Preparation of Alcalase-catalyzed casein plasteins in the presence of proline addition and the ACE-inhibitory activity of the plasteins in vitro

Casein hydrolysates were prepared by hydrolysis of casein with alkaline protease Alcalase for 6 h and showed the highest ACE-inhibitory activity in vitro with an IC₅₀ value of 47.1 μg mL⁻¹. Casein hydrolysates prepared were subjected to Alcalase-catalyzed plastein reaction in the presence or absence of proline addition to prepare casein plasteins. Some optimal reaction conditions of plastein reaction in the presence of proline addition were studied using response surface methodology with the decrease in free amino groups in the casein plasteins as response. When the concentration of casein hydrolysates was fixed at 35% (w w⁻¹) and reaction time at 6 h, the optimal conditions were reaction temperature 48 °C, addition level of proline 0.54 mol/mol free amino groups of casein hydrolysates and addition level of Alcalase 9.5 kU g⁻¹ proteins. With these conditions, the maximal decrease in free amino groups in casein plasteins was 195.7 μmol g⁻¹ proteins. The ACE-inhibitory activities of twelve casein plasteins in vitro, prepared in the presence or absence of proline addition with different reaction extents, were evaluated and compared. The results showed that the ACE-inhibitory activity of the casein plasteins prepared in the presence of proline addition changed irregularly, different to that of the casein plasteins prepared in the absence of proline addition, and might relate to the different linking of proline to the peptides in casein hydrolysates during plastein reaction. When the casein plasteins prepared in the presence of proline addition had a decrease in free amino groups 195.7 μmol g⁻¹ proteins, the IC₅₀ value of the casein plasteins was lowered to 0.2 μg mL⁻¹.     

Modification of Soybean Protein Hydrolysates by Alcalase-Catalyzed Plastein Reaction and the ACE-Inhibitory Activity of the Modified Product In Vitro

Soybean protein hydrolysates were prepared by hydrolyzing soybean protein isolates with a protease alcalase to a degree of hydrolysis of 16.6%, and then modified by alcalase-catalyzed plastein reaction to reveal the impact of plastein reaction on the ACE-inhibitory activity of the modified product in vitro. The suitable conditions of plastein reaction of soybean protein hydrolysates were selected based on the results of response surface methodology with the decreased amount of the free amino groups of the modified product as response. When reaction temperature was fixed at 30°C, the selected conditions were as follows: concentration of soybean protein hydrolysates of 45% (w/w), addition level of alcalase of 275 U/g peptides, and reaction time of 3 to 4 h. Soybean protein hydrolysates and eight modified products were evaluated for their ACE-inhibitory activities in vitro. The assay results highlighted that plastein reaction improved the ACE-inhibitory activity of the modified product. The IC₅₀ of the modified products ranged from 0.64 to 1.11 mg/mL, while that of soybean protein hydrolysates was 1.45 mg/mL. The decreased amount of the free amino groups of the modified product showed influence on the ACE-inhibitory activity in vitro. Analysis results from size exclusion chromatography confirmed that some plasteins with higher molecular weights were formed in the modified product. Our results showed that alcalase-catalyzed plastein reaction could be applied as a potential approach to enhance the ACE-inhibitory activity of soybean protein hydrolysates in vitro.     

Angiotensin-converting enzyme inhibitory activity of milk protein hydrolysates: Effect of substrate,enzyme and time of hydrolysis

Nine milk protein substrates were hydrolysed in vitro with five proteases for various times (0, 3, 6, and 24 h), and the angiotensin-converting enzyme (ACE)-inhibitory activity of hydrolysates was assessed. Overall, the casein substrates gave rise to hydrolysates with significantly higher ACE-inhibitory activity than the whey protein (WP) substrates (85% vs. 79%). No significant difference between 3 and 24 h of hydrolysis was found. A reasonable correlation was found between the ACE inhibition of the 6 h hydrolysates determined in vitro and estimated by in silico modelling. The highest ACE-inhibitory activity was found in hydrolysates made with thermolysin followed by proteinase K, trypsin, pepsin and Bacillus licheniformis protease. The IC₅₀ values for thermolysin hydrolysates of caseins and WPs were 45–83 and 90–400 μg mL⁻¹, respectively, with α-lactalbumin giving the highest inhibitory activity. Thermolysin, proteinase K and trypsin were useful for the release of highly potent ACE-inhibitory peptides from both WPs and caseins.     

