Characterisation of trypsin purified from the viscera of Tunisian barbel (Barbus callensis) and its application for recovery of carotenoproteins from shrimp wastes

Trypsin was purified from the viscera of barbel by precipitation using ammonium sulphate (0-80%), Sephadex G-100, and Mono Q-Sepharose ion exchange chromatography. The trypsin was purified 27-fold, with 79 U/mg specific activity and 31% recovery. The enzyme had a molecular weight of 24 kDa; purified trypsin appeared as a single band on native-PAGE. The optimum pH and temperature for enzyme activity were pH 10.0 and 55 °C with BAPNA used as a substrate. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECTPYSQ. The Michaelis-Menten constant (K_m) and catalytic constant (k_cat) values of the enzyme were 0.018 mM and 1.21 s⁻¹, respectively. The study also investigated the effects of purified trypsin on the recovery of carotenoproteins from shrimp (Parapenaeus longirostris) shells through hydrolysis using 1.0 U barbel trypsin/g shrimp shells for 1 h at 30 °C. The freeze-dried carotenoproteins recovered contained 71.09% protein, 16.47% lipid, 7.78% ash, and 1.79% chitin.     

Anticoagulant activities of goby muscle protein hydrolysates

The anticoagulant activities of protein hydrolysates prepared from goby muscle by treatment with various bacterial alkaline proteases were investigated. All proteases exhibited varying degrees of hydrolysis (DH) and all goby protein hydrolysates (GPHs) caused a significant prolongation of both the thrombin time (TT) and the activated partial thromboplastin time (APTT). The hydrolysate generated by the crude protease from Bacillus licheniformis NH1 displayed the highest anticoagulant activity, and the higher TT (about 32 s) at a concentration of 5 mg/mL was obtained with hydrolysate having a DH of 8.86%. This hydrolysate was then fractionated by size exclusion chromatography on a Sephadex G-25 column into five major fractions (F1–F5). Fraction F2, which exhibited the highest anticoagulant activity, was then fractionated by reversed-phase high-performance liquid chromatography. The molecular masses and amino acid sequences of four peptides in peptide sub-fraction F2–6, which exhibited the highest anticoagulant activity, were determined using ESI-MS and ESI-MS/MS, respectively. The structures of these peptides were identified as Leu-Cys-Arg, His-Cys-Phe, Cys-Leu-Cys-Arg and Leu-Cys-Arg-Arg.     

Antibacterial activity of Thai edible plants against gastrointestinal pathogenic bacteria and isolation of a new broad spectrum antibacterial polyisoprenylated benzophenone,chamuangone

Twenty-two edible plant extracts were subjected to evaluation of their antibacterial activity against some gastrointestinal pathogenic bacteria, including Escherichiacoli, Salmonella typhimurium, Salmonella typhi, Shigella sonnei and Helicobacter pylori using the disc diffusion and broth microdilution methods. Sixteen of the plant extracts exhibited antibacterial activity against one or more tested bacteria. Only Garcinia cowa leaf extracts exhibited antibacterial activity against all tested bacteria. Purification of the ethyl acetate extract of G. cowa leaves using an antimicrobial assay-guided isolation afforded a new polyprenylated benzophenone, chamuangone, that exhibited satisfactory antibacterial activity against Streptococcus pyogenes (minimum inhibitory concentration (MIC) 7.8 μg/ml), Streptococcus viridans and H. pylori (MICs 15.6 μg/ml), and Staphylococcus aureus, Bacillus subtilis and Enterococcus sp. (MICs 31.2 μg/ml).     

Antibacterial activity of the extracts from the fruit rinds of Garcinia cowa and Garcinia pedunculata against food borne pathogens and spoilage bacteria

The crude hexane and chloroform extracts from the fruit rinds of Garcinia cowa and Garcinia pedunculata were studied for their antibacterial activity against some foodborne pathogens and spoilage bacteria such as Bacillus cereus, Bacillus coagulans, Bacillus subtilis, Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentrations (MICs) of the extracts determined by the agar dilution method were ranging from 15 to 500 μg/ml and 300 to 1250 μg/ml for G. cowa and G. pedunculata, respectively. However, the hexane and chloroform extracts from the fruit rinds of G. cowa exhibited marked inhibitory effect against all the test organisms and were more effective than that of G. pedunculata extracts. The antibacterial activity of all the extracts was more pronounced against the tested Gram-positive bacteria than the tested Gram-negative bacterium. Furthermore, this study is the first report on the in vitro antibacterial activity of extracts from the fruit rinds of G. cowa and G. pedunculata.     

