Emulsification of chemical and enzymatic hydrolysates of beta-lactoglobulin: characterization of the peptides adsorbed at the interface

Bovine beta-Lactoglobulin (BLG) was cleaved by BNPS-skatole (2-(2'-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine), trypsin, or pepsin in 40% ethanol before emulsification with hexadecane in order to characterize the peptides active at the interfaces. The total digests and the different phases obtained after emulsification were analyzed by RP-HPLC to separate the peptides according to their gradual order on a hydrophilicity-to-hydrophobicity scale. In each case, hydrophobic peptides were recovered in the creamed phase and characterized by mass spectrometry and sequencing. After tryptic hydrolysis, short peptides were identified at the interfacial layer as fragments S21-L32, V41-L57, V41-K60, and W61-K70 linked to L149-I162 by a C66-C160 bond. It indicates that the hydrophilic/hydrophobic distribution of the amino acids in the sequence of the fragments is more relevant to adsorption than the length of the peptide. BNPS-skatole and peptic hydrolysis produced larger hydrophobic peptides which were also recovered in the creamed phase of the emulsion and characterized.     

Foaming characteristics of chemical and enzymatic hydrolysates of bovine β‐lactoglobulin

The foaming properties of bovine β‐lactoglobulin (BLG( BNPS‐skatole (2‐(2′‐nitrophenylsulfenyl)‐3‐methyl‐3′‐bromoindolenine) (BNPS( trypsin (T) and pepsin in 25, 30 and 40% ethanol (P25, P30, P40) hydrolysates were investigated in the 0.2 to 1 mg/ml range. Foaming capacity and foam stability were assessed in terms of drained liquid volume and foam volume. Foam texture was analyzed from video images obtained during foam decay. The foaming capacity of BNPS, P30 and P40 was similar to that of BLG and greater than that of T or P25. All hydrolysates except BNPS were less stable than BLG at all concentrations tested. This result was insured by texture analysis. Principal component analysis confirmed the distribution of the samples into three groups based on their increasing stability: (i): P25, (ii): P30 and P40, and (iii): BLG and BNPS. Tryptic hydrolysate had the poorest foaming properties. The results are considered in relation to the molecular characteristics of the peptides, particularly their size and hydrophobicity.     

Determination of reaction kinetics of hydrolysis of tilapia (Oreochromis niloticus) protein for manipulating production of bioactive peptides with antioxidant activity,angiotensin‐I‐converting enzyme inhibitory activity and Ca‐binding properties

To manipulate enzymatic hydrolysis of tilapia (Oreochromis niloticus) muscle protein for production of bioactive peptides, its reaction kinetics was intensively studied. The study showed that the production of peptides with different bioactive properties including antioxidant activity, angiotensin‐I‐converting enzyme (ACE) inhibition and Ca‐binding property and their kinetics were affected by the degree of hydrolysis and substrate concentration. A comparative study on reaction kinetics found that the kinetic parameters for the production of each bioactive peptide are unique, that is, the maximum initial velocity, V_max, for hydrolysis of protein was as high as 1.07 mg mL^?1 min^?1, but that for the production of peptides with antioxidant activity and Ca‐binding property were very low, range of 7.14–66.7 μg mL^?1 min^?1, and that for the production of peptides with ACE inhibitory activity was the lowest, at 2.57 μg mL^?1 min^?1. This knowledge of reaction kinetics of protein hydrolysis would be useful for manipulating and optimising the production of peptides with desired bioactive properties.     

Partial characterization of β‐ d‐xylosidase from wheat malts

Arabinoxylans (AXs) from wheat malts potentially affect beer quality and production. β‐ d ‐Xylosidase is a key enzyme that degrades the main chains of AXs to produce xylose. This study performed a partial characterization of β‐ d ‐xylosidase from wheat malts. The optimal temperature was 70 °C and the enzyme exhibited excellent thermostability, that is, residual activities were 92.6% at 60 °C for 1 h. The enzyme was stable over a pH range of 3.0–6.0 and showed optimum activity at pH 3.5 and 4.5. Kinetic parameters K_m and V_max of wheat malt β‐ d ‐xylosidase against p‐nitrophenyl‐xyloside were 1.74 mmol L⁻¹ and 0.76 m m min⁻¹, respectively. The enzyme activity was severely inhibited by Cu²⁺, moderately inhibited by Mn²⁺, Mg²⁺, Al³⁺, Ca²⁺, Ba²⁺ and Na⁺ and mildly inhibited by Fe³⁺ and Fe²⁺. The partial enzymatic characterization achieved in this study can be used as a theoretical basis for purifying β‐ d ‐xylosidase from wheat malts. Copyright © 2015 The Institute of Brewing & Distilling     

