Lipase-assisted generation of 2-methyl-3-furanthiol and 2-furfurylthiol from thioacetates

Enzymatic hydrolysis of S-3-(2-methylfuryl) thioacetate and S-2-furfuryl thioacetate using lipase from Candida rugosa produced 2-methyl-3-furanthiol and 2-furfurylthiol, respectively. When reactions were carried out at room temperature and pH 5.8, 2-methyl-3-furanthiol was produced in a optimal yield of 88% after 15 min of reaction, whereas 2-furfurylthiol was obtained in a yield of 80% after 1 h of reaction time. Enzymatic hydrolysis was also performed in n-hexane, n-pentane, and water/propylene glycol mixture. The reaction rates in these media were slower as compared to those in aqueous medium; however, the reaction yields were quite similar. As expected, the stability of the generated 2-methyl-3-furanthiol and 2-furfurylthiol was better in n-hexane, n-pentane, and the water/propylene glycol mixture as compared to that in water or phosphate buffer.     

Lunasin and Bowman-Birk protease inhibitor concentrations of protein extracts from enzyme-assisted aqueous extraction of soybeans

Lunasin and Bowman-Birk protease inhibitor (BBI) are two soybean peptides to which health-promoting properties have been attributed. Concentrations of these peptides were determined in skim fractions produced by enzyme-assisted aqueous extraction processing (EAEP) of extruded full-fat soybean flakes (an alternative to extracting oil from soybeans with hexane) and compared with similar extracts from hexane-defatted soybean meal. Oil and protein were extracted by using countercurrent two-stage EAEP of soybeans at 1:6 solids-to-liquid ratio, 50 °C, pH 9.0, and 120 rpm for 1 h. Protein-rich skim fractions were produced from extruded full-fat soybean flakes using different enzyme strategies in EAEP: 0.5% protease (wt/g extruded flakes) used in both extraction stages; 0.5% protease used only in the second extraction stage; no enzyme used in either extraction stage. Countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes was used as a control. Protein extraction yields increased from 66% to 89-96% when using countercurrent two-stage EAEP with extruded full-fat flakes compared to 85% when using countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes. Extruding full-fat soybean flakes reduced BBI activity. Enzymatic hydrolysis reduced BBI contents of EAEP skims. Lunasin, however, was more resistant to both enzymatic hydrolysis and heat denaturation. Although using enzymes in both EAEP extraction stages yielded the highest protein and oil extractions, reducing enzyme use to only the second stage preserved much of the BBI and Lunasin.     

Inhibition of lipid oxidation in cooked beef patties by hydrolyzed potato protein is related to its reducing and radical scavenging ability

Protein hydrolysates were prepared by limited alcalase hydrolysis (0.5, 1, and 6 h, corresponding to degrees of hydrolysis of 0.72, 1.9, and 2.3, respectively) of heat-coagulated potato protein. The hydrolysates were characterized for peptide composition, ferric reducing/antioxidant power (FRAP), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging activity, and Fe2+- and Cu2+-chelation capacity. Hydrolyzed and intact proteins were formulated (4%, w/w) into beef patties to determine in situ antioxidant efficacy. Thiobarbituric acid-reactive substances (TBARS) and peroxide value (PV) formed in cooked and PVC-packaged patties during storage (4 degrees C, 0-7 days) were analyzed. Hydrolysis increased the protein solubility by 14-19-fold and produced numerous short peptides (< 6 kDa). The FRAP values of the protein sample (23 micromol/g) increased markedly after hydrolysis but were similar between the three hydrolysates (597-643 micromol/g). Similarly, the ABTS radical-scavenging activity also was increased by hydrolysis and was the greatest for the 1-h hydrolysate. Hydrolysis increased the Cu2+-chelation activity but decreased the Fe2+-chelation ability of the protein. The production of PV in patties after 7 days of storage was lowered 44.9% and 74.5% (P < 0.05), and that of TBARS was reduced 40.9% and 50.3% (P < 0.05), by intact and hydrolyzed proteins, respectively.     

