Purification and characterization of angiotensin I converting enzyme inhibitory peptides from the rotifer, Brachionus rotundiformis

Angiotensin I converting enzyme (ACE) inhibitory peptide was isolated from the marine rotifer, Brachionus rotundiformis. ACE inhibitory peptides were separated from rotifer hydrolysate prepared by Alcalase, α-chymotrypsin, Neutrase, papain, and trypsin. The Alcalase hydrolysate had the highest ACE inhibitory activity compared to the other hydrolysates. The IC₅₀ value of Alcalase hydrolysate for ACE inhibitory activity was 0.63 mg/ml. We attempted to isolate ACE inhibitory peptides from Alcalase prepared rotifer hydrolysate using gel filtration on a Sephadex G-25 column and high performance liquid chromatography on an ODS column. The IC₅₀ value of purified ACE inhibitory peptide was 9.64 μM, and Lineweaver–Burk plots suggest that the peptide purified from rotifer protein acts as a competitive inhibitor against ACE. Amino acid sequence of the peptide was identified as Asp-Asp-Thr-Gly-His-Asp-Phe-Glu-Asp-Thr-Gly-Glu-Ala-Met, with a molecular weight 1538 Da. The results of this study suggest that peptides derived from rotifers may be beneficial as anti-hypertension compounds in functional foods resource.     

鹿茸血肽疏水性分离及抗氧 化和ACE抑制作用的研究

实验采用DA201-C型大孔吸附树脂对Alcalase蛋白酶水解鹿茸血3 h的水解液进行吸附,采用25%、50%、75%、100%乙醇分级洗脱,收集各组分进行氨基酸组成分析,发现各洗脱组分具有不同疏水性值,同时测定各组分的血管紧张素转化酶(ACE)抑制活性和二苯代苦味肼基自由基(DPPH·)清除活性:75%乙醇洗脱组分的ACE抑制活性最高,为54.71%,且ACE抑制活性与组分疏水性值显著相关;100%乙醇洗脱组分的DPPH清除率最高.     

Protein hydrolysates from Bluefin Tuna (Thunnus thynnus) heads as influenced by the extent of enzymatic hydrolysis

Functional properties and antioxidant activities of protein hydrolysates from tuna (Thunnus thynnus) heads (THPHs), with different degrees of hydrolysis, obtained by treatment with Bacillus mojavensis A21 alkaline proteases and Alcalase, were investigated. Protein content of all freeze-dried THPHs ranged from 73.74 ± 0.5 to 78.56 ± 1.2%. The THPHs had excellent solubility, compared to untreated tuna head proteins and possessed interfacial properties, which were governed by their concentrations. Similarly, at a degree of hydrolysis (DH) of 12 and 15%, > 90% nitrogen solubility was observed at all experimental pH values tested. The emulsifying activity index (EAI) and emulsion stability index (ESI) of both hydrolysates at different DHs decreased (p < 0.05) with increasing DH. At low DH (5%), hydrolysates exhibited strong emulsifying properties. All THPHs produced by the A21 proteases generally showed higher antioxidative activity than that of the Alcalase protein hydrolysates. The highest DPPH radical-scavenging activity (78 ± 2.1% at 3 mg/mL) was obtained with a DH of 15%. The IC₅₀ value for the β-carotene bleaching assay was 0.5 ± 0.03 mg/mL. Alcalase (DH = 12%) and A21 (DH = 15%) protein hydrolysates contained glutamic acid/glutamine and arginine as the major amino acids, followed by lysine, aspartic acid/ asparagine, histidine, valine, phenylalanine, and leucine. In addition, the THPHs had a high percentage of essential amino acids, which made up 50.52 and 50.47%, of the protein hydrolysates obtained by the Alcalase and A21 proteases, respectively. Therefore, THPHs can be used as a promising source of functional peptides with antioxidant properties.     

