Improving antioxidant activities of whey protein hydrolysates obtained by thermal preheat treatment of pepsin,trypsin, alcalase and flavourzyme

Antioxidant activity of whey protein concentrate (WPC) hydrolysates was evaluated. Hydrolysates were obtained by pepsin, trypsin, alcalase and flavourzyme enzymatic reaction and preheat treatment of 95 °C for 5 or 10 min. The degree of hydrolysis (DH) was determined by 2,4,6‐trinitrobenzene sulphonic acid method, and antioxidant properties were determined by three spectrophotometric methods: ferricyanide method, ferric reducing/antioxidant power assay and diphenyl‐picryl hydrazinyl radical‐scavenging activity. For all the enzymes, briefly preheat treatment (95 °C/5 min) increased DH of WPC. Alcalase hydrolysates showed the highest antioxidant activity by three methods. The changes in antioxidant activity was coincidental with the changes in DH (R² = 0.988). Hydrolysates analysed by polyacrylamide gel electrophoresis and high performance liquid chromatography indicated that the α‐La was hydrolysed completely by pepsin, trypsin and alcalase and was resistant to flavourzyme to some extent; β‐lactoglobulin was only completely hydrolysed by trypsin and alcalase. Results indicated that antioxidant activity of hydrolysates was greatly related to the exposure of amino acid residues.     

Antioxidant,immunomodulatory and antiproliferative effects of gelatin hydrolysates from seabass (Lates calcarifer) skins

This study investigated the antioxidant, immunomodulatory and antiproliferative potentials of gelatin hydrolysates from seabass skins in cell model systems. Gelatin hydrolysates were extracted from seabass skins using different processes and enzyme concentrations. The ability of the hydrolysates to protect against H₂O₂‐induced DNA damage was assessed on U937 cells using the Comet assay, and one of the samples showed DNA protective effects. All samples showed immunomodulatory potential by significantly (P < 0.05) reducing interleukin‐6 (IL‐6) and IL‐1β production in lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophage cells. Antiproliferative activities of seabass skin hydrolysates were measured using human colon cancer (Caco‐2) and liver cancer (HepG2) cell lines as the model cell cultures. The inhibition of cell proliferation of Caco‐2 and HepG2 cancer cells occurred in a dose‐dependent manner at concentrations of 1–25 mg mL^?1. Therefore, seabass skin hydrolysates prepared using an appropriate process could serve as a potential functional food ingredient with various health benefits.     

In vitro antioxidant and immunomodulatory activity of transglutaminase-treated sodium caseinate hydrolysates

Sodium caseinate (NaCN) was incubated prior to and after hydrolysis with a microbial transglutaminase (TGase) and hydrolysed with Prolyve 1000. The resultant hydrolysates were tested for their immunomodulatory and antioxidant activity. TGase-treated hydrolysates significantly reduced (p < 0.05) the production of IL-6 at 0.5 and 1 mg mL⁻¹ and the non-TGase treated hydrolysate reduced the production of IL-6 at 1 mg mL⁻¹ in concanavalin (ConA) stimulated Jurkat T cells. None of the samples had an effect on IL-2. The hydrolysates showed higher oxygen radical absorbance capacity assay and ferric reducing antioxidant power activity than unhydrolysed NaCN, but no significant (p > 0.05) differences were found between the TGase-treated and non-TGase-treated samples. In the presence of hydrogen peroxide, the non-TGase-treated sample exhibited the highest DNA protective effect in U937 cells. These findings suggest that NaCN derived hydrolysates with and without treatment with TGase may exert specific antioxidant, genoprotective and anti-inflammatory effects.     

