不同蛋白酶水解酪蛋白及其对产物功能性质的影响

采用Alcalase 2．4L和Protamex两种蛋白酶分别水解酪蛋白酸钠（蛋白质含量88．03％）至5％、10％、15％和20％等不同的水解度（DH），并对酪蛋白酸钠及填水解产物的各种功能性质进行了分析测定。结果表明：酪蛋白酸钠经水解后，蛋白质、水分和灰分含量发生变化，游离氨基量增加且增加与DH相关；水解产物中的多肽分子量较小，平均分子量小于8103D，并且分子量随DH的增大而减小，在DH为15％和20％的水解产物中多肽分子量均低于5043D：水解产物的溶解性随DH的增大而增强，在pH4．0～5．0、DH10％～20％的范围内产物溶解度84．8％～98％，说明在等电点条件下，酪蛋白酸钠水解后溶解性得到改善：与酪蛋白酸钠相比，水解产物的乳化性和起泡性减弱；不同水解产物的氨基酸组成差异不是很大，与酪蛋白酸钠也很接近。     

Proteolysis characteristics of sarcoplasmic, myofibrillar, and stromal proteins separated from grass carp and antioxidant properties of their hydrolysates

Proteolysis of grass carp sarcoplasmic, myofibrillar, and stromal proteins by 5 commercial proteases were studied. Sarcoplasmic and myofibrillar protein could be well hydrolyzed by Alcalase 2.4 L to reach high protein recoveries (PR) (71.86±2.46 and 80.77±3.05%, respectively), while the maximum PR for stromal protein was only 42.83±2.84%. However, stromal hydrolysates, containing mostly 6–10 kDa fraction, exhibited higher ·OH scavenging activities due to its high content of antioxidant-assisting amino acids. Alcalase 2.4 L and pancreatin 6.0, which produced hydrolysates with relative high degree of hydrolysis (DH), were used for further hydrolysis of whole grass carp protein with the assistance of response surface methodology (RSM). The results showed that serine proteases (Alcalase 2.4 L and pancreatin 6.0) could produce sarcoplasmic, myofibrillar, or stromal hydrolysates with relatively high PR, DH, and strong ·OH scavenging activity, which may be used to prepare antioxidant hydrolysates from grass carp.     

鳙鱼鱼肉蛋白的酶解及其对功能性质的影响

为了改善鳙鱼鱼肉蛋白(BCMP)的功能性质以扩大其在食品工业中的应用,以鳙鱼为原料制备了鳙鱼鱼肉蛋白,并利用碱性蛋白酶Alcalase2.4L对其进行水解,得到了3种不同水解度(DH4.5%、DH9.0%、DH13.5%)的酶解物。研究了BCMP及其酶解物的功能性质,包括溶解性、持水性、持油性、乳化性、起泡性。结果表明,与原鳙鱼鱼肉蛋白相比,酶解物的功能性质除持油性以外均有不同程度的提高。此外,DH4.5%的酶解物乳化性和起泡性最高,过度水解(DH9.0%、DH13.5%)反而造成乳化性和起泡性下降。     

两种酶水解鲢鱼蛋白产物功能性质研究

采用Alcalase 2.4L和Flavourzyme 500L蛋白酶对鲢鱼肉进行水解,研究了水解产物的水解度与体系的pH值对其功能性质的影响。水解产物的溶解度在pH4时最低,碱性时具有较高的溶解度;浊度随pH值变化趋势与溶解度相反,浊度越大,溶解度越小。随水解度的增加,水解产物的乳化活性指数、乳化稳定性、起泡性和泡沫稳定性减小(p<0.05);在水解度相同情况下,水解产物的功能性质取决于所用酶的种类。结果表明,鲢鱼肉蛋白水解产物的功能性质受其水解度和所用酶种类的影响。     

