Influence of enzyme treatment of rice bran on gamma-aminobutyric acid synthesis by Lacto bacillus

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system. The production GABA from the cheaper glutamic acid is a valuable process. Rice bran is a potential source of glutamic acid decarboxylase which was therefore selected for production of gamma-aminobutyric acid (GABA) using lactic acid bacteria (Lactobacillus brevis (Lb.brevis) VTCC-B397). The influences of treatment of defatted rice bran with α-amylase, Alcalase (Al), Flavourzyme (Fl) and their combinations at different concentration ratios on the GABA synthesis were examined. With α-amylase impacts, concentration of GABA (6.41 mg mL⁻¹) was about 2.38 times higher than the control sample (2.69 mg mL⁻¹) under hydrolysis conditions of pH 6.5, temperature of 95 °C and α-amylase concentration of 0.15% (v/w) for 45 min. The α-amylase hydrolysis as prerequisite for protein hydrolysis using Al: Fl (ratio Al: Fl of 3: 7, concentration of 2% (w/w), pH = 8, temperature of 50 °C) reached the highest GABA synthesis efficiency, at 8.43 mg mL⁻¹. Results showed that the enzyme treatment positively affected the fermentation using Lb. brevis for GABA biosynthesis from defatted rice bran.     

Isolation of bovine serum albumin from whey using affinity chromatography

The adsorption of bovine serum albumin (BSA) to a chromatography resin with immobilised llama antibody fragments as affinity ligands was investigated. The maximum adsorption capacity of the affinity resin was 21.6 mg mL⁻¹ with a Langmuir equilibrium constant of 20.4 mg mg⁻¹. Using packed bed chromatography, BSA was adsorbed from pure BSA solutions. Recovery was achieved by desorption at pH 3. In experiments with initial BSA concentrations of 1 mg mL⁻¹ and 0.1 mg mL⁻¹, BSA could be concentrated to 6.9 and 7.7 mg mL⁻¹, respectively. BSA was also isolated from filtered bovine cheese whey containing less than 0.1 mg mL⁻¹ BSA. The purified BSA was in this case concentrated to 7.4 mg mL⁻¹ BSA. The presence of other components in the feedstock did not alter the adsorption capacity of the affinity resin. The results show that high recovery combined with high purity can be obtained using affinity chromatography.     

Selective separation and characterisation of dual ACE and DPP-IV inhibitory peptides from rainbow trout (Oncorhynchus mykiss) protein hydrolysates

Microwave pretreatment and hydrolysis were applied to rainbow trout (Oncorhynchus mykiss) by-products to produce bioactive peptides with dual in vitro angiotensin-I converting enzyme (ACE) and dipeptidyl-peptidase IV (DPP-IV) inhibitory activities. Peptides were fractionated using the single step electrodialysis with ultrafiltration membrane (EDUF). Concentration of cationic peptides (CP) increased in the recovery solution, reaching 125 μg mL⁻¹ after a 4-h treatment with migration rate of 15.68 ± 2.98 g m⁻² h. CP fractions displayed ACE and DPP-IV I inhibitory properties, with IC₅₀ values of 0.0036 mg mL⁻¹ and 1.23 mg mL⁻¹ respectively. The bioactivity was attributed to the low molecular weight peptides (300–500 Da) recovered. CP exhibited non-competitive inhibition patterns for ACE and DPP-IV, which were dose dependent. These results showed that bioactive peptides can successfully be separated from complex hydrolysate mixtures by EDUF. The fractionated peptides can serve as potential functional food ingredients or nutraceuticals for the management of hypertension and diabetes.     

Pancreatin-induced coalescence of oil-in-water emulsions in an in vitro duodenal model

The behaviour of cationic lactoferrin-stabilized and anionic β-lactoglobulin (β-lg)-stabilized oil-in-water emulsions (20.0% (w/w) soy oil, 1.0% (w/w) protein) in the presence of simulated intestinal fluid (SIF) containing physiological concentrations of pancreatin (0.0–10.0 mg mL^?1) and/or bile salts (0.0–25.0 mg mL^?1) at 37 °C, pH 7.5 and inorganic salts (39 mm K₂HPO₄, 150 mm NaCl and 30 mm CaCl₂) was investigated. Both emulsions showed a significant degree of coalescence and fatty acid release on mixing with SIF. Appreciably negative ζ-potential values (≥?50 mV) for both types of emulsion droplet at the highest pancreatin/bile salts concentration could be attributed to displacement of and/or binding to the interfacial proteins by bile salts, together with interfacial proteolysis by pancreatin, which enhanced the potential for lipase to act on the hydrophobic lipid core, thus generating free fatty acids and possibly mono- and/or diglycerides at the droplet surface.     