Purification and characterisation of antioxidative peptides from unfractionated rice bran protein hydrolysates

Crude rice bran protein (CRBP) was prepared by alkaline extraction and then treated with 0.6 m HCl to remove phytic acid. The phytate‐free rice bran protein (PFRBP) was hydrolysed with proteases M, N, S, P and pepsin under optimal conditions. Hydrolysates obtained from various hydrolysis periods were subjected to analysis for their degree of hydrolysis (DH) and functional properties. The hydrolysates were fractionated by reversed‐phase column chromatography on Kaseigel ODS resin (120–140 μm) using a stepwise gradient of aqueous ethanol, and their activities were measured. The 40% ethanol fraction of protease P 4 h‐hydrolysate was separated by successive reversed‐phase high‐performance liquid chromatography and the amino acid sequences of isolated antioxidative peptides were determined by a protein sequencer and matrix‐assisted laser desorption ionisation‐time of flight mass spectrometry. Crude rice bran protein had higher antioxidative activity than PFRBP, due to the presence of phytic acid. Phytate contents of rice bran, CRBP and PFRBP were 2.5%, 1.42% and 0%, respectively. The activity of PFRBP increased upon protease digestion. Protease M hydrolysates showed the highest DH, but the lowest antioxidative activity. Hydrolysates with DH below 10% had higher antioxidative activity than those above 20%. This result indicates that the antioxidative activity of the hydrolysates is inherent to their characteristics amino acid sequences of peptides depending on the protease specificities.     

Antioxidant activities of pepsin hydrolysates of water‐ and salt‐soluble protein extracted from pork hams

The objective of this study was to evaluate the antioxidant activities of pepsin‐digested water‐soluble protein (WSP) and salt‐soluble protein (SSP) extracted from pork ham. WSP and SSP were hydrolysed by pepsin for 2–10 h with 2‐h increments. The protein hydrolysates by pepsin were analysed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, and reducing power of the hydrolysates was measured. In addition, antioxidant activity of the hydrolysates was determined using linoleic acid emulsion system. Protein bands with molecular weight higher than 7 kDa in WSP and SSP were completely hydrolysed by pepsin after 2 h of digestion time. WSP hydrolysates (WSPH) and SSP hydrolysates (SSPH) had higher ferric reducing power than controls (WSP and SSP without pepsin digestion). Reducing powers of WSPH were higher than those of SSPH, regardless of digestion time. The oxidative activity of linoleic acid was predominantly inhibited by the addition of WSPH and SSPH, especially 0.5% protein hydrolysate. These results indicate that several functional peptides of pork protein digested by pepsin might have antioxidant activity, and thus they may be used as an antioxidant agent in muscle food system.     

In vitro antioxidant activity of protein hydrolysates prepared from corn gluten meal

BACKGROUND: Corn gluten meal (CGM), a major by‐product of corn wet milling, is mainly used as forage in China. Because of its particular amino acid composition, in which there are large amounts of hydrophobic amino acids such as leucine, alanine and phenylalanine, CGM protein was thought to be a good resource to obtain antioxidant peptides. CGM protein was hydrolysed with a biochemical grade alcalase and the derived hydrolysates were assessed for their antioxidant properties in different in vitro assay systems, including inhibiting activity on lipid peroxidation, by reducing power, hydroxyl radical scavenging activity, and DPPH radical scavenging activity. The effects of concentration and molecular weight (MW) of hydrolysates on antioxidant activity were investigated. RESULTS: The results showed that CGM hydrolysates were effective antioxidants, and there was a dose‐dependent relationship between hydrolysate concentration and antioxidant activity; the highest antioxidant activity was found in peptides 500–1500 Da, and the antioxidant activity of peptides below 500 Da or peptides above 1500 Da were all lower than that of total hydrolysates. CONCLUSIONS: The finding showed that the antioxidant activity of CGM hydrolysates was related to molecular weight and hydrolysate concentration, and the active antioxidant fraction should be in the peptides fraction of 500–1500 Da. CGM protein hydrolysates can be a source of natural antioxidant and used as a food additive. Copyright © 2008 Society of Chemical Industry     

The effect of lactic acid bacteria fermentation on the antioxidant activity of wheat gluten pancreatin hydrolysates

