Heat-induced aggregation of β-lactoglobulin A and B with α-lactalbumin

β-Lactoglobulin A and β-lactoglobulin B were heated at 75°C in the absence and presence of α-lactalbumin, and the aggregation products were characterized by size exclusion chromatography in combination with multi-angle laser light scattering and electrophoretic techniques. α-Lactalbumin did not form aggregates when heated alone, but in admixture with β-lactoglobulin it was incorporated into both the disulphide-bonded and the hydrophobically associated aggregates as well as forming α-lactalbumin dimers and other oligomers. The presence of α-lactalbumin diminished the proportion of smaller aggregates and increased the number of very large aggregates within both variant protein mixtures. In the presence of α-lactalbumin, β-lactoglobulin A was converted into a series of disulphide-bonded and the hydrophobically associated aggregates more slowly, but with a greater proportion of hydrophobically associated aggregates, than β-lactoglobulin B. These patterns are similar to that when β-lactoglobulin A or B are heated on their own. These and other results indicate that the mechanism of aggregation of α-lactalbumin/β-lactoglobulin mixtures is governed by β-lactoglobulin.     

Effects of γ-irradiation on molar mass and properties of Konjac mannan

Konjac mannan (KM) is a water-soluble glucomannan with high molar mass. Here, the effects of γ-irradiation on the structure of KM, its viscosity, molar mass distribution and the state of sorbed water were studied after irradiation at reduced pressure. These changes were investigated using ESR, FT-IR, UV, viscometer, SEC–MALS and DSC. Free radical yields increased with absorbed dose. Irradiation led to chain scission, but introduced no significant new chemical groups into the structure, apart from a small increase in content of carbonyl groups. The intrinsic viscosity, molar mass and radius of gyration decreased rapidly with increasing dose up to 10 kGy and then at a slower rate. The Mark–Houwink–Sakurada equation for the KM gave [η]=5.30×10⁻⁴ M^0.78. The α value showed that KM molecules are solvated in the form of random coils in water. There are three types of sorbed water in irradiated KM as in the original KM. There is no significant change in water binding ability for KM with M_W greater than 2×10⁵.     

Susceptibility of equine κ- and β-caseins to hydrolysis by chymosin

Equine whole casein was hydrolysed by chymosin and some peptides generated were characterised by microsequencing after reversed-phase high performance liquid chromatography or sodium dodecyl sulphate polyacrylamide gel electrophoresis. β-Casein was readily hydrolysed into amino- and carboxy-terminal fragments after cleavage of the Leu₁₉₀–Tyr₁₉₁ bond. These two fragments seemed to be resistant to further chymosin hydrolysis on incubation for up to 24 h. Equine κ-casein was purified by affinity chromatography on immobilised wheat germ agglutinin. O-Glycosylated κ-casein was found to represent less than 6.8% of the equine casein components. Equine κ-casein was also hydrolysed and para-κ-casein and glycomacropeptide were generated after cleavage of the Phe₉₇–Ile₉₈ bond.     

Comparative behaviour of goat β and αs1-caseins at the air–water interface and in solution

Here we present a comparative study of caprine β- and α_s1-caseins behaviours at the air–water interface and in solution. Both caseins were purified from the milk of a single goat homozygous at the α_s1- and β-Cn loci, with a high degree of purity (98%). Physical measurements (ellipsometry, surface pressure and surface rheology) were performed at the air–water interface, whereas SAXS measurements were performed on casein solutions. Our results clearly show that self-organizations, both at the air–water interface and in solution are different for β- and α_s1-caseins. β-casein is unfolded in solution and forms a network at the interface, while α_s1-casein forms compact objects in solution and is organised in fluid domains at the interface. We also show that the presence of Ca²⁺ in the subphase strongly disturbs the interfacial layer formed by the caseins. It is elsewhere worth noting that in solution, the aggregation of α_s1-casein induced by calcium ions is associated with a pronounced change in the molecular structural organisation of the protein, which seems to adopt, in these conditions, an unfolded structure.     