Characterization of an α‐amylase from sorghum (Sorghum bicolor) obtained under optimized conditions

Sorghum malt α‐amylase can compete with bacterial α‐amylase in industrial applications, if sufficiently stable and produced in a large enough quantity. Conditions for maximal α‐amylase production in sorghum malt and the physico‐chemical properties of the α‐amylase so produced are reported in this study. Sorghum grains were steeped in buffers with varying pH (4.0–8.0) for 24 h, at room temperature, and germinated for another 48 h to obtain the green malt. The buffer that induced the highest quantity of α‐amylase was chosen as the optimal pH and served as the medium for further steeping experiments conducted at different temperatures (10, 20, 30, 40, 50 and 60°C). The α‐amylase activity in the extract was determined in order to obtain the optimum temperature for α‐amylase induction at this particular pH. For the purpose of comparison, the α‐amylase produced at the optimum pH and temperature was purified to apparent homogeneity by a combination of ion‐exchange and size‐exclusion chromatography, and further characterized. Eight‐fold higher α‐amylase activity was induced in pH 6.5 buffer at 20°C compared with water, the traditional steeping medium. The K_m and V_max were estimated to be 1.092 ± 0.05 mg mL^?1 and 3516 ± 1.981 units min^?1, respectively. The activation energy of the purified amylase for starch hydrolysis was 6.2 kcal K^?1 mol^?1. Chlorides of calcium and manganese served as good activators, whereas CuSO₄ inhibited the enzyme with a 42% loss in activity at 312 mm salt concentration. Copyright © 2012 The Institute of Brewing & Distilling     

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8–11

A protease from sorghum malt variety KSV8–11 was purified by a combination of dialysis against 4 M sucrose, ion‐exchange chromatography on Q‐Sepharose (Fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 5‐fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg^?1 protein. SDS‐PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag⁺, Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺, iodoacetic acid (IAA) and p‐chloromercuribenzoate (p‐CMB), stimulated by Cu²⁺, Sr²⁺, phenylmethylsulfonyl‐fluoride (PMSF) and 2‐mercaptoethanol (2‐ME) while Mn²⁺ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K_m of 18 mg·mL^?1 and a V_max of 11.1 μmol · mL^?1 · min^?1 with casein as substrate.     

A preliminary study of monosaccharide composition and α‐glucosidase inhibitory effect of polysaccharides from pumpkin (Cucurbita moschata) fruit

In this work, crude polysaccharide extracts were extracted from pumpkin (Cucurbita moschata) fruit by hot water. After removal of proteins and purification, polysaccharides of pumpkin fruit (PP1‐1) were subjected to structural identification. Gas chromatography analysis indicated that PP1‐1 comprised of galactose (86.4%), and glucose (13.6%). The molecular weight of PP1‐1 was measured to be 0.87 × 10⁴ Da by gel permeation chromatography. The inhibitory kinetic evaluation showed that it was non‐competitive inhibition of PP1‐1 on the α‐glucosidase‐catalysed hydrolysis of PNPG. The Michaelis–Menten constant (K_m) was 0.106 m , and the inhibitory constants (K_i), 0.435 mg.     

HPLC analysis for trans-vitamin K1 and dihydro-vitamin K1 in margarines and margarine-like products using the C30 stationary phase

A C₃₀ column successfully separated the cis and trans isomers of vitamin K₁ in margarines, reduced-fat margarine-like products and in their ingredient oils. We also measured the compound 2′3′-dihydro-vitamin K₁, a derivative formed during hydrogenation of oils containing vitamin K₁. We compared an enzymatic procedure, currently under AOAC collaborative study for milk and infant formulas, with a more direct extraction method in analyzing margarines and margarine-like products. Both methods have good precision and were applicable to the majority of products examined. Margarines or margarine-like products identifying liquid soy-bean oil, hydrogenated soybean oil or liquid canola oil as their primary ingredients contained about 50–160 μg vitamin K₁/100 g. Blends of sunflower and soybean oils contained <50 μg vitamin K₁/100 g. Hardened or “stick” margarines contained more 2′3′-dihydro-vitamin K₁ than “soft” or “tub” products (122–285 μg/100 g vs 38–131 μg/100 g, respectively). Eight of 18 products (44%) contained 10% or more of the Reference Daily Intake for vitamin K₁ per serving. Higher-fat margarines contained more vitamin K₁ than their lower-fat counterparts.     