The antioxidant activities of the fermented wheat gluten hydrolysates with different fermentation times were investigated to elucidate the impact of lactic acid bacteria (LAB) fermentation on the wheat gluten hydrolysates. Prior to LAB fermentation, wheat gluten was deamidated by hydrochloric acid and then hydrolysed by pancreatin to 12 and 24 h, respectively. Results showed that LAB fermentation had significant impacts on the enzymatic efficiency and antioxidant activities of wheat gluten. The degree of hydrolysis and protein recovery of hydrolysates gradually increased and then reached maximum values, respectively, when fermenting with LAB for 36 h. The hydrolysis degree and protein recovery of fermented pancreatin 24‐h hydrolysates were larger than that of the fermented pancreatin 12‐h hydrolysates during the whole fermentation. The antioxidant activity analysis revealed a marked increase and improvement in the scavenging activities of 1,1‐Diphenyl‐2‐picrylhydrazyl·radicals, hydroxyl radicals and oxygen radical absorbance capacity, while the scavenging activities of ABTS⁺ radical decreased as the fermentation time extended. The antioxidant activities of pancreatin 24‐h hydrolysates were higher than that of the pancreatin 12‐h hydrolysates during the whole LAB fermentation.     

Fractionation and characterisation for antioxidant activity of hydrolysed whey protein

Whey protein isolate (WPI) was hydrolysed for 1 h using Alcalase, Protamex and Flavourzyme. Native WPI, hydrolysed WPI and two commercial WPI hydrolysates were subjected to fractionation by size exclusion chromatography. Antioxidant activity of WPI fractions was measured with a liposome‐oxidising system (50 µM FeCl₃/0.1 µM ascorbate, pH 7.0). Lipid oxidation was measured as thiobarbituric acid‐reactive substances (TBARS). Gel electrophoresis and amino acid analysis were run to identify the peptide composition. The influence of amino acid composition on antioxidant activity was evaluated using multivariate analysis methods (correlation analysis, principal component analysis, multiple linear regression and discriminant analysis). TBARS assays indicated the presence of antioxidant activity in all protein fractions, including non‐hydrolysed WPI. For native and hydrolysed WPI samples the first fraction (> 45 kDa) showed a higher TBARS inhibition effect (24–27%) when compared with lower‐molecular‐weight fractions and hydrolysate mixtures. In contrast, for commercial WPI hydrolysates a higher inhibitory effect was found in most of the lower‐molecular‐weight fractions (30–55%). The ability of WPI fractions to delay lipid oxidation was found to be related to the prevalence of histidine and hydrophobic amino acids. Copyright © 2004 Society of Chemical Industry     

Purification and characterization of antioxidative peptides derived from rice bran protein hydrolysates

Rice bran protein fraction (RBPF)—albumin, globulin, glutelin and prolamin were hydrolyzed with proteases M, N, P, S and pepsin under their optimal conditions for 24 h. Hydrolysates of various hydrolysis periods were collected and subjected to peptide mapping and the antioxidative activity measured by the 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) method. Protease M hydrolysates showed high degree of hydrolysis (DH), but low antioxidative activity. On the contrary, pepsin hydrolysates showed low DH with high activity. Albumin and globulin hydrolysates had higher DH values, but lower values for glutelin and prolamin. The globulin hydrolysate (Opep2) from 2 h-pepsin hydrolysis was separated by using three consecutive purification steps with RP-HPLC. Nineteen antioxidative peptides were isolated and their amino acid sequences were determined by a gas-phase protein sequencer and MALDI-TOF mass spectrometry. These peptides were composed of 6–30 amino acid residues with molecular masses ranging from 670–3,611 Da. Tyr-Leu-Ala-Gly-Met-Asn had the highest antioxidative activity among them.     

Antioxidant, antihypertensive and antimicrobial properties of ovine milk caseinate hydrolyzed with a microbial protease

BACKGROUND: Bioactive peptides might be released from precursor proteins through enzymatic hydrolysis. These molecules could be potentially employed in health and food products. In this investigation, ovine milk caseinate hydrolysates obtained with a novel microbial protease derived from Bacillus sp. P7 were evaluated for antioxidant, antimicrobial, and angiotensin I‐converting enzyme (ACE)‐inhibitory activities. RESULTS: Antioxidant activity measured by the 2,2′‐azino‐bis‐(3‐ethylbenzothiazoline)‐6‐sulfonic acid method increased with hydrolysis time up to 2 h, remaining stable for up to 4 h. Hydrolysates showed low 2,2‐diphenyl‐1‐picrylhydrazyl radical‐scavenging abilities, with higher activity (31%) reached after 1 h of hydrolysis. Fe²⁺‐chelating ability was maximum for 0.5 h hydrolysates (83.3%), decreasing thereafter; and the higher reducing power was observed after 1 h of hydrolysis. ACE‐inhibitory activity was observed to increase up to 2 h of hydrolysis (94% of inhibition), declining afterwards. 3 h hydrolysates were shown to inhibit the growth of Bacillus cereus, Corynebacterium fimi, Aspergillus fumigatus, and Penicillium expansum. CONCLUSION: Ovine caseinate hydrolyzed with Bacillus sp. P7 protease presented antioxidant, antihypertensive, and antimicrobial activities. Hydrolysis time was observed to affect the evaluated bioactivities. Such hydrolysates might have potential applications in the food industry. Copyright © 2011 Society of Chemical Industry     