Selectivity and antimicrobial action of bovine lactoferrin derived peptides against wine lactic acid bacteria

In this study, the antibacterial activities of a bovine Lactoferrin pepsin hydrolysate (LFH) and a synthetic peptide derived from bovine lactoferricin (LfcinB_17–31) have been evaluated against Oenococcus oeni and three additional lactic acid bacteria (LAB) known to cause spoilage during winemaking processes. Inhibition of bacterial growth was demonstrated in vitro in synthetic broth media (MRS) for both LFH and LfcinB_17–31. The bactericidal activity of the synthetic peptide was also assayed and found to vary depending on the bacterial species and the matrix in which exposure to peptide occurred (either MRS broth or white must). Specificity of LfcinB_17–31 for Lactobacillus brevis, Pediococcus damnosus, and O. oeni was demonstrated in must fermentation experiments in which these three LAB co-existed with the winemaking Saccharomyces cerevisiae T73 in the presence of the peptide. Finally, fermentation experiments also showed that LfcinB_17–31 at inhibitory concentrations did not alter either fermentation kinetics or specific enological parameters.     

Recently isolated antidiabetic hydrolysates and peptides from multiple food sources: a review

Abstract

Diabetes, a metabolic syndrome of global importance has been on a progressive rise in recent years. Several pharmacological approaches have been made, which have proved effective, but with underlying side effects. Bioactive hydrolysates (BHs) and peptides (BPs) from food sources, however, have shown the relative advantage of imparting less adverse effects. Furthermore, BHs and BPs from food have been discovered to impart their antidiabetic potentials through one or more mechanisms such as inhibition of digestive enzymes, inhibition of the antigenic enzyme – Dipeptyl peptidase IV (DPP-IV), decrease in blood glucose levels and increase in insulin uptake. Several plants and animal sources have been used as protein sources for the isolation of antidiabetic hydrolysates and peptides through different mechanisms and analytical techniques. This review integrates recent research information about several popular and unconventional food sources of BHs and BPs, their isolation techniques, antidiabetic effects and protein profiles. In addition, the fractionation technique(s) employed in each study and inhibition potentials of BHs and BPs are reviewed. This article is intended to supplement accessible scholarly literature and intellectual awareness on the subject of food-oriented approach for the management of diabetes.     

菜籽源锌螯合肽的制备、纯化和结构解析

采用碱性蛋白酶水解菜籽蛋白制备水解物,运用固定金属亲和层析(IMAC-Zn2+)和葡聚糖凝胶G25(Sephadex G25)层析分离纯化;利用反相高效液相色谱(RP-HPLC)和电喷雾电离质谱(ESI-MS)鉴定序列;采用EDTA络合滴定法(ECT)、双硫腙比色法(DCM)、原子吸收光谱(AAS)和电感耦合等离子体原子发射光谱法(ICP-AES)检测锌螯合率。试验表明:菜籽蛋白的4 h水解物锌螯合潜力最佳;分离该水解物获得3个肽组分(I,Ⅱ和Ⅲ),其中Ⅱ和Ⅲ具有锌螯合能力;进一步分离后分别获得3个(Ⅱ-1,Ⅱ-2,Ⅱ-3)和2个肽组分(Ⅲ-1和Ⅲ-2),组分Ⅱ-3的锌螯合率最高,高于同浓度的谷胱甘肽(GSH)(P0.05);鉴定Ⅱ-3序列获得4条肽,即Ala-Arg(AR),Asn-Ser-Met(NSM),Gly-Lys-Arg(GKR)和Glu-Pro-SerHis(EPSH)。其中NSM的锌螯合率最高,来源于菜籽蛋白NADH-enoyl-ACP reductase Chain A序列中的296-298氨基酸残基。因此,菜籽蛋白可以作为微量元素螯合肽的优良来源。     

Affinity purification and characterisation of chelating peptides from chickpea protein hydrolysates

A chickpea protein hydrolysate produced with pepsin and pancreatin was used for the affinity purification of chickpea chelating peptides. Three chelating peptide fractions were obtained after affinity chromatography with immobilised copper. These peptide fractions showed a higher chelating activity and histidine contents than the original protein hydrolysate. Chelating activity was positively correlated with the histidine content of the purified fractions. Different subfractions were also obtained after gel filtration chromatography from the affinity purified peptide fractions. Some of these subfractions showed a higher chelating activity and histidine contents than the original fractions. These results suggest that a combination of high His contents, around 20–30%, and small peptide size provide the best chelating activities. Thus sequential purification with affinity and gel filtration chromatography is a useful procedure for the purification of chickpea peptides with high chelating activity. These results show that a range of chelating peptides are generated during digestion of the chickpea proteins that, after metal chelation, may prevent the generation of reactive oxygen species (ROS) and favour metal absorption.     

