High hydrostatic pressure assisted enzymatic hydrolysis of whey proteins

Whey proteins, due to their high nutritional value, are generally hydrolyzed to reduce the allergenicity and used as ingredients in many special products, such as infant formulae, geriatric products, highly energetic supplements or dietetic foods or in foods produced to prevent nutrition related diseases, like food intolerances and allergies.The aim of this work was to assess the applicability of innovative technologies, such as high hydrostatic pressure (HHP) processes, to assist the enzymatic hydrolysis of target proteins, namely whey protein concentrate (WPC-80), in order to modify their antigenicity. Experiments were carried out to verify the effectiveness of HHP technology to accelerate whey protein hydrolysis reaction with selected enzymes (α-chymotrypsin, bromelain), and to affect the protein allergenic power. To this purpose, different HHP treatments were carried out at several pressure levels (100, 200, 300 and 400 MPa) and the untreated whey protein samples were used as control. A defined enzyme/substrate ratio of 1/10 w/w was used in the experiments, while the treatment time was changed from 0 to 30 min (0, 5, 15, or 30 min).The experimental data demonstrated that High Hydrostatic Pressure (HHP) induced WPC-80 unfolding at the highest value of the pressure applied (400 MPa) as indicated by the higher exposure of free sulfhydryl groups. When HHP was used in combination with enzymatic hydrolysis, the degree of hydrolysis increased not only with the pressure level applied but also with the processing time. These results suggested that, even if the exposure of hidden epitopes upon protein unfolding increased the antigenicity of whey proteins, further peptide bonds cleavage also took place after hydrolysis. This effect could modify whey proteins antigenic sequences, and thus their antigenic power.     

分步酶解法制备黄浆水活性肽

黄浆水是传统豆制品点脑成型过程压榨出的废弃物,富含低聚糖、蛋白质等营养成分。该研究通过比较酶种类及用量、酶解温度、酶解时间对黄浆水蛋白质水解度的影响,采用正交试验优化获得黄浆水短肽最佳分步酶解工艺：（1）酸性蛋白酶加酶量2 000 U/g,pH 4.0,温度55 ℃,水解2 h;（2）中性蛋白酶8 000 U/g,pH 6.0,温度50 ℃,水解6 h。在此条件下进行验证,水解度可达25.95%,血管紧张素转化酶体外抑制活性达92.0%。采用酸性蛋白酶和中性蛋白酶分步酶解黄浆水制备短肽,制备条件温和,水解度高,可为豆制品加工废弃物的高值化利用奠定基础。     

Immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins

Whey contains a number of immunomodulatory peptides that are naturally present or that are part of the primary sequence of whey proteins. These peptide sequences can be released during digestion in the gut and can also be produced by in vitro enzymatic hydrolysis. This paper reviews the existing literature on the effect of whey peptides on the specific (lymphocyte activation and proliferation, antibody production, cytokine expression) and non-specific (functions of macrophages, granulocytes and natural killer cells) immune responses. Major discrepancies in the effect of some peptides on lymphocyte proliferation have been reported while the more limited literature on antibody production is less controversial. The effects of whey peptides on hypersensitivity, induction of oral tolerance and response to infections and diseases are also reviewed. The in vitro and in vivo data on the immunomodulatory potential of whey peptides are relatively abundant. However, the potential of whey peptides as effective health ingredients remains to be demonstrated, which may be due to the lack of clinical evidence and the limitations of some in vivo models.     

Effects of combined high pressure and enzymatic treatments on the hydrolysis and immunoreactivity of dairy whey proteins

The effect of high-pressure (HP) treatment on the hydrolysis of dairy whey proteins by trypsin, chymotrypsin and pepsin was analysed. Isostatic pressure (100–300 MPa for 15 min at 37 °C) was applied to the protein substrate prior to its enzymatic hydrolysis. Digestion was also conducted at atmospheric pressure (0.1 MPa) and under high pressure. The extent of hydrolysis was measured by the o-phthaldialdehyde method, the peptide profile was analysed by reverse-phase high performance liquid chromatography (RP-HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the residual immunochemical reactivity was assessed by an ELISA test using a pool of seven sera from children allergic to bovine milk, an individual serum also positive (positive control) and two sera from non-allergic children (negative controls). The high pressure increased the degree of hydrolysis by the three enzymes used. Chymotrypsin and trypsin showed the highest proteolysis at 100 and 200 MPa followed by pepsin at 300 MPa. The β-lactoglobulin was hydrolysed by trypsin and chymotrypsin at atmospheric and at high pressures, whereas the pepsin only hydrolysed this protein under high pressure. Pepsin and trypsin hydrolysed α-lactalbumin in all cases. In contrast, this protein was not digested by chymotrypsin, irrespective of the pressure applied. An important decrease of immunochemical reactivity was found for pepsin and trypsin hydrolysates obtained under high pressure. The pool of seven sera detected immunoreactivity in the products of chymotrypsin hydrolysis under high pressure, which was not detected when the serum of one patient was used. The results suggest that dairy whey hydrolysates obtained by pepsin and trypsin in combination with HP treatment could be used as a source of peptides in hypo-allergenic infant formulae.     