Bitterness in Bacillus proteinase hydrolysates of whey proteins

Whey protein concentrate (WPC) hydrolysates were generated with three commercially available Bacillus proteinase preparations (pH 7.0, 50 °C, 20% (w/v) WPC). Alcalase 2.4L hydrolysates were more bitter than Prolyve 1000 and Corolase 7089 hydrolysates when the proteinase activities were included at equivalent high and low addition levels. A glutamyl endopeptidase (GE) activity present in Alcalase was not detected in the Prolyve and Corolase preparations. Hydrolysate bitterness significantly increased when GE activity was included during Prolyve hydrolysis of WPC, indicating that inclusion of the GE activity was linked with the higher bitterness in Alcalase hydrolysates. A peptide present at higher levels in Prolyve compared to Alcalase hydrolysates was identified by mass spectrometry as β-lactoglobulin f(43–57). Hydrolysis of this peptide by GE was shown to release fragments with increased average hydrophobicity (Q-value). This may, in part, explain the higher level of bitterness associated with Alcalase compared to Prolyve hydrolysates of WPC.     

不同苦荞蛋白酶解产物抗氧化活性研究

以苦荞蛋白作为底物,采用碱性蛋白酶Alcalase 2.4 L、木瓜蛋白酶、胃蛋白酶、胰蛋白酶以及胃蛋白酶加胰蛋白酶模拟体内蛋白消化,制备苦荞麦蛋白水解物。采用DPPH及ABTS~+·法比较不同的蛋白水解物与水解前苦荞蛋白的体外抗氧化活性。结果表明:不同蛋白酶水解产物水解度由高到低的顺序为:碱性蛋白酶胃蛋白酶~胰蛋白酶胃蛋白酶木瓜蛋白酶胰蛋白酶,其中碱性蛋白酶水解苦荞蛋白水解度达29.95%。苦荞蛋白本身具有一定的抗氧化能力,其中DPPH清除率及ABTS~+·清除率最高分别达71.91%及11.25%,但均显著低于阳性对照Vc。随着水解程度的增加,苦荞蛋白水解产物抗氧化能力逐渐增强。其中,以碱性蛋白酶酶解产物抗氧化活性最高,其DPPH清除率及ABTS~+·清除率最高分别为91.65%(0.5 mg/mL)及16.67%(1 mg/mL),均显著高于原苦荞蛋白。其中,碱性蛋白酶水解产物的DPPH自由基清除率在高浓度(0.5mg/mL)条件下,与阳性对照Vc持平。同时碱性蛋白酶酶解产物抗氧化性(DPPH清除率及ABTS~+·清除率)显著优于其他蛋白酶解产物。因此,苦荞麦蛋白采用碱性蛋白酶解制备苦荞水解产物可作为天然的抗氧化剂。     

Biochemical properties and antioxidant activity of myofibrillar protein hydrolysates obtained from patin (Pangasius sutchi)

Antioxidant activities of myofibrillar protein hydrolysates (MPH) prepared from patin (Pangasius sutchi) using papain and Alcalase^® 2.4 L with different degrees of hydrolysis (DH) were investigated. With a DH of 65.83%, the hydrolysate prepared with papain exhibited the maximum of 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical‐scavenging activity (71.14%) with a reducing power of 0.310. At a concentration of 1 mg mL^?1, the papain‐MPH exhibited a Trolox equivalent antioxidant capacity (TEAC) of 70.50 ± 1.22 μmol g^?1 protein. With a DH of 83.6%, the Alcalase‐MPH had the highest metal‐chelating activity. Low molecular weight peptides showed higher antioxidant activities than high molecular weight peptides. Both papain‐MPH and Alcalase‐MPH contained high amounts of the essential amino acids (48.71% and 48.10%, respectively) with glutamic acid, aspartic acid and lysine as the dominant amino acids. These results suggest that the protein hydrolysates derived from patin may be used as an antioxidative ingredient in both functional food and nutraceutical applications.     