Production of hydrolysate with antioxidative activity by enzymatic hydrolysis of extruded corn gluten

Hydrolysate of extruded corn gluten with higher solubility and antioxidative property was prepared. Extrusion and starch removal of corn gluten were applied as pretreatment before enzymatic hydrolysis by Alcalase. The amylase hydrolysis of starch at 70°C for 3 h resulted in the removal of the starch from the extruded corn gluten. The best hydrolysis results can be obtained by conducting the hydrolysis at 60°C with water addition 20 g/g protein, enzyme addition 0.048 Ansen units/g protein, pH 8.5, and 120 min. Degree of hydrolysis of extruded and nonextruded corn gluten reached 39.54 and 31.16%, respectively, under the optimal condition. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the optimal hydrolysate revealed that proteolysis of extruded corn gluten was more extensive than proteolysis of its counterpart which was not subjected to extrusion. The molecular weight of the peptides in the optimal hydrolysate was mainly over 3,710–660 Da as determined by gel filtration chromatography. The hydrolysates displayed good solubility and antioxidative activity. The separation profile of the hydrolysate on an ion exchange chromatography of Q-Sepharose Fast Flow showed that many kinds of peptides had antioxidative effect. A new peptide with antioxidative activity was purified, and its amino acid sequence was Phe-Pro-Leu-Glu-Met-Met-Pro-Phe, which was identified by Q-TOF2 mass spectrometry.     

Debittering effect of Actinomucor elegans peptidases on soybean protein hydrolysates

Effects of the enzymes in Actinomucor elegans extract and the enzyme Alcalase 2.4L on debittering the soybean protein hydrolysates were investigated. When the protein was treated only with the latter, a strong bitterness formed; but it decreased if the protein was treated with both the enzymes. The more the enzymes were used, weaker was the bitterness tasted. SDS-PAGE profile and ESI-MS spectrum of the hydrolysates evidenced that the Alcalase could convert the protein into peptides rapidly, while the enzymes in the A. elegans extract were able to further degrade some peptides which were difficult or unable to be hydrolyzed by the Alcalase. Further systematic analysis of the peptidases showed that the Alcalase exhibited a significant endopeptidase activity towards NBZ-Phe-pNA substrate (p < 0.01), whereas many exopeptidases in the A. elegans extract had the carboxypeptidase activity towards N-CBZ-Ile-Leu (p < 0.01). It is concluded that those exopeptidases presented in the A. elegans extract can benefit by decreasing the bitterness of the soybean protein hydroysate. They are also capable of being used with the Alcalase in a single-step enzymatic reaction to prepare the bitterless protein hydrolysate, which may be an efficient application for food industry.     

菜籽蛋白酶解液制备N-酰化肽的研究(Ⅱ)——蛋白水解率对N-酰化肽性能的影响

用不同水解率的水解液合成了N 酰化肽 ,对比了它们的表面活性 ,由碱性蛋白酶水解菜籽蛋白 ,当水解率为 30 .6%时 ,所合成的N 酰化肽性能比水解率为 1 5.7%、2 0 .3%时要好 ,用SephadexG 1 5分析了水解液中肽分子量的分布情况 ,发现较佳水解率的水解液中 80 %的肽分子量小于 1 50 0Da。     

Influence of the rapeseed protein hydrolysis process on CHO cell growth

Different protein hydrolysates were prepared from enzymatic hydrolyses of a rapeseed isolate (>90% protein content) using different commercial enzymes of non-animal origin. The extent of hydrolysis was controlled to produce hydrolysates corresponding to various degrees of hydrolysis (DH) from 5 to 30. These hydrolysates were characterized according to their solubility and size peptide pattern. Different growth behaviours of Chinese Hamster Ovary cells were observed when these various hydrolysates were added in serum-free medium containing transferrin, albumin and insulin. Hydrolysates from low degree of hydrolysis generally did not exhibit significant positive effect on cell growth; conversely hydrolysates from extensive hydrolysis, corresponding to a major low molecular size peptides content, usually allowed an increase of the maximal cell density. However, depending on the enzyme used, the supplementation with hydrolysates corresponding to a high degree of hydrolysis and composed of at least 70% peptides with a molecular size under 1kDa, led to different maximal cell density values, indicating the importance of enzyme specificity and consequently the nature of the released peptides. This result showed that the positive influence of the rapeseed hydrolysates on cell growth was not only due to a nutritional support tied to the addition of small peptides but may be related to the presence of peptides exhibiting growth or survival factor effects. Furthermore, total substitution of proteins (transferrin, albumin and insulin) in the cell culture medium by some rapeseed hydrolysates appeared to be a promising alternative to improve the cell growth in protein-free media.     