High pressure thermal denaturation kinetics of whey proteins

Pressure processing of foodstuff has been applied to produce or modify proteinaceous gel structures. In real pressure processing the treatment is non-isothermal, due to the adiabatic nature of the process and the heat loss from the product to the vessel. In order to estimate the effect of pressurization on milk constituents pressure and temperature dependent kinetics were determined separately from each other. In a detailed kinetic study whey protein isolate was treated under isobaric (200 to 800 MPa) and isothermal conditions (-2 to 70 degrees C), and the resulting degree of denaturation of beta-lactoglobulin A and B and alpha-lactalbumin was analysed. Kinetic parameters of denaturation were estimated using a one step non-linear regression method which allowed a global fit of the whole data set. The isobaric isothermal denaturation of beta-lactoglobulin and alpha-lactalbumin was found to follow third and second order kinetics, respectively. Isothermal pressure denaturation of both beta-lactoglobulin fractions do not differ significantly and were characterized by an activation volume decreasing with increasing temperature from -10 to about -30 ml mol(-1), which demonstrates that the denaturation rate is accelerated with increasing temperature. The activation energy of about 70 to 100 kJ mol(-1) obtained for beta-lactoglobulin A and B is not dependent to a great extent on the pressure which indicates that above 200 MPa denaturation rate is limited by the aggregation rate while pressure forces unfolding of the molecule.     

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.     

Assessment of the residual immunoreactivity of soybean whey hydrolysates obtained by combined enzymatic proteolysis and high pressure

Soybean (Glycine max) whey was hydrolyzed for 15 min using three food-grade proteases (Alcalase, Neutrase, Corolase PN-L) at atmospheric pressure (0.1 MPa) and under high pressure (HP) at 100, 200, and 300 MPa. All hydrolysates were analyzed by SDS-PAGE and their residual immunoreactivity was assessed by immunoblotting using the sera from children allergic to soybean. As shown in SDS-PAGE, Alcalase, Neutrase, and Corolase PN-L produced different patterns of hydrolysis. Each enzyme showed a similar proteolytic activity at atmospheric pressure and at 100 MPa, while an increased degree of hydrolysis was observed at 200 and 300 MPa. No residual antigenicity was observed in the hydrolysates obtained by Alcalase and Corolase PN-L in all considered conditions of hydrolysis. A positive reaction associated with a band having molecular weight of about 70 kDa was observed in the immunoblotting of the hydrolysates obtained with Neutrase at 0.1, 100, and 200 MPa, while no antigenicity was detected for those samples obtained under high pressure, at 300 MPa. These results suggest that high pressure combined with suitable enzymatic activity could be a useful tool for obtaining hydrolysates with low immunoreactivity to be used in special foods (hypoallergenic foods).     

Separation of iron-binding protein from whey through enzymatic hydrolysis

Whey protein concentrate (WPC) was hydrolyzed by nine proteolytic enzymes to examine the effectiveness of the hydrolysates to bind iron. Degree of WPC hydrolysis was higher with pancreatin (13.91%), alcalase (13.60%), and flavourzyme (12.80%) compared with other enzymes (esperase, neutrase, papain, pepsin, protease and trypsin). Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high performance liquid chromatography analyses revealed maximum hydrolysis of α-LA and β-LG with alcalase. Molecular masses of peptides derived from alcalase hydrolysate were smaller than 6.5 kDa. Iron-binding by alcalase hydrolysate was the highest (97.6%) of all other hydrolysates. Using ion-exchange chromatography alcalase hydrolysate was eluted at a 0.25 m NaCl gradient concentration with higher iron-binding ability. This eluted fraction had higher Lys (18.09%), Ala (17.24%), and Phe (16.58%) contents. Alcalase showed noticeably better effectiveness than other enzymes to produce a hydrolysate for the separation of iron-binding peptides derived from WPC.     