三种氨基酸添加下酶法修饰酪蛋白水解物的ACE抑制活性

采用碱性蛋白酶水解酪蛋白,制备水解度为12.4%、IC50为42.19μg/mL的酪蛋白水解物。在添加外源氨基酸的情况下对水解物进行类蛋白反应修饰,并响应面法研究氨基酸添加量、酶添加量、反应温度及3种氨基酸的影响。结果表明:氨基酸添加量、反应温度、氨基酸种类对修饰反应影响显著,而酶添加量的影响不大;分别添加苯丙氨酸、亮氨酸、缬氨酸制备3个酪蛋白水解物修饰产物,其IC50降低至21.03～25.13μg/mL,表明添加外源氨基酸可提高修饰产物的体外ACE抑制活性,但添加不同氨基酸的影响不显著。     

Coupled Neutrase–Catalyzed Plastein Reaction Mediated the ACE-Inhibitory Activity In Vitro of Casein Hydrolysates Prepared by Alcalase

Casein hydrolysates with a degree of hydrolysis of 13.5% were prepared by hydrolyzing casein with an alkaline protease Alcalase, and showed ACE-inhibition in vitro with an IC₅₀ value of 45.2 μg/mL. The hydrolysates were modified by plastein reaction catalyzed by a neutral protease Neutrase to reveal the impact of the coupled Neutrase-catalyzed plastein reaction on the ACE-inhibition of the casein hydrolysates. The effects of addition level of Neutrase, substrate concentration, reaction temperature, and time on the plastein reaction of the casein hydrolysates were studied with the varying amount of free amino groups of the modified hydrolysates as index. The results illustrated that the amount of free amino groups of the modified hydrolysates increased in all occasions, and the addition level of Neutrase, substrate concentration, and reaction time had a clear impact on the plastein reaction. Six modified hydrolysates were prepared at a substrate concentration of 40% (by weight), Neutrase addition level of 3 kU/g peptides, reaction temperature of 35°C, and different reaction time. The assay results highlighted that the coupled Neutrase-catalyzed plastein reaction improved the ACE-inhibition of six modified hydrolysates with IC₅₀ values ranging from 15.6 to 20.0 μg/mL. Size exclusion chromatography analysis showed that some plasteins with a molecular weight of about 68 kDa existed in the modified hydrolysates. The results also demonstrated that it was the coupled Neutrase-catalyzed plastein reaction but not further hydrolysis of casein hydrolysates that enhanced the ACE-inhibition of the modified casein hydrolysates.     

Properties of thermostable extracellular FOS-producing fructofuranosidase from <Emphasis Type="Italic">Cryptococcus</Emphasis> sp.

The present work was devoted to investigations concerning the fructooligosaccharide producing activity of Cryptococcus sp. LEB-V2 (Laboratory of Bioprocess Engineering, Unicamp, Brazil) and its extracellular fructofuranosidase. After cell separation, the enzyme was purified by ethanol precipitation and anion exchange chromatography. The enzyme showed both fructofuranosidase (FA) and fructosyl transferase (FTA) activity. With sucrose as substrate, the data failed to fit the Michaelis–Menten behaviour, showing a substrate inhibitory model. The K _m, K _i and v _max values were shown to be 64 mM, 3 M and 159.6 μmol mL⁻¹ min⁻¹ for FA and 131 mM, 1.6 M and 377.8 μmol mL⁻¹ min⁻¹ for FTA, respectively. The optimum pH and temperature were found to be around 4.0 and 65 °C, while the best stability was achieved at pH 4.5 and temperatures below 60 °C, for both the FA and FTA. Despite the strong FA activity, the high transfructosylating activity allowed for good FOS production from sucrose (35% yield).     

