature, and time. In addition, the production and change of p-coumaric acid glucoside, ferulic acid glucoside and their methyl esters and p-coumaric acid, ferulic acid, and their methyl esters during the process of hydrolysis was also investigated.     

脱酰胺与双酶协同作用提高小麦面筋蛋白酶解效率

为了探讨了不同脱酰胺处理和双酶协同作用方式对小麦面筋蛋白酶解效率及其产物抗氧化活性的影响,该文研究了小麦面筋蛋白在各种预处理方式和酶解条件下的蛋白回收率、水解度、抗氧化性能及肽分子量分布情况。结果显示,单独热处理(90℃,30 min)小麦面筋蛋白对其酶解效率无显著影响,而采用添加0.5 mol/L柠檬酸溶液进行热处理(质量分数为5%,90℃,30 min)可显著(P0.05)提高其蛋白回收率。此外,酶制剂添加顺序及双酶共同水解作用时间对酶解效率均具有较大影响:加入谷氨酰胺酶预先水解对小麦面筋蛋白的深度水解有促进作用;一定时间内的双酶协同作用有利于酶解的进行,但较长时间的双酶作用反而会抑制酶解效率。采用谷氨酰胺酶(质量分数为0.2%)对经柠檬酸加热处理的小麦面筋蛋白作用12 h后再加入胰酶(质量分数为0.6%)共同作用7 h可使蛋白回收率达70.74%,水解度达到9.88%;另外,酶解产物的自由基清除能力ABTS+(2,2’-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)+)值与氧化自由基吸收能力(ORAC,oxygen radical absorbance capacity)值分别达到478.95 mmol/g和213.85μmol/g,提示该酶解产物是一种潜在优秀食品抗氧化剂。研究结果可为拓宽小麦面筋蛋白的应用领域,以及高效制备抗氧化活性肽提供方法和理论指导。     

Mild acetosolv process to fractionate bamboo for the biorefinery: structural and antioxidant properties of the dissolved lignin

Fractionation of lignocellulosic material into its constitutive components is of vital importance for the production of biofuels as well as other value-added chemicals. The conventional acetosolv processes are mainly focused on the production of pulp from woody lignocelluloses. In this study, a mild acetosolv process was developed to fractionate bamboo under atmospheric pressure to obtain cellulosic pulp, water-soluble fraction, and acetic acid lignin. The structural features of the lignins obtained under various conditions were characterized with elemental analysis, sugar analysis, alkaline nitrobenzene oxidation, gel permeation chromatography (GPC), (1)H nuclear magnetic resonance ((1)H NMR), and heteronuclear single-quantum coherence (HSQC) spectroscopy. As compared to milled wood lignin (MWL) of bamboo, acetic acid lignins had low impurities (carbohydrates 2.48-4.56%) mainly due to the cleavage of linkages between lignin and carbohydrates. In addition, acetic acid lignins showed a low proportion of syringyl (S) units. Due to the cleavage of linkages between lignin units, acetic acid lignins had weight-average molecular weights ranging from 4870 to 5210 g/mol, less than half that of MWL (13000 g/mol). In addition, acetic acid lignins showed stronger antioxidant activity mainly due to the significant increase of free phenolic hydroxyls. The lignins obtained with such low impurities, high free phenolic hydroxyls, and medium molecular weights are promising feedstocks to replace petroleum chemicals.     

Pectic methyl and nonmethyl esters in potato cell walls.

Because pectins are released from potatoes and other plants under conditions that cleave ester linkages, it has been suggested that there are other galaturonoyl ester cross-links between pectin chains in addition to the known non-cross-linking methyl esters. A microscale titration method and a copper binding method were developed for the measurement of total polymer carboxyl (essentially pectic) ester content in potato cell walls. Relative to the uronic acid content of the cell walls, the degree of total esterification was 57-58%. Comparison with levels of methanol released on ester hydrolysis allowed nonmethyl uronoyl esters to be estimated to be 14-15% relative to total uronic acid. The possibility of nonmethyl-esterified linkages being formed in potato cell walls by a side-reaction catalyzed by pectin methyl esterase (PME) was investigated, but no increase in nonmethyl-esterified pectin was observed under conditions where pectin was being effectively de-esterified by endogenous PME activity.     

Microassay for rapid screening of alpha-amylase activity

A microassay was developed for measuring the activity of alpha-amylases in the nanogram enzyme concentration range, based on the use of dye-labeled cross-linked starch as the substrate, and the release of soluble colored fragments formed in enzyme hydrolysis. Reaction conditions were optimized to generate a linear correlation between the increase in absorbance and a reaction time of 0-10 min, as well as enzyme concentrations in the range of 0-50 ng. A standard curve for the conversion of absorbance to enzyme activity units was constructed. The protocol developed was applied to monitoring the production of ultralow concentrations of recombinant barley alpha-amylase in yeast cells.     

