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).     

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.     

Rheological properties of soy protein hydrolysates obtained from limited enzymatic hydrolysis

Soy protein products hexane-defatted soy flour, extruded-expelled soy flour, soy protein concentrate and soy protein isolate, were modified by using the enzyme bromelain to 2% and 4% degrees of hydrolysis (DH). Peptide profiles, water solubility, and rheological properties including dynamic shear, large deformation, and apparent viscosities of resulting hydrolysates were determined. Protein subunits profiles for the hydrolysed isolates and concentrates were extensively altered by the treatment while only minor changes were observed for the hydrolysed flours. Water solubility profiles of all hydrolysates in the pH range of 3.0-7.0 were enhanced by hydrolysis. For the unhydrolysed controls, the isolate had the highest storage modulus (G′), followed by the concentrate, the extruded-expelled flour and the hexane-defatted flour. The hydrolysates retained some of their gelling ability even though the losses in storage modulus (G′) were substantial. After heating step to 95 °C, the G′ values of all substrates at 25 °C decreased with increase in DH. Texture profile analyses of the soy protein gels were also lower in hardness after hydrolysis. The Power Law model provided excellent fit to hydrolysate dispersions flow (R²>0.99). Hydrolysis decreased the consistency coefficients of dispersion and increased flow behavior index resulting in thinner dispersions. These results suggest that limited protease hydrolysis of various soy protein meals with bromelain produce soy protein ingredients with modified rheological properties.     

Proteolytic pattern, antigenicity, and serum immunoglobulin E binding of beta-lactoglobulin hydrolysates obtained by pepsin and high-pressure treatments

This study evaluates the use of high pressure to enhance pepsin hydrolysis of β-lactoglobulin (β-LG). The protein was subjected to high pressure before and during the proteolytic process. Analysis of remnant β-LG, identification of the peptides produced, and evaluation of antigenicity (binding to commercial antibodies) and binding to IgE of allergic patients’ sera were conducted in the hydrolysates. The results showed that the application of high pressure before the enzyme treatment slightly improved the proteolytic process but did not reduce the antigenicity or IgE binding of the hydrolysates. The application of high pressure during the enzymatic treatment enhanced the production of large intermediate fragments that were further proteolysed to smaller fragments as proteolysis proceeded for longer periods. At 400 MPa, all the intact protein was removed in minutes, simultaneously decreasing its antigenicity and serum IgE binding properties. However, for considerable reduction of the antigenicity and IgE binding of β-LG, extending the incubation time with the enzyme was needed to reduce the amount of potentially allergenic intermediate peptides. Changes of β-LG under pressure at acidic pH are discussed.     

Hemoglobin hydrolysates from porcine blood obtained through enzymatic hydrolysis assisted by high hydrostatic pressure processing

The purpose of this work was to obtain discoloured hemoglobin (Hb) hydrolysates from porcine red blood cells fraction by combining enzymatic hydrolysis and high hydrostatic pressure (HHP) treatments. Samples of Hb were submitted to treatments of enzymatic hydrolysis by trypsin (EC 3.4.21.4) or pepsin (EC 3.4.23.1), under controlled conditions of temperature and pH, for 2, 6 and 24 h, and pressurization at 400 MPa, at 20 °C for 15 min, after the addition of the proteases. To evaluate the effects of the proteolysis reactions, the effectiveness of discolouration, the extent of hydrolysis as the percentage of non-protein nitrogen, and the SDS-PAGE electrophoretic profiles of the hydrolysates were determined. The results showed that HHP had a significant influence on enzymatic hydrolysis of Hb by trypsin, whereas did not improve the extent of proteolysis in the case of pepsin. The main conclusion derived from this study is that pressurization of Hb samples after the addition of trypsin enhanced enzymatic hydrolysis of the Hb, hence permitting a decrease in the incubation time.

Industrial relevance

The present study is focussed on assessing the effects of enzymatic hydrolysis with trypsin or pepsin assisted by HHP treatment on the discolouration of Hb from porcine blood. HHP treatment was able to increase the activity of trypsin and to enhance discolouration effectiveness of Hb with pepsin. The results from this work corroborate the potential of the application of HHP processing combined with enzymatic treatments on the development of food ingredients from porcine blood.     