Evaluation of Functional Properties in Protein Hydrolysates from Bluewing Searobin (Prionotus punctatus) Obtained with Different Microbial Enzymes

Enzymatic hydrolysis of proteins from low commercial value fish could be produced for uses like functional ingredients in a wide and always increasing zone of application in different food products. The objective of this work was to evaluate the functional properties and the amino acid profile of enzymatic hydrolysates from Bluewing searobin (Prionotus punctatus), using two microbial enzymes, Alcalase and Flavourzyme. The enzymatic hydrolysate obtained through the addition of the enzyme Alcalase reached the maximum solubility (42%) at pH 9, water holding capacity (WHC) of 2.4 g_water  g_protein ⁻¹, 4.5 g_oil g_protein ⁻¹ of oil holding capacity (OHC) and an emulsifying activity index (EAI) of 54 m² g_solids ⁻¹ at pH 3. On the other hand, the hydrolysate obtained from Flavourzyme attained 38% of solubility at pH 9, 3.7 g_water  g_protein ⁻¹ and 5.5 g_oil g_protein ⁻¹ for the holding capacities, and an EAI of 71 m² g_solids ⁻¹ at pH 11. The hydrolysate with Flavourzyme produced best results for WHC, OHC, and EAI because it had solubility lower than the hydrolysate of Alcalase. The hydrolysate produced by Alcalase had a higher amino acid content compared with Flavourzyme’s hydrolysate. However, both showed a good essential amino acid amounts. In general, these results indicate the potential utilization of the hydrolysate from Bluewing searobin in food formulations for the direct human consumption.     

Potential ACE-inhibitory activity and nanoLC-MS/MS sequencing of peptides derived from aflatoxin contaminated peanut meal

Our lab has developed a process for sequestering aflatoxin from contaminated peanut meal (PM) using commercial bentonite clays while protein is simultaneously extracted and hydrolyzed by a commercial protease. The objectives of this study were to sequence generated peptides and evaluate their potential ACE-inhibitory properties. Aflatoxin in the unprocessed PM was 610 μg kg⁻¹ compared to 9.7 μg kg⁻¹ on a dry weight basis in the 120 min hydrolysate. This hydrolysate displayed significant ACE-inhibitory activity with an IC₅₀ of 295.1 μg mL⁻¹. Ultrafiltration and size exclusion chromatography (SEC) improved the ACE-inhibitory properties, with the SEC fraction containing the smallest peptides having an IC₅₀ = 44.4 μg mL⁻¹. Additionally, 271 unique peptides were identified by nanoLC-MS/MS, of which 147 belonged to major seed storage proteins. This advanced characterization data will ultimately allow for more efficient production of hydrolysates with ACE-inhibitory activity or other bioactivities of interest from PM.     

Production of Cell Membrane-Bound α- and β-Glucosidase by Lactobacillus acidophilus

Hydrolytic enzymes, viz. α- and β-glucosidase, were produced from indigenous isolate, Lactobacillus acidophilus, isolated from fermented Eleusine coracana. Production of these enzymes was enhanced by optimizing media using one factor at a time followed by response surface methodology. The optimized media resulted in a 2.5- and 2.1-fold increase in α- and β-glucosidase production compared with their production in basal MRS medium. Localization studies indicated 80% of the total activity to be present in the cell membrane-bound fraction. Lack of sufficient release of these enzymes using various physical, chemical, and enzymatic methods confirmed their unique characteristic of being tightly cell membrane bound. Enzyme characterization revealed that both α- and β-glucosidase exhibited optimum catalytic activity at 50 °C and pH 6.0 and 5.0, respectively. K _m and V _max of α-glucosidase were 4.31 mM and 149 μmol min⁻¹ mL⁻¹ for p-nitrophenyl-α-d-glucopyranoside as substrate and 3.8 mM and 120 μmol min⁻¹ mL⁻¹ for β-glucosidase using p-nitrophenyl-β-d-glucopyranoside as the substrate.     