Cloning and characterization of an antifungal class III chitinase from suspension-cultured bamboo ( Bambusa oldhamii ) cells

A class III chitinase cDNA (BoChi3-1) was cloned using a cDNA library from suspension-cultured bamboo ( Bambusa oldhamii ) cells and then transformed into yeast ( Pichia pastoris X-33) for expression. Two recombinant chitinases with molecular masses of 28.3 and 35.7 kDa, respectively, were purified from the yeast's culture broth to electrophoretic homogeneity using sequential ammonium sulfate fractionation, Phenyl-Sepharose hydrophobic interaction chromatography, and Con A-Sepharose chromatography steps. N-Terminal sequencing and immunoblotting revealed that both recombinant chitinases were encoded by BoChi3-1, whereas SDS-PAGE and glycoprotein staining showed that the 35.7 kDa isoform (35.7 kDa BoCHI3-1) was glycosylated and the 28.3 kDa isoform (28.3 kDa BoCHI3-1) was not. For hydrolysis of ethylene glycol chitin (EGC), the optimal pH values were 3 and 4 for 35.7 and 28.3 kDa BoCHI3-1, respectively; the optimal temperatures were 80 and 70 degrees C, and the K(m) values were 1.35 and 0.65 mg/mL. The purified 35.7 kDa BoCHI3-1 hydrolyzed EGC more efficiently than the 28.3 kDa isoform, as compared with their specific activity and activation energy. Both recombinant BoCHI3-1 isoforms showed antifungal activity against Scolecobasidium longiphorum and displayed remarkable thermal (up to 70 degrees C) and storage (up to a year at 4 degrees C) stabilities.     

Biochemical properties of recombinant leucine aminopeptidase II from Bacillus stearothermophilus and potential applications in the hydrolysis of Chinese anchovy (Engraulis japonicus) proteins

The effects of various factors on the activity and conformation of recombinant leucine aminopeptidase II (rLAP II) from Bacillus stearothermophilus and its potential utilization in the hydrolysis of anchovy proteins were investigated. The optimal temperature and pH of rLAP II were 55 °C and 8.0 in phosphate buffer, and its activity was strongly stimulated by Co(2+). Conformational studies indicated that maintaining the α-helical structure had a critical effect on rLAP II activity. rLAP II was used to hydrolyze anchovy proteins, and it exhibited high specificity for peptides with molecular weight between 6000 and 1000 Da and positive coordination with endogenous enzymes and commercial Flavourzyme. Its use will enhance protein hydrolysis in species of aquatic animals. rLAP II could potentially be used to remove bitterness in the protein hydrolysis industry.     

Antioxidant phenols in barley (Hordeum vulgare L.) flour: comparative spectrophotometric study among extraction methods of free and bound phenolic compounds

Phenolic compounds are found in both free and bound forms in cereals. The majority is in the insoluble bound form, that is, bound to cell wall material, such as ferulic acid and its derivatives. The antioxidant properties of the phenolic compounds in grains are associated with the health benefits of grains and grain products. The extraction capacity of several solvent mixtures, for extracting free phenols from barley flours, and the possibility of employing a rapid automated solvent extraction method were studied. The extraction yield of each method was evaluated by correlating several spectrophotometric indices (absorption at 280, 320, and 370 nm and total phenolic compounds by the Folin-Ciocalteu method) with the antioxidant activities of the barley extracts (scavenging activity by the 2,2-diphenyl-1-picrylhydrazyl method). Interesting results were obtained when ethanol and acetone-based extraction mixtures were employed to extract free phenols. A comparison was made between alkaline and acid hydrolysis. The extraction yield of bound phenolic compounds increased when the digestion time for alkaline hydrolysis was prolonged.     

超声辅助竹节虾头酶解及抗氧化肽分离研究

为了实现竹节虾加工副产物的高值化利用,以竹节虾加工废弃物中的虾头副产物为原料,以水解度和DPPH清除率作为评价指标,采用中性蛋白酶酶解,通过响应面法优化超声辅助酶解工艺,并依次通过超滤、凝胶层析色谱和反相高效液相色谱等分离方法,从竹节虾虾头酶解产物中分离制备抗氧化肽,采用超高压液相色谱串联质谱联用技术对肽的结构进行表征。结果表明,在中性蛋白酶添加量为3 000 U·g~(-1)、p H值7.0条件下,最佳超声辅助酶解工艺参数为超声时间41 min,超声温度55℃,超声频率22 k Hz,料液比1∶9(w/v),在此条件下获得的酶解产物DPPH清除率达69.50%。当水解时间为0.5~2.5 h时,超声辅助酶解的酶解产物水解度和DPPH清除率比非超声辅助酶解工艺分别高17.95%和18.83%,该工艺缩短了酶解时间,节约了能耗。酶解产物经超滤初步分离发现,相对分子质量在3k Da(SHP4)的组分具有显著的抗氧化活性。用凝胶层析法进一步分离纯化SHP4组分后得到4个峰,其中SHP4-II的DPPH清除率最高;SHP4-II通过反相高效液相纯化后也得到4个主要肽峰,在多肽含量为1.5 mg·m L~(-1)时,SHP4-II-4的DPPH清除率最高,达到85.69%,并且具有较好的分离度,质谱分析发现,该抗氧化肽的结构为Gly-Asn-Gly-Leu-Pro(455.99 Da)。本研究结果为虾肽抗氧化保健食品的研发提供了一定的科学依据。     

Quantitative determination of hydroxycinnamic acids in wheat, rice, rye, and barley straws, maize stems, oil palm frond fiber, and fast-growing poplar wood.