Trypsin Hydrolysed Protein Fractions as Radical Scavengers and Anti-bacterial Agents from <Emphasis Type="Italic">Ficus deltoidea</Emphasis>

Different molecular sizes of protein hydrolysates were prepared from the crude protein extract of Ficus deltoidea using the technique of membrane ultrafiltration after trypsin hydrolysis. Gel electrophoretic images shows the presence of 12, 8, 7 and 7 protein bands for the protein fractions prepared from the molecular weight cut-off of 3, 10, 30 and 100 kDa, respectively. The protein hydrolysates were found to have higher radical scavenging activity than those unhydrolysed fractions at the similar molecular size. They exhibited significant differences in the radical scavenging activities based on one-way analysis of variance, except for the protein hydrolysates of 30 and 100 kDa. The smallest protein hydrolysates, 3 kDa appeared to have the comparable activity (30%) with bovine serum albumin as a positive control in this study. Similarly, the 3 kDa protein hydrolysates achieved the highest inhibitory activity (87.5%) against Pseudomonas aeruginosa at the concentration of 128 µg/mL. The protein hydrolysates were found to be more effective against gram negative bacteria (P. aeruginosa and Escherichia coli) because of lower minimum inhibitory concentration (MIC) and effective inhibitory concentration at 50% (EC₅₀) than gram positive bacterium (Staphylococcus aureus). Trypsin catalysed hydrolysis seemed to improve the anti-bacterial activity of protein hydrolysates in a bacterial strain dependent manner. The MIC could achieve 1–55 µg/mL at different molecular sizes of protein fractions. Mass spectra matching revealed that 26% of 226 identified proteins belonged to the category of plant defensive proteins in stress management and metal handling.     

Production of angiotensin I-converting enzyme inhibitory peptides from defatted canola meal

To simplify the method of ACE-inhibitory peptide production, defatted canola meal was subjected to enzymatic proteolysis. Alcalase 2.4L and protease M “Amano” were found to be the most efficient enzymes in releasing ACE-inhibitory peptides from canola proteins among 13 tested enzymes. The IC₅₀ values of canola protein hydrolysates ranged from 18.1 to 82.5 μg protein/mL. Differences in ACE-inhibitory activities of various protein hydrolysates reflected varied enzyme specificities. A positive correlation was determined between ACE-inhibitory activity and the degree of hydrolysis (r = 0.5916, p < 0.001). Ion-exchange chromatography of canola protein hydrolysate increased the protein content greater than 95% without loss of ACE-inhibitory activity. This fraction was resistant to the degradation of gastrointestinal enzyme and ACE during in vitro incubation and may be a useful ingredient in the formulation of hypotensive functional food products.     

The high expression of Aspergillus pseudoglaucus protease in Escherichia coli for hydrolysis of soy protein and milk protein

Abstract

The hydrolysates of soy protein and milk protein are nutritional and functional food ingredients. Aspergillus pseudoglaucus aspergillopepsin I (App) is an acidic protease, including signal peptide, propeptide, and catalytic domain. Here, we cloned the catalytic domain App with or without propeptide in Escherichia coli. The results showed that the App without propeptide was not expressed or did not exhibit activity and App with propeptide (proApp) was highly expressed with a specific activity of 903?U/mg. Moreover, the denaturation temperature of proApp was 4.1?°C higher than App’s. The proApp showed 104?U/mg and 252?U/mg hydrolysis activities towards soy protein and milk protein under acidic conditions. By RP-HPLC analysis, the peptides obtained from the hydrolysates of soy protein and milk protein were hydrophilic peptides. This work first demonstrates efficient proteolysis of soy protein and milk protein through the functional expression of full-length proApp, which will likely have valuable industrial applications.     