Improving antioxidant activities of whey protein hydrolysates obtained by thermal preheat treatment of pepsin,trypsin, alcalase and flavourzyme

Antioxidant activity of whey protein concentrate (WPC) hydrolysates was evaluated. Hydrolysates were obtained by pepsin, trypsin, alcalase and flavourzyme enzymatic reaction and preheat treatment of 95 °C for 5 or 10 min. The degree of hydrolysis (DH) was determined by 2,4,6‐trinitrobenzene sulphonic acid method, and antioxidant properties were determined by three spectrophotometric methods: ferricyanide method, ferric reducing/antioxidant power assay and diphenyl‐picryl hydrazinyl radical‐scavenging activity. For all the enzymes, briefly preheat treatment (95 °C/5 min) increased DH of WPC. Alcalase hydrolysates showed the highest antioxidant activity by three methods. The changes in antioxidant activity was coincidental with the changes in DH (R² = 0.988). Hydrolysates analysed by polyacrylamide gel electrophoresis and high performance liquid chromatography indicated that the α‐La was hydrolysed completely by pepsin, trypsin and alcalase and was resistant to flavourzyme to some extent; β‐lactoglobulin was only completely hydrolysed by trypsin and alcalase. Results indicated that antioxidant activity of hydrolysates was greatly related to the exposure of amino acid residues.     

Antioxidative and functional properties of protein hydrolysate from defatted skipjack (Katsuwonous pelamis) roe

Antioxidative and functional properties of protein hydrolysate from defatted skipjack (Katsuwonous pelamis) roe, hydrolysed by Alcalase 2.4 L (RPH) with different degrees of hydrolysis (DH) at various concentrations were examined. As DH increased, the reduction of DPPH, ABTS radicals scavenging activities and reducing power were noticeable (p < 0.05). The increases in metal chelating activity and superoxide scavenging activity were attained with increasing DH (p < 0.05). However, chelating activity gradually decreased at DH above 30%. All activities except superoxide anion radical scavenging activity increased as the concentration of hydrolysate increased (p < 0.05). Hydrolysis using Alcalase could increase protein solubility to above 80% over a wide pH range (2–10). The highest emulsion ability index (EAI) and foam stability (FS) of hydrolysates were observed at low DH (5%) (p < 0.05). Concentrations of hydrolysates determined interfacial properties differently, depending on DH. The molecular weight distribution of RPH with 5%DH (RPH5) was determined using Sephadex G-75 column. Two major peaks with the molecular weight of 57.8 and 5.5 kDa were obtained. Fraction with MW of 5.5 had the strongest metal chelating activity and ABTS radical scavenging activity. The results reveal that protein hydrolysates from defatted skipjack roe could be used as food additives possessing both antioxidant activity and functional properties.     

乙醇对谷朊粉酶解产物的苦味影响研究

为了探寻降低谷朊粉酶解产物苦味的方法,通过添加不同浓度的乙醇(5%、10%和15%),研究其对碱性蛋白酶酶解产物苦味值、游离氨基酸和相对分子质量分布的影响,以及在乙醇存在下碱性蛋白酶、中性蛋白酶以及风味蛋白酶3种复配酶解体系所制得的酶解产物的苦味值和相对分子质量分布变化。结果表明:加入乙醇后,在相同的水解度下(DH),碱性蛋白酶的酶解产物苦味值降低,并与加入乙醇的浓度呈负相关。添加乙醇后,相对分子质量小于1 000的组分含量显著(P0.05)降低,游离疏水性氨基酸(Pro,Ile,Phe和Met)显著(P0.05)增加。添加乙醇后,3种复配酶解体系酶解产物苦味降低,相对分子质量小于1 000的组分含量显著(P0.05)降低。与添加碱性蛋白酶单一酶解相比,在相同的水解度下3种酶复配酶解产物的苦味值进一步降低。添加低浓度乙醇对3种酶的活性影响较小。     