Effect of heat and enzymatic treatment on the antihypertensive activity of whey protein hydrolysates

The influence of heat and enzymatic treatments on the hypotensive activity of hydrolysates derived from whey protein isolate was examined. The whey protein isolate (WPI) was previously denatured at 65 or 95 °C and hydrolyzed using the enzymes Alcalase, α-chymotrypsin or Proteomix. The hydrolysates thus obtained were characterized and studied with regard to their angiotensin converting enzyme (ACE) inhibitory activity and hypotensive activity in spontaneously hypertensive rats (SHR). The enzyme α-chymotrypsin was found to produce hydrolysates with the highest ACE inhibitory activity. The hydrolysate that most effectively reduced blood pressure in SHR was obtained from WPI previously denatured at 65 °C and treated with the enzyme Alcalase. The hydrolysate with the highest ACE inhibitory activity was able to reduce the arterial blood pressure of the animals only after intraperitoneal administration, suggesting an interference of gastrointestinal enzymes in the absorption of active peptides from this hydrolysate.     

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％。     

Combined effects of high-pressure and enzymatic treatments on the hydrolysis of chickpea protein isolates and antioxidant activity of the hydrolysates

A chickpea protein isolate (CPI) was pretreated before hydrolysis under a pressure that varied between 100 and 600MPa. The hydrolysis rate increased significantly with pressure above 300MPa. At 40min, the DH of the control was 15.3%, while the DH of the CPI treated at 300MPa was 18.5%, which reached 23.74% post treatment at 400MPa. The pretreatment of CPI above 300MPa enhanced the superoxide anion capturing rate of enzymatic hydrolysis. Pretreatment at 400MPa significantly reduced the hydrolysis time with the release of antioxidant peptides. While hydrolysis by Alcalase during treatment at high pressure (100-300MPa) significantly increased the degree of hydrolysis (DH), its maximum value peaked after hydrolysis at 200MPa for 30min. In addition, hydrolysates obtained at high pressure (100-300MPa) had a higher superoxide anion capturing rate. High-pressure treatment at 200MPa for 20min resulted in products with high antioxidative activity. The molecular-weight (MW) determination of the enzymatic hydrolysates indicated that hydrolysis at high pressure could significantly increase the amount of low-molecular-weight peptides.     

Chemical characterisation and determination of sensory attributes of hydrolysates produced by enzymatic hydrolysis of whey proteins following a novel integrative process

The overall aim of this work was to characterise the major angiotensin-converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins, through the application of a novel integrative process. This process consisted of the combination of adsorption and microfiltration within a stirred cell unit for the selective immobilisation of β-lactoglobulin and casein-derived peptides (CDP) from whey. The adsorbed proteins were hydrolysed in situ, which resulted in the separation of peptide products from the substrate and fractionation of peptides. Two different hydrolysates were produced: (i) from CDP (IC₅₀ = 287 μg/mL) and (ii) from β-lactoglobulin (IC₅₀ = 128 μg/mL). The well-known antihypertensive peptide IPP and several novel peptides that have structural similarities with reported ACE inhibitory peptides were identified and characterised in both hydrolysates. Furthermore, the hydrolysates were assessed for bitterness. No significant difference was found between the bitterness of the control (milk with no hydrolysate) and hydrolysate samples at different concentrations (at, below and above the IC₅₀).     

酶解对牛乳酪蛋白抗原性的影响

采用5种常用酶对酪蛋白进行酶解,用间接竞争性ELISA方法测定酪蛋白水解物的残留抗原性.结果表明,采用酶解的方法能够有效降低酪蛋白的抗原性,但是不能够将抗原表位完全除去,酶解产物仍保留有一部分抗原性.不同的酶降低酪蛋白抗原性的程度不同,碱性蛋白酶降低酪蛋白抗原性的效果最佳,但是酶解物苦味较重,木瓜蛋白酶既能有效降低酪蛋白的抗原性,酶解物又具有较好风味.     

Antioxidant and antihypertensive protein hydrolysates produced from tuna liver by enzymatic hydrolysis

In order to better utilize a fish by-product, the enzymatic hydrolysis of tuna liver was performed using commercially available proteases such as Flavourzyme, Alcalase, Protamex, and Neutrase. The hydrolysates were prepared as both first step hydrolysates and second step hydrolysates. The molecular weight distribution of the hydrolysates was determined by size exclusion chromatography and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, which analyzed a representative hydrolysate type with a weight range of 1000–3000 Da. The antioxidant activities of the tuna liver hydrolysates against 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical, and hydrogen peroxide, along with a reducing power assay, showed that the second step hydrolysates had more potent antioxidant activity than the first step hydrolysates. However, the first step hydrolysates exhibited more effective chelating activity. Furthermore, the protection ability of the hydrolysates toward hydroxyl radical-induced oxidative DNA damage was evaluated by measuring the conversion of supercoiled pBR322 plasmid DNA to the open circular form. In addition, an angiotensin I-converting enzyme (ACE) inhibition assay revealed that all hydrolysates had similar ACE-inhibitory properties. These findings suggest that tuna liver hydrolysates may be a beneficial ingredient to use in functional foods.     