Bowman-Birk and Kunitz Protease Inhibitors among Antinutrients and Bioactives Modified by Germination and Hydrolysis in Brazilian Soybean Cultivar BRS 133

Soybean contains constituents that have antinutritional and bioactive properties. Enzymatic hydrolysis and germination can enhance the biological activity of these compounds in soybean. The objective of this study was to investigate the effect of germination, Alcalase (protease) hydrolysis, and their combination on the concentrations of antinutritional and bioactive compounds in Brazilian soybean cultivar BRS 133. A combination of germination and Alcalase hydrolysis resulted in the degradation of Bowman-Birk inhibitor (BBI), Kunitz trypsin inhibitor (KTI), and lunasin by 96.9, 97.8, and 38.4%. Lectin was not affected by any of the processing treatments when compared to nongerminated and nonhydrolyzed soy protein extract. Total isoflavones (ISF) and total saponins (SAP) increased by 16.2 and 28.7%, respectively, after 18 h of germination, while Alcalase hydrolysis led to the reduction of these compounds. A significant correlation was found between concentrations of BBI and KTI, BBI and lunasin, BBI and ISF, KTI and lunasin, KTI and ISF, KTI and SAP, lunasin and ISF, and ISF and SAP. Germination and Alcalase hydrolysis interacted in reducing BBI, ISF, and SAP. This study presents a process of preparing soy flour ingredients with lower concentrations of antinutritional factors and with biologically active constituents, important for the promotion of health associated with soybean consumption. In conclusion, 18 h of germination and 3 h of Alcalase hydrolysis is recommended for elimination of protease inhibitors, while bioactives are maintained by at least 50% of their original concentrations.     

Preparation and Functional Properties of Gluten Hydrolysates with Wheat‐Bug (Eurygaster spp.) Protease

Suni‐bug (Eurygaster spp.) enzyme was partially purified from bug‐damaged wheat and used to prepare gluten hydrolysates at 3% and 5% degree of hydrolysis (DH). Functional properties of gluten and gluten hydrolysates were determined at 0.2% (w/v) protein concentration and pH 2–10. Gluten solubility after enzymatic hydrolysis increased significantly (P < 0.05) up to 89.1, 89.6, and 95.0% at pH 7, 8, and 10, respectively. Emulsion stability (ES) of gluten hydrolysates improved at neutral and alkaline pH (P < 0.05) and emulsifying capacity (EC) increased significantly (P < 0.05) except at pH 10. Foaming capacity (FC) values of gluten hydrolysates were significantly higher (P < 0.05) at pH 6, 7, 8; foam stability (FS) values of gluten hydrolysates were significantly higher (P < 0.05) at pH 6 and 7. Enzymatic modification of gluten by wheat‐bug enzyme resulted in hydrolysates with higher antioxidant activity compared to gluten. Significant correlations (P < 0.001) were found between solubility and EC, ES, FC, and FS values of gluten and its hydrolysates with 3% and 5% DH.     

Nutritional Enhancement of Soy Meal via Aspergillus oryzae Solid‐State Fermentation

Antinutritional factors in soy meal (SM) include trypsin inhibitor, galactooligosaccharides (GOSs), structural polysaccharides, and large‐molecular‐weight protein. These antinutritional factors limit the usage of SM for young monogastric animals. Aspergillus oryzae solid‐state fermentation was applied to eliminate these factors, and changes in physical and chemical characteristics of SM were investigated. A. oryzae–treated SM was more nutrient dependent than oxygen dependent, which was illustrated by scanning electron microscopy. After 36 h of fermentation, the concentration of GOSs (raffinose, stachyose, and verbascose) and trypsin inhibitor decreased from an initial value of 9.48 mmol/100 g to a nondetectable level. Structural polysaccharides decreased by 59% (w/w), and the degree of hydrolysis of SM protein increased from an initial value of 0.9 to 7% (w/w) through the seven‐day fermentation. Fermentation also modified nutritional factors. Protein content increased from 50.47 to 58.93% (w/w) after 36 h of fermentation. Amino acid contents were significantly enhanced. The research thoroughly studied the A. oryzae solid‐state fermentation of SM, and the resulting premium product could provide a better protein source for monogastric animals.     

Glass transition and water effects on sucrose inversion by invertase in a lactose-sucrose system

Enzymatic changes are often detrimental to quality of low-moisture foods. In the present study, effects of glass transition and water on sucrose inversion in a lactose-sucrose food model were investigated. Amorphous samples were produced by freeze-drying lactose-sucrose (2:1)-invertase (20 mg invertase/49.4 g of carbohydrate) dissolved in distilled water. Sorption isotherms were determined gravimetrically at 24 degrees C. Sucrose hydrolysis was determined by monitoring glucose content using a test kit and the amounts of fructose, glucose, and sucrose using HPLC. The glass transition temperatures, T(g), at various water contents were measured using differential scanning calorimetry (DSC). The BET and the GAB sorption models were fitted to experimental data up to a(w) 0.444 and 0.538, respectively. Water sorption and DSC results suggested time-dependent crystallization of sugars at a(w) 0.444 and above. Significant sucrose hydrolysis occurred only above T(g), concomitantly with crystallization. Sucrose hydrolysis and crystallization were not likely in glassy materials.     

