CHARACTERIZATION OF PHYTASE ACTIVITY IN LUPIN SEED

Lupin phytase (myo-inositol hexaphosphate phosphohydrolase) and the degradation of its substrate phytic acid were studied during seed germination. Phytase and acid phosphatase activities were detected in nongerminated seeds and increased from days 1 to 8 of germination, while the concentration of phytic acid decreased during the same period. Phytase was extracted with 2% CaCl₂ solution and partially purified by ammonium sulfate fractionation and HPLC-gel permeation chromatography. Substrate specificites were determined using pnitrophenylphosphate or phytic acid. The pH and temperature optima for the fraction obtained by gel permeation were 5.0 and 50C respectively, while the Km and V _max values were 0.33 mM and 0.13 μmol·mL^?1·min^?1, respectively. It was not possible to separate phytase from phosphatase by use of gel permeation chromatography due to similarities in apparent molecular mass, however resolution of the two enzymes was achieved by DEAE-cellulose chromatography.     

Antioxidant and anticancer activities of enzymatic hydrolysates of solitary tunicate (<Emphasis Type="Italic">Styela clava</Emphasis>)

The antioxidant and anticancer activities of solitary tunicate (Styela clava) hydrolysate manufactured with different proteases (Alcalase 2.4 L FG, Thermoase PC10F, and pepsin) and optimized by response surface methodology (RSM) were investigated. The hydrolysate produced by Alcalase generally had greater antioxidant and anticancer activities. Two major fractions, F₁ and F₂, were produced by applying Alcalase hydrolysates to Sephacryl S-100 HR gel filtration chromatography. Fraction F₂ with a lower molecular weight (3.6±0.1 kDa) had higher ABTS and DPPH radical scavenging activities than F₁ (5.0±1.0 kDa), with IC₅₀ values of 11.4 and 227.5 μg/mL, respectively. Moreover, fraction F₂ had higher anticancer activitiy, with IC₅₀ values of 577.1, 1,163.3, and 887.2 μg/mL against AGS, DLD-1, and HeLa cells, respectively. Fraction F₂ was also rich in tyrosine, lysine, arginine, phenylalanine, and histidine. These results suggest that solitary tunicate hydrolysates have significant health-promoting effects with excellent antioxidant and anticancer activities.     

Canola Phytase: Isolation and Characterization

Two phytase isoenzymes were isolated from 8-day germinated canola cv Regent. Gel filtration chromatography of an ammonium sulfate fractionated extract on Sephadex G-100 produced one peak with phytase activity. The phytase fraction was separated into two isoenzymes by DEAE-cellulose chromatography. The optimum pH was 4.5–5.0 and 5.0 for the phytase isoenzymes 1 and 2, respectively. Both isoenzymes exhibited maximum activity at 50°C. Km values at pH 5.0 were 0.36 and 0.25 mM for phytase 1 and 2 isoenzymes, respectively, while molecular weight determination showed both fraction were identical with a molecular weight of 70,100 ± 4,000 daltons.     

In-vitro and in-vivo dephosphorylation of rapeseed meal by means of phytate-degrading enzymes derived from Aspergillus Niger

A meal of ‘double low’ rapeseed (var ‘Jantar’) was subjected to phytate hydrolysis using enzyme preparations derived from a mycelium of Aspergillus niger which contained phytase (EC 3.1.3.8) and acid phosphatase (EC 3.1.3.2) activities. The complete conversion of myo-inositol hexa- and pentaphosphates present in rapeseed meal to lower phosphate esters of myo-inositol was accomplished at 40°C, a pH value of 4.5, phytase dosage (in phytase units (PhytU)) 0.1 PhytU g^?1 accompanied by acid phosphatase activity 37.1 units g^?1, in 1 h. Under these conditions, complete dephosphorylation was observed in 4 h. Decreasing the pH value to 3.0 caused a rise in the amount of inorganic phosphorus released, while increasing to 5.5 resulted in substantial reduction in the reaction rate. Purification of phytase to a specific activity 0.375 PhytU mg^?1 of protein exhibited a negative influence upon the yield of rapeseed dephosphorylation. The substitution of calcium phosphate for a preparation of phytase in feed containing rapeseed meal did not cause significant differences in the body weight gain or in tibia mineralisation of broilers (Gains galus, ‘Astra B’).     