Novel casein-derived peptides with antihypertensive activity

In this study, we report novel casein-derived peptide sequences with angiotensin converting enzyme (ACE)-inhibitory activity and antihypertensive activity demonstrated in spontaneously hypertensive rats (SHR). The peptides were obtained by enzymatic hydrolysis of total isoelectric casein with pepsin. To identify ACE-inhibitory peptides, the casein hydrolysate was fractionated by semi-preparative high performance liquid chromatography, and 44 (CN) peptides contained in the active fractions were sequenced by using an ion trap mass spectrometer. Among the identified peptides, three sequences, that corresponded to α_s1-CN f(90–94) (RYLGY), α_s1-CN f(143–149) (AYFYPEL), and α_S2-CN f(89–95) (YQKFPQY), showed IC₅₀ values as low as 0.71 μm, 6.58 μm, and 20.08 μm, respectively. These three peptides also exerted antihypertensive activity when they were orally administered to SHR at a dose of 5 mg kg⁻¹ of body weight. The activity of peptides RYLGY and AYFYPEL in SHR was similar to that found for tripeptide VPP when orally administered at the same dose.     

Structural characterization of barley β-glucan extracted using a novel fractionation technique

A barley β-glucan concentrate prepared according to a novel technology was further purified and subjected to detailed structural characterization by NMR spectroscopy. β-Glucan was hydrolysed with β-glucan-4-glucanohydrolase (lichenase). Fractions of hydrolysate were collected using an HPLC-fraction collector. Intact β-glucan and the major fractions collected were subjected to MALDI-TOF–MS and NMR analyses. The two major oligosaccharides produced by lichenase hydrolysis of purified barley β-glucan were identified as β-d-Glc p-(1 → 4)- β-d-Glc p-(1 → 3)-β-d-Glc p and β-d-Glc p-(1 → 4)-β-d-Glc p-(1 → 4)-β-d-Glc p-(1 → 3)-β-d-Glc p based on ¹³C and ¹H NMR data. Spectrums were similar to those documented for barley β-glucan in the literature.     

Release of β-casomorphins 5 and 7 during simulated gastro-intestinal digestion of bovine β-casein variants and milk-based infant formulas

The release of β-casomorphin-5 (BCM5) and β-casomorphin-7 (BCM7) was investigated during simulated gastro-intestinal digestion (SGID) of bovine β-casein variants (n = 3), commercial milk-based infant formulas (n = 6) and experimental infant formulas (n = 3). SGID included pepsin digestion at pH 2.0, 3.0 and 4.0 and further hydrolysis with Corolase PP™. β-Casein (β-CN) variants were extracted from raw milks coming from cows of Holstein-Friesian and Jersey breeds. Genomic DNA was isolated from milk and the β-CN genotype was determined by a PCR-based method. Phenotype at protein level was determined by capillary zone electrophoresis in order to ascertain the level of gene expression. Recognition and quantification of BCMs involved HPLC coupled to tandem MS. Regardless of the pH, BCM7 generated from variants A1 and B of β-CN (5–176 mmol/mol casein) the highest amount being released during SGID of form B. As expected, the peptide was not released from variant A2 at any steps of SGID. BCM5 was not formed in hydrolysates irrespective of either the genetic variant or the pH value during SGID. Variants A1, A2 and B of β-CN were present in all the commercial infant formulae (IFs) submitted to SGID. Accordingly, 16–297 nmol BCM7 were released from 800 ml IF, i.e. the daily recommended intake for infant. Industrial indirect-UHT treatments (156 °C × 6–9 s) did not modify release of BCM7 and, during SGID, comparable peptide amounts formed in raw formulation and final heat-treated IFs.     

Susceptibility of an industrial α-lactalbumin concentrate to cross-linking by microbial transglutaminase

The susceptibility of an industrial α-lactalbumin concentrate to cross-linking with a microbial transglutaminase from Streptoverticillium mobaraense was investigated. At a protein concentration of 0.5% w v⁻¹, the maximum cross-linking was observed at 50°C, pH 5 and at 5 h of incubation time. Results from sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis showed that most of the monomeric form of α-lactalbumin was converted to polymers too large to move into the gel matrix. Addition of ethylenediamine tetraacetic acid or SDS prior to the incubation of protein–enzyme mixture, further enhanced the transglutaminase reaction with the industrial α-lactalbumin. Results from reverse phase chromatography indicated that cross-linking caused a broadening of the α-lactalbumin peak with little change in the average hydrophobicity of the protein. In contrast to the reported results on pure α-lactalbumin, the industrial α-lactalbumin concentrate showed considerable cross-linking with transglutaminase even without the reduction of the disulphide bonds. This difference was attributed to the partially unfolded secondary structures in the industrial α-lactalbumin concentrate.     