酶法制备不同水解度高温豆粕水解产物的理化特性研究

采用Alcalase酶和木瓜蛋白酶分别对高温大豆粕进行酶解,通过控制酶解反应得到水解度为5%、10%和15%的6种水解产物,研究两种酶对不同水解度的水解产物理化特性的影响。结果表明,Alcalase酶和木瓜蛋白酶均可产生6种不同分子量范围的水解产物,但各部分比例具有显著差异(P0.5),其平均分子量随水解度的增加逐渐减少,Alcalase酶的水解产物中小于2562 Da小分子量肽所占比例更高。豆粕蛋白的疏水基团在酶解反应中发生暴露与断裂的数量差,导致其表面疏水性随水解度增加呈现先下降再上升的变化,即水解度为10%的表面疏水性最低。zeta电势的绝对值随水解度不断上升,分子间的斥力增大,相同水解度下两种酶对zeta电势的影响并不显著。此外,在pH值为3、5、7和9时,水解产物的溶解性随着水解度的增加而逐渐增高,乳化活性和乳化稳定性则逐渐降低。     

Characteristics of Bellamya purificata snail foot protein and enzymatic hydrolysates

The foot muscle protein of Bellamya purificata (mud snail, named Luosi in Chinese) was investigated. Its conformation change and increase in solubility were researched during enzymatic hydrolysis. The protein conformation was looser following an increase in pH (from 10 to 12), while the β-sheet was the main conformation at pH 12. Blending, ultrasonic extraction, ultradispersing and alkaline treatment increased the solubility of the foot muscle protein. The effects of several proteases on its hydrolysis were compared and Proleather FG-F was chosen. The relative molecular mass distribution, the free amino acids (FAA) content and the angiotensin-I converting enzyme (ACE) inhibitory activity of the hydrolysates were quantitatively analyzed and compared. In the Proleather FG-F hydrolysates, the percentage of the peptides with molecular weight between 150 and 2000 Da were 84.65%, much more than that in the Alcalase 2.4L hydrolysates (68.44%). Proleather FG-F released much less FAA (5.80%), than Alcalase 2.4L (17.01%). The IC₅₀ of the Proleather FG-F hydrolysate was 0.69 mg/ml, whereas for the Alcalase 2.4L hydrolysate the value was 3.30 mg/ml. Finally, response surface methodology (RSM) was used to optimize the factors (pH, enzyme: substrate ratio- E/S- and temperature) affecting Proleather FG-F hydrolysis.     

Immunoreactive properties of peptide fractions of cow whey milk proteins after enzymatic hydrolysis

Commercial whey protein concentrate (WPC) was hydrolysed with either Alcalase 2.4 FG (Novo Nordisk), or papain (Sigma) (in one‐step process) or with two enzymes (in two‐step process) to determine the changes in the immunoreactivity of α‐lactalbumin and β‐lactoglobulin. Enzymatic hydrolysis of WPC was performed by pH‐stat method. Hydrolysates were analysed using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, immunoblotting and size‐exclusion chromatography (SE‐HPLC). Immunoreactive properties of peptide fractions separated from the hydrolysates by fast protein liquid chromatography (FPLC) were determined using dot‐immunobinding and enzyme‐linked immunosorbent assay (ELISA) methods. Finally the sensory analysis was used to confirm organoleptic changes resulting from the application of different enzymes. The ‘two‐step’ process was observed to be the most effective however allergenic epitopes were still present, as it was found by ELISA with anti‐α‐la and anti‐β‐lg antibodies. The addition of papain as the second enzyme in the hydrolysis process contributed to the improvement of the sensory properties of WPC hydrolysate as compared with the Alcalase hydrolysate. Alcalase‐papain partially hydrolysated WPC can be found a promising base for production of the tolerogenic formula.     

Enzymatic preparation and functional properties of wheat gluten hydrolysates

The water-insolublity of wheat gluten is one of the major limitations for its more extensive use in food processing. Wheat gluten was enzymatically hydrolyzed by several commercially available proteases (Pancreatin Trypsin 6.0S, Porcine pepsin, Pancreatin and Alcalase 2.4L) with protein recovery varying from 42.5 ± 0.7% to 81.3 ± 0.1%. The hydrolytic efficiency of these proteases on wheat gluten was also compared. Alcalase served best for the preparation of wheat gluten hydrolysates (WGHs). Thus, Alcalase-assisted hydrolysates of wheat gluten (AWGHs) with different degrees of hydrolysis (DH 5.0, 10.0 and 15.0%) were further assessed for their functionalities. All the AWGHs had excellent solubility (>60%) over a pH range of 2–12. The emulsifying and foaming properties of AWGH with relatively low DH (5.0%) were remarkably higher compared to the original gluten. However, extensive hydrolysis of gluten resulted in remarkable reduction in emulsifying and foaming properties.     