Antioxidant effectiveness of coffee extracts and selected constituents in cell‐free systems and human colon cell lines

Scope: Epidemiological studies suggest that coffee can reduce the risk of degenerative diseases such as diabetes type 2, cardiovascular disease and cancer. These beneficial effects have partly been attributed to the antioxidant activity of coffee. We determined composition and antioxidant potential of differentially roasted coffee extracts and investigated the impact of selected original constituents and roast products. Methods and results: Parameters studied were direct antioxidant activity (trolox equivalent antioxidant capacity/oxygen radical absorbing capacity), cellular reactive oxygen species (ROS) level, DNA damage and protein expression of NAD(P)H: quinone oxidoreductase, γ‐glutamylcysteine ligase and glutathione reductase in HT‐29/Caco‐2 cells at 24‐h incubation. All extracts showed distinct direct antioxidant activity: medium roasts>light roast AB1 (caffeoylquinic acid (CQA)‐rich Arabica Brazil extract); dark roast AB2 (N‐methylpyridinium (NMP)‐rich Arabica Brazil extract), and diminished t‐butylhydroperoxide‐induced ROS level in HT‐29 cells (AB2>medium roasts>AB1). NAD(P)H:quinone oxidoreductase 1 expression and γ‐glutamylcysteine ligase expression were distinctly induced by AB1 and 5‐CQA, but not by AB2 and NMP. 5‐CQA and caffeic acid exhibited highest trolox equivalent antioxidant capacity/oxygen radical absorbing capacity values (5‐CQA: 1.3/3.5 mM and caffeic acid: 1.3/3.9 mM trolox); ROS level was distinctly diminished by 5‐CQA (≥3 μM), catechol (30 μM) and trigonelline (≥30 μM), whereas menadione‐induced DNA damage in Caco‐2 cells was reduced by NMP compounds (1–30 μM). Conclusion: The results emphasize that both original constituents and roast products contribute to the cellular antioxidant effectiveness of coffee.     

Immunoreactive properties of peptide fractions of cow whey milk proteins after enzymatic hydrolysis

Commercial whey protein concentrate (WPC) was hydrolysed with either Alcalase 2.4 FG (Novo Nordisk), or papain (Sigma) (in one‐step process) or with two enzymes (in two‐step process) to determine the changes in the immunoreactivity of α‐lactalbumin and β‐lactoglobulin. Enzymatic hydrolysis of WPC was performed by pH‐stat method. Hydrolysates were analysed using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, immunoblotting and size‐exclusion chromatography (SE‐HPLC). Immunoreactive properties of peptide fractions separated from the hydrolysates by fast protein liquid chromatography (FPLC) were determined using dot‐immunobinding and enzyme‐linked immunosorbent assay (ELISA) methods. Finally the sensory analysis was used to confirm organoleptic changes resulting from the application of different enzymes. The ‘two‐step’ process was observed to be the most effective however allergenic epitopes were still present, as it was found by ELISA with anti‐α‐la and anti‐β‐lg antibodies. The addition of papain as the second enzyme in the hydrolysis process contributed to the improvement of the sensory properties of WPC hydrolysate as compared with the Alcalase hydrolysate. Alcalase‐papain partially hydrolysated WPC can be found a promising base for production of the tolerogenic formula.     