Characterization of α-carrageenan solution behavior by field-flow fractionation and multiangle light scattering

The salt mediated molecular conformation change of alpha (α)-carrageenan was studied in 0.1 M solutions of NaCl, NaI, and KCl. Asymmetric Field-Flow Fractionation with multiangle laser light scattering (AFFF/MALLS) detection was used to determine the average molecular weight, radius of gyration, and hydrodynamic radius which were in turn used to calculate the molecular density. In the presence of 0.1 M NaCl, an inert salt that does not promote gelation, α-carrageenan has a denser structure compared to κ-carrageenan of a similar molecular weight. A distinct and dramatic increase in the molecular weight (factor of 2) was observed for α-carrageenan in 0.1 M NaI compared to 0.1 M NaCl. This combined with only a slight change in the radius of gyration, suggests intermolecular interaction to a more compact structure (e.g., coaxial helices). A similar increase in molecular weight is observed in 0.1 M KCl, accompanied with an approximate 50% increase in the radius of gyration as well as an increase in polydispersity. This may also be attributed to intermolecular interaction with helix formation (coaxial or lateral) or may be due to K⁺ cations interacting with naturally occurring residual ι-carrageenan in the sample. As previously reported for other carrageenans the random coil to helix transition of α-carrageenan appears to be stabilized by K⁺ cation or I⁻ anion in an aqueous environment.     

Cross-linking of β-casein by Trichoderma reesei tyrosinase and Streptoverticillium mobaraense transglutaminase followed by SEC–MALLS

Enzymatic modification of proteins, in order to produce functional materials such as hydrogels, adhesives and films via cross-linked networks or scaffolds of proteins, is a constantly evolving technology to create tailored micro- and nanostructured materials for food, cosmetic, and medical applications. For the successful utilization of oxidoreductases or transferases such as tyrosinases and transglutaminases, respectively, it is crucial to understand the action of these enzymes on protein substrates. In this study, cross-linking of the milk protein β-casein by Trichoderma reesei tyrosinase (TrTyr) was studied using size-exclusion chromatography (SEC) equipped with multi-angle light scattering (MALLS) and ultraviolet/visible (UV/Vis) detectors in order to determine the molecular mass (MM), radius of gyration (R_G) and degree of polymerization (DP) of the reaction products. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect early polymerization states. The widely used Streptoverticillium mobaraense transglutaminase (Tgase) was used for comparison to tyrosinase from T. reesei. The results showed that cross-linking of β-casein by these two different types of enzymes resulted in the formation of polymerized reaction products with MM ranging from 500 to 1700 kg mol⁻¹ depending on the enzyme dosage and incubation time. The DP varied from 21 to 71, respectively. In the case of TrTyr the polymerized reaction products were slightly colored, and formation of the covalent cross-linking of β-casein could be monitored by UV/Vis as a function of incubation time.     

Influence of complexing carboxymethylcellulose on the thermostability and gelation of α-lactalbumin and β-lactoglobulin

Thermostability and gelation of the main proteins of whey, α-lactalbumin (α-lac) and β-lactoglobulin (β-lg) recovered by selective complexation with carboxymethylcellulose (CMC) was studied to evaluate its functionality in food systems. Their behavior was compared to the non-complexed proteins. Both complexes showed a maximum stability at pH 4, that is close to the pH of obtention of β-lg/CMC coacervate (pH 4) and α-lac/CMC coacervate (pH 3.2). Protein complexation increased the thermostability of β-lg by approximately 6–8 °C and that of α-lac by approximately 26 °C due to immobilization of protein molecules in a complex, mainly by electrostatic interactions and because of different amounts of bound polysaccharide. The denaturation enthalpy of complexed proteins markedly decreased as compared to free proteins. Storage modulus (G′) and loss modulus (G″) were recorded to reflect the structure development during heating β-lg/CMC and α-lac/CMC complexes at different pH values. β-lg/CMC complex at 20 wt% was a viscoelastic liquid at pH values within 2 and 8 but upon heating turned to a particulate viscoelastic gel. However, α-lac/CMC complex formed before heating opaque, large visible white particulate aggregates that sticked together to give a solid viscoelastic structure that was not further modified by thermal processing.     