Activities and sequences of the angiotensin I‐converting enzyme (ACE) inhibitory peptides obtained from the digested lentil (Lens culinaris) globulins

The aim of this study was the identification of potentially bioaccessible ACE‐inhibitory peptides obtained by in vitro gastrointestinal digestion of lentil globulins. ACE‐inhibitory peptides were purified by ion exchange chromatography and gel filtration. After the first step of purification, three peptide fractions with potential antihypertensive properties were obtained and the highest inhibitory activity was determined for the fraction 5 (IC₅₀ = 0.02 mg mL^?1). This fraction was separated on Sephadex G10, and six peptide fractions were obtained. The peptides of fraction (5‐F) with the highest potential antihypertensive activity (IC₅₀ = 0.13 mg mL^?1) were identified using ESI‐MS/MS. The sequences of peptides were KLRT, TLHGMV and VNRLM. Based on Lineweaver–Burk plots for the fraction 5‐F, the kinetic parameters as K_m (1.24 mm ), V_max (0.012 U min^?1), K_i (0.12 mg mL^?1) and mode of inhibition were determined.     

Antioxidant activities of enzymatic‐hydrolysed proteins of dromedary (Camelus dromedarius) colostrum

This work investigated the antioxidant activities of dromedary colostrum proteins before and after hydrolysis by pepsin, trypsin, α‐chymotrypsin, pancreatin and papain. The enzymatic hydrolysis affected the degrees of hydrolysis, electrophoretic profiles, molecular weight distribution and hydrophobic/hydrophilic properties of the generated peptides. The antioxidant activities were evaluated using four antioxidant assays, including 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) radical‐scavenging activities, ferric reducing power and ferrous ion chelating activity. Interestingly, the antioxidant activities of dromedary colostrum proteins were enhanced after enzymatic hydrolysis. The highest antioxidant potential was obtained by pancreatic hydrolysates (P ≤ 0.05). These results suggest that dromedary colostrum protein hydrolysates are an important source of natural antioxidant peptides.     

Studies on the interaction of ‐epigallocatechin‐3‐gallate from green tea with bovine β‐lactoglobulin by spectroscopic methods and docking

The binding interaction between‐epigallocatechin‐3‐gallate (EGCG) and bovine β‐lactoglobulin (βLG) was thoroughly studied by fluorescence, circular dichroism (CD) and protein–ligand docking. Fluorescence data revealed that the fluorescence quenching of βLG by EGCG was the result of the formation of a complex of βLG–EGCG. The binding constants and thermodynamic parameters at two different temperatures and the binding force were determined. The binding interaction between EGCG and βLG was mainly hydrophobic and the complex was stabilised by hydrogen bonding. The results suggested that βLG in complex with EGCG changes its native conformation. Furthermore, preheat treatment (90 °C, 120 °C) and emulsifier (sucrose fatty acid ester) all boosted the binding constants (K_a) and the binding site values (n) of the βLG‐EGCG complex. This study provided important insight into the mechanism of binding interactions of green tea flavonoids with milk protein.     