非离子表面活性剂PEG对棉秆木质纤维素酶解的影响

为了提高纤维素的水解作用,减少酶的用量,该文研究不同分子量的聚乙二醇(PEG,poly ethylene glycol)表面活性剂对棉秆木质纤维素水解复合酶的影响,研究不同分子量的PEG对还原糖、纤维素酶、木质素酶、蛋白吸附率、酶动力学特征的影响。结果表明,2.5g/L添加量的PEG 2000、PEG 4000、PEG 6000和PEG 8000均能提高棉秆木质纤维素水解酶的活力,增加纤维素的转化率,减少蛋白的非特异性吸附率,提高棉秆木质素和纤维素的降解率,提高最大反应速度和降低酶与底物的亲和力,其中PEG 6000效果最好。3.0g/L的PEG 6000添加量对纤维素的转化率最高,达到58.12%(P<0.05)。对于吸附率,2.0、3.0、4.0、5.0g/L的添加量与对照相比差异显著(P<0.05),且相互之间差异不显著(P>0.05)。添加3.0g/L PEG 6000的表面活性剂能保持上清液中较高的蛋白浓度,水解3h后,没有添加表面活性剂的处理,纤维滤纸酶酶活从0.1245FPU/mL下降到0.012FPU/mL,下降了90.37%,添加PEG 6000后,FPA酶的活性恢复到0.041FPU/mL;水解48h,添加PEG 6000和不添加PEG 6000纤维素的转化率分别达到65.71%和54.68%,添加PEG 6000提高纤维素转化率20.18%。结果表明,PEG6000可以提高纤维素的转化率,为工业生产实践提供了参考。     

Enzymatic hydrolysis of biological and environmental samples as pretreatment for analysis

Four commercially available proteases were tested, in conjunction with a lipase, for efficacy in hydrolyzing 3 tissue substrates: cod fillet, chicken egg, and bovine liver. Enzymatic hydrolysis of tissues minimizes the formation of emulsions during liquid-liquid extraction and does not accelerate the decomposition of acid- or base-labile analytes. Recovery of hexane and benzene phases from the hydrolysates was also evaluated. Protease from Streptomyces griseus combined with lipase from Candida cylindracea (available commercially) produced the highest percent hydrolysis (relative to classical acid hydrolysis) in all 3 tested tissues (60-95%) and the greatest recovery of hexane (100%) and benzene (92-100%) solvent phases.     

Protease Treatment for the Stabilization of Rice Bran: Effects on Lipase Activity,Antioxidants, and Lipid Stability

Rice bran lipid is rapidly made rancid by endogenous lipase enzymes. To inactivate rice bran lipase, an enzymatic hydrolytic method was developed and then compared with the thermal method. The efficiency of five proteolytic enzymes including trypsin, chymotrypsin, papain, bromelain, and Flavourzyme enzyme to stabilize rice bran was investigated. Moreover, the antioxidant content and storage stability of enzymatically stabilized rice bran (ESRB), thermally stabilized rice bran (TSRB), and raw rice bran (RRB) were studied. Trypsin, chymotrypsin, and papain showed a higher rate of hydrolysis than bromelain and Flavourzyme enzyme. After 120 min of hydrolysis, rice bran hydrolyzed by trypsin, chymotrypsin, and papain inactivated 80, 86, and 79% of lipase activity, respectively, whereas lipase activity of rice bran hydrolyzed by bromelain and Flavourzyme enzyme was higher than the initial rice bran. With a similar lipase inactivation level, the cheaper papain was used to produce ESRB. Total phenolics content of ESRB was 52.89 and 94.10% higher than in RRB and TSRB, respectively. In addition, γ‐oryzanol content in ESRB was 2.23‐ and 2.05‐fold of that in RRB and TSRB, respectively. Lipase activity of RRB increased throughout the two months of storage, whereas no change in lipase activity was observed in ESRB and TSRB. At the end of storage, free fatty acid contents of RRB, TSRB, and ESRB were 15.30, 4.67, and 3.92%, respectively. We propose enzymatic hydrolysis by papain for stabilization of rice bran with high antioxidant content and storage stability.     