大豆蛋白酶解的研究

用几种常用蛋白酶对大豆蛋白进行酶解,利用均匀设计安排试验,确定各种酶的最佳酶解条件,并以水解度（DH）为考察标准,选出水解度最大的酶,确定其最佳加酶量和酶解时间。     

限制性酶解乳清蛋白功能性质研究

探究乳清蛋白在碱性蛋白酶限制性水解下功能性质变化。以乳清蛋白的溶解性,乳化性、乳化稳定性,起泡性、起泡稳定性为考察指标,确定乳清蛋白的等电点及分析不同水解度下乳清蛋白功能性质在p H调控下的变化。结果表明:乳清蛋白的等电点为4.8。乳清蛋白进行限制性酶解后功能性质有了很大提高,其中溶解性在DH14、p H10下达到最大值,较原蛋白提高了14.55%;起泡性在DH14、p H4下达到最大值,较原蛋白提高了107.5%;起泡稳定性在DH4、p H4下达到最大值,比原蛋白提高了8.66%;乳化性在DH14、p H12下达到最大值,比原蛋白提高了56.1%;乳化稳定性DH4、p H12下达到最大值,比原蛋白提高了50.42%。      

大豆浓缩蛋白的酶水解改性的研究

以产品纯度高、反应温和的酶水解作为大豆浓缩蛋白的改性方法,以氮溶解指数为考察指标,通过木瓜蛋白酶和1398中性蛋白酶单酶水解和双酶混合水解大豆浓缩蛋白单因素和正交试验进行分析比较,表明双酶混合水解效果优于单酶水解,其最佳工艺条件为:底物浓度为6.5%、pH为6.5、温度为55℃、反应时间为3 h、混合酶浓度为5 000 U/g,大豆浓缩蛋白的氮溶解指数可以恢复到81.94%,具有较高的工业化应用前景。     

酶解制备苷元型大豆异黄酮

以大豆异黄酮糖苷为原料,酶解制备苷元型大豆异黄酮。以水解率和苷元得率为指标对几种来源的β-葡萄糖苷酶、β-半乳糖苷酶、纤维素酶进行筛选,确定最适酶解用酶。通过单因素实验对酶添加量、底物质量浓度、酶解温度、pH、酶解时间进行优化。结果表明,最佳酶解工艺条件为：采用β-葡萄糖苷酶（300 U/g）,酶添加量7%,底物质量浓度1.6 mg/mL,酶解温度56 ℃,pH 4.8,酶解时间6 h。在最佳工艺条件下,大豆异黄酮糖苷的水解率及苷元得率分别达到96.84%和99.74%。     

Immunomodulatory properties of the milk whey products obtained by enzymatic and microbial hydrolysis

The objective of this study was to investigate the in vitro immunomodulatory effect of milk whey through enzymatic hydrolysis, microbial fermentation and two‐stage hydrolytic reactions (enzymatic and microbial reactions) by analysing cytokine profiles. Results indicated that the milk whey sample fermented by Lactobacillus kefiranofaciens M1 and two‐stage hydrolytic reactions (hydrolysed by Alcalase and then fermented by L. kefiranofaciens M1) could significantly induce the production of tumour necrosis factor (TNF)‐α and IL‐12 compared with the milk whey control and enzymatic treatments. Further characterisation of the immunomodulatory factor by membrane filtration and mutanolysin hydrolysation, the stimulatory activity for IL‐12 and TNF‐α production was found to be reduced and to be correlated positively with the cell wall components in L. kefiranofaciens M1. In addition, Th2‐polarised splenocytes revealed that L. kefiranofaciens M1 had both IL‐12 inducing and IL‐4 repressing activities. These results suggested that L. kefiranofaciens M1 could direct the Th1/Th2 balance toward Th1.     

酶解对乳清蛋白抗原性影响的研究

研究了酶解对乳清蛋白抗原性的影响。选择了7种常见蛋白酶在同一水解模式下水解乳清蛋白，用竞争ELISA法测定水解物的残留抗原性，从而间接测定其过敏性变化。结果表明，酶解能有效降低乳蛋白抗原性，但水解物仍能与特异抗体反应，保留一部分抗原性。不同酶对乳清蛋白过敏原的影响不同，酶的特异性对乳清蛋白水解物的抗原性有较大的影响，碱性蛋白酶降低乳蛋白抗原性的效果最佳，对抗β-乳球蛋白（β-LG）和抗α-乳白蛋白（α-LA）抗体的抗原性分别降低了50．02％和99．72％。     

酶法水解生产大豆多肽研究

本文研究了Alcalase酶对大豆蛋白的有限水解作用 ,分析了酶加量、pH值、温度、底物浓度、反应时间等因素对大豆蛋白酶水解的影响 ,确定了Alcalase蛋白酶水解大豆蛋白的较佳条件范围 ;同时研究了Flavourzyme酶对大豆多肽的水解作用 ,及对大豆多肽风味的影响 ,提出了采用Alcalase酶和Flavourzyme酶双酶法分步酶解工艺来生产低苦味大豆多肽的方法     