STABILITY AND ENZYMATIC PROPERTIES OF β-GALACTOSIDASE FROM KLUYVEROMYCES FRAGILIS

Kluyveromyces fragilis β-galactosidase purified to electrophoretic, chromatographic and immunochemical homogeneity was used. The enzyme specifically required potassium ions for stability; MnCl₂ increased the stability. The enzyme was maximally stable at pH 6.5 to 7.5; stability was markedly less at pH's below 6.5 and above pH 8.5 at 37°C. Temperature denaturation followed first order kinetics with an activation energy for denaturation of 56 kcal/mol. Maximum activity was achieved in the presence of 5mM KCl. In potassium phosphate buffer, the enzyme was further activated by Mn²⁺, Mg²⁺, Co²⁺ and Zn²⁺; Mn²⁺, at 0.1 mM, gave the highest activation. None of these ions activated the enzyme in Tris buffer and> 0.1 mM Zn²⁺ caused complete loss of activity. Activity was completely inhibited by ethylenediaminetetraacetate and partially restored by addition of MnCl₂. p-Chloromercuribenzoate caused rapid loss of activity which could be restored by dithiothreitol. Iodoacetamide, N-ethylmaleimide and sodium tetrathionate did not inactivate the enzyme. The enzyme was specific for β-galactosides. K_m's for o-nitrophenyl β-D-galactopyranoside and lactose were 2.72 and 13.9 mM, respectively, at pH 6.6 D-Galactono-1, 4-lactone was a good competitive inhibitor (K_i=0.17 mM). pH optimum for hydrolysis of o-nitrophenyl β-D-galactopyranoside was 6.2–6.4. V_max for this substrate was dependent on two ionizable groups of pK_a of 6.13 and 6.51 while V_max/K_m was dependent on two ionizable groups of pK_a of 6.39 and 7.23. Activation energy for hydrolysis of o-nitrophenyl β-D-galactopyranoside at pH 7.0 was 9.1 kcal/mol in the range 20–40°C.     

Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on l‐arabinose and d‐xylose

Genes encoding l ‐arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on l ‐arabinose was dependent on a functioning l ‐arabinose transporter, or by screening a differential display library, respectively. These transporters also transport d ‐xylose and were designated KmAXT1 (arabinose–xylose transporter) and PgAXT1, respectively. Transport assays using l ‐arabinose showed that KmAxt1p has K_m 263 mm and V_max 57 nm /mg/min, and PgAxt1p has K_m 0.13 mm and V_max 18 nm /mg/min. Glucose, galactose and xylose significantly inhibit l ‐arabinose transport by both transporters. Transport assays using d ‐xylose showed that KmAxt1p has K_m 27 mm and V_max 3.8 nm /mg/min, and PgAxt1p has K_m 65 mm and V_max 8.7 nm /mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed K_m 371 mm and V_max 341 nm /mg/min for l ‐arabinose, and K_m 25 mm and V_max 76 nm /mg/min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both l ‐arabinose and d ‐xylose, one scenario for the complete usage of biomass‐derived pentose sugars would require only the low‐affinity, high‐throughput transporter Gal2p and one additional high‐affinity general pentose transporter, rather than dedicated d ‐xylose or l ‐arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering. Accession Nos: KmAXT1: GZ791039; PgAXT1: GZ791040 Copyright © 2015 John Wiley & Sons, Ltd.     

Purification and functional characterisation of an α‐l‐rhamnosidase from Penicillium citrinum MTCC‐3565

An extracellular α‐l ‐rhamnosidase from Penicillium citrinum MTCC‐3565 has purified to homogeneity from its culture filtrate using ethanol precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 45.0 kDa in SDS‐PAGE analysis showing the purity of the enzyme preparation. The native PAGE analysis showed the monomeric nature of the purified enzyme. Using p‐nitrophenyl α‐l ‐rhamnopyranoside as substrate, K_m and V_max values of the enzyme were 0.30 mm and 27.0 μm min mg^?1, respectively. The k_cat value was 20.1 s giving k_cat/K_m value of 67.0 mm s^?1 for the same substrate. The pH and temperature optima of the enzyme were 8.5 and 50 °C, respectively. The activation energy for the thermal denaturation of the enzyme was 29.9 KJ mol^?1. The α‐l ‐rhamnosidase was able to hydrolyse naringin, rutin and hesperidin and liberated l ‐rhamnose, indicating that the purified enzyme can be used for the preparation of α‐l ‐rhamnose and pharmaceutically important compounds by derhamnosylation of natural glycosides containing terminal α‐l ‐rhamnose. The α‐l ‐rhamnosidase was active at the level of ethanol concentration present in wine, indicating that it can be used for improving wine aroma.     