The enhancement of gastrointestinal digestibility of β-LG by dynamic high-pressure microfluidization to reduce its antigenicity

Dynamic high-pressure microfluidization (DHPM) can increase the antigenicity of β-lactoglobulin (β-LG) in previous researches. We observed in this study that the antigenicity of DHPM-modified β-LG at pressures of 0.1, 80 and 160 MPa after in vitro digestion declined from 13.41 to 12.27 and 7.19 μg mL⁻¹, gradually. The enhancement of gastrointestinal digestibility of β-LG was related to the aggregation state and conformational changes induced by DHPM and was reflected by the electrophoretic bands of low molecular weight (5 and 10 kDa) shown in electrophoresis, the reduction of particle size, the generation of smaller peptide fragments (m/z 837.444, 955.492 and 1215.616) detected by mass spectrum and the increase in surface hydrophobicity. These changes contributed to the decrease in antigenicity. Simultaneously, a schematic diagram was proposed to demonstrate the conceivable mechanism of antigenicity changes of modified β-LG after in vitro digestion.     

Effects of simulated gastrointestinal digestion on the physicochemical properties,erythrocyte haemolysis inhibitory ability and chemical antioxidant activity of mulberry leaf protein and its hydrolysates

Proteins and peptides must be degraded and modified in the gastrointestinal (GI) tract prior to absorption; this process changes their physicochemical and biological properties. Mulberry leaf protein (MP) and its hydrolysates (HMP) have favourable antioxidant activities. To investigate, in vitro and separately, the effects of GI digestion and intestinal digestion on the stability of MP and HMP, we monitored the changes in secondary structures, amino acids, molecular weights and antioxidant activities. We found that MP was more hydrolysed by pepsin than by pancreatin, unlike HMP. The final digests of MP and HMP were mainly composed of polypeptides (0.5–6.5 kDa) and oligopeptides (<0.5 kDa), respectively. The GI digestion influenced MP and HMP differently; GI digestion increased the antioxidant efficiency of MP and decreased that of HMP. For the intestinal digests, the antioxidant activities of MP and HMP also differed. The 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) and 2,2′‐azinobis (3‐ethylbenzothiazoline‐6‐sulphonic acid) diammonium salt (ABTS) radical quenching abilities of MP and HMP at 1 mg mL^?1 were comparable to or exceeded that of L‐glutathione (GSH) and 6‐hydroxy‐2,5,7,8‐tetramethychroman‐2‐carboxylic acid (trolox). Meanwhile, the erythrocyte haemolysis rates of MP, HMP and their GI products at 0.05–1.0 mg mL^?1 were significantly lower than that of the 2,2′‐azobis (isobutyramidine) dihydrochloride (AAPH) control. Both MP and HMP can be used as natural antioxidants and may promote digestive health.     

Dietary interference on the oxidation and hydrolysis of liposomes during in vitro digestion

In this study, the influence of dietary compounds, including antioxidants (ethylenediaminetetraacid, ascorbic acid, catechin), chitosan and lactoferrin, on the stability of liposomes during in vitro adult and infant digestion was investigated in terms of formation of thiobarbituric acid-reactive substances (TBARS) and free fatty acid release. Liposomes were more sensitive to degradation in adults than in infants, suggesting lower pH (1.5) and higher concentration of pancreatic enzymes (3.2 mg mL⁻¹) and bile salts (5 mg mL⁻¹) that indicated greater damage to the structure of liposomes. Compared to ethylenediaminetetraacid and ascorbic acid, catechin presented the most apparent protective effect against liposomal oxidation by scavenging free radicals. Chitosan promoted the formation of TBARS, while lactoferrin facilitated the hydrolysis rate of liposomes under both adult and infant conditions. This study provided an insight into the development of healthcare products and functional foods related to liposomes for special populations.     

