Use of macroporous adsorption resin for simultaneous desalting and debittering of whey protein hydrolysates

Whey protein isolate (WPI) was hydrolysed to whey protein hydrolysates (WPH) of degree of hydrolysis equal to 15% using Protease N ‘Amano’ G (IUB 3.4.24.28) in a batch reactor at 55 °C and pH 7.0 according to the pH‐stat procedure. Ash was removed by adsorbing WPH onto macroporous adsorption resins (MAR). Following rinsing with deionised water, desorption was achieved by washing with 20%, 40% and 75% alcohol (v v^?1) to obtain the three fractions HS20, HS40 and HS75. Ash reduced from 15.71% (WPH) to 4.38% (HS20), 2.02% (HS40) and 2.38% (HS75). Similarly, the protein content was enriched from a low of 64.89% (WPH) to 94.74% (HS20), 95.32% (HS40) and 92.00% (HS75). The fractions were analysed for surface hydrophobicity (SH_o), angiotensin‐I converting enzyme (ACE) inhibition, emulsifying activity index, total amino acids composition and molecular weight distribution. Fraction HS75 was objectionably bitter, showed superior ACE inhibition (lowest IC₅₀), had the highest content of hydrophobic and essential amino acids and contained about 71% of <600 Da with no fractions exceeding 4142 Da. Desorption with alcohol weakened the hydrophobic interaction forces between the peptides and resins and hence eluted the peptides, with the bitter HS75 being extracted.     

Use of response surface methodology to optimise the hydrolysis of whey protein isolate in a tangential flow filter membrane reactor

Response surface methodology (RSM) was used to investigate the effects of substrate concentration (S), enzyme concentration (E) and initial permeate flow rate, (J_i), on permeate flux behaviour in a 10 kDa nominal molecular weight cut-off (NMWCO) tangential flow filter (TFF) enzyme membrane reactor (EMR) during 3 h hydrolysis of whey protein isolate (WPI) using Protease N Amano (IUB 3.4.24.28, Bacillus subtilis) at pH 7.0 and 45 °C. The average residual permeate flow rate (J_residual), residual enzyme activity (A_residual) and product recovered in permeate designated as apparent sieving (S_apparent) were monitored. The quadratic model regression equations obtained revealed that all the three factors had significant but dissimilar influences on permeate flux behaviour. J_residual, S_apparent and A_residual increased with increasing E, A_residual decreased with increasing J_i and there was substrate inhibition at low E. The optimised factors were S = 4.72% (w/v), E = 0.055% (w/v; hence E/S ≈ 1% w/w) and J_i = 6.91 mL/min (approximately 0.7% reactor volume per minute). The optimised values were 87.24%, 52.37% and 35.08% for J_residual, A_residual and S_apparent, respectively. The actual values for the responses agreed well with the predicted values implying that RSM is suitable for EMR optimisation. Covariance values showed that J_residual and S_apparent increased concomitantly while A_residual decreased with increasing S_apparent and J_residual.     

Hydrolysis of β-lactoglobulin by trypsin under acidic pH and analysis of the hydrolysates with MALDI–TOF–MS/MS

Trypsin (EC 3.4.21.4) hydrolysis of food proteins are done at the optimum pH (7.8) and temperature (37 °C). Little information is available on the effect of sub-optimal conditions on hydrolysis. Bovine β-lactoglobulin (β-Lg) was hydrolysed by trypsin under acidic pH (pH 4–7) between 20 and 60 °C and the substrate concentration from 2.5% to 15% (w/v) and compared with hydrolysis at pH 7.8 and 37 °C. Aliquots were taken at different times (t = 0 up to 10 min). Samples were analysed using matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI–TOF–MS/MS) with α-cyano-4-hydroxycinnamic acid (HCCA) and 2,5-dihydroxyacetophenone (DHAP) matrices. Hydrolysis patterns of β-Lg were generally similar at pH 7.8, 7, 6 and 5 while at pH 4 fewer peptides were detected except a unique fragment f(136–141). The different cleavage sites of β-Lg showed low resistance to trypsin at optimum conditions and pH 7 while being random and simultaneous. At lower pH, some cleavage sites showed increased resistance, while hydrolysis was relatively slow and ordered. Initial attack by trypsin occurred at Arg⁴⁰–Val⁴¹, Lys¹⁴¹–Ala¹⁴² and Arg¹⁴⁸–Leu¹⁴⁹ resistance was at Lys⁶⁰–Trp⁶¹, Arg¹²⁴–Thr¹²⁵ and Lys¹³⁵–Phe¹³⁶. Five domains were identified based on β-Lg resistance to trypsin in the order f(1–40) < f(41–75) < f(76–91) > f(92–138) > f(139–162). Results suggest that hydrolysis away from trypsin optimum offer better hydrolysis process control and different peptides. This strategy may be used to protect target bioactive or precursor peptides, or avoid the production of unwanted peptides.     