Purification,characterisation and application of α‐l‐rhamnosidase from Penicillium citrinum MTCC‐8897

An α‐l ‐rhamnosidase secreted by Penicillium citrinum MTCC‐8897 has been purified to homogeneity from the culture filtrate of the fungal strain using ammonium sulphate precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The sodium dodecyl sulphate/polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass 51.0 kDa. The native polyacrylamide gel electrophoresis also gave a single protein band confirming the enzyme purity. The K_m and V_max values of the enzyme for p‐nitrophenyl α‐l ‐rhamnopyranoside were 0.36 mm and 22.54 μmole min^?1 mg^?1, respectively, and k_cat value was 17.1 s^?1 giving k_cat/K_m value of 4.75 × 10⁴ m ^?1 s^?1. The pH and temperature optima of the enzyme were 7.0 and 60 °C, respectively. The purified enzyme liberated l ‐rhamnose from naringin, rutin, hesperidin and wine, indicating that it has biotechnological application potential for the preparation of l ‐rhamnose and other pharmaceutically important compounds from natural glycosides containing terminal α‐l ‐rhamnose and also in the enhancement of wine aroma.     

Phytosterols in banana (Musa spp.) flower inhibit α‐glucosidase and α‐amylase hydrolysations and glycation reaction

Three phytosterols were isolated from Musa spp. flowers for evaluating their capabilities in inhibiting glucosidase and amylase activities and glycation of protein and sugar. The three phytosterols were identified as β‐sitosterol (PS1), 31‐norcyclolaudenone (PS2) and (24R)‐4α, 14α, 4‐trimethyl‐5α‐cholesta‐8, 25(27)‐dien‐3β‐ol (PS3). IC₅₀ values (the concentration of inhibiting 50% of enzyme activity) of PS1, PS2 and PS3 against α‐glucosidase were 283.67, 11.33 and 43.10 μg mL^?1, respectively. For inhibition of α‐amylase, the IC₅₀ values of PS1, PS2 and PS3 were 52.55, 76.25 and 532.02 μg mL^?1, respectively. PS1 was an uncompetitive inhibitor against α‐amylase with K_m at 5.51 μg mL^?1, while PS2 and PS3 exhibited a mixed‐type inhibition with K_m at 52.36 and 2.49 μg mL^?1, respectively. PS1 and PS2 also significantly inhibited the formation of advanced glycation end products (AGEs) in a BSA–fructose model. The results suggest that banana flower could possess the capability in prevention of the diseases associated with abnormal blood sugar and AGEs levels, such as diabetes.     

An α‐l‐rhamnosidase from Aspergillus awamori MTCC‐2879 and its role in debittering of orange juice

The extracellular α‐l ‐rhamnosidase has been purified by growing a new fungal strain Aspergillus awamori MTCC‐2879 in the liquid culture growth medium containing orange peel. The purification procedure involved ultrafiltration using PM‐10 membrane and anion‐exchange chromatography on diethyl amino ethyl cellulose. The purified enzyme gave single protein band in SDS‐PAGE analysis corresponding to molecular mass 75.0 kDa. The native PAGE analysis of the purified enzyme also gave a single protein band, confirming the purity of the enzyme. The K_m and V_max values of the enzyme for p‐nitrophenyl‐α‐l ‐rhamnopyranoside were 0.62 mm and 27.06 μmole min^?1 mg^?1, respectively, yielding k_cat and k_cat/k_m values 39.90 s^?1 and 54.70 mm ^?1 s^?1, respectively. The enzyme had an optimum pH of 7.0 and optimum temperature of 60 °C. The activation energy for the thermal denaturation of the enzyme was 35.65 kJ^?1 mol^?1 K^?1. The purified enzyme can be used for specifically cleaving terminal α‐l ‐rhamnose from the natural glycosides, thereby contributing to the preparation of pharmaceutically important compounds like prunin and l ‐rhamnose.     