Properties of <Emphasis Type="Italic">Aspergillus subolivaceus</Emphasis> free and immobilized dextranase

Aspergillus subolivaceus dextranase is immobilized on several carriers by entrapment and covalent binding with cross-linking. Dextranase immobilized on BSA with a cross-linking agent shows the highest activity and considerable immobilization yield (66.7%). The optimum pH of the immobilized enzyme is shifted to pH 6.0 as compared with the free enzyme (pH 5.5). The optimum temperature of the reaction is resulted at 60 °C for both free and immobilized enzyme. Thermal and pH stability are significantly improved by the immobilization process. The calculated K _m of the immobilized dextranase (14.24 mg mL⁻¹) is higher than that of the free dextranase (11.47 mg mL⁻¹), while V _max of the immobilized enzyme (2.80 U μg protein⁻¹) is lower than that of the free dextranase (11.75 U μg protein⁻¹). The immobilized enzyme was able to retain 76% of the initial catalytic activity after 5.0 cycles.     

Effects of Maillard reaction conditions on the antigenicity of α-lactalbumin and β-lactoglobulin in whey protein conjugated with maltose

The effects of Maillard reaction conditions (weight ratio of protein to sugar, temperature and time) on the antigenicity of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) in conjugates of whey protein isolate (WPI) with maltose were investigated. Response surface methodology was used to establish models to predict the antigenicity of α-LA and β-LG and find an optimal reaction condition under which the antigenicity of α-LA and β-LG reduces to minimum value. Conjugating WPI with maltose was an effective way to reduce the antigenicity of α-LA and β-LG. The antigenicity of α-LA decreased from 32.25 μg mL⁻¹ to 10.91 μg mL⁻¹. And the antigenicity of β-LG decreased from 272.4 μg mL⁻¹ to 38.17 μg mL⁻¹. Temperature had the greatest effect on the antigenicity of α-LA, while weight ratio of WPI to maltose was the most significant factor on the antigenicity of β-LG.     

Glutaminase‐induced deamidation and hydrolysis of casein and metal‐chelating or ACE‐inhibitory activity of the hydrolysates in vitro

Glutaminase (EC 3.5.1.2) was applied in this work to induce deamidation and hydrolysis of casein. Some reaction conditions based on casein deamidation were studied. Three casein hydrolysates with degree of deamidation of 2.8%, 5.8% and 8.5%, or degree of hydrolysis of 2.5%, 3.4% and 4.9%, respectively, were prepared at casein concentration 5% (w/v), glutaminase addition level 400 U kg^?1 casein, reaction temperature 37 °C and reaction times 6, 12 and 24 h, respectively. Evaluation results showed that when iron (II) was added at 60 μm , iron (II)‐chelating powers of three hydrolysates were 41.1, 45.4 and 55.3%, while that of original casein and EDTA were 36.1 and 13.6%. Calcium (II)‐chelating power of three hydrolysates was 1.23, 1.41 and 1.49 mmol g^?1 casein, whereas that of original casein was 1.05 mmol g^?1 casein. Three hydrolysates also had ACE‐inhibitory activity in vitro, with IC₅₀ values from 0.75 to 2.34 mg mL^?1.     

Determination and Evaluation of the Mineral Composition of Chinese Cabbage (Beta vulgaris)

Inductively coupled plasma optical emission spectrometry was used to determine the elements present in Chinese cabbage (Beta vulgaris). The accuracy of the method was confirmed by analysis of a certified reference material of spinach leaves. The study involved 57 samples that were collected in 13 Brazilian cities. Average concentrations of elements found per gram of Chinese cabbage were as follows: 3.44 mg g⁻¹ sodium, 5.09 mg g⁻¹ potassium, 1.25 mg g⁻¹ phosphorous, 0.85 mg g⁻¹ calcium, 0.49 mg g⁻¹ magnesium, 2.79 μg g⁻¹ manganese, 9.50 μg g⁻¹ iron, 0.74 μg g⁻¹ copper, 14.28 μg g⁻¹ zinc, and 6.44 μg g⁻¹ strontium. Principal component analysis and hierarchical cluster analysis demonstrated that there is no systematic difference in the mineral composition between the cabbage samples that were analyzed.     