Enzymatic Milling of Corn: Optimization of Soaking,Grinding, and Enzyme Incubation Steps

Enzymatic milling is a modified wet‐milling process that uses proteases to significantly reduce the total processing time during corn wet milling and eliminates the need for sulfur dioxide as a processing agent. To optimize the overall enzymatic milling procedure and minimize the amount of enzyme, a series of experiments were done to determine the best first grind parameters and the optimal enzyme additions. The yields for germ, germ quality, and starch recovery were used for evaluation of first grind and enzyme addition, respectively. The specific processing conditions evaluated were the soaking time and first grind parameters. After soaking and first grind optimization, enzyme concentration and pH determinations were evaluated using bromelain as an example. The first grind procedure was optimized by evaluating a combination of different soaking and grinding conditions followed by a fixed enzyme addition and incubation step. The pH profile of bromelain for enzymatic milling was evaluated for pH 3.5–6.5 and the optimum was determined to be pH 5.0. Enzyme addition was then evaluated using the optimized first grind conditions and bromelain additions with 0–1.9 g of enzyme (based on protein)/kg of corn. Results showed that the minimum addition of bromelain to reach starch yields equivalent to conventional yields were ≈0.4 g of protein/kg of corn. This amount is significantly less than what was previously used and reported.     

Evaluation of oligosaccharide synthesis from lactose and lactulose using β-galactosidases from Kluyveromyces isolated from artisanal cheeses

The β-galactosidase activity of 15 Kluyveromyces strains isolated from cheese belonging to Kluyveromyces lactis and Kluyveromyces marxianus species was tested for the production of oligosaccharides derived from lactose (GOS) and lactulose (OsLu). All Kluyveromyces crude cell extracts (CEEs) produced GOS, such as 6-galactobiose and 3'-, 4'-, and 6'-galactosyl-lactose. At 4 h of reaction, the main trisaccharide formed was 6'-galactosyl-lactose (20 g/100 g of total carbohydrates). The formation of OsLu was also observed by all CEEs tested, with 6-galactobiose, 6'-galactosyl-lactulose, and 1-galactosyl-lactulose being found in all of the reaction mixtures. The synthesis of trisaccharides predominated over other oligosaccharides. K. marxianus strain O3 produced the highest yields of GOS and OsLu after 4 h of reaction, reaching 42 g/100 g of total carbohydrates (corresponding to 80% lactose hydrolysis) and 45 g/100 g of total carbohydrates (corresponding to 87% lactulose hydrolysis), respectively. Therefore, the present study contributes to a better insight into dairy Kluyveromyces β-galactosidases and shows the feasibility of these enzymes to transglycosylate lactose and lactulose, producing high yields of prebiotic oligosaccharides.     

Enzymatic production of xylooligosaccharides from cotton stalks

Xylooligosaccharide (XO) production was performed from xylan, which was obtained by alkali extraction from cotton stalk, a major agricultural waste in Turkey. Enzymatic hydrolysis was selected to prevent byproduct formation such as xylose and furfural. Xylan was hydrolyzed using a commercial xylanase preparation, and the effects of pH, temperature, hydrolysis period, and substrate and enzyme concentrations on the XO yield and degree of polymerization (DP) were investigated. Cotton stalk contains about 21% xylan, the composition of which was determined as 84% xylose, 7% glucose, and 9% uronic acid after complete acid hydrolysis. XOs in the DP range of 2-7 (X6 approximately X5>X2>X3) were obtained with minor quantities of xylose in all of the hydrolysis conditions used. Although after 24 h of hydrolysis at 40 degrees C, the yield was about 53%, the XO production rate leveled off after 8-24 h of hydrolysis. XO yield was affected by all of the parameters investigated; however, none of them affected the DP of the end product significantly, except the hydrolysis period. Enzyme hydrolysis was maintained by the addition of fresh substrate after 72 h of hydrolysis, indicating the persistence of enzyme activity. The optimal hydrolysis conditions were determined as 40 degrees C, pH 5.4, and 2% xylan. The obtained product was fractionated via ultrafiltration by using 10, 3, and 1 kDa membranes. Complete removal of xylanase and unhydrolyzed xylan was achieved without losing any oligosaccharides having DP 5 or smaller by 10 kDa membrane. After a two-step membrane processing, a permeate containing mostly oligosaccharides was obtained.     