Cytoprotective effects of anthocyanins and other phenolic fractions of Boysenberry and blackcurrant on dopamine and amyloid β‐induced oxidative stress in transfected COS‐7 cells

There is growing interest both from consumers and researchers in the role that berries play in human health. In the experiments reported here, we assessed the ability of anthocyanins and phenolic fractions of Boysenberry and blackcurrant to ameliorate the deleterious effect of the amyloid β_25–35 (100 µmol L^?1, 24 h) and dopamine (1 mmol L^?1, 4 h) on calcium buffering (recovery) of M1 muscarinic receptor‐transfected COS‐7 cells. Cell viability was also studied. Our results demonstrate that extracts of Boysenberry and blackcurrant showed significant protective effect and restored the calcium buffering ability of cells that had been subjected to oxidative stress induced by dopamine and the amyloid β_25–35. Blackcurrant polyphenolics showed slightly higher protective effect against dopamine, whereas Boysenberry polyphenolics had a higher effect against the amyloid β_25–35. In viability studies, all extracts showed significant protective effects against dopamine and amyloid β_25–35‐induced cytotoxicity. Our results provide further evidence for the protective effects of berries against the neurotoxic effect of dopamine and amyloid β_25–35 in brain cells. Copyright © 2007 Society of Chemical Industry     

Angiotensin‐I converting enzyme inhibitory and antioxidant activities of peptide fractions extracted by ultrafiltration of cowpea Vigna unguiculata hydrolysates

BACKGROUND: Enzymatic proteolysis of food proteins is used to produce peptide fractions with the potential to act as physiological modulators. Fractionation of these proteins by ultrafiltration results in fractions rich in small peptides with the potential to act as functional food ingredients. The present study investigated the angiotensin‐I converting enzyme (ACE‐I) inhibitory and antioxidant activities for hydrolysates produced by hydrolyzing Vigna unguiculata protein extract as well as ultrafiltered peptide fractions from these hydrolysates. RESULTS: Alcalase^®, Flavourzyme^® and pepsin–pancreatin were used to produce extensively hydrolyzed V. unguiculata protein extract. Degree of hydrolysis (DH) differed between the enzymatic systems and ranged from 35.7% to 58.8%. Fractionation increased in vitro biological activities in the peptide fractions, with IC₅₀ (hydrolysate concentration in µg protein mL^?1 required to produce 50% ACE inhibition) value ranges of 24.3–123 (Alcalase hydrolysate, AH), 0.04–170.6 (Flavourzyme hydrolysate; FH) and 44.7–112 (pepsin–pancreatin hydrolysate, PPH) µg mL^?1, and TEAC (Trolox equivalent antioxidant coefficient) value ranges of 303.2–1457 (AH), 357.4–10 211 (FH) and 267.1–2830.4 (PPH) mmol L^?1 mg^?1 protein. CONCLUSION: The results indicate the possibility of obtaining bioactive peptides from V. unguiculata proteins by means of a controlled protein hydrolysis using Alcalase^®, Flavourzyme^® and pepsin–pancreatin. The V. unguiculata protein hydrolysates and their corresponding ultrafiltered peptide fractions might be utilized for physiologically functional foods with antihypertensive and antioxidant activities. Copyright © 2010 Society of Chemical Industry     

Effects of Sugars on the Formation of Nanometer‐Sized Droplets of Vegetable Oil by an Isothermal Low‐Energy Emulsification Method

Effects of sugars on the formation of nanometer‐sized oil droplets induced by the addition of vegetable oil to aqueous dispersions of polyoxyethylene sorbitan monooleate (MOPS, Tween 80) at 25 °C without the application of intensive mechanical energy were investigated. Phase diagrams were constructed using polarized light microscopy and small angle X‐ray scattering (SAXS) to elucidate the relationship between the type of phases involved in the process of emulsification and the droplet size in the resulting emulsions. Nanometer‐sized oil droplets as small as 220 nm in diameter were obtained when the sponge phase (L₃) was formed at first, followed by the phase transition to coexisting multiple phases including the micellar cubic phase (I₁) with increasing vegetable oil content. Sugars expanded the area of the sponge phase toward lower MOPS contents, enabling the formation of nano‐emulsions from a wider range of the initial composition. The area of the sponge phase increased in the order of d ‐fructose ≈ d ‐glucose < sucrose < d ‐maltose, consistent with the order of the literature value of the mean number of equatorial hydroxyl groups per sugar molecule and that of the hydration number of sugar that represents the average number of water molecules forming a complex with a single molecule of sugar in aqueous solution. The present results confirm that sugars facilitate the formation of nano‐emulsions using the isothermal low‐energy emulsification method, presumably due to their abilities to shift the effective hydrophile–lipophile balance (HLB) of the surfactant toward the hydrophobic side.