Effect of dynamic high-pressure microfluidization at different temperatures on the antigenic response of bovine β-lactoglobulin

The antigenic response of β-lactoglobulin (β-Lg), treated by dynamic high-pressure microfluidization (DHPM) at different temperatures, was determined by an indirect competitive enzyme-linked immunosorbent assay using polyclonal antibodies from rabbit serum. DHPM treatment causes changes in the protein structure and may influence the antigenicity of β-Lg. DHPM treatment of β-Lg at 90 °C showed significant effects with the antigenic response of 5.2 μg mL⁻¹ (untreated), 45 μg mL⁻¹ (40 MPa), 79 μg mL⁻¹ (80 MPa), 132 μg mL⁻¹ (120 MPa), and 158 μg mL⁻¹ (160 MPa). In combination with temperature treatment (70–90 °C), the antigenic response enhanced as the temperature increased at 160 MPa. The β-Lg antigenicities were about 14, 108, and 158 μg mL⁻¹ at 70, 80, and 90 °C, respectively. However, the influence of DHPM pressures on the antigenic response of β-Lg standards was different. DHPM modified β-Lg standards showed a remarkable increase in antigenicity when treated to 80 MPa. Above 80 MPa, the antigenic response decreased.     

Angiotensin I-converting enzyme inhibitory activity of enzymatic hydrolysates of goat milk protein fractions

Casein and whey protein fractions from goat milk were hydrolysed by subtilisin and trypsin, individually and in combination, to release angiotensin converting enzyme (ACE)-inhibitory peptides. Selected hydrolysates were fractionated by size exclusion chromatography (SEC) and further characterised. The highest ACE-inhibitory activity was obtained from the casein fraction hydrolysed by the combination of enzymes. SEC presented 4 fractions with fraction F2 (<2.3 kDa) containing the highest concentration of peptides and the highest activity. F2 contained a number of peptides not previously identified from caprine caseins but with structural similarity to other ACE-inhibitory peptides. The most active fraction in relation to protein content was F4 with IC₅₀ between 9.3 and 5.1 μg mL⁻¹. This fraction contained a compound tentatively identified as WY, an active dipeptide not previously reported from caseins. The high inhibitory capacity of these fractions points towards the advantage of implementing a membrane process to concentrate the most active peptides.     

酪蛋白水解物的酶法修饰与ACE抑制活性变化

利用枯草杆菌碱性蛋白酶水解酪蛋白制备酪蛋白水解物,其水解度为11.2%,IC50为47.1μg/mL。再应用相同的酶对酪蛋白水解物进行类蛋白反应修饰,考察底物浓度、温度和酶添加量对类蛋白反应的影响,并制备5个不同的修饰产物测定其ACE抑制活性和IC50值。结果表明,修饰产物的ACE抑制活性随修饰程度(游离氨基减少量)的增加而提高,并且都高于未经修饰的酪蛋白水解物。当游离氨基减少量为154.65μmol/g(蛋白)时,修饰产物的IC50值可降至0.6μg/mL。毛细管电泳分析结果显示类蛋白修饰后水解物的多肽组成情况发生明显变化。研究结果证明酪蛋白水解物的ACE抑制活性可以通过类蛋白反应的修饰作用而提高。     

Ace Inhibition and Enzymatic Resistance In Vitro of a Casein Hydrolysate Subjected to Plastein Reaction in the Presence of Extrinsic Proline and Ethanol- or Methanol-Water Fractionation

Casein was hydrolyzed by alcalase to a degree of hydrolysis of 10.9% to obtain a hydrolysate having ACE-inhibition in vitro with an IC₅₀ value of 52.6 μg/mL. The prepared hydrolysate was modified by alcalase-catalyzed plastein reaction with extrinsic proline added at 0.4 mol/mol free amino groups (on the basis of the hydrolysate), and fractionated by ethanol- or methanol-water solvents in proportions of 3:7, 5:5, or 7:3 (v/v), respectively. With the decrease of free amino groups of the modified hydrolysate as the response, the optimized plastein reaction conditions were alcalase addition of 3.1 kU/g peptides, substrate concentration of 50% (w/v), and reaction temperature of 25°C. Four modified hydrolysates prepared with different reaction times exhibited higher ACE-inhibitory activities than the original hydrolysate. The evaluation results showed that solvent fractionation of the modified hydrolysate with the maximum activity (IC₅₀ = 13.0 μg/mL) yielded the separated soluble fraction's higher activity but the precipitate fraction's lower one. Further enzymatic digestion of the modified hydrolysate with the maximum activity and its two fractionated products by four proteases in vitro caused damage to the activities, but the residual activities of the final digests were higher than that of the original hydrolysate, indicating that the plastein reaction could confer casein hydrolysate protease resistance.     

