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.     

Fractionation and identification of ACE-inhibitory peptides from α-lactalbumin and β-casein produced by thermolysin-catalysed hydrolysis

The thermolysin catalysed hydrolysates of α-lactalbumin and β-casein were fractionated by size-exclusion chromatography (SEC) and reversed-phase high performance liquid chromatography (RP-HPLC) in order to identify the peptides responsible for the high ACE-inhibitory activity of these hydrolysates. The SEC fractionation separated many co-eluting peptides into different fractions allowing individual peptides to be isolated in one or two subsequent semi-preparative RP-HPLC fractionation steps. Five potent ACE-inhibitory peptides from α-lactalbumin were isolated. They all contained the C-terminal sequence -PEW, corresponding to amino acid residues 24–26 in α-lactalbumin, and had IC₅₀ values of 1–5 μm. From one SEC fraction of the β-casein hydrolysate two potent ACE-inhibitory peptides were isolated and identified as f58-76 and f59-76 of β-casein A2. They both contained IPP as the C-terminal sequence and had IC₅₀ values of 4 and 5 μm. From another SEC fraction a new but less ACE-inhibitory peptide from β-casein was identified (f192–196; LYQQP).     

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.     

Xyloglucan and β-glucans: origins and destinations

In ultrastructural studies, cellulose microfibrils can be seen to form an integral part of the primary wall framework along with xyloglucan, which is firmly bound to the wall matrix. Callose (1, 3-β-glucan) is not part of the wall proper but is deposited after wounding between wall and plasmamembrane in the periplasmic space. Of these extracellular end products, only xyloglucan has been detected intracellularly. The four enzymes required for xyloglucan assembly have all been found in Golgi and pulse-chase experiments in vivo show that xyloglucan molecules are completed in Golgi before being secreted via dictyosome vesicles. Cellulose and 1,3-β-glucan, in contrast, both appear to be formed at the cell surface. Plant membrane preparations assayed immediately after isolation can form a small amount of 1,4-β-linked product from UDP-glucose, which has a relatively low mol. wt equivalent to dextran 70 000. Only a few glucose units appear to be added to an endogenous acceptor. However, plasmamembrane, isolated from protoplasts after a period of digestion in a mixture of hydrolases, shows no capacity to form 1,4-β-glucan, but instead forms 1,3-β-glucan with a high mol. wt. The available data are consistent with the possibility that 1,4-β-glucan synthase is converted to l,3-β-glucan synthase in isolated (perturbed) membranes and in wounded tissues. The question of particular current interest is whether such a conversion takes place in a reversible manner, in which event conditions may be found to isolate 1,3-β-glucan synthase and reconstitute it into a complex that regains the capacity to form 1,4-β-glucan. This may never be possible, however, if 1,4-β-glucan linkages are differentially susceptible to decay in the presence of proteases while the capacity to form 1, 3-β-linkages is stimulated. It may be, therefore, that if membranes were isolated under conditions that completely inhibited endogenous proteases, the yield of 1,4-β-glucan synthase would be maximized and the development of 1,3-β-glucan synthase activity minimized.     

Effect of inulin on the rheological properties of silken tofu coagulated with glucono-δ-lactone

This study investigated the rheological properties of inulin-containing silken tofu coagulated with glucono-δ-lactone (GDL) upon heating. Inulin (Raftiline^® HP-gel) was added to a soy protein isolate-enriched cooked soymilk at 0%, 1%, 2%, 3% and 4% (w/v) levels along with 0.4% (w/v) GDL to prepare acid-induced silken tofu. Gelation was induced by heating the soymilk mixture from 20 to 90 °C at a constant rate (1 °C/min) or isothermally at 90 °C for 30 min. The gelling properties were measured with dynamic small-deformation mechanical analysis and static large-deformation compression tests. The rheological changes in soymilk during gelation were dependent upon both the pH decline (hydrolysis of GDL) and the specific temperature of heating. Control samples heated to 50 °C, with the pH lowered to 5.95, started to gel, showing a rapid increase in storage (G′) and loss (G″) moduli afterwards. The addition of 2% inulin lowered the on-set gelling temperature by 2.8 °C and improved (P < 0.05) both rheological parameters of the tofu gel as well as hardness and rupture force (textural profile analysis) of the formed silken tofu. The results indicated that inulin enhances the viscoelastic properties of GDL-coagulated silken tofu, and the textural effect of inulin is an added benefit to its current application mainly as a prebiotical ingredient in food.     