Impact of the environmental conditions and substrate pre-treatment on whey protein hydrolysis: A review

Proteins in solution are subject to myriad forces stemming from interactions with each other as well as with the solvent media. The role of the environmental conditions, namely pH, temperature, ionic strength remains under-estimated yet it impacts protein conformations and consequently its interaction with, and susceptibility to, the enzyme. Enzymes, being proteins are also amenable to the environmental conditions because they are either activated or denatured depending on the choice of the conditions. Furthermore, enzyme specificity is restricted to a narrow regime of optimal conditions while opportunities outside the optimum conditions remain untapped. In addition, the composition of protein substrate (whether mixed or single purified) have been underestimated in previous studies. In addition, protein pre-treatment methods like heat denaturation prior to hydrolysis is a complex phenomenon whose progression is influenced by the environmental conditions including the presence or absence of sugars like lactose, ionic strength, purity of the protein, and the molecular structure of the mixed proteins particularly presence of free thiol groups.

In this review, we revisit protein hydrolysis with a focus on the impact of the hydrolysis environment and show that preference of peptide bonds and/or one protein over another during hydrolysis is driven by the environmental conditions. Likewise, heat-denaturing is a process which is dependent on not only the environment but the presence or absence of other proteins.     

Alpha‐Lactalbumin: Its Production Technologies and Bioactive Peptides

ABSTRACT: Alpha‐lactalbumin (α‐La), a globular protein found in all mammalian milk, has been used as an ingredient in infant formulas. The protein can be isolated from milk using chromatography/gel filtration, membrane separation, enzyme hydrolysis, and precipitation/aggregation technologies. α‐La is appreciated as a source of peptides with antitumor and apoptosis, antiulcerative, immune modulating, antimicrobial, antiviral, antihypertensive, opioid, mineral binding, and antioxidative bioactivities, which may be utilized in the production of functional foods. Nanotubes formed by the protein could find applications in foods and pharmaceuticals, and understanding its amyloid fibrils is important in drawing strategies for controlling amyloidal diseases. Bioactive peptides in α‐La are released during the fermentation or ripening of dairy products by starter and nonstarter microorganisms and during digestion by gastric enzymes. Bioactive peptides are also produced by deliberate hydrolysis of α‐La using animal, microbial, or plant proteases. The occurrence, structure, and production technologies of α‐La and its bioactive peptides are reviewed.     

大孔吸附树脂提取苦味乳清蛋白水解物及其功能性质和生物活性的研究

用生物蛋白酶NAmano G水解乳清分离蛋白（WPI），水解过程中因不断加碱引入的过多的灰分用大孔吸附树脂除去，吸附的水解产物用20％（HS20）、40％（HS40）和75％（HS75）乙醇洗脱，根据洗脱所用的乙醇浓度的不同可以将洗脱的产品分为HS20、HS40、HS75三个组分，分析这三组分的表面疏水性（SH0），血管紧张素一转换酶（ACE）的抑制作用，乳化特性（EAI），氨基酸组分和分予量分布。结果表明HS75具有最好的ACE抑制作用，疏水性氨基酸含量最高，相对分子质量小于600Da占7l％，不存在相对分子质量超过4142Da的组分。盐几乎完全从大孔吸附树脂吸附物中被除去且水解物回收率高。乙醇解吸可能改变了溶剂的相互作用，削弱了多肽链和树脂之间的疏水相互作用，从而使水解物从吸附的树脂上被洗脱。     

Influence of buffer type and concentration on the peptide composition of trypsin hydrolysates of β-lactoglobulin

Proteins acquire conformation in aqueous media due to the influence of the buffer salt composition and concentration. Such influence may have impact on the enzyme–substrate interaction and somehow steer the enzyme attack properties, leading to release of dissimilar products. Our group has sought to investigate the influence of the hydrolysis environment on the trypsinolysis of a model protein, β-lactoglobulin (β-Lg). This work was aimed at investigating the effect of different buffers and their concentrations on the trypsinolysis patterns of β-Lg. The traditional NaOH-buffered water, in comparison to Tris–HCl and potassium-phosphate buffer at 62.5 mM–1.0 M were used at pH 8.5 for the pH drop and pH 7.8 for the hydrolysis. Bovine trypsin (EC 3.4.21.4) was used at an enzyme-to-substrate ratio of 1%. The samples were analysed for mass composition, using LC-ESI-TOF/MS and MALDI-TOF/MS for monitoring time-dependence of peptide evolution. In all buffer types and concentrations, peptides f(1–8), f(15–40, f(125–138) and f(142–148) were detected, implicating ease of hydrolysis of the terminal regions of β-Lg. A peptide from f(9–14), with sequence Gly-Leu-Asp-Ile-Gln-Lys, was detected at >0.5 M Tris–HCl only, while peptide f(71–75) was unique to <125 mM Tris–HCl and >250 mM potassium-phosphate buffer. Hydrolysis under buffer produced trypsin-specific peptides, numerous chymotrypsin-like non-specific peptides but no disulphide-linked peptides. Trypsinolysis shifted to the N-terminal region of lysine under some conditions. Hydrolysis under buffer holds potential for the avoidance of some peptides with undesirable characteristics while preserving a diversity of different peptides with possible bioactive properties.     