Antioxidant and metal chelating activities of Phaseolus vulgaris L. var. Jamapa protein isolates, phaseolin and lectin hydrolysates

A search in a database of potential bioactive short sequences in food proteins reveals that bioactive peptides with a variety of beneficial effects for cardiovascular health are present in the sequence of common bean proteins, including bioactive sequences with antioxidant properties. A protein isolate, the storage protein phaseolin and a lectin extract from Phaseolus vulgaris L. var. Jamapa, were hydrolyzed by treatment with pepsin and pancreatin in order to investigate the possible release of peptides with antioxidant and metal chelating properties. Antioxidant activity was determined in Caco-2 cells exposed to a free radical generator, and iron and copper chelating activities were determined using colorimetric methods. The highest antioxidant activity, 71% inhibition, was found in the hydrolyzed protein isolate. Copper and iron chelating activities were highest in the lectin and phaseolin hydrolysates, 53% and 81%, respectively. Thus, experimental data indicates, as suggested by the database search, that antioxidant peptides are abundant in pepsin-pancreatin hydrolysates, which may represent a valuable health-promoting property in common bean.     

Functional properties and in vitro antioxidant activity of roe protein hydrolysates of Channa striatus and Labeo rohita

Bioactive roe protein hydrolysates were prepared from Channa striatus (CRPH) and Labeo rohita (LRPH) and their functional and in vitro antioxidant properties evaluated. The degree of hydrolysis was 28.41% at 60min in channa and 18.85% in labeo roe concentrates at 90min. The yields of protein hydrolysates were 24.15% and 12.45% for channa and labeo roe protein concentrates, respectively. The protein content was identical (58%) in both roe protein hydrolysates. Protein solubility in channa was higher (90.48%) when compared to labeo (50.6%) at pH 12. Higher oil absorption capacity and foam stability were observed in CRPH and higher emulsifying capacity was found in LRPH. Smaller peptides of 12kDa were noted in both CRPH and LRPH. In vitro antioxidant activity was higher in CRPH than in LRPH as seen from DPPH radical scavenging and ferric reducing power.     

Antioxidant and metal chelating activities of peptide fractions from phaseolin and bean protein hydrolysates

Bean protein isolate and phaseolin were hydrolysed using pepsin and pancreatin, and the resulting hydrolysates were filtered through a 1kDa cut-off membrane and fractionated by size exclusion chromatography. Three fractions corresponding to MW 0.7-1.0kDa, 0.43-0.7kDa and <0.43kDa (A1, A2, and A3 for protein isolate fractions, and B1, B2, and B3 for phaseolin fractions) were assayed for antioxidant and metal chelating activity and they were also subjected to amino acid and SDS-PAGE analysis. Fractions A1 and B1 had the highest copper chelating activity (78% and 82%, respectively), while iron chelating activity was the highest in fractions A1 and B3 (36% and 16%, respectively). Fractions A2 and B3 had the highest antioxidant activity as determined by inhibition of reducing power and β-carotene bleaching, while the highest ABTS radical scavenging activity was found in A3 and B3. Thus, fractions coming from the isolate and phaseolin had similar activities except for iron chelation, suggesting that phaseolin is the major contributor to the antioxidant and copper chelating activities of the hydrolysed protein isolate.     

Antioxidant and free radical-scavenging activities of smooth hound (Mustelus mustelus) muscle protein hydrolysates obtained by gastrointestinal proteases

We have investigated the antioxidative activity of five hydrolysates from smooth hound (Mustelus mustelus) meat obtained by various gastrointestinal proteases: crude enzyme extract, low molecular weight (LMW) alkaline protease and trypsin-like protease from M. mustelus intestine, pepsin from M. mustelus stomach, and bovine trypsin.     

Protein hydrolysates from meriga (Cirrhinus mrigala) egg and evaluation of their functional properties

Protein hydrolysates from underutilised meriga (Cirrhinus mrigala) fish egg were prepared by using commercial Alcalase and papain enzymes. The degree of hydrolysis was 62% for Alcalase and 17.1% for papain, after 90 min digestion at 50–55 and 60–65 °C, respectively. The protein content of Alcalase-produced hydrolysate was higher (85%) than that of papain hydrolysate (70%) (p < 0.05). Hydrolysis by both enzymes increased protein solubility of fish egg protein hydrolysates to above 72.4% over a wide pH range (2–12). Results showed that the hydrolysates had good fat absorption capacity (0.9 and 1.0 g/g sample), foam capacity (70% and 25%) and emulsifying capacity (4.25 and 5.98 ml/g hydrolysate), respectively for Alcalase and papain protein hydrolysates. Gel filtration chromatograms and SDS–PAGE analysis indicated the distribution of smaller peptides. These results suggested that fish egg protein hydrolysates could be useful in the food industry.     

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.     