Amino acid composition,antioxidant and functional properties of protein hydrolysates from the roe of rainbow trout (Oncorhynchus mykiss)

A fish roe protein hydrolysate from rainbow trout (Oncorhynchus mykiss) trout roe protein hydrolysates (TRH) was produced by pepsin and Alcalase. Proximate, amino acid compositions, protein digestibility and molecular mass distribution of the hydrolysates were determined. The degree of hydrolysis was found to be 44.08% and 27.62% (pepsin and Alcalase, respectively). The two hydrolysates contained a high amount of essential amino acids (33.53% Alcalase–29.39% pepsin). The results showed that TRH by different enzymes is a good source of the leucine and lysine amino acids. The pepsin produced a white powder with higher brightness (L* = 89.50). Alcalase hydrolysate was brownish yellow in colour (L* = 52.85, a* = 10.30, b* = 26.25). The hydrolysates represented excellent antioxidant activities in various concentrations. TRHs showed a good foaming and emulsification properties. The results thus revealed that protein hydrolysates from rainbow trout roe could be used as food additives possessing essential amino acids and antioxidant activity.     

Proteinase and exopeptidase hydrolysis of whey protein: Comparison of the TNBS,OPA and pH stat methods for quantification of degree of hydrolysis

Whey protein hydrolysates were generated with Alcalase 2.4L and Debitrase HYW20 which are proteinase and exopeptidase enriched enzyme preparations, respectively. Degree of hydrolysis (DH) values were quantified with the TNBS, OPA and pH stat methods. Poor correlation was observed between the three methods for DH values in Debitrase HYW20 hydrolysates. For Alcalase 2.4L hydrolysates, the OPA method gave DH values that were approximately 15% lower than the pH stat, whereas TNBS DH values were similar to the pH stat method. As whey proteins are relatively rich in cysteine, a weak and unstable reaction between OPA and cysteine was thought to contribute to the under-estimation of DH in whey protein hydrolysates. Since TNBS reacts strongly with cysteine and TNBS DH values were unaffected by the type of enzyme preparation used to generate the hydrolysate, the TNBS method was deemed most suitable for the quantification of DH in whey protein hydrolysates.     

Physicochemical and antioxidant properties of buckwheat (Fagopyrum esculentum Moench) protein hydrolysates

The enzymatic hydrolysis of common buckwheat (Fagopyrum esculentum Moench) protein isolate (BPI) by Alcalase and some physiochemical and antioxidant properties of the resulting hydrolysates were characterised. The hydrolysis resulted in remarkable decrease in the globulins or protein aggregates and concomitant increase in peptide fragments. The surface hydrophobicity of the hydrolysates decreased with increasing degree of hydrolysis (DH) and reached a minimum at DH 15%, but increased at further hydrolysis, whereas their amino acid compositions were unchanged. The polyphenol content of the hydrolysates gradually decreased with DH increasing from 0% to 15%, while it on the contrary increased upon further hydrolysis. The hydrolysates exhibited excellent antioxidant activities, including DPPH radical scavenging ability, reducing power and ability to inhibit linoleic acid peroxidation. The antioxidant activities of these hydrolysates were closely related to their polyphenol contents. The results indicated that polyphenol-rich buckwheat proteins are unique protein materials for the production of the hydrolysates with good nutritional and antioxidant properties.     