Quantification of major camel milk proteins by capillary electrophoresis

Proteins from dromedary camel milk (CM) produced in Europe were separated and quantified by capillary electrophoresis (CE). CE analysis showed that camel milk lacks β-lactoglobulin and consists of high concentration of α-lactalbumin (2.01 ± 0.02 mg mL⁻¹), lactoferrin (1.74 ± 0.06 mg mL⁻¹) and serum albumin (0.46 ± 0.01 mg mL⁻¹). Among caseins, the concentration of β-casein (12.78 ± 0.92 mg mL⁻¹) was found the highest followed by α-casein (2.89 ± 0.29 mg mL⁻¹) while κ-casein represented only minor amount (1.67 ± 0.01 mg mL⁻¹). These results were in agreement with sodium dodecyl sulphate-polyacrylamide gel electrophoresis patterns. Overall, CE offers a quick and reliable method for the determination of major CM proteins, which may be responsible for the many nutritional and health properties of CM.     

Mixed fermentation of jujube juice (Ziziphus jujuba Mill.) with L. rhamnosus GG and L. plantarum-1: effects on the quality and stability

This study was conducted aiming at improving the quality of jujube juice by mixed fermentation of lactobacilli. Selection of favourable lactobacilli and addition of nitrogen sources were explored as an efficient method to improve the viability of probiotic in jujube juice. After fermentation, the viability increased to 9.15 ± 0.10 Log CFU mL⁻¹, while the content of lactic acid increased to 5.61 ± 0.03 mg mL⁻¹. The total phenolic and total flavonoid content were 2663.03 ± 11.95 μg mL⁻¹ and 163.95 ± 0.47 μg mL⁻¹ respectively. Moreover, the stability of fermented jujube juice during refrigeration was investigated, which showed that the viability dropped to 8.84 ± 0.6 Log CFU mL⁻¹ and the concentration of lactic acid slowly increased to 6.51 ± 0.04 mg mL⁻¹; the ABTS value showed a 4.26% reduction and FRAP value did not significantly (P < 0.05) change during refrigerated storage. In addition to the existing knowledge, our data aid to the future applications of the jujube as a potential ingredient in novel probiotic foods formulation.     

Anti‐acetylcholinesterase,antidiabetic, anti‐inflammatory,antityrosinase and antixanthine oxidase activities of Moroccan propolis

Biological properties of Moroccan propolis have been scarcely studied. In the present work, the total phenols and flavonoids from 21 samples of propolis collected in different places of Morocco or 3 supplied in the market were determined, as well as the in vitro capacity for inhibiting the activities of acetylcholinesterase, α‐glucosidase, α‐amylase, lipoxygenase, tyrosinase, xanthine oxidase and hyaluronidase. The results showed that samples 1 (region Fez‐Boulemane, Sefrou city) (IC₅₀ = 0.065, 0.006, 0.020, 0.050, 0.014 mg mL^?1) and 23 (marketed) (IC₅₀ = 0.018, 0.002, 0.046, 0.037, 0.008 mg mL^?1) had the best in vitro capacity for inhibiting the α‐amylase, α‐glucosidase, lipoxygenase, tyrosinase and xanthine oxidase activities, respectively. A negative correlation between IC₅₀ values and concentration of phenols, flavones and flavanones was found. These activities corresponded to the generally higher amounts of phenols and flavonoids. In the same region, propolis samples have dissimilar phenol content and enzyme inhibitory activities.     

Variation in in vitro digestibility of barley protein.

In vitro digestibility of protein was measured with pepsin/pancreatin in 321 spring barley lines grown in the field. The variation in digestibility was far less than the variation in the protein content. A small environmental influence on the digestibility was found. Two entries had slightly impaired digestibilities; these findings were partially verified in a repeated field trial, but were not confirmed in vivo. In vitro digestibilities of barleys grown in pots at various N-levels were positively correlated with protein or hordein content. In vitro digestibility was negatively correlated with the fibre content in decortification fractions of Bomi and the high-lysine mutant Risø 1508. The digestibility was positively correlated with the hordein concentration for the Bomi fractions, but not for the 1508 fractions.     