Precipitation of β-carotene microparticles from SEDS technique using supercritical CO2

This work investigates the application of the Solution-Enhanced Dispersion by supercritical fluids technique for the precipitation of β-carotene. The effect of pressure (8.0–12.0 MPa), temperature (293–313 K) anti-solvent flow rate (20–40 mL/min), solution flow rate (1–4 mL/min) and concentration of β-carotene in the dichloromethane solution (4 and 8 mg/mL) on the precipitation yield, particle morphology and particle size and size distribution was examined. Precipitated powders presented mean particle size varying from 3.2 μm to 96.8 μm with morphology of β-carotene microparticles changing from plate-like to leaf-like particles. The statistical analysis of the experimental results revealed that pressure, organic solution concentration and CO₂ flow rate had a significant effect on particle size. The precipitation yield was observed to be within the range of 71–94% and was statistically influenced by system temperature and pressure, and anti-solvent flow rate.     

Release and identification of angiotensin-converting enzyme-inhibitory peptides as influenced by ripening temperatures and probiotic adjuncts in Cheddar cheeses

The aim of the study was to examine the release of angiotensin-converting enzyme (ACE)-inhibitory peptides in Cheddar cheeses made with starter lactococci and Bifidobacterium longum 1941, B. animalis subsp. lactis LAFTI^® B94, Lactobacillus casei 279, Lb. casei LAFTI^® L26, Lb. acidophilus 4962 or Lb. acidophilusLAFTI^® L10 during ripening at 4 and 8 °C for 24 weeks. ACE-inhibitory activity of the cheeses was maximum at 24 weeks. Cheeses made with the addition of Lb. casei 279, Lb. casei LAFTI^® L26 or Lb. acidophilus LAFTI^® L10 had significantly higher (P < 0.05) ACE-inhibitory activity than those without any probiotic adjunct after 24 weeks at 4 and 8 °C. The IC₅₀ of cheeses ripened at 4 °C was not significantly different (P > 0.05) to that ripened at 8 °C. The lowest value of the IC₅₀ (0.13 mg mL⁻¹) and therefore the highest ACE-inhibitory activity corresponded to the cheese with the addition of Lb. acidophilus LAFTI^® L10. Several ACE-inhibitory peptides were identified as κ-CN (f 96–102), α_s1-CN (f 1–9), α_s1-CN (f 1–7), α_s1-CN (f 1–6), α_s1-CN (f 24–32) and β-CN (f 193–209). Most of the ACE-inhibitory peptides accumulated at the early stage of ripening, and as proteolysis proceeded, some of the peptides were hydrolyzed into smaller peptides.     

Hydrolysis of β-glucosyl ester linkage of p-hydroxybenzoyl β- -glucose, a chemically synthesized glucoside, by β-glucosidases

To investigate the hydrolysis of glucosyl esters by β-glucosidase, p-hydroxybenzoyl β- -glucose (pHBG) was chemically synthesized. The hydrolytic activity of some β-glucosidases for pHBG was compared to that for p-nitrophenyl β-glucoside (pNPG). The Clavibacter michiganense and Flavobacterium johnsonae enzymes could hydrolyze pHBG and steviol glycosides which are natural glucosyl esters. The commercial β-glucosidase originating from Caldocellum saccharolyticum also hydrolyzes pHBG despite having no activity for steviol glycosides. The β-glucosidase from Aspergillus niger cleaved the glucosyl ester linkage much more weakly than the glucosidic linkage. The pH-activity profile for the hydrolysis of pHBG was similar to that of pNPG by the C. saccharolyticum β-glucosidase. The similar profiles for these substrates suggested that the active site for the glucosyl ester chemically resembles that for glucoside with respect to catalysis. Kinetic analysis of the C. saccharolyticum β-glucosidase for mixed substrates of pHBG and pNPG showed that the hydrolysis of pHBG competed with that of pNPG. This result indicated that there is only one active site for both the glucosyl ester and glucoside. Mass spectroscopic analysis of the hydrolysates of pHBG in H₂¹⁸O suggested that β-glucosidase hydrolyzes glucosyl esters between the anomeric carbon of glucose and the carbonyl oxygen, not between the carbonyl carbon and the carbonyl oxygen.     