Isolation of individual hop iso-α-acids stereoisomers by β-cyclodextrin

Individual iso-α-acids that are responsible for the bitter taste of beer need to be isolated because these acids are required as reference standards in quantitative analysis and when studying the parameters which effect the quality of beer. However, these pure compounds are very expensive, due to inefficient isolation methods. In this study a new isolation method has been developed, in order to reduce the isolation cost. β-Cyclodextrin has been used for the isolation of trans- and cis-iso-α-acids. The separation from the mixture of stereoisomers was achieved by complexation, using ethanol:water (1:2, v/v) as a solvent at a temperature of 50 °C for 30 min. The molar ratio of iso-α-acids sample to β-cyclodextrin for complexation was 1:1. Precipitation time varied between 9 h and 2 days, depending on the iso-α-acid. Release of the guest from the cyclodextrin complex was successfully accomplished by elution with methanol.     

Electrostatic effects on β-lactoglobulin transitions during heat denaturation as studied by differential scanning calorimetry

This work comprises the study of the thermal treatment of β-lg and its denaturation as a function of pH and ionic strength followed by differential scanning calorimetry. The concentration of protein was 14 (w/v)% in order to study the behaviour of highly concentrated β-lg solutions during heating. The denaturation temperature of β-lg was dependent on both pH and ionic strength, meaning that electrostatic interactions between protein monomers in the native state were important for the denaturation of β-lg. The thermograms from the calorimetric measurements also revealed that the quarternary structure of β-lg at pH-values close to the isoelectric point was influenced by the presence of salt and the nature of the salt (NaCl, KI and LiI). Small exotherms emerged in the thermograms at the low temperature side of the denaturation temperature for β-lg. The presences of these exotherms are probably caused by restructuring of the quarternary structure of native β-lg prior to denaturation, due to dissociation into smaller entities and possible also formation of a liquid crystalline-like structure in the highly concentrated protein solution. The present study provides a contribution to the understanding of the importance of the electrostatic interactions between native β-lg molecules and how different salts and ionic strengths affect the denaturation properties of the protein in concentrated systems.     

Whey protein concentrate/λ-carrageenan systems: Effect of processing parameters on the dynamics of gelation and gel properties

The thermal characteristics, dynamics of gelation and gel properties of commercial whey protein concentrate (WPC), WPC/λ-carrageenan (λ-C) mixtures (M) and WPC/λ-C spray-dried mixtures (DM) have been characterized. In a second stage, the effect of the gelling variables (T, pH, total solid content) on gelation and textural properties of DM was evaluated through a Doehlert uniform shell design.The presence of λ-C either in mixtures (M) or in DM promoted the WPC gelation at lower concentration (8%). M showed higher rates of formation and better gel properties (higher hardness, adhesiveness, springiness and cohesiveness) than DM.Nevertheless, when the effects of pH (6.0–7.0), heating temperature (75–90 °C) and total solid content (12–20 wt%) on gelation dynamics and gel properties of DM were studied, gels with a wide range of rheological and textural properties were obtained. While pH did not affect the gelation dynamics, it had some effect on rheological and textural properties. Total solid content and heating temperature were the most important variables for the dynamics of gelation (gelation rate (1/t_gel), gelation temperature (T_gel), rate constant of gel structure development (k_G), elastic modulus after cooling (G′_c) and textural parameters (hardness, springiness and cohesiveness).     

Complex coacervation between β-lactoglobulin and acacia gum in aqueous medium

The compatibility of β-lactoglobulin (β-lg) and acacia gum in aqueous medium was investigated as a function of the pH (3.6–5.0), the protein to polysaccharide weight ratio (50:1–1:20) and the total biopolymer concentration (0.1–5 wt%). The ternary phase diagrams obtained at low ionic strengths (0.005–10.7 mM) typically accounted for phase separation through complex coacervation. Thus a drop-shaped two-phase region was anchored in the water-rich corner. The electrostatic nature of the interactions between the two biopolymers was pointed out according to the pH dependence of the two-phase region's breadth. Following the absorbance of the mixtures at 650 nm, the influence of the protein to polysaccharide ratio was also demonstrated. Electrophoretic mobility (μ_E) measurements and chemical analyses of separated phases revealed the formation of soluble and insoluble coacervates and complexes. A remarkable value of the protein to polysaccharide weight ratio (2:1) at pH 4.2 gave the same protein to polysaccharide (Pr:Ps) ratio in the two phases after 2 days, implying that electrostatic interactions are maximum between β-lg and acacia gum. The increase of the total biopolymer concentration reduced the influence of pH and protein to polysaccharide ratio. Also, the increase of the pH close to the β-lg IEP reduced the influence of the total biopolymer concentration and Pr:Ps ratio. As the biopolymer content was increased at pH 3.6 and 4.2, the relative β-lg solubility increased probably because of the self-suppression of complex coacervation.     