大豆秸秆纤维素酶水解条件的研究

为了从大豆秸秆中提取生物降解性塑料的原料-乳酸对经粉碎及氨处理的大豆秸秆纤维素酶水解条件进行了研究。研究结果表明,酶水解最佳工艺条件为pH4.8,温度45℃,反应时间28h,底物浓度5%,酶用量为450IU/g(秸秆)。对酶解液成分进行了分析,酶解液主要由葡萄糖、木糖、纤维二糖组成,为下一步酶解液乳酸发酵实验提供了理论参考。     

High pressure microfluidization treatment of heat denatured whey proteins for improved functionality

Solutions (5% protein) of a whey protein concentrate (WPC) in fresh acid whey or in water, as well as the fresh whey alone, were adjusted to pH 5.8, 4.8 or 3.8, heat treated at 90 °C for 10 min and further exposed to high pressure (150 MPa) microfluidization treatment. The volumes of sediment after centrifugation were recorded as a measure of the degree of insolubility of the proteins. Microfluidization disrupted the heat-induced aggregates into non-sedimenting whey protein polymers so that in some cases, especially at pH 3.8, the products studied were almost completely resistant to sedimentation after the microfluidization treatments. Heat denatured/microfluidized whey proteins reaggregated upon subsequent heating, with the pH having a major impact on the amount of sediment produced. Microfluidization of aqueous WPC solutions heat-treated before spray- or freeze-drying substantially increased the solubility of the powders upon reconstitution. Heat-induced viscoelastic gels were produced from freeze-dried microfluidized samples processed at pH 3.8 and reconstituted to solutions containing 12% (w/w) protein.     

酶解乳清蛋白制备降胆固醇活性肽的工艺研究

利用生物酶技术酶解乳清蛋白,制备具有降胆固醇活性的多肽,通过响应面试验、二次旋转回归设计建立回归模型,以胆固醇胶束溶解度抑制率为评价指标,对乳清蛋白的酶解条件进行了优化。结果表明,乳清蛋白的最佳酶解条件为：酶解时间8.5 h、酶解温度55 ℃、酶解pH 8.0、加酶量4.7%、乳清蛋白含量5.8%,在此条件下,酶解乳清蛋白得到的活性肽的胆固醇胶束溶解度抑制率为18.21%。     

Effect of whey protein enzymatic hydrolysis on the rheological properties of acid-induced gels

A protein dispersion blend of β-lactoglobulin and α-lactalbumin was heat-denatured at pH 7.5, hydrolyzed by α-chymotrypsin and then acidified with glucono-δ-lactone to form gels at room temperature. Heat treatment induced the formation of whey protein polymers with high concentration of reactive thiol groups (37 μmol/g). The reactive thiol group concentration was reduced by half after 40 min enzymatic hydrolysis. It was further reduced after enzyme thermal deactivation. During acidification, the first sign of aggregation for hydrolyzed polymers occurred earlier than for non hydrolyzed polymers. Increasing the hydrolysis duration up to 30 min resulted in more turbid gels characterized by an open microstructure. Elastic and viscous moduli were both reduced, while the relaxation coefficient and the stress decay rate constants were increased by increasing the hydrolysis duration. After one week storage at 5 °C, the hardness of gels made from hydrolyzed polymers increased by more than 50%. The effect of polymer hydrolysis on acid-induced gelation is discussed in relation to the availability and reactivity of thiol groups during gel formation and storage.     

High pressure treatment of bovine milk: effects on casein micelles and whey proteins

Effects of high pressure (HP) on average casein micelle size and denaturation of alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) in raw skim bovine milk were studied over a range of conditions. Micelle size was not influenced by treatment at pressures <200 MPa, but treatment at 250 MPa increased micelle size by approximately 25%, while treatment at > or = 300 MPa irreversibly reduced it to approximately 50% of that in untreated milk. The increase in micelle size after treatment at 250 MPa was greater with increasing treatment time and temperature and milk pH. Treatment times > or = 2 min at 400 MPa resulted in similar levels of micelle disruption, but increasing milk pH to 7.0 partially stabilised micelles against HP-induced disruption. Denaturation of alpha-la did not occur < or = 400 MPa, whereas beta-lg was denatured at pressures >100 MPa. Denaturation of alpha-la and beta-lg increased with increasing pressure, treatment time and temperature and milk pH. The majority of denatured beta-lg was apparently associated with casein micelles. These effects of HP on casein micelles and whey proteins in milk may have significant implications for properties of products made from HP-treated milk.