Impact of milieu conditions on the α-lactalbumin glycosylation in the dry state

Maillard reaction is influenced by protein and sugar properties, water activity (a_w) as well as the glycosylation time and temperature. The aim of this work was to investigate the influence of environmental parameters on the glycosylation reaction kinetics and to develop a technology platform for protein glycosylation as a possible substrate pre-treatment. The glycosylation reaction of bovine α-lactalbumin (α-La) was performed with lactose and maltodextrin in the dry-state at 40, 50 or 60 °C performed at a_w of 0.33, 0.44 or 0.58 for reaction times of 8, 24 or 48 h. The degree of glycosylation (DG) was determined as the loss of lysine using the ortho-phthalaldehyde (OPA) method. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) with Coomassie and glycoprotein staining was also performed. The reaction with lactose reached higher DG values in all cases as compared to reactions with maltodextrin (maximum DG of 85% and 31%, respectively, at a_w = 0.58 after 48 h). Lactosylation kinetics showed that the second order rate constants increased with increasing temperature and were highest at a_w = 0.58 in all cases. The activation energies were determined as 97.1 ± 37.7, 193.9 ± 9.1 and 136.6 ± 15.6 kJ/mol for a_w = 0.33, 0.44 and 0.58, respectively and showed an increasing trend with increasing temperature. Glycosylation of α-La offers a new process for improvement of functional properties as well as being a substrate pre-treatment process to control enzymatic digestion in order to generate tailor-made peptides as food additives with important health benefits like probiotics due to glycoprotein resistance to further enzyme hydrolysis.     

Comparison of a modified spectrophotometric and the pH-stat methods for determination of the degree of hydrolysis of whey proteins hydrolysed in a tangential-flow filter membrane reactor

A procedure was developed to determine the degree of hydrolysis (DH) of whey protein hydrolysates (WPH) during hydrolysis in either 3 kDa or 10 kDa tangential-flow filter (TFF) enzymatic membrane reactors (EMR). Protease N Amano G (IUB 3.4.24.28, Bacillus subtilis) was used to hydrolyse an initial 5% (w v^?1) aqueous solution of whey protein isolate (86.98% protein) at pH 7.0 and 55 °C with continuous recirculation and simultaneous removal of hydrolysates through the TFF, in single- or two-stage operation. The DH in the permeate and the retentate were determined as the concentration of the free α-NH₂ using 2,4,6-trinitrobenzene 1-sulphonic acid (TNBS) and compared to the pH-stat method. In the new method, the DH of the permeate, the retentate and for the total EMR process could be quantified together or independently. The pH-stat method exaggerated the DH in the EMR because of the leakage of the alkali. The TNBS method was more reliable for DH estimation in the EMR.     

Influence of temperature and degree of hydrolysis on the peptide composition of trypsin hydrolysates of β-lactoglobulin: Analysis by LC–ESI-TOF/MS

Enzymatic hydrolysis of proteins is influenced, either positively or negatively, by the hydrolysis conditions, temperature, enzyme concentration and pH, as well as substrate pre-treatments, e.g. heat-denaturing, glyco-conjugation and/or cross-linking. Purified bovine β-lactoglobulin (96.0% nitrogen) was hydrolysed using trypsin (EC 3.4.21.4, bovine pancrease) at between 30 and 50 °C to degrees of hydrolysis (DHs) between 1 and ∼9.0%. The time taken to reach the desired DH varied greatly, being shortest at 45 and 50 °C and longest at 30 °C. The hydrolysates were analysed by tandem liquid chromatography–electrospray ionisation time-of-flight mass spectra (LC–ESI-TOF/MS) and results showed that the detectable peptides, at both 30 °C and 35 °C, were similar at DH 1%. However, not only were the detectable peptides produced at 40–50 °C different from those produced at lower temperatures, but the trypsin released peptides due to non-specific hydrolysis of β-Lg. The pattern resembled a shift of trypsinolysis towards chymotrypsinolysis, probably due to steric ‘stretching’ and increase of the catalytic pocket, thus allowing bulky amino acids to be processed. Hydrolysis at 30 °C to DH 5% and 10% also led to the release of peptides due to non-specific cleavage by trypsin. These results indicate that trypsin could only release peptides in a predictable manner at temperatures near, but lower than, the declared optimum of 37 °C. Above this temperature and above DH 5–10% at 30 °C, hydrolysis followed a mixed trypsin- and chymotrypsin-like activity. Lys–Pro, Lys–Ile(–Pro) and Lys–Phe bonds remained stable to trypsin at all temperatures. Some peptides with a high content of hydrophobic amino acids were undetected by ESI-TOF/MS, probably due to their poor ionisation.