Effect of bioactive peptides isolated from yeastolate,lactalbumin and NZCase in the insect cell growth

In this study, we have described the biological activity of various hydrolysates and its effect on cell growth, growth rate and doubling time. A potent cell culture enhancer factor was observed in the yeastolate hydrolysates, mainly in the protein fractions with low molecular weight. In this case, a growth enhancer of 60.66% was obtained. Despite a lower efficiency of crude lactalbumin hydrolysates (14%), when lactalbumin and yeastolate were added together to the culture, the cell yields were of 102%, showing a synergic effect. Nevertheless, sub fraction from LMW, of lactalbumin, obtained by Sephadex G-10 gel filtration chromatography showed a higher positive effect (23.3%) than low molecular weight fraction of lactalbumin without this chromatography step (11.3%). It is suggested that low molecular weight lactalbumin could have some inhibitory protein. On the other hand, NZCase low molecular weight showed a positive effect of 29.33%, while its sub fractions showed a negative effect of 5.5%. With these data we can suggest that these hydrolysates could be an important element to design new media, serum free, being helpful in protein recombinant production.     

Antioxidant peptides isolated from the marine rotifer,Brachionus rotundiformis

Protein derived from the rotifer Brachionus rotundiformis was hydrolyzed using different proteases (Alcalase, α-chymotrypsin, Neutrase, papain, pepsin and trypsin) for production of antioxidant peptide. Antioxidant activities of hydrolysates were evaluated using DPPH radical scavenging activity. Peptic hydrolysate exhibited the highest antioxidative activity compared to other hydrolysates. To identify antioxidant peptides, peptic hydrolysate was purified using consecutive chromatographic methods, and antioxidant peptides were identified to be Leu-Leu-Gly-Pro-Gly-Leu-Thr-Asn-His-Ala (1076 Da), and Asp-Leu-Gly-Leu-Gly-Leu-Pro-Gly-Ala-His (1033 Da) by Q-TOF ESI mass spectroscopy. EC₅₀ values of purified peptides were 189.8 and 167.7 μM, respectively. Antioxidant activities of peptides purified from the rotifer protein hydrolysate were evaluated, with results showing that peptides significantly quenched free radicals.     

Opposite Contributions of Glycinin- and β-Conglycinin-Derived Peptides to the Aggregation Behavior of Soy Protein Isolate Hydrolysates

The aggregation behavior as a function of pH was studied for hydrolysates obtained by hydrolysis of soy protein isolate (SPI) and glycinin- and β-conglycinin-rich protein fractions with subtilisin Carlsberg. The substrates were hydrolyzed up to degrees of hydrolysis (DH) of 2.2% and 6.5%. Compared with nonhydrolyzed SPI, a decrease in solubility was observed for the hydrolysates of SPI [0.8% (w/v) protein, I = 0.03 M] around neutral pH. At pH 8.0, glycinin hydrolysates had a much lower solubility (∼43% and 60%, respectively, for DH 2.2% and 6.5%) than SPI and β-conglycinin-derived hydrolysates, which were almost completely soluble. Peptides that aggregated were all larger than 5 kDa, and as estimated by size-exclusion chromatography their composition was almost independent of the aggregation pH. The solubility of hydrolysates of SPIs with a varying glycinin and β-conglycinin composition showed that glycinin-derived peptides are the driving force for the lower solubility of SPI hydrolysates. The solubility of SPI hydrolysates at pH 8.0 was shown not to be the sum of that of glycinin and β-conglycinin hydrolysates. Assuming that the separate hydrolysis of glycinin and β-conglycinin did not differ from that in the mixture (SPI), this indicates that β-conglycinin-derived peptides have the ability to inhibit glycinin-derived peptide aggregation.     