Optimisation, by response surface methodology, of degree of hydrolysis and antioxidant and ACE-inhibitory activities of whey protein hydrolysates obtained with cardoon extract

The hydrolysis of bovine whey protein concentrate (WPC), α-lactalbumin (α-La) and caseinomacropeptide (CMP), by aqueous extracts of Cynara cardunculus, was optimized using response surface methodology. Degree of hydrolysis (DH), angiotensin-converting enzyme (ACE)-inhibitory activity and antioxidant activity were used as objective functions, and hydrolysis time and enzyme/substrate ratio as manipulated parameters. The model was statistically appropriate to describe ACE-inhibitory activity of hydrolysates from WPC and α-La, but not from CMP. Maximum DH was 18% and 9%, for WPC and α-La, respectively. 50% ACE-inhibition was produced by 105.4 (total fraction) and 25.6 μg mL⁻¹ (<3 kDa fraction) for WPC, and 47.6 (total fraction) and 22.5 μg mL⁻¹ (<3 kDa fraction) for α-La. Major peptides of fractions exhibiting ACE-inhibition were sequenced. The antioxidant activities of WPC and α-La were 0.96 ± 0.08 and 1.12 ± 0.13 μmol trolox equivalent per mg hydrolysed protein, respectively.     

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.     

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.     

Food sensitization of guinea pigs with enzymatic hydrolysates of milk whey proteins

Adult guinea pigs received intragastrically solutions of milk whey protein concentrate (CWPM), its enzymatic hydrolysate (HWPM) and peptide fraction (PF) obtained by means of HWPM ultrafiltration. Milk protein antigen content of CWPM, HWPM and PF was preliminary characterized with two-dimensional and rocket immunoelectrophoresis. Undigested beta-lactoglobulin (beta LG) absorbed from the gut, was measured three hours after the administration by means of competitive radioimmunoassay. Administration of proteins was repeated twice with daily intervals. Twenty one days later the levels of circulating anti-beta LG antibodies were measured by Farr's method and then the reaction of active anaphylactic shock was triggered by intravenous infusion of whole skimmed milk. It was established that while antigens including beta LG content decreased in the rank of CWPM-HWPM-PF the absorption of beta LG into the blood serum and the violence of acute anaphylaxis decreased also. The largest level of specific antibodies to beta LG was detected in animals that received the hydrolized protein the fact being connected with the presence of aggregated beta LG in this preparation. The employment of ultrafiltration of enzymatic hydrolyzate leads to elimination of the majority of noninactivated antigens from the filtrate, consequence of this being the low level of animal sensitization by this preparation. It has been concluded that ultrafiltration technology has good advances in manufacturing hypoallergenic milk formulae.     

牡蛎酶解过程的成分变化及脱腥初步研究

对牡蛎原浆液、酶解液及其浓缩液的基本营养成分进行分析比较,分别采用了活性炭吸附、β-环状糊精包埋法和酵母发酵法对牡蛎酶解液进行脱腥,研究了温度,时间,pH和脱腥剂用量等对脱腥效果的影响. 结果表明:牡蛎蛋白质、水分含量分别为9. 46%和81. 42%:酶解后,蛋白质和水分含量分别为7. 36%和83. 18%;酶解液浓缩后,蛋白质含量高达52. 55%. 活性炭脱腥效果最优:用量为0. 5%,在pH7. 0、30℃条件下吸附0. 5 h,酶解液基本无腥味,蛋白质回收率可达84. 65%. 促进牡蛎酶解液的推广应用.     