Preparation and characterization of molecular weight standards of low polydispersity from oat and barley (1→3)(1→4)-β- -glucan

Purified (1→3)(1→4)-β- -glucans (β-glucans) from oat and barley with broad molecular weight (MW) distribution were separated into seven fractions using gradient precipitation with ammonium sulfate (NH₄)₂SO₄. The MW of each fraction decreased consecutively with the concentration of (NH₄)₂SO₄ at which it was precipitated. The MW distribution of each fraction was much narrower compared to the parent sample and is comparable to commercially available pullulan MW standards. To determine whether the fractionation process was separating sub-fractions of different structure, the original β-glucan sample and each fraction were hydrolyzed by a (1→3)(1→4)-D-β-glucan-4-glucanohydrolase (lichenase, E.C.3.2.1.73) and the liberated oligosaccharides were analyzed by high performance anion exchange chromatography. The analysis revealed no differences in oligosaccharide pattern (DP 2–9) derived from each fraction and the parent sample. In particular, the tri/tetra oligosaccharide ratio remained constant for all fractions, indicating no fractionation based on structural features had taken place. The effect of starting β-glucan concentration on the fractionation process was studied. The results showed that it was possible to achieve good separation at overlapping parameter c[η] lower than 3.5. Further increase in starting β-glucan concentration hindered clear separation of the fractions. Temperature also affected the fractionation efficiency. The higher the temperature, the lower the amount of (NH₄)₂SO₄ that was necessary to precipitate the samples of same MW. A Mark Houwink relationship was derived from the measured MW and intrinsic viscosity for fractions from oat and barley, respectively.     

Structural modification and characterization of rice starch treated by Thermus aquaticus 4-α-glucanotransferase

Rice starch was modified using Thermus aquaticus 4-α-glucanotransferase (TAαGTase) in this study. The changes in the molecular structure and the effect on the starch retrogradation by TAαGTase treatment were investigated on isolated rice starch. By treating TAαGTase, molecular weight profile of amylopectins shifted to higher elution time from 1.0 × 10⁸ to 2.4 × 10⁷ or 0.8 × 10⁷, depending on the level of enzyme dosage. Meanwhile, there were huge increases in the proportions of content corresponding to amylose size and even smaller molecules. On treating with TAαGTase, short branch chains (DP 1–8) increased, and longer branch chains (>DP 19) increased significantly as well, with a broader distribution up to DP 46 compared to the control rice starch. Amylose content decreased from 30.0 to 21.8–23.7%. This indicated that the amylose could be transferred to the amylopectin branch chain by the disproportionation of TAαGTase, resulting in lowering the amylose content and the formation of amylopectin with a broader branch-chain length distribution. TAαGTase modified rice starch showed that X-ray diffraction pattern of the B-type crystalline even before cold storage, and that a variety of cyclic glucans (DP 5–19) were produced by enzymatic reaction. In particular, the accelerated rate of starch retrogradation was clearly observed compared to the control due to an overall increase in the number of elongated long-branch chains, decrease in the amount of amylose–lipid complex, and the possible synergistic effects of these factors.     