Covalent immobilization of alkaline proteinase on amino-functionalized magnetic nanoparticles and application in soy protein hydrolysis

Magnetic nanoparticles (MNPs) were synthesized and surface modified with (3-Aminopropyl)triethoxysilane (APTES). The alkaline proteinase (AP) was covalently immobilized on the APTES-modified MNPs through glutaraldehyde linkage. The resulting AP-loaded MNPs have an average size of 84 nm in aqueous solution, and a magnetization of 40 emu/g, endowing the immobilized enzyme with excellent magnetic responsively and dispersity. The maximum amount of AP and catalytic activity immobilized 1.0 mg MNPs was 120 μg and 25.3 units, respectively. Immobilized AP showed maximum activity at pH 10.0 and 50°C. Compared with free enzyme, the immobilized AP exhibited better storage stability. Moreover, immobilized AP can be reused 10 times and still maintained about 50% of its initial activity. The degree of hydrolysis of soy protein hydrolysates for immobilized AP could reach 19.0%, which was closer to the value of free enzyme. The molecular weight (M.W.) analysis showed that the soy protein was hydrolyzed successfully into small peptides of two main fractions with an average M.W. of 742 and 2126 Da. This study indicated that the immobilized AP could be used to hydrolyze continuously soy protein for potential industry application. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2756, 2019.     

Hydrolysis of rapeseed protein isolates: Kinetics,characterization and functional properties of hydrolysates

Two rapeseed protein isolates corresponding to albumins and globulins, respectively, were produced from an industrial defatted rapeseed meal. A pilot-scale process of protein extraction has been developed to remove major anti-nutritional compounds, easy to scale-up and using recyclable solvents. The kinetics of the hydrolyses of these two protein substrates using Alcalase 2.4L^® were compared by the measurement of the degree of hydrolysis (DH) when varying the initial proteins and enzyme concentrations. The globulins isolate was hydrolysed more efficiently than the albumins isolate mainly due to the compact and globular conformation of albumin (napin). Kinetic parameters have been determined for both substrates and a log-linear relation has been established between the DH values at a definite time and the initial enzyme/substrate ratio. Such relationships allow an effective monitoring of hydrolysis process since the hydrolysates analysis using reverse-phase chromatography coupled with mass spectrometry revealed that peptide maps corresponding to peptides of molecular weight inferior to 1 kDa are similar at a specific DH, independently of the reaction temperature and initial concentrations of substrate and enzyme. Thus, it is demonstrated that the DH is the sole parameter needed to control the physico-chemical properties and consequently the functionalities (solubility, foaming and emulsifying properties) which depend on the nature of peptides present in the hydrolysates.     

脑蛋白粉的制备及其酶解方法的研究

研究了以鲜猪脑制备蛋白粉并进行酶水解的方法 ;脑蛋白粉质量、不同酶和加酶方法对水解结果的影响 ;分析了水解液中游离氨基酸和低分子肽谱 ,并与国内外类似研究进行了比较。结果表明 :丙酮、乙醚沉淀制得的脑蛋白粉含氮 9.0 %～ 1 1 .5% ,脂肪 2 .5%～ 4 .0 % ,适于酶解 ;水解液含有 1 6种游离氨基酸 ,总量达 3 2 .4 4mg/ml和 4个分子量小于 1万的肽。提出精制脑蛋白粉进行水解、减压浓缩、超滤是提高水解液有效成分的技术措施。     

Analysis of novel angiotensin-I-converting enzyme inhibitory peptides from protease-hydrolyzed marine shrimp Acetes chinensis.