Preparation and characterization of papain-modified sesame (Sesamum indicum L.) protein isolates

Defatted sesame meal ( approximately 40-50% protein content) is very important as a protein source for human consumption due to the presence of sulfur-containing amino acids, mainly methionine. Sesame protein isolate (SPI) is produced from dehulled, defatted sesame meal and used as a starting material to produce protein hydrolysate by papain. Protein solubility at different pH values, emulsifying properties in terms of emulsion activity index (EAI) and emulsion stability index (ESI), foaming properties in terms of foam capacity (FC) and foam stability (FS), and molecular weight distribution of the SPI hydrolysates were investigated. Within 10 min of hydrolysis, the maximum cleavage of peptide bonds occurred as observed from the degree of hydrolysis. Protein hydrolysates have better functional properties than the original SPI. Significant increase in protein solubility, EAI, and ESI were observed. The greatest increase in solubility was observed between pH 5.0 and 7.0. The molecular weight of the hydrolysates was also reduced significantly during hydrolysis. These improved functional properties of different protein hydrolysates would make them useful products, especially in the food, pharmaceutical, and related industries.     

Hydrolysis of the amyloid prion protein and nonpathogenic meat and bone meal by anaerobic thermophilic prokaryotes and streptomyces subspecies

Transmissible spongiform encephalopathies are caused by accumulation of highly resistant misfolded amyloid prion protein PrPres and can be initiated by penetration of such pathogen molecules from infected tissue to intact organism. Decontamination of animal meal containing amyloid prion protein is proposed thanks to the use of proteolytic enzymes secreted by thermophilic bacteria Thermoanaerobacter, Thermosipho, and Thermococcus subsp. and mesophilic soil bacteria Streptomyces subsp. Keratins alpha and beta, which resemble amyloid structures, were used as the substrates for the screening for microorganisms able to grow on keratins and producing efficient proteases specific for hydrolysis of beta-sheeted proteic structures, hence amyloids. Secretion of keratin-degrading proteases was evidenced by a zymogram method. Enzymes from thermophilic strains VC13, VC15, and S290 and Streptomyces subsp. S6 were strongly active against amyloid recombinant ovine prion protein and animal meal proteins. The studied proteases displayed broad primary specificities hydrolyzing low molecular mass peptide model substrates. Strong amyloidolytic activity of detected proteases was confirmed by experiments of hydrolysis of PrPres in SAFs produced from brain homogenates of mice infected with the 6PB1 BSE strain. The proteases from Thermoanaerobacter subsp. S290 and Streptomyces subsp. S6 are the best candidates for neutralization/elimination of amyloids in meat and bone meal and other protein-containing substances and materials.     

Evaluation of detoxification methods on toxic and antinutritional composition and nutritional quality of proteins in Jatropha curcas meal

The Jatropha curcas meal was detoxified by different methods, and the effect of detoxification was evaluated in this study. The method that hydrolysis of enzymes (cellulase plus pectinase) followed by washing with ethanol (65%) had a significant (p < 0.05) effect on the toxin, antinutritional components, and nutritional quality of proteins. After this treatment, the phorbolesters (PEs) were decreased by 100%. The antinutritional components (phytates, tannins, saponins, protease inhibitor, and lectin activities) were decreased to tolerable levels, which were lower than those in soybean meal. The crude protein in detoxified meal was 74.68%, and the total content of amino acids was 66.87 g/100 g of dry matter. The in vitro protein digestibility (IVPD) increased from 82.14 to 92.37%. The pepsin-insoluble nitrogen was only 4.57% of the total nitrogen, and about 90% of the protein was true protein. The protein-digestibility-corrected amino acid score (PDCAAS) of the meal was 0.75. The results showed that this treatment was a promising way to detoxify J. curcas meal, and the nutritional quality of detoxified meal can be simultaneously enriched and improved.     

Differences in Starch Granule Composition and Structure Influence In Vitro Enzymatic Hydrolysis of Grain Meal and Extracted Starch in Two Classes of Canadian Wheat (Triticum aestivum L.)