Extracts of Mexican Oregano (<Emphasis Type="Italic">Lippia berlandieri</Emphasis> Schauer) with Antioxidant and Antimicrobial Activity

Antimicrobial activity of fractions obtained from Mexican oregano (Lippia berlandieri Schauer) chloroform extract was tested by growth inhibition against Escherichia coli, Staphylococcus aureus and Bacillus cereus, and antioxidant capacity was tested by inhibition of linoleic acid oxidation. Fractions were obtained by differences in polarity or structure (phenolic and non-phenolic fraction). Gram-positive organisms were more susceptible to Mexican oregano extracts. Fraction 3 (by polarity) and phenolic fractions I, II, III, IV and V were the extracts with higher antimicrobial activity. The non-phenolic fraction had effect against B. cereus. Polarity fraction 5 and phenolic Fraction II had a high antioxidant capacity; a 0.08% concentration of fraction 5 had a similar effect as butylated hydroxytoluene at 0.01% concentration. Fractions of Mexican oregano with different polarity and functional groups had antioxidant and antimicrobial activity and can be used in a variety of applications.     

Isolation of antimicrobial compounds from aniseed and techno-economic feasibility report for industrial-scale application

This work evaluates the antimicrobial activity of the fractionated aqueous extract obtained from the aerial parts of aniseed. Fifteen fractions were collected by column chromatography using ethanol and water as mobile phases. All fractions were tested as potential antimicrobial agents against three common food-spoilage microorganisms: a yeast (Saccharomyces cerevisiae), a mold (Aspergillus awamori) and a bacterium (Lactobacillus delbrueckii subsp. bulgaricus). Fraction 2 exhibited the highest antimicrobial activity in both types of susceptibility test, and therefore, it was further studied for the identification of bioactive compounds. GC-MS analysis of Fraction 2 revealed the presence of threo-Anethole glycol. Based on laboratory-scale experiments, a process flowsheet for potential industrial-scale application is proposed combined with a techno-economic feasibility report. Fraction 2 with the detected compound threo-Anethole glycol showed promising results encouraging the attempts for industrial-scale application of antimicrobial compounds produced from aniseed.     

Phenolic profiles of andean mashua (Tropaeolum tuberosum Ruíz & Pavón) tubers: Identification by HPLC-DAD and evaluation of their antioxidant activity

Qualitative high performance liquid chromatography with diode array detection (HPLC-DAD) was performed to characterize non-anthocyanin phenolic compounds in three different coloured mashua genotypes. The ORAC antioxidant activity contribution in the tubers related to the type of phenolic compounds present was also evaluated. Phenolic compounds were analysed by separating them into four main fractions: fraction I obtained by means of a liquid–liquid partition with ethyl acetate and fractions II, III and IV obtained by elution on a Sephadex LH-20 column. Fraction I revealed the presence of gallic acid, gallocatechin, procyanidin B₂ and epigallocatechin. Other phenolic compounds such as hydroxycinnamic and hydroxybenzoic acid derivatives, rutin and/or myricetin derivatives were also present in fraction I. Fraction II was mainly composed of epicatechin, hydroxycinnamic and hydroxybenzoic acid derivatives. Fraction III presented mainly anthocyanins for the purple coloured mashua tubers and rutin, hydroxycinnamic acid and hydroxybenzoic acid derivatives for the yellow coloured genotype. Fraction IV was composed of proanthocyanidins. Alkaline and acid hydrolysis of the different fractions revealed the presence of gallocatechin, epicatechin, p-coumaric acid, o-coumaric acid, cinnamic acid, protocatechuic acid, rutin and quercetin as the main phenolic moieties present. The proanthocyanidin fractions were the major contributors to the ORAC antioxidant activity of the mashua tubers for two of the three genotypes (34.7–39.2%). The results obtained in the present study confirm that mashua tubers constitute a promising source of antioxidant phenolics and could potentially be considered as a functional food with beneficial health effects.     

Anticancer,antioxidant, and antimicrobial activities of anemone (Anemone cathayensis)

This study was performed to evaluate the anticancer, antioxidant, and antimicrobial activities of fractions and isolated compounds from anemone (Anemone cathayensis). Fourteen compounds were isolated from extracts. Anticancer activities of fractions and compounds were determined by MTT assay, and all tested fractions showed inhibition activity on human breast cancer cells (MDA-MB-231). The fraction 6 displayed the strongest anticancer activity, and inhibition percent was 50.32%. The antioxidant effect of fractions was evaluated by using DPPH scavenging assays. Fraction 5 had a higher DPPH radical scavenging activity with low IC₅₀ value of 30.578 μg/mL. The antimicrobial activity of the fractions was evaluated against 3 microorganisms using the agar well diffusion method. The fractions also showed moderate antimicrobial activity. These results suggest that anemone could hold a good potential source for human health.     