Calcium and chitosan-mediated clustering of whey protein particles for tuning their colloidal stability and flow behaviour

Whey protein microgel (WPM) particles were prepared by heating whey protein dispersions (40 mg mL⁻¹) at pH 5.8 and 80 °C for 15 min. Supplementation with 5 mm CaCl₂ at pH 6.0 increased the hydrodynamic diameter of WPMs, but did not influence the ζ-potential of the particles. The latter was attributed to clustering of WPMs by Ca²⁺ bridges. Supplementing WPM suspension with chitosan (0.5 mg mL⁻¹) at pH 5.0 caused formation of micron-sized clusters and a progressive increase in suspension viscosity over the subsequent 48 h. The latter was ascribed to development of a complexed lattice that inhibited sedimentation of WPMs. Immediately after chitosan supplementation, the WPM suspension changed from Newtonian to shear-thinning fluid. Fourier transform infra-red spectroscopy results suggested that microgelation proceeded by formation of inter-molecular β-sheets and loss of α-helix structure. It also indicated that chitosan may interact with WPMs at pH 6.0 through its acetyl groups.     

Iron‐encapsulated cold‐set whey protein isolate gel powder – Part 1: Optimisation of preparation conditions and in vitro evaluation

A central composite design with a quadratic model was used to investigate the effects of three independent variables involved in the synthesis of iron‐encapsulated cold‐set whey protein isolate gel (WPI) on encapsulation efficiency (EE) and L*, a*, b* colour characteristics. The optimal conditions for maximum EE with minimum colour alteration were determined as 6.8% WPI, 18.8 mM iron and pH 7. In an in vitro gastrointestinal assay, only about 28% of the encapsulated iron was released in the gastric condition (with pepsin at pH 1.2), compared to 95% in the intestinal condition (with pancreatin at pH 7.5).     

Angiotensin-I-converting enzyme (ACE)-inhibitory peptides from Thai jasmine rice bran protein hydrolysates

Rice bran protein hydrolysate (<50 kDa RBPH) from Thai jasmine variety demonstrating a high Angiotensin I converting enzyme (ACE) inhibitory activity was purified and characterised. ACE inhibitory peptides were obtained from a two-step purification process: gel filtration and preparative reverse-phase high-performance chromatography (RP-HPLC) and then identified by mass spectrometer hybrid quadrupole-time-of-flight. A novel peptide GSGYF in the RBPH was firstly identified and found to have a partial sequence homology of Oryza sativa Japonica Group. This sequence was further synthesised to exhibit as good an inhibition potency with IC₅₀ value of 2.11 µg mL⁻¹ as Captopril (1.15 µg mL⁻¹). The cytotoxicity test revealed that this RBPH is non-toxic against Vero cells. In addition, the <50 kDa RBPH was resistant to in vitro digestion by pepsin and trypsin. These findings suggest that the RBPH containing ACE inhibitory peptides is likely to be safer and healthier than synthetic drugs and can be an effective food supplement for lowering blood pressure.     

Binding affinity between dietary polyphenols and β-lactoglobulin negatively correlates with the protein susceptibility to digestion and total antioxidant activity of complexes formed

Non-covalent interactions between β-lactoglobulin (BLG) and polyphenol extracts of teas, coffee and cocoa were studied by fluorescence and CD spectroscopy at pH values of the gastrointestinal tract (GIT). The biological implications of non-covalent binding of polyphenols to BLG were investigated by in vitro pepsin and pancreatin digestibility assay and ABTS radical scavenging activity of complexes formed. The polyphenol–BLG systems were stable at pH values of the GIT. The most profound effect of pH on binding affinity was observed for polyphenol extracts rich in phenolic acids. Stronger non-covalent interactions delayed pepsin and pancreatin digestion of BLG and induced β-sheet to α-helix transition at neutral pH. All polyphenols tested protected protein secondary structure at an extremely acidic pH of 1.2. A positive correlation was found between the strength of protein–polyphenol interactions and (a) half time of protein decay in gastric conditions (R² = 0.85), (b) masking of total antioxidant capacity of protein–polyphenol complexes (R² = 0.95).     