桑叶蛋白抗氧化肽的酶法制备

为了提高桑叶蛋白MLP的抗氧化活性,用碱性蛋白酶Alcalase、复合蛋白酶Protamex、木瓜蛋白酶Papain、风味蛋白酶Flavourzyme、中性蛋白酶Neutrase及胰蛋白酶Trypsin等6种蛋白酶对MLP进行单酶酶解及双酶、三酶复合酶解,并对酶解前后的化学组成、分子量分布、多肽得率、氨基酸组成、自由基清除能力、还原能力等进行对比分析。结果表明,MLP主要由分子量大于6.5 ku的大分子肽及蛋白质组成,酶解物则主要由分子量为0.3~0.6 ku的小肽及0.6~6.5 ku的多肽组成;相较于过度酶解,适度酶解能更好的改善MLP的抗氧化活性;多肽得率与自由基清除能力显著正相关(r=0.916~0.985);6种蛋白酶中,碱性蛋白酶、中性蛋白酶及复合蛋白酶的酶解物抗氧化活性显著优于MLP及其他三种蛋白酶解物;中性蛋白酶单独酶解物的抗氧化活性显著优于双酶、三酶复合酶解物。对中性蛋白酶的单酶酶解条件进行优化,结果表明底物浓度为20 mg/mL,E/S为1%(W/W),用中性蛋白酶酶解2 h所得的酶解物(NH)的抗氧化活性最高,后期研究中可选用中性蛋白酶制备桑叶蛋白抗氧化肽。NH或许可以作为食品中较有潜力的抗氧化剂。     

Radical scavenging properties of protein hydrolysates from Jumbo flying squid (Dosidicus eschrichitii Steenstrup) skin gelatin

Gelatin extracted from Jumbo flying squid skin was hydrolyzed with serials of protease. The scavenging effects of gelatin hydrolysates on superoxide and hydroxyl radical were assessed by chemiluminescence analysis. Properase E and pepsin have shown efficient hydrolysis of squid skin gelatin to obtain high radical scavenging activity peptides. The conditions chosen for enzymic hydrolysis of squid skin gelatin with properase E were pH 9.0, 45 °C, 3 h reaction time, and enzyme‐to‐substrate ratio of 1:50. Hydrolysates combining properase E and pepsin showed the best radical scavenging activity. For fragmented hydrolysates by ultrafiltration, fractions UF‐2 (M_w < 2000 Da) had high yield and radical scavenging activity. Copyright © 2006 Society of Chemical Industry     

Biological detoxification of waste house wood hydrolysate using Ureibacillus thermosphaericus for bioethanol production

Hydrolysates of lignocelluloses hydrolyzed by diluted sulfuric acid contain toxic compounds that inhibit ethanol production by Saccharomyces cerevisiae and the ethanologenic recombinant Escherichia coli KO11. We investigated the biological detoxification of a hydrolysate of waste house wood (WHW) by a thermophilic bacterium, Ureibacillus thermosphaericus. When the hydrolysate was treated with this bacterium at 50 degrees C for 24 h, the ethanol production rate by S. cerevisiae increased markedly and was comparable to that for the hydrolysate treated with an excess amount of calcium hydroxide (overliming). Chromatographic analysis of synthetic hydrolysates containing furfural or 5-hydroxymethyl furfural that are considered to be major toxic compounds in hydrolysates revealed that U. thermosphaericus degrades these compounds. In the WHW hydrolysates, however, the concentrations of these compounds were not decreased markedly by the bacterium. These results suggest that the bacterium degrades minor but more toxic compounds or phenolic compounds in the WHW hydrolysates. The combination of bacterial and overliming treatments of hydrolysates minimized significantly the decrease in ethanol production rate by E. coli KO11 as fermentation proceeded. Because the bacterium grows rapidly and does not consume sugars, our biological detoxification should be useful for bioethanol production from acid hydrolysates of lignocelluloses.     