A new method has been developed for the quantitative determination of hydroxycinnamic acids participating in ester or ether linkages to the cell wall polymers. The method is based on mild alkaline hydrolysis followed by acid hydrolysis or mild alkaline hydrolysis, which partially removed esterified phenolic acids, and high-temperature concentrated alkaline treatment, which cleaved both the ester and ether linkages. It was found that traditional mild alkaline hydrolysis and acid hydrolysis released only part of the ester- and ether-linked phenolic acids, respectively. Approximately half (44.0-47.9%) of the total ester-linked p-coumaric acid and 18.2-32.6% of the total esterified ferulic acid remained ester-linked to the mild alkali-soluble lignin polymers, and 55.0-72.0% of the total ether-linked p-coumaric acid and 37.5-53.8% of the total ether-linked ferulic acid remained ether-linked to the solubilized lignin molecules after the acid hydrolysis. To correct this, a second mild alkaline hydrolysis of the alkali-soluble lignin preparations and acid hydrolysis of the solubilized lignin fractions, obtained from the first acid hydrolysis of the cell wall materials, was investigated. On the basis of this new method, a majority of the cell wall p-coumaric acid (55.8-81.5%) was found to be ester-linked to cell wall components, mainly to lignin, and about half of the cell wall ferulic acid is etherified through its phenolic oxygen to the cell wall lignin component, whereas the remainder is esterified to the cell wall hemicelluloses and/or lignin in different plant materials.     

Effect of pectolytic enzyme preparations on the phenolic composition and antioxidant activity of asparagus juice

Commercial pectolytic enzymes were investigated for their influence on phenolics and antioxidant activities of asparagus juice. The antioxidant activity of asparagus juice was analyzed according to 2,2'-diphenyl-l-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) methods. The enzymes, with the exception of pectinase from Rhizopus sp., contained rutinase, which hydrolyzed rutin to quercetin. Asparagus juice treated with Viscozyme had the highest quercetin content without exhibiting a significant increase in the antioxidant activity. For a pectinase from Aspergillus niger, the antioxidant activity of asparagus juice was markedly reduced. Caution should be paid in the selection of pectolytic enzyme preparations for production of antioxidant activity-rich juice.     

鳙鱼皮水解物美拉德反应产物抗氧化活性研究

为进一步利用鱼类加工副产物,采用微波技术预处理鳙鱼皮,碱性蛋白酶酶解制备鳙鱼皮水解物(FSH),将其与葡萄糖、木糖和核糖进行美拉德反应,评价美拉德反应产物的抗氧化活性和挥发性风味物质。结果表明,美拉德反应显著提高了鳙鱼皮水解物的抗氧化活性,核糖美拉德反应产物的抗氧化活性最高,在135℃、35 min条件下,核糖美拉德反应产物的总氧化活性为2 156 μmol·L^-1 FeSO₄,DPPH自由基清除能力达到96.53%,超氧阴离子自由基清除能力达到88.08%。采用固相微萃取-气相色谱-质谱联用(SPME-GC-MS)分析美拉德反应产物的挥发性风味物质发现,呈肉香味的吡嗪、呋喃类杂环化合物相对含量增加,从而改善了鳙鱼皮水解物的风味。本研究结果为制备食品天然抗氧化剂提供了理论依据,为鱼类加工副产物高值化开发与利用提供了参考。     

Antioxidant activity of the main bioactive derivatives from oleuropein hydrolysis by hyperthermophilic beta-glycosidase.

The main reaction products obtainable by the hydrolysis of commercially available oleuropein by hyperthermophilic beta-glycosidase were purified and structurally characterized by UV and 1H and 13C NMR analyses. Their antioxidant activity, in particular their capacity to inhibit the fatty acid peroxidation rate, was studied. The molecular structures assigned revealed the presence of two elenolic acid forms presenting different antioxidant abilities closely correlated to their molecular structures, as well as an unstable elenolate which is a rearrangement product of the oleuropein aglycon. This molecule, under the reaction conditions (pH 7.0, 60 degrees C) required for beta-glycosidase activity, rapidly gives rise to 3,4-dihydroxy-phenylethanol (hydroxytyrosol).