Effect of fermentation by Bacillus subtilis on antioxidant and cytotoxic activities of black rice bran

Black rice bran was fermented with Bacillus subtilis KU3 isolated from Korean traditional food, Kimchi. Antioxidant and cytotoxic activities of the fermented black rice bran were investigated. Total phenolic and anthocyanin contents decreased from 171.54 mg GAE g^?1 and 2.31 mg g^?1 to 139.13 mg GAE g^?1 and 2.12 mg g^?1, respectively, after fermentation. Antioxidant activities determined by 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging, β‐carotene bleaching and ferric thiocyanate assay were correlated with total phenolic and anthocyanin contents. Non‐fermented black rice bran extract (NFBE) showed greater antioxidant activities than fermented black rice bran extract (FBE). Cytotoxic activities measured by MTT assay showed that both NFBE and FBE had over 50% activities. The cytotoxic activities of FBE against MCF‐7 and HeLa cells were 71.65% and 68.07%, respectively, at 8.0 mg mL^?1, but those of NFBE were lower than 50%. These results suggested that the cytotoxic activity of black rice bran improved through fermentation, while antioxidant activity reduced.     

Effects of selected processing treatments on antigenicity of banana prawn (Fenneropenaeus merguiensis) tropomyosin

Different processing techniques were assessed to attenuate the antigenicity of banana prawn tropomyosin (Fenneropenaeus merguiensis). Frying and boiling at different temperatures, acid treatment using different acids and storage at −20 °C up to 3 months were investigated. Untreated prawn sample was used as a control. Frying significantly increased antigenicity (6–8 times) in temperature-dependent manner, whereas the trend was fundamentally reversed with boiling. Boiling at 121 °C resulted in the lowest antigenicity (12.99 mg mL⁻¹) among all heat-treated samples, yet higher than the control (5.06 mg mL⁻¹). Freezing had initially very minor impact, although prolonged storage at −20 °C increased antigenicity slightly (2.29 mg mL⁻¹) compared with control. Antigenicity was impacted the most by a reduction in pH independent on the type of acid as both acetic and HCl acids significantly reduced antigenicity of tropomyosin by ~90% compared with control. This could be considered as a new approach to processing that may potentially reduce tropomyosin-derived antigenicity in prawns and prawn products. This study also confirmed the actual molecular weight of banana prawn tropomyosin was 37 kDa implementing a new methodology using unstained gel.     

In vitro digestibility of protein from barley and other cereals.

An in vitro method for measuring barley protein digestibility is presented. Samples were first incubated with pepsin in HCl; pancreatin was then added concomitantly with a bacteriostatic borate buffer. After TCA-precipitation, soluble nitrogen was measured. The digestion was unaffected by accumulated free amino acids. There were no free amino acids following pepsin treatment, but the essential ones were well liberated by pancreatin. Results for barley grown in the field or in pots, and for decortified barley fractions agreed with true digestibility values determined with rats. Of these samples, the field-grown barleys per se differed too little for the accuracy to be confirmed. The other cereals tested, oats, rye, maize, wheat, and rice, gave unsatisfactory results with pepsin/pancreatin, and also with pepsin, pancreatin, or pronase used separately. The ranking of the cereals according to in vitro digestibility depended on the type of enzyme and on the enzyme-to-substrate ratio.     

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     

Boarfish (Capros aper) protein hydrolysate has potent insulinotropic and GLP-1 secretory activity in vitro and acute glucose lowering effects in mice

The anti-diabetic actions of a boarfish protein hydrolysate (BPH) were investigated in cultured cells and mice. A boarfish (Capros aper) muscle protein hydrolysate was generated using the enzymes Alcalase 2.4 L and Flavourzyme 500 L. Furthermore, the BPH was subjected to simulated gastrointestinal digestion (SGID). BPH and SGID samples (0.01–2.5 mg mL⁻¹) were tested in vitro for DPP-IV inhibition and insulin and GLP-1 secretory activity from BRIN-BD11 and GLUTag cells, respectively. The BPH and SGID samples, caused a dose-dependent increase (4.2 to 5.3-fold, P < 0.001) in insulin secretion from BRIN-BD11 cells and inhibited DPP-IV activity (IC₅₀ 1.18 ± 0.04 and 1.21 ± 0.04 mg mL⁻¹), respectively. The SGID sample produced a 1.3-fold (P < 0.01) increase in GLP-1 secretion. An oral glucose tolerance test (OGTT) was conducted in healthy mice (n = 8), with or without BPH (50 mg/kg bodyweight). BPH mediated an increase in plasma insulin levels (AUC_{(0–120 min),} P < 0.05) and a consequent reduction in blood glucose concentration (P < 0.01), after OGTT in mice versus controls. The BPH showed potent anti-diabetic actions in cells and improved glucose tolerance in mice.