Biochemical,biological and histological evaluation of some culinary and medicinal herbs grown under greenhouse and field conditions

BACKGROUND: Increasing evidence supports the potential health benefits of herbal extracts displaying antioxidant, anti‐inflammatory and antitumour activities. Environment can have a pronounced effect on phenolic content and antioxidant capacity. The objectives of this study were to evaluate the total phenolic contents and antioxidant capacities of five different herbs grown under greenhouse and field conditions and to assess their potential anti‐inflammatory effects. RESULTS: High total polyphenolic (TPP) content (measured by the Folin‐Ciocalteu reagent method) and high Trolox equivalent antioxidant capacity (TEAC) were observed in all herbs evaluated. Leaves from thyme, sage, spearmint and peppermint grown in the greenhouse showed significantly higher TPP content and TEAC than those grown under field conditions, with a threefold difference being observed in peppermint. Rosemary, spearmint and peppermint extracts showed stronger inhibition of cyclooxygenase COX‐2 than of COX‐1. CONCLUSION: The results show that producing herbs under greenhouse conditions can improve their biological activities by increasing TPP contents and antioxidant capacities. The selective inhibition of COX‐2 activity by rosemary, spearmint and peppermint suggests that they may be useful as anti‐inflammatory agents with fewer side effects than regular non‐steroidal drugs. Copyright © 2010 Society of Chemical Industry     

Comparison of bioactive peptides prepared from sheep cheese whey using a food‐grade bacterial and a fungal protease preparation

Novel bacterial (HT) and fungal (FPII) food‐grade protease preparations were evaluated for their ability to hydrolyse sheep cheese whey (SCW) and the generation of bioactive peptides. Both protease preparations hydrolysed the whey proteins to small peptides over 24‐h hydrolysis time, but the time course hydrolysis profiles were different as evaluated by SDS‐PAGE. The HT whey hydrolysate had considerably higher antioxidant and angiotensin‐I converting enzyme (ACE)‐inhibitor activity than the FPII hydrolysate. Neither hydrolysate was cytotoxic towards Vero cells. OFFGEL electrophoresis of the small peptide pool fraction (<15 amino acids) of each hydrolysate indicated differences in the pI distribution of the bioactive peptides. This likely reflects the diverse hydrolytic specificity of the proteases. Although the antioxidant activity of both hydrolysates was not significantly affected by simulated gastrointestinal digestion, the loss of ACE‐inhibitor activity was greater with the FPII hydrolysate.     

In vitro antioxidant activities of hydrolysates obtained from Iranian wild almond (Amygdalus scoparia) protein by several enzymes

A protein extract from wild almond was hydrolysed using five different enzymes (pepsin, trypsin, chymotrypsin, alcalase and flavourzyme). The hydrolysates were then assayed for their antioxidant activities. The highest extent of proteolysis was obtained with alcalase (0.35; determined as the change in the absorbance at 340 nm, ΔA₃₄₀) and the lowest was with pepsin (ΔA₃₄₀ = 0.12). Radical scavenging activities obtained by 2, 2′‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulphonic acid) and ferric‐reducing abilities of the hydrolysates demonstrated that the hydrolysate from alcalase had significantly (P < 0.05) greater antioxidant activity. Analysis of the molecular weight distributions showed that peptides produced by alcalase were smaller than those produced by chymotrypsin, trypsin and flavourzyme. Based on the current study, the hydrolysates produced by alcalase can be suggested as potential antioxidant agents in food industry and for use in functional foods.     

Effect of bovine colostrum on the growth inhibition and differentiation of human leukemic U937 cells

BACKGROUND: Colostrum is the breast milk of female mammals produced within in a short time after giving birth. It is thought to protect neonates from infection as well as to facilitate immune system maturation. In this study the effect of bovine colostrum on the proliferation and differentiation of human leukemic U937 cells was investigated to understand more about its immunomodulatory activity. RESULTS: Human mononuclear cells (MNCs) were stimulated with bovine colostrum (CS) and then filtered to obtain a conditioned medium (CM) (CS‐MNC‐CM). CS‐MNC‐CMs prepared with day 1 to day 4 colostrums inhibited U937 cells by 39.4–64.4%. The expressions of surface markers CD11b and CD14 on U937 cells in the treated groups were 30.6–33.5% and 40.0–42.6% respectively. High levels of cytokines IL‐1β, IFN‐γ and TNF‐α were detected in CS‐MNC‐CMs. CONCLUSION: Evidence indicates that colostrums stimulate human MNCs to secrete cytokines IL‐1β, IFN‐γ and TNF‐α which subsequently inhibit the growth of U937 cells and induce their differentiation into mature monocytes and macrophages. There is a possibility for bovine colostrum to be processed into an anti‐leukemia ingredient for use in health foods. Copyright © 2009 Society of Chemical Industry