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.     

Kinetic studies of the thermal inactivation of plasmin in acid or sweet whey

The thermal behaviour of the milk alkaline proteinase, plasmin, was studied in acid and sweet (rennet) whey; indigenous (bovine) plasmin was studied in the former system, but endogenous porcine plasmin was added in the latter, due to the very low levels of residual plasmin. The inactivation of plasmin in both systems followed first-order inactivation kinetics, which was consistent with previous observations of plasmin inactivation in milk and model milk systems. The thermal inactivation of plasmin in acid whey (D_{90 °C}=108±29 min, z=24.5±1.2 °C) was much slower than in the sweet whey system (D_{90 °C}=0.021±0.006 min, z=7.3±0.3 °C). Similarly, denaturation of β-lactoglobulin (β-lg) followed a first-order inactivation profile and this protein was also more heat stable in acid whey (D_{90 °C}=86±76 min, z=13.7±1.5 °C) than sweet whey (D_{90 °C}=0.81±0.29 min, z=9.1±0.5 °C). While it is possible that the increased heat stability of plasmin in acid whey is due to reduced sulphydryl/disulphide interchange reactions between plasmin and β-lg, it also appears that structural changes in the plasmin molecule were a significant contributory effect on the thermal stability of plasmin in this system. Increasing the pH of acid whey decreased the heat stability of plasmin. However, adjusting the pH of sweet whey had little effect on the heat stability of plasmin. Overall, severe heat treatments may be required to ensure inactivation of the enzyme in acid whey, but a balance is required between reducing the activity of plasmin and maintaining the functionality of whey proteins as food ingredients.     

Microencapsulation of shiitake (Lentinula edodes) essential oil by complex coacervation: formation,rheological property,oxidative stability and odour attenuation effect

Complex coacervation of gelatin (GE) with carboxymethylcellulose (CMC) and the microencapsulation of shiitake essential oil (SEO) using the GE‐CMC coacervate were investigated. The ζ‐potential and coacervate yield data showed that the optimal complexation pH and CMC/GE ratio were 4.0 and 0.15 g g^?1, respectively. At this condition, the coacervate yield was 85.35 ± 4.89% and ζ‐potential was almost zero. The SEO contained microcapsules fabricated by the GE‐CMC coacervate were also electrostatic inducted formation, and the highest encapsulation efficiency was shown to be 85.75 ± 2.89%. The rheological measurement indicated that the GE‐CMC coacervated microcapsules had a viscoelastic solid behaviour (G’ > G”) which was resulted mainly from the interactions between GE molecules and CMC chains. The improved oxidative stability and odour attenuation effect of the GE‐CMC coacervated microcapsules were believed to be attributed to the increased protection against oxidation and flavour release by providing a physical barrier after complex coacervation.     

Protein–polysaccharide interactions: The determination of the osmotic second virial coefficients in aqueous solutions of β-lactoglobulin and dextran

Solutions containing dextran and solutions containing mixtures of dextran +β-lactoglobulin are studied by membrane osmometry. The low concentration range of these solutions is considered. From the measured osmotic pressures the virial coefficients are obtained. These are analyzed using the osmotic virial coefficient of β-lactoglobulin solutions published earlier by us [Schaink, H.M., & Smit, J.A.M. (2000). Determination of the osmotic second virial coefficient and the dimerization of beta-lactoglobulin in aqueous solutions with added salt at the isoelectric point. PCCP, 2, 1537–1541]. The second cross-virial coefficient A₁₂ is found to be positive indicating a repulsive and probably mainly steric interaction between neutral in nature dextran and and practically uncharged β-lactoglobulin (pH=5.18). The measurements show that the β-lactoglobulin has only a small tendency to form multimers in the presence of dextran. The phase diagram of solutions of dextran+Whey Protein Isolate (appr. 60% β-lactoglobulin) is also presented. The McMillan–Mayer equation of state that considers only the second virial coefficients is found to be unreliable for the extrapolation up to the concentrations at which phase separation is expected.     

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.     

Impact of cosolvents on formation and properties of biopolymer nanoparticles formed by heat treatment of β-lactoglobulin–Pectin complexes

The purpose of this study was to determine the influence of neutral cosolvents on the formation and properties of biopolymer nanoparticles formed by thermal treatment of protein–polysaccharide electrostatic complexes. Biopolