Exploring the Milk-Clotting and Proteolytic Activities in Different Tissues of Vallesia glabra: a New Source of Plant Proteolytic Enzymes.

Proteolytic enzymes are widely distributed in nature, playing essential roles in important biological functions. Recently, the use of plant proteases at the industrial level has mainly increased in the food industry (e.g., cheesemaking, meat tenderizing, and protein hydrolysate production). Current technological and scientific advances in the detection and characterization of proteolytic enzymes have encouraged the search for new natural sources. Thus, this work aimed to explore the milk-clotting and proteolytic properties of different tissues of Vallesia glabra. Aqueous extracts from the leaves, fruits, and seeds of V. glabra presented different protein profiles, proteolytic activity, and milk-clotting activity. The milk-clotting activity increased with temperature (30-65 °C), but this activity was higher in leaf (0.20 MCU/mL) compared with that in fruit and seed extracts (0.12 and 0.11 MCU/mL, respectively) at 50 °C. Proteolytic activity in the extracts assayed at different pH (2.5-12.0) suggested the presence of different types of active proteases, with maximum activity at acidic conditions (4.0-4.5). Inhibitory studies indicated that major activity in V. glabra extracts is related to cysteine proteases; however, the presence of serine, aspartic, and metalloproteases was also evident. The hydrolytic profile of caseins indicated that V. glabra leaves could be used as a rennet substitute in cheesemaking, representing a new and promising source of proteolytic enzymes.

In Vitro Antithrombotic and Hypocholesterolemic Activities of Milk Fermented with Specific Strains of Lactococcus lactis.

Milk fermented with specific lactic acid bacteria (LAB) was reported to be a rich source of metabolites, such as peptides with different biological activities that may have a positive effect on cardiovascular health. Thus, in this study, the antithrombotic and hypocholesterolemic activities of fermented milk with specific strains of Lactococcus lactis were investigated before and after exposure to a simulated gastrointestinal digestion (SGD) model. The inhibition of thrombin-induced fibrin polymerization (IC50 peptide concentration necessary to inhibit thrombin activity by 50%), anticoagulant activity, inhibition of micellar solubility of cholesterol and bile acid binding capacity of water soluble fractions (WSF) <3 kDa from fermented milk were evaluated. Results indicated that the WSF from fermented milk with Lc-572 showed antithrombotic (IC50 = 0.049 mg/mL) and hypocholesterolemic (55% inhibition of micellar solubility of cholesterol and 27% bile acid binding capacity) activities. Meanwhile, fermented milk with Lc-571 showed mainly antithrombotic activity (IC50 = 0.045 mg/mL). On the other hand, fermented milk with Lc-600 presented mainly hypocholesterolemic activity (31.4% inhibition of micellar solubility of and 70% bile acid binding capacity). Moreover, biological activities were not lost after simulated gastrointestinal digestion conditions. Thus, fermented milk with these specific L. lactis strains show potential for the development of functional foods.

Effect of Amidated Low-Methoxyl Pectin on Physicochemical Characteristics of Jumbo Squid (Dosidicus gigas) Mantle Muscle Gels.

Jumbo squid (Dosidicus gigas) muscle proteins show low functionality with limited use in gel products. This work aims to assess the influence of adding the natural and commercially available fibre, amidated low-methoxyl pectin (at 0.5, 1.0, 1.5, 2.0 and 3.0%), on the physicochemical and functional characteristics of jumbo squid (Dosidicus gigas) mantle muscle gels. The addition of 0.5% fibre showed an immediate effect on the gel texture profile analysis, improving hardness (p<0.05) from (3.4±0.7) N of the control (no added fibre) to (5.2±0.9) N, and increasing elasticity (p≥0.05). Shear force was significant only at 3.0% fibre addition. Water holding capacity also improved (p<0.05) with fibre addition (from 75% in the control to 90-95% after the treatments). Whiteness was affected (p<0.05) when 3.0% fibre was added. Differential scanning calorimetry showed two endothermic transition peaks in the gels. The second peak (actin) increased (p<0.05) by 1-2 °C with fibre addition. Therefore, the present study demonstrates that amidated low-methoxyl pectin (0.5-3.0%) is an excellent ingredient to improve jumbo squid mantle muscle protein functionality, increasing the gel texture and water retention characteristics.

Comparison of the milk-clotting properties of three plant extracts.

Several proteases from plant sources have been proposed as milk coagulants, however, limited research has been done on their milk-clotting properties. The effect of temperature on the milk-clotting activity of kiwi fruit, melon and ginger extracts was evaluated, as well as the effects of the different extracts on curd properties. Melon extracts showed high milk-clotting activity over a broad temperature range (45-75 °C) while kiwi fruit and ginger extracts showed high activity over a narrower temperature range, with a maximum at 40 and 63 °C, respectively. Curds produced using kiwi extracts had textural properties comparable with those obtained using commercial rennet, while melon extracts produced a fragile gel and low curd yield. The milk-clotting behavior of the three plant extracts was related to the protease specificity present in these extracts. The kiwi proteases displayed chymosin-like properties and thus hold the best potential for use as a milk coagulant in cheese production.

Structure-function characterization of the recombinant aspartic proteinase A1 from Arabidopsis thaliana.