Acetes chinensis is an underutilized shrimp species thriving in the Bo Hai Gulf of China. In a previous study, we had used the protease from Bacillus sp. SM98011 to digest this kind of shrimp and found that the oligopeptide-enriched hydrolysate possessed antioxidant activity and high angiotensin I-converting enzyme (ACE) inhibitory activity with an IC50 value of 0.97 mg/ml. In this paper, by ultrafiltration, gel permeation chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC), five peptides with high ACE inhibitory activity were purified from the shrimp hydrolysates and their sequences were identified by amino acid composition analysis and molecular weight (MW) analysis. Three of them, FCVLRP (a), IFVPAF (f) and KPPETV (j), were novel ACE inhibitory peptides. Their IC50 values were 12.3 microM, 3.4 microM and 24.1 microM, respectively, and their recoveries were 30 mg/100 g (solid basis of shrimp), 19 mg/100 g and 33 mg/100 g, respectively. Lineweaver-Burk plots for the three novel peptides showed that they are all competitive inhibitors. To test the ACE inhibitory activity of peptide a, f, j after they were digested by digestive enzymes in vivo, 12 derived peptides from FCVLRP and IFVPAF were synthesized based on their amino acid sequences and the cleavage sites of digestive enzymes. No digestive enzyme cleavage site was found in KPPETV. The IC50 values of the derived peptides were determined and the result showed that except for VPAF, FC and FCVL, the ACE inhibitory activity of the other nine derived peptides did not significantly change when compared with their original peptides. Surprisingly, five peptides had lower IC50 values than their original peptides, particularly for RP (IC50 value = 0.39 microM), which is about 30 times lower than its original peptide and almost the lowest IC50 value for ACE inhibitory peptides reported. Therefore, the novel peptides identified from A. chinensis hydrolysates probably still maintain a high ACE inhibitory activity even if they are digested in vivo. This is the first report about novel ACE inhibitory peptides from hydrolysates of marine shrimp A. chinensis. The novel peptides from hydrolysate of A. chinensis and some of their derived peptides with high ACE inhibitory activity probably have potential in the treatment of hypertension or in clinical nutrition.     

Chemometric analysis of soy protein hydrolysates used in animal cell culture for IgG production – An untargeted metabolomics approach

Soy protein hydrolysates are used as the most cost effective medium supplement to enhance cell growth and recombinant protein productivity in cell cultures. Such hydrolysates contain diverse classes of compounds, such as peptides, carbohydrates and phenolic compounds. To identify if specific compounds dominate the functionality of hydrolysates in cell cultures, thirty samples of hydrolysates with different cell culture performances were analyzed for chemical composition using an untargeted metabolomics approach. Out of 410 detected compounds, 157 were annotated. Most of the remaining 253 compounds were identified as peptides, but could not be annotated exactly. All compounds were quantified relatively, based on their average signal intensities. The cell growth and total immunoglobulin (IgG) production, relative to the CD medium (100%), ranged from 148 to 438% and 117 to 283%, respectively. Using bootstrapped stepwise regression (BSR), the compounds with the highest inclusion frequency were identified. The most important compound, i.e. phenyllactate and ferulate explained 29% and 30% of the variance for cell growth and total IgG production, respectively. Surprisingly, all compounds identified in the BSR showed a positive correlation with cell growth and total IgG production. This knowledge can be applied to monitor the production and accumulation of these compounds during the production process of hydrolysates. Consequently, the processing conditions can be modulated to produce soy protein hydrolysates with enhanced and consistent cell culture performance.     

Influence of protein and carbohydrate contents of soy protein hydrolysates on cell density and IgG production in animal cell cultures

The variety of compounds present in chemically defined media as well as media supplements makes it difficult to use a mechanistic approach to study the effect of supplement composition on culture functionality. Typical supplements, such as soy protein hydrolysates contain peptides, amino acids, carbohydrates, isoflavones, and saponins. To study the relative contribution of these compound classes, a set of hydrolysates were produced, containing 58‐83% proteinaceous material and 5‐21% carbohydrates. While the content of the different compounds classes varied, the composition (e.g., peptide profiles, carbohydrate composition) did not vary in hydrolysates. The hydrolysates were supplemented to a chemically defined medium in cell culture, based on equal weight and on equal protein levels. The latter showed that an increase in the carbohydrate concentration significantly (P value < 0.004) increased integral viable cell density (IVCD) (R = 0.7) and decreased total IgG (R = ?0.7) and specific IgG production (R = ?0.9). The extrapolation of effects of protein concentration showed that an increase in protein concentration increased total and specific IgG production and suppressed IVCD. In addition to proteins and carbohydrates, the functionality of soy protein hydrolysates may be modulated by the presence of other minor compounds. In the current study, the large differences in the balance between total proteins and total carbohydrates in the supplemented media seem to be a main factor influencing the balance between the viable cell density, total IgG, and specific IgG production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1396–1405, 2015