Pectic polysaccharides of mango (Mangifera indica L): structural studies

Pectic polymers of raw and ripe mango fruits revealed several distinct fractions upon diethylaminoethyl‐cellulose chromatography. Fraction I, the arabinogalactan, had a relative ratio of 3:1 for galactose and arabinose, while the two rhamnogalacturonans (fractions II and III) were composed of galacturonic acid, arabinose, galactose and rhamnose in the relative ratios of 69:15:14:2 and 62:10:23:4, respectively. Specific rotation, FTIR, GC‐MS and NMR data revealed fraction I to be a 1→5‐α‐L arabinofuranan linked to the main chain of 1→4‐β‐D ‐galactopyranan through 1→3 linkages. The carboxyl‐reduced fraction II had 1→4‐α‐D galactopyranuronic acid residues in the main chain, which were further involved in branching through rhamnose. The striking drop in their abundance and M_r at the end of ripening, denoting depolymerization in situ, is discussed in the context of textural softening. Copyright © 2004 Society of Chemical Industry     

Reducing,Radical Scavenging,and Chelation Properties of Fermented Soy Protein Meal Hydrolysate by Lactobacillus plantarum LP6

Fermented soybean protein meal hydrolysate (FSPMH) was fractionated by gel filtration on Sephadex G-15. The fractions obtained were subjected to various antioxidant assays, amino acid and molecular weight determinations. Among the seven fractions, the highest antioxidant activity was found in fraction F2, with significant differences (P < 0.01) 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, hydroxyl radicals (^.OH) scavenging and Cu²⁺ chelating activity. Fraction F2 exhibited scavenging of DPPH (59.43%), ^.OH (72.80%) and 44.47% Cu²⁺ chelating activity. All other fractions showed variable activities in different assays. Amino acid analyses of F2 fraction with the strongest antioxidant activity also had the highest percentage of related antioxidative amino acids content (Histidine 3.46, Serine 5.78, Valine 4.08 and Lysine 11.49 g/100 g protein) compared with other six fractions. The molecular weight distribution of F2 was found to vary from 170 to 1500 Da.     

ISOLATION AND ANALYSIS OF A NOVEL ACIDIC POLYSACCHARIDE WITH GLUCOKINASE-STIMULATING ACTIVITY FROM COARSE GREEN TEA

Water‐soluble polysaccharides from coarse green tea were separated by anion‐exchange chromatography into five fractions (fraction A [FA], fraction B, fraction C [FC], fraction D and fraction E). Two of these fractions, FA and FC, contained significant glucokinase‐stimulating activity (P < 0.05). The major component, FC, showed the most activity, and thus, was further purified by gel filtration chromatography, thereby obtaining fraction C‐1 (FC‐1) and fraction C‐2 (FC‐2). The biological activity of the two fractions was investigated, and FC‐1 displayed higher glucokinase‐stimulating activity (P < 0.01). Chemical tests combined with IR and UV spectroscopy revealed that FC‐1 is an acidic polysaccharide without conjugation to protein. Sugars of FC‐1 are composed of rhamnose, arabinose, mannose, glucose and galactose in the ratio of 12.57:22.95:4.4:39.34:20.77. Uronic acid analysis by ion chromatography showed that FC‐1 contains 8% galacturonic acid, and its molecular weight was estimated to be approximately 6 × 10⁴ using a Sephacryl S200 column. These results are different from the observations previously reported, therefore suggesting that FC‐1 is a novel polysaccharide.     

Isolation and identification of antioxidative peptides from hydrolysate of threadfin bream surimi processing byproduct

The objective of this study was to determine the antioxidant activity and possible mode of action of partially purified peptides derived from threadfin bream surimi byproduct. The frame, bone and skin (FBS) byproduct, which was obtained from a deboning process, was hydrolyzed by Virgibacillus sp. SK33 proteinase and fractionated using anion exchange and size exclusion chromatography. Three fractions, i.e., B1, B2 and B3, were obtained, and the amino acid sequences of the peptides in all 3 fractions were determined using LC-MS/MS. Fractions B2 and B3 contained higher amounts of Trp, Met, Cys and Tyr residues than fraction B1. Fraction B3 exhibited high ABTS radical scavenging activity and ferric reducing antioxidant power (FRAP), while metal chelation and hydroxyl radical scavenging ability were principle modes of action of peptides in fraction B2 and B3. In addition, fraction B1 and a synthetic peptide selected from the pooled de novo peptides of fraction B3, FLGSFLYEYSR, had a cellular radical scavenging effect when HepG2 cells were treated with H₂O₂.     