罗非鱼鱼皮鱼鳞蛋白的酶解及超滤分离

采用Alcalase2.4L、Protamex、Papain、PTN6.0S和Neutrase酶解罗非鱼鱼皮鱼鳞蛋白,探讨了超滤对酶解液分子质量分布和氨基酸组成的影响。结果表明,Alcalase2.4L、PTN6.0S酶解产物的蛋白质回收率、肽得率和水解度均较高,Alcalase2.4L∶PTN6.0S为1∶2添加时酶解效果最好,蛋白回收率为96.37%,水解度为13.24%,肽得率为83.13%。经超滤处理后,3000 u以下分子质量的肽段达到97.73%,比超滤前增加了5.37%,总氨基酸含量由84076.12 mg/100 g增加到97234.79 mg/100 g。     

Enzymatic hydrolysis of heated whey: iron-binding ability of peptides and antigenic protein fractions

This study evaluated the influence of various enzymes on the hydrolysis of whey protein concentrate (WPC) to reduce its antigenic fractions and to quantify the peptides having iron-binding ability in its hydrolysates. Heated (for 10 min at 100°C) WPC (2% protein solution) was incubated with 2% each of Alcalase, Flavourzyme, papain, and trypsin for 30, 60, 90, 120, 150, 180, and 240 min at 50°C. The highest hydrolysis of WPC was observed after 240 min of incubation with Alcalase (12.4%), followed by Flavourzyme (12.0%), trypsin (10.4%), and papain (8.53%). The nonprotein nitrogen contents of WPC hydrolysate followed the hydrolytic pattern of whey. The major antigenic fractions (β-lactoglobulin) in WPC were degraded within 60 min of its incubation with Alcalase, Flavourzyme, or papain. Chromatograms of enzymatic hydrolysates of heated WPC also indicated complete degradation of β-lactoglobulin, α-lactalbumin, and BSA. The highest iron solubility was noticed in hydrolysates derived with Alcalase (95%), followed by those produced with trypsin (90%), papain (87%), and Flavourzyme (81%). Eluted fraction 1 (F-1) and fraction 2 (F-2) were the respective peaks for the 0.25 and 0.5 M NaCl chromatographic step gradient for analysis of hydrolysates. Iron-binding ability was noticeably higher in F-1 than in F-2 of all hydrolysates of WPC. The highest iron contents in F-1 were observed in WPC hydrolysates derived with Alcalase (0.2 mg/kg), followed by hydrolysates derived with Flavourzyme (0.14 mg/kg), trypsin (0.14 mg/kg), and papain (0.08 mg/kg). Iron concentrations in the F-2 fraction of all enzymatic hydrolysates of WPC were low and ranged from 0.03 to 0.05 mg/kg. Fraction 1 may describe a new class of iron chelates based on the reaction of FeSO₄·7H₂O with a mixture of peptides obtained by the enzymatic hydrolysis of WPC. The chromatogram of Alcalase F-1 indicated numerous small peaks of shorter wavelengths, which probably indicated a variety of new peptides with greater ability to bind with iron. Alcalase F-1 had higher Ala (18.38%), Lys (17.97%), and Phe (16.58%) concentrations, whereas the presence of Pro, Gly, and Tyr was not detected. Alcalase was more effective than other enzymes at producing a hydrolysate for the separation of iron-binding peptides derived from WPC.     

Enzymatic hydrolysis of wheat gluten by proteases and properties of the resulting hydrolysates

The insolubility of gluten in aqueous solutions is one of the major limitations for its more extensive use in food processing. Wheat gluten was enzymatically hydrolyzed by several commercially available proteases (Alcalase 2.4L, PTN 6.0S, Pepsin, Pancreatin, Neutrase and Protamex™) with protein recovery of 81.3%, 42.5%, 53.3%, 61.6%, 46.3% and 43.8%, respectively. The hydrolytic efficiency of these proteases on wheat gluten was also compared. Alcalase served best for the preparation of wheat gluten hydrolysates with the maximum degree of hydrolysis (DH) 15.8%. Subsequently, the solubility of wheat gluten hydrolysates (WGHs) obtained with those enzymes was comparably evaluated. The products had excellent solubility (>60%) over a pH range of 2–12. The molecular weight distribution of WGHs was further determined by SDS-PAGE and size exclusion chromatography on Sephadex G-15. The results showed that with the increasing of DH values, there occurred a large amount of smaller polypeptides.