Aspartic proteinases (APs) are involved in several physiological processes in plants, including protein processing, senescence, and stress response and share many structural and functional features with mammalian and microbial APs. The heterodimeric aspartic proteinase A1 from Arabidopsis thaliana (AtAP A1) was the first acid protease identified in this model plant, however, little information exists regarding its structure function characteristics. Circular dichroism analysis indicated that recombinant AtAP A1 contained an higher alpha-helical content than most APs which was attributed to the presence of a sequence known as the plant specific insert in the mature enzyme. rAtAP A1 was stable over a broad pH range (pH 3-8) with the highest stability at pH 5-6, where 70-80% of the activity was retained after 1 month at 37 degrees C. Using calorimetry, a melting point of 79.6 degrees C was observed at pH 5.3. Cleavage profiles of insulin beta-chain indicated that the enzyme exhibited a higher specificity as compared to other plant APs, with a high preference for the Leu(15)-Tyr(16) peptide bond. Molecular modeling of AtAP A1 indicated that exposed histidine residues and their interaction with nearby charged groups may explain the pH stability of rAtAP A1.

Expression and characterization of the recombinant aspartic proteinase A1 from Arabidopsis thaliana.

The present study reports the recombinant expression, purification, and partial characterization of a typical aspartic proteinase from Arabidopsis thaliana (AtAP A1). The cDNA encoding the precursor of AtAP A1 was expressed as a functional protein using the yeast Pichia pastoris. The mature form of the rAtAP A1 was found to be a heterodimeric glycosylated protein with a molecular mass of 47kDa consisting of heavy and light chain components, approx. 32 and 16kDa, respectively, linked by disulfide bonds. Glycosylation occurred via the plant specific insert in the light chain. The catalytic properties of the rAtAP A1 were similar to other plant aspartic proteinases with activity in acid pH range, maximal activity at pH 4.0, K(m) of 44 microM, and k(cat) of 55 s(-1) using a synthetic substrate. The enzyme was inhibited by pepstatin A.

Capillary electrophoresis for the analysis of meat authenticity.

In this overview, different meat authenticity issues are presented, as well as a wide variety of methods available for meat authentication. Unlike chromatographic, traditional gel electrophoretic, or immunological methods, which have been routinely used in analytical laboratories, the application of capillary electrophoresis (CE) is relatively new in solving meat authentication issues. Several unique CE applications based on meat protein fingerprinting are discussed for the analysis of meat species in unheated meat products. For protein data interpretation, pattern recognition is used to account for the natural variability present within the same meat species. While gel DNA-based methods are widely used for determining meat species in heat processed products, few DNA-based methods utilizing CE have been reported. Moreover, the methods reported are qualitative or semiquantitative. Thus, the need for quantitative competitive PCR CE methods in the determination of meat species is addressed. For the determination of meat extenders, CE methods were either protein-based or based on specific markers. Polyphenols are used as specific markers for soy detection and hydroxyproline is used as a specific marker for collagen determination. Finally, the potential of electrophoretically mediated miroanalysis (EMMA) for the detection of meat that may have been previously frozen and retailed as "fresh" is highlighted.

New capillary electrophoresis method for the determination of furosine in dairy products.

A new capillary electrophoresis (CE) method was established for the quantitative determination of furosine in dairy products. Sample preparation and suitable electrophoretic conditions allowed accurate and reproducible quantitation of furosine in dairy products. Sample preparation consisted of drying hydrolyzed samples, redissolving them in 0.2 M NaOH, and purifying them by solid-phase extraction. The electrophoretic separation was carried out in an uncoated capillary maintained at 30 degrees C using 0.1 M phosphate buffer containing the additive hexadecyl trimethylammonium bromide (HDTAB, 1.2 mM) (pH 7.0) under 10 kV voltage and reverse polarity. Coefficients of variation of less than 2.25% for migration time and 5.80% for peak areas indicated that the technique was reproducible. The calibration curve followed a linear relationship with a highly significant (p < 0.01) coefficient of multiple determination (R (2) = 0.997). The limit of quantitation was 0.5 ppm, a concentration that corresponds to 4.5 mg/100 g of protein in milk samples. Furosine concentration (mg/100 g of protein) ranges of different dairy products (raw, pasteurized, UHT, and evaporated milks and yogurt) agreed with ranges previously reported. Therefore, the CE method presented is a suitable technique for the routine assessment of furosine in dairy products.

Determination of beta-hydroxyacyl CoA-dehydrogenase activity in meat by electrophoretically mediated microanalysis.

The combined use of an in-tube enzyme assay and capillary electrophoresis for determining beta-hydroxyacyl CoA-dehydrogenase (beta-HADH) activity in meat was investigated. Beta-HADH is a significant mitochondrial enzyme in food muscle; thus, the determination of its activity is important in food analysis. The enzymatic assay and the separation of the reaction products were carried out by electrophoretically mediated microanalysis (EMMA) using a plug-plug reaction mode at variable potential. For the quantification of beta-HADH activity, the rate of conversion of reduced beta-nicotinamide adenine dinucleotide (NADH) to beta-nicotinamide adenine dinucleotide (NAD+) was calculated by determining NAD+ at 260 nm. A calibration curve for NAD+ concentration versus normalized areas showed a highly significant (p < 0.001) linear relationship (R2 = 0.993). Accurate quantification of beta-HADH activity was achieved since on-line monitoring allowed us to account for the NAD+ produced from NADH degradation by applying a correction factor. An average reaction time of 0.66 +/- 0.06 sec was determined for a protein concentration in the range of 0.1-0.5 mg protein/mL. The assay was reproducible since coefficients of variation of less than 6.2% were calculated for triplicate analyses.