Starch granule composition and amylopectin structure affect starch digestibility, an important factor influencing wheat grain utilization for human food consumption. Six bread wheat cultivars with four belonging to the Canada Western Red Spring (CWRS) and two Canada Prairie Spring Red (CPSR) market classes were analyzed for the relationship between their grain constituents and in vitro enzymatic hydrolysis of starch. CPSR cultivars had higher starch and amylose concentrations compared with CWRS cultivars, which had a higher protein concentration. Starch granule size distribution did not differ among the genotypes, except AC Foremost, which had significantly (P < 0.05) higher volume percent of B‐type starch granules (≈15%) and lower volume percent of A‐type starch granules (≈9%) compared with other cultivars. Fluorophore‐assisted capillary electrophoresis revealed a lower content of R‐IV (DP 15–18, ≈6%) and a higher content of R‐VII (DP 37–45, ≈7%) chains in the CPSR cultivars compared with the CWRS cultivars. Starch in vitro enzymatic hydrolysis showed that compared with CWRS cultivars, the two CPSR cultivars had reduced amounts of readily digestible starch and higher amounts of slowly digestible starch and resistant starch. Consequently, the two CPSR cultivars also showed lower hydrolysis indexes in grain meal as well as extracted starch. CPSR cultivars, with higher starch and amylose concentrations, as well as a higher content of long chains of amylopectin, showed a reduced starch in vitro enzymatic hydrolysis rate.     

Release of D-xylose from wheat straw by acid and xylanase hydrolysis and purification of xylitol

Xylitol is a valuable sweetener produced from xylose-rich biomass. Our objective was to optimize conditions for maximum release of D-xylose from wheat straw by acid or enzyme hydrolysis with minimal release of other monosaccharides, and to purify xylitol from three other alditols. Ground straw was treated with 10 parts of 0.2-0.4 M sulfuric acid at 110-130 degrees C for 15-45 min or at reflux with 0.75-1.25 M sulfuric acid for 1.5-3 h. Under optimum conditions of either 0.3 M acid at 123 degrees C for 28 min or 1.0 M acid at 100 degrees C for 3 h, 18 or 19% of D-xylose plus approximately 6% other sugars were produced from straw (dry basis). A 16% yield of D-xylose plus 6% other sugars was obtained when hydrothermally (10% straw, 160 degrees C, 1 h) treated straw was incubated with a commercial xylanase. The lack of enzyme specificity for D-xylose release was attributed to the autohydrolysis of polysaccharides during the pretreatment plus slow hydrolysis of cellulose during enzyme digestion. Xylitol with a purity of 95% was obtained in 10% yield from straw after the reduction of an acid-hydrolyzate followed by fractional crystallization. Purification of the mixture of four alditols by open-column chromatography on a strongly basic anion-exchange resin in hydroxide form gave 7% xylitol crystals with a purity of 99%.     

A novel defensin encoded by a mungbean cDNA exhibits insecticidal activity against bruchid

A cDNA encoding a small cysteine-rich protein designated VrCRP was isolated from a bruchid-resistant mungbean. VrCRP encodes a protein of 73 amino acids containing a 27 amino acid signal peptide and 8 cysteines. On the basis of the amino acid sequence similarity and conserved residues, it is suggested that VrCRP is a member of the plant defensin family. VrCRP protein was obtained by overexpression of VrCRP with a truncated signal peptide in an IMPACT system. Artificial seeds containing 0.2% (w/w) of the purified VrCRP-TSP were lethal to larvae of the bruchid Callosobruchus chinensis. VrCRP is apparently the first reported plant defensin exhibiting in vitro insecticidal activity against C. chinensis.     

Purification of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide with an antihypertensive effect from loach (Misgurnus anguillicaudatus)

To isolate and characterize novel angiotensin I-converting enzyme (ACE) inhibitory peptide from loach (Misgurnus anguillicaudatus), six proteases, pepsin, α-chymotrypsin, bromelain, papain, alcalase, and Neutrase, were used to hydrolyze loach protein. The hydrolysate (LPH) generated by bromelain [ratio of enzyme to substrate, 3:1000 (w/w)] was found to have the highest ACE inhibitory activity (IC(50), 613.2 ± 8.3 μg/mL). Therefore, it was treated by ultrafiltration to afford fraction of LPH-IV (MW < 2.5 kDa) with an IC(50) of 231.2 ± 3.8 μg/mL, having higher activity than the other fractions. Then, LPH-IV was isolated and purified by consecutive purification steps of gel filtration chromatography and reverse-phase high-performance liquid chromatography to afford a purified peptide with an IC(50) of 18.2 ± 0.9 μg/mL, an increase of 33.7-fold in ACE inhibitory activity as compared with that of LPH. The purified peptide was identified as Ala-His-Leu-Leu (452 Da) by Q-TOF mass spectrometry and amino acid analyzer. An antihypertensive effect in spontaneously hypertensive rats revealed that oral administration of LPH-IV could decrease systolic blood pressure significantly.