Gel filtration fractionation of winged bean (Psophocarpus tetragonolobus) proteins

Winged bean proteins were extracted by a pH 9.5, 0.3M borate extraction medium. The extracted proteins were fractionated by Sepharose CL-6B gel filtration chromatography into four fractions. Fraction 2 (F2, mol. wt 200 000) and Fraction 3 (F3, mol. wt 35 000) are the two major protein fractions. F3 was further resolved into Fraction 3A (F3A, mol. wt 44 160) and Fraction 3B (F3B, mol. wt 23 700) by Sephadex G-75 gel filtration chromatography. Both F2 and F3 fractions were heterogeneous when examined by polyacrylamide gel electrophoresis method. There are some varietal variations in the distribution of winged bean protein fractions recovered by gel filtration chromatography. Trypsin inhibitor and chymotrypsin inhibitor activity were found only in F3B, whereas haemagglutinating activity was found only in F3A.     

Purification and properties of a phytase from Candida krusei WZ-001

A phytase from Candida krusei WZ-001 isolated from soil was purified to electrophoretic homogeneity by ion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The phytase is composed of two different subunits with molecular masses of 116 kDa and 31 kDa on SDS-PAGE (or 120 kDa and 30 kDa on gel chromatography), with the larger subunit having a glycosylation rate of around 35%. The phytase has an optimum pH of 4.6, an optimum temperature of 40 degrees C and a pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, p-chroloromercuribenzoate (pCMB) and phenylmethylsulfonyl fluoride (PMSF). The phytase displayed a broad substrate specificity and the K(m) for phytate was 0.03 mM. Phytate was sequentially hydrolyzed by the phytase. Furthermore, 1D and 2D NMR analyses and bioassay of myoinositol indicated that the end hydrolysis product of phytate was myoinositol 2-monophosphate.     

Control of amylase and protease activities in a phytase preparation by ampholyte-free preparative isoelectric focusing for unrefined cereal-containing bread

Direct addition of a food additive-grade Aspergillus niger phytase preparation to 30% brown rice flour-added bread ingredients reduced the bread myo-inositol hexaphosphate (IP₆) content. The phytase preparation had protease and amylase activities. Addition of the crude phytase preparation induced bread crust collapse. Protease-free high- and low-amylase phytase fractions were prepared using preparative ampholyte-free isoelectric focusing (autofocusing). Addition of the protease-free low-amylase phytase fraction did not significantly affect loaf volume and did not collapse the crust. Addition of the protease-free high-amylase phytase fraction significantly increased loaf volume approximately 1.5× in the presence of amylase at 193–1029 U. However, addition of 1029 and 2058 U of amylase induced partial and whole bread crust collapse, respectively. Therefore, removal of protease and control of amylase activities in the phytase preparation are crucial in the preparation of brown rice flour-added bread with a low myo-inositol phosphate (IP₆) and good swelling property.     

Contribution of Leu and Hyp residues to antioxidant and ACE-inhibitory activities of peptide sequences isolated from squid gelatin hydrolysate

Squid gelatin obtained from inner and outer tunics was hydrolysed with Alcalase to isolate antioxidant peptide sequences. The ACE-inhibitory activity of the isolated peptides was also evaluated. After fractionation by ultrafiltration and size-exclusion chromatography into four fractions, the antioxidant activity of the peptide fractions was determined by radical scavenging ability and ferric reducing power. Fraction FIII showed the highest antioxidant activity, although slight differences could be expected in the antioxidant activity of the different fractions based on the amino acid composition. FIII was subjected to liquid chromatography and tandem mass spectrometry (LC–MS/MS) and two major compounds were identified: the compound with m/z 952.42, which could be mostly comprised by the carbohydrate fucose, and the peptide with m/z 1410.63. Three possible sequences were proposed and synthesised for this peptide, and the contribution of Leu or Hyp residues to the antioxidant and ACE-inhibitory activities of the resulting sequence was evaluated. The presence of Leu residues in the peptide sequence in replacement of Hyp seems to play an important role in the antioxidant and ACE-inhibitory activity.     

Isolation and characterization of 1,4-β-glucan 4-glucanohydrolases (EC 3.2.1.4) from a technical Trichoderma viride cellulase

The following enzyme activities were detected in a Trichoderma viride cellulase (Röhm 2230 B): 1,4-β-d-glucan cellobiohydrolase (C₁), 1,4-β-d-glucan 4-glucanohydrolase (C_x), β-glucosidase, β-galactosidase, polygalacturonase, proteinase, xylanase, amylase, esterase and ‘polyphenoloxidase’. Isolation of cellulolytic enzymes was performed starting with adsorption chromatography on Avicel SF, leading to separation of more than 90% of non-cellulolytic enzymes and 96% of β-glucosidase activity (= fraction A). In fraction A, 30% of the C_x activity was determined whereas, in a separated fraction, B, the remaining C_x and the total C₁ activity was established. Further fractionation of B using ion-exchange and gel chromatography resulted in the separation of three purified enzyme fractions, PI to PIII, with endo C_x activities. Additionally, C₁ activity was found in PIII. PI-PIII were characterized by means of their pH optima, isoelectric points and molecular weights.     