Study of a microwave digestion method for total arsenic determination in marine mussels by electrothermal atomic absorption spectrometry: application to samples from the Ria de Arousa

Arsenic determination in mussel tissue was performed by electrothermal atomic absorption spectrometry (ETAAS) with Zeeman background correction and using iridium as a chemical modifier. Samples were digested by microwave heating using a mixture of nitric and sulphuric acids. This mixture makes possible the destruction of organoarsenic compounds, specifically arsenobetaine, prior to the graphite furnace determination. Optimum pyrolysis and atomization temperatures were 1,100 and 1,800 °C, respectively. The method was precise (with RSD% < 10), accurate (study of a certified reference material: 18.4 ± 1.4 μg As g⁻¹ vs. a certified content: 18.0 ± 1.1 μg As g⁻¹; recoveries between 90 and 104%) and sensitive (LOD 0.21 μg g⁻¹ on a dry weight basis). The method was applied to the determination of arsenic in aquaculture mussels collected in four sampling campaigns from the productive Ría de Arousa (estuary sited in Galicia, NW of Spain).     

Determination of Copper,Cobalt, Lead,and Iron in Table Salt by FAAS After Separation Using Violuric Acid and Multiwalled Carbon Nanotubes

A separation–enrichment technique for the determination of trace amounts of copper(II), cobalt(II), lead(II), and iron(III) as violuric acid chelates on multiwalled carbon nanotubes at pH 6.0 was established. Analytes were determined by flame atomic absorption spectrometry. The effects of some analytical parameters like pH, amounts of violuric acid, flow rates, eluent type, and sample volume were investigated. The influences of the matrix ions were also investigated. The relative standard deviations for analyte elements were below 10%. The quantification limits of the analyte ions were found as 0.36 μg g⁻¹ for copper, 0.43 μg g⁻¹ for lead, 0.15 μg g⁻¹ for cobalt, and 0.38 μg g⁻¹ for iron. The accuracy of presented method was checked by the analysis of TMDA 54.4 fortified lake water, NIST SRM 1515 apple leave, and HR-1 Humber river sediment certified reference materials. The method was applied to analyte contents of table salt samples from different origin. The levels of iron in the analyzed table salt samples were found in the range of 1.6–6.4 μg g⁻¹, while lead was found in only one sample as 5.0 μg g⁻¹. In all other samples, cobalt, lead, and copper were found below the quantification limits of the analytes.     

Antioxidant properties of whey protein hydrolysates as measured by three methods

Four microbial proteases (Alcalase, Flavourzyme, Neutrase and Protamex) were used for the preparation of whey protein hydrolysates. The aim of this research was to find out whether these hydrolysates can be used as a source of whey derived antioxidants. Hydrolyzed samples, including their unhydrolyzed protein solutions were tested by the ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) decolorization assay, by the total radical-trapping potential method and by the assay of liposomes peroxidation (fluorescence photometry). Antioxidant properties were enhanced by hydrolysis in most of cases. Alcalase hydrolysates were found as the most effective antioxidants as determined by ABTS assay (~50% of antioxidant activity at 0.1 mg ml⁻¹ of hydrolysate in reaction) and fluorescence photometry. Liposomes were oxidized ~50% less (1.1 μM of α-tocopherol equivalent) with Alcalase hydrolysates additive (at 5.85 mg ml⁻¹ of hydrolysate in reaction). Hydrolysates did not inhibit the oxidation of liposomes at concentrations below 1.0 mg ml⁻¹ in reaction. On the contrary, results of total trapping potential method did not agree with findings observed in other tests. In this assay, Neutrase hydrolysates showed the best antioxidant properties. Pro-oxidant properties were observed in solutions containing (prior to the enzyme Protamex addition only) intact whey protein as determined by the measurement of the liposome peroxidation. The ABTS assay was optimized for the evaluation of the antioxidant activity in whey protein hydrolysates. The reaction time should be prolonged to avoid underestimation of the antioxidant activity.