Angiotensin-I converting enzyme inhibitory and antioxidant activities of protein hydrolysates from Phaseolus lunatus and Phaseolus vulgaris seeds

Phaseolus lunatus and Phaseolus vulgaris protein concentrates were hydrolyzed with the enzymes Alcalase^® and Flavourzyme^® at different reaction times, and the angiotensin-I converting enzyme (ACE-I) inhibitory activity, antioxidant properties and amino acid composition measured in the hydrolysates. With Alcalase^®, the highest degree of hydrolysis (DH) in P. lunatus was 37.94% at 45 min, and in P. vulgaris was 49.48% at 30 min. With Flavourzyme^®, the highest DH's were 22.03% and 26.05%, respectively, both at 90 min. ACE-I inhibitory activity in the Alcalase^® hydrolysates was IC₅₀ = 0.056 mg mL⁻¹ for P. lunatus at 90 min, and IC₅₀ = 0.061 mg mL⁻¹ for P. vulgaris at 60 min. In the Flavourzyme^® hydrolysates this activity was IC₅₀ = 0.0069 mg mL⁻¹ for P. lunatus at 90 min and IC₅₀ = 0.127 mg mL⁻¹ for P. vulgaris at 45 min. In SDS-PAGE, the hydrolysates exhibited low molecular weight bands. Antioxidant activity was 11.55 mmol L⁻¹ TEAC mg⁻¹ protein for P. lunatus with Flavourzyme^® at 90 min and 10.09 mmol L⁻¹ TEAC mg⁻¹ protein for P. vulgaris with Alcalase^® at 60 min. Amino acid composition exhibited high amino acid hydrophobic residues content.     

Optimization of the production of Momordica charantia L. Var. abbreviata Ser. protein hydrolysates with hypoglycemic effect using Alcalase

Momordica charantia L. Var. abbreviata Ser. protein was hydrolyzed using six different proteases. The results showed Alcalase 2.4L to have the best hydrolyzing capacity, followed by Pancreatin. In addition, Alcalase hydrolysate had stronger hypoglycemic effect than that of Pancreatin hydrolysate at the same dose. Alcalase was chosen to produce M. charantia L. Var. abbreviata Ser. protein hydrolysates (MCPHs) with hypoglycemic effect. Response surface methodology (RSM) was applied to optimize the hydrolysis conditions using Alcalase. Model equation was proposed with regard to the effect of enzyme/substrate ratio, pH and temperature. The optimum values for enzyme/substrate ratio, pH and temperature were found to be 2.37%, 9.2 and 57 °C respectively.     

Purification of angiotensin I-converting enzyme-inhibitory peptides from the enzymatic hydrolysate of defatted canola meal

Defatted canola meals from seeds of different processing origins were hydrolyzed by Alcalase to give hydrolysates that inhibited angiotensin converting enzyme (ACE) activity. Heat treated meals yielded protein hydrolysates with 50% ACE-inhibitory concentrations of 27.1 and 28.6 μg protein/ml compared with 35.7 and 44.3 μg protein/ml for the none-heat treated meals. Separation of the hydrolysate on a Sephadex G-15 gel permeation column (GPC) yielded a fraction with an IC₅₀ value of 2.3 μg protein/ml. Amino acid analysis showed that the GPC fraction contained 45% content of aromatic amino acids in comparison to 8.5% of the hydrolysate. Two peptides, Val-Ser-Val (IC₅₀ = 0.15 μM) and Phe-Leu (IC₅₀ = 1.33 μM) were purified, and located in the primary structure of canola napin and cruciferin native proteins. The results suggest that canola protein hydrolysate is a potential ingredient for the formulation of hypotensive functional foods.     

Bactericidal activities against pathogenic bacteria by selected constituents of plant extracts in carrot broth

HPLC-DAD analysis provided evidence for the certain identification of some constituents of hydroalcoholic extracts from Lithospermum erythrorhizon, Rheum palmatum, Thymus vulgaris, Lippia citriodora, and a mixture of Rosmarinus officinalis, Salvia lavandulifolia and Thymus mastichina. Their inhibitory and bactericidal activities in vitro against Escherichia coli and Staphylococcus aureus were evaluated either in Luria–Bertani (LB) broth or a model food system, Tyndallised carrot broth (TCB). Shikonin exhibited high inhibitory activity against both microorganisms, but to a lesser extent than the basic unit naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Rhein and carnosic acid showed noticeable inhibitory effects on S. aureus. In inoculated TCB containing 15 mg/L naphthazarin or 47 mg/L shikonin there was about 6 log reductions in E. coli counts, while 20 mg/L naphthazarin, 26.4 mg/L rhein, 45 mg/L shikonin or 68.7 mg/L carnosic acid caused 1.6, 2.4, 3.1 and 3.1 log reductions in S. aureus inocula, respectively. Log reductions obtained for S. aureus in TCB compared unfavourably with those found in LB broth.