紫苏籽粕蛋白源抗氧化肽的纯化、结构鉴定及体外抗氧化活性

通过单因素和响应面实验对紫苏籽粕蛋白的闪提工艺进行了优化,将所获蛋白通过碱性蛋白酶酶解,酶解物依次通过超滤和柱层析进行分离纯化,并对抗氧化活性最高的多肽进行液质分析。结果表明,紫苏籽粕蛋白提取的最优工艺参数为：料液比1︰20(g/mL)、闪提pH 10、闪提时间30 s和闪提转速3000 rpm。蛋白酶解物经超滤分离后,其分子量小于3kDa的组分具有较高的抗氧化活性、该组分经DEAE-32离子交换色谱分离后, 得到D1、D2、D3共3个组分,其中D1的抗氧化活性最好,D1经Sephadex G-25凝胶层析分离纯化后,富集得到G1和G2两个组分,组分G1抗氧化活性最高。抗氧化肽G1对DPPH、ABTS、O_2^-、.OH 自由基清除率及还原力依次为98.97%、85.11%、91.83%、93.40%、0.477;通过LC-MS-MS鉴定,抗氧化活性肽G1组分主要由15个氨基酸组成,荷质比m/z为672.39,分子质量为1718.82 Da,其氨基酸序列为Glu－Met－Pro－Tur－lle－Ala－Ser－Met－Gly－lle－Tyr－Val－Val－Ser－Lys。     

The effect of germination on the phytase activity,phytate and total phosphorus contents of some Nigerian‐grown grain legumes

BACKGROUND: Grain legumes are under‐exploited as possible sources of phytase for the poultry industry. The current study was conducted to assess the effect of germination on phytase activities, phytate and total phosphorus content in samples of Nigerian‐grown grain legumes. The legumes screened were African yambean (AYB, Sphenostylis stenocarpa), lima bean (Phaseolus lunatus), pigeon pea (Cajanus cajan), cowpea (Vigna unguiculata) and groundnut (Arachis hypogea). RESULTS: Phytase activity was low in AYB, lima bean and pigeon pea but high in cowpea and groundnut. Phytate content ranged between 3.01 g kg^?1 and 8.95 g kg^?1 while total phosphorus content ranged between 2.63 g kg^?1 and 5.93 g kg^?1. The grain legumes with higher phytase activity recorded the lowest phytate and phosphorus content. During germination there was an initial 4‐fold to 35‐fold increase in phytase activity after 6–7 days of germination followed by a decrease until 10 days (P < 0.05). The increase in phytase activity during germination was accompanied by a significant reduction in phytate (P < 0.05) and a small but significant increase in total phosphorus. CONCLUSION: The increase in phytase activity and the accompanying decrease in phytate content could have a positive implication for the nutrition of poultry and ruminants and for the environment. Copyright © 2010 Society of Chemical Industry     

Purification and identification of ACE inhibitory peptides from Haruan (Channa striatus) myofibrillar protein hydrolysate using HPLC–ESI-TOF MS/MS

Haruan myofibrillar protein was hydrolysed with proteinase K and thermolysin to isolate Angiotensin converting enzyme (ACE) inhibitory peptides. The thermolysin hydrolysate of myofibrillar protein with the highest ACE inhibition activity (IC₅₀ = 0.033 mg/ml) was fractionated by ultrafiltration and size exclusion chromatography to three fractions. Fraction F2 with higher ACE inhibitory activity was separated into five fractions (A–E) using reversed-phased high performance liquid chromatography (RP-HPLC). Fraction C showed 81% inhibition activity and was subjected to HPLC coupled to electrospray ionisation-time-of-flight mass spectrometry (ESI-TOF MS/MS). Two peptide sequences for the most abundant fragments were identified as VPAAPPK (IC₅₀ = 0.45 μM) at 791.155 m/z and NGTWFEPP (IC₅₀ = 0.63 μM) at 1085.841 m/z. The presence of two proline residues at the C-terminal sequence is responsible for the high ACE inhibitory activity of these peptides. The results suggest that Haruan meat protein hydrolysate is a potent ACE inhibitor and may be used to decrease blood pressure.