Antibody binding and functional properties of whey protein hydrolysates obtained under high pressure

This paper examines the potential of high hydrostatic pressure to produce whey protein hydrolysates that combine low immunoglobulin (Ig)G- and IgE-binding with acceptable functional properties, with the aim to produce milk-based ingredients with reduced potential allergenicity that could be used in hypoallergenic foods. Treatment with pepsin and chymotrypsin under high pressure produced, in minutes, hydrolysates in which α-lactalbumin and β-lactoglobulin were totally proteolysed, giving rise to large and hydrophobic peptides. Such hydrolysates presented reduced antigenicity and human IgE-binding properties. The hydrolysates obtained with pepsin at 400 MPa showed improved heat stability, particularly at a pH, close to the isoelectric point of the whey proteins, and their emulsion activity indexes at pH 7.0 were superior to those of the untreated whey proteins. These results suggest that the peptides present retained low antigenicity together with sufficient capacity to form emulsions.     

风味蛋白酶水解制备豌豆蛋白抗氧化酶解产物工艺条件优化

采用风味蛋白酶水解豌豆蛋白,对制备豌豆蛋白酶解产物清除DPPH.清除率进行研究;通过单因素实验和响应面回归分析,得到制备酶水解产物最佳酶解工艺条件:酶解温度48.8℃、酶解时间3.1 h、pH 5.5、加酶量4.0%、底物浓度6.0%,在此条件下,风味蛋白酶酶解产物对DPPH.清除率为60.10%。     

Biologically active peptides obtained by enzymatic hydrolysis of Adzuki bean seeds

This study investigated the antioxidant and antihypertensive activities of peptides obtained from protein fractions of Adzuki bean seeds. Peptides were obtained by the use of hydrolytic enzymes in vitro under gastrointestinal conditions. A determination was made of the activity of the peptide inhibitors of the angiotensin I converting enzyme (ACE), and the antiradical and ion chelating activity of peptides from different protein fractions. The highest peptide levels after the absorption process (<7 kDa) were noted in the albumin fraction (50.69 μg/ml). Furthermore, it was found that peptides from the prolamin fraction were characterised by the highest antiradical activity and ACE inhibitory activity (IC₅₀ = 0.17 mg/ml). Peptides obtained from the globulin fraction showed the highest ability to chelate iron ions, and peptides from the glutelin fraction were characterised as being the most effective in the chelation of copper ions.     

Peptic and tryptic hydrolysis of native and heated whey protein to reduce its antigenicity

This study examined the effects of enzymes on the production and antigenicity of native and heated whey protein concentrate (WPC) hydrolysates. Native and heated (10 min at 100°C) WPC (2% protein solution) were incubated at 50°C for 30, 60, 90, and 120 min with 0.1, 0.5, and 1% pepsin and then with 0.1, 0.5, and 1% trypsin on a protein-equivalent basis. A greater degree of hydrolysis was achieved and greater nonprotein nitrogen concentrations were obtained in heated WPC than in native WPC at all incubation times. Hydrolysis of WPC was increased with an increasing level of enzymes and higher incubation times. The highest hydrolysis (25.23%) was observed in heated WPC incubated with 1% pepsin and then with 1% trypsin for 120 min. High molecular weight bands, such as BSA, were completely eliminated from sodium dodecyl sulfate-PAGE of both native and heated WPC hydrolysates produced with pepsin for the 30-min incubation. The α-lactalbumin in native WPC was slightly degraded when incubated with 0.1% pepsin and then with 0.1% trypsin; however, it was almost completely hydrolyzed within 60 min of incubation with 0.5% pepsin and then with 0.5% trypsin. Incubation of native WPC with 1% pepsin and then with 1% trypsin for 30 min completely removed the BSA and α-lactalbumin. The β-lactoglobulin in native WPC was not affected by the pepsin and trypsin treatments. The β-lactoglobulin in heated WPC was partially hydrolyzed by the 0.1 and 0.5% pepsin and trypsin treatments and was completely degraded by the 1% pepsin and trypsin treatment. Antigenicity reversibly mimicked the hydrolysis of WPC and the removal of β-lactoglobulin from hydrolysates. Antigenicity in heated and native WPC was reduced with an increasing level of enzymes. A low antigenic response was observed in heated WPC compared with native WPC. The lowest antigenicity was observed when heated WPC was incubated with 1% pepsin and then with 1% trypsin. These results suggested that incubation of heated WPC with 1% pepsin and then with 1% trypsin was the most effective for producing low-antigenic hydrolysates by WPC hydrolysis and obtaining low molecular weight small peptides. Further research is warranted to identify the low molecular weight small peptides in the WPC hydrolysates produced by pepsin and trypsin, which may enhance the use of whey.