Purification of angiotensin I-converting enzyme-inhibitory peptides from the enzymatic hydrolysate of defatted canola meal

Defatted canola meals from seeds of different processing origins were hydrolyzed by Alcalase to give hydrolysates that inhibited angiotensin converting enzyme (ACE) activity. Heat treated meals yielded protein hydrolysates with 50% ACE-inhibitory concentrations of 27.1 and 28.6 μg protein/ml compared with 35.7 and 44.3 μg protein/ml for the none-heat treated meals. Separation of the hydrolysate on a Sephadex G-15 gel permeation column (GPC) yielded a fraction with an IC₅₀ value of 2.3 μg protein/ml. Amino acid analysis showed that the GPC fraction contained 45% content of aromatic amino acids in comparison to 8.5% of the hydrolysate. Two peptides, Val-Ser-Val (IC₅₀ = 0.15 μM) and Phe-Leu (IC₅₀ = 1.33 μM) were purified, and located in the primary structure of canola napin and cruciferin native proteins. The results suggest that canola protein hydrolysate is a potential ingredient for the formulation of hypotensive functional foods.     

Structural and mechanical characterization of κ/ι-hybrid carrageenan gels in potassium salt using Fourier Transform rheology

The mechanical and structural properties of κ/ι-hybrid carrageenan gels obtained at various concentrations in the presence of 0.1 m KCl were studied with Fourier Transform rheology (FTR) and cryoSEM imaging. FTR data show that gels formed at concentration below 1.25 wt% exhibit a strain hardening behavior. The strain hardening is characterized by a quadratic increase of the scaled third harmonic with the strain and a third harmonic phase angle of zero degree. Both features are weakly depending on the concentration and conform to predictions from a strain hardening model devised for fractal colloidal gels. However, the phase angle of the third harmonic reveals that κ/ι-hybrid carrageenan gels obtained at higher concentrations show shear thinning behavior. Colloidal gel models used to extract structural information from the concentration scaling of gel equilibrium shear modulus G₀ and the strain dependence of FTR parameters suggest that κ/ι-hybrid carrageenan gels are built from aggregating rod-like strands (with fractal dimension x = 1.13) which essentially stretch under increasing strain. The mechanically relevant structural parameters fairly match the gel fractal dimension (d = 1.66) obtained from the cryoSEM analysis.     

Effects of different food commodities on larval development and α-amylase activity of Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae)

The present work was undertaken to study the influence of four commodities (wheat flour, dates, sorghum and barley) on Plodia interpunctella post-embryonic development. Larval weight, larval mortality, pupation and adult emergence were recorded. The study also aimed to find out the effect of these commodities on protein and glycogen production as well as on α-amylase activity. Results indicated that the weight of fourth instar larvae placed on dates increased gradually. Percentage mortality was low. Pupation and adult emergence were delayed. In contrast, the weight of larvae placed on wheat flour, sorghum or barley remained low. Pupation and adult emergence occurred sooner than among those placed on dates and the percentage mortality was highest for larvae placed on barley. Results also showed that protein content and α-amylase activity were lower for larvae placed on dates than for those placed on other commodities. The biochemical composition of different commodities showed that dates are a rich source of glucose, while their protein and starch contents were very low as compared to the other commodities. In contrast, wheat flour, sorghum and barley contained large amounts of starch and protein and low amounts of glucose. Thus, the reduction in α-amylase activity was probably due to the high levels of glucose in dates.     

Potential ACE-inhibitory activity and nanoLC-MS/MS sequencing of peptides derived from aflatoxin contaminated peanut meal

Our lab has developed a process for sequestering aflatoxin from contaminated peanut meal (PM) using commercial bentonite clays while protein is simultaneously extracted and hydrolyzed by a commercial protease. The objectives of this study were to sequence generated peptides and evaluate their potential ACE-inhibitory properties. Aflatoxin in the unprocessed PM was 610 μg kg⁻¹ compared to 9.7 μg kg⁻¹ on a dry weight basis in the 120 min hydrolysate. This hydrolysate displayed significant ACE-inhibitory activity with an IC₅₀ of 295.1 μg mL⁻¹. Ultrafiltration and size exclusion chromatography (SEC) improved the ACE-inhibitory properties, with the SEC fraction containing the smallest peptides having an IC₅₀ = 44.4 μg mL⁻¹. Additionally, 271 unique peptides were identified by nanoLC-MS/MS, of which 147 belonged to major seed storage proteins. This advanced characterization data will ultimately allow for more efficient production of hydrolysates with ACE-inhibitory activity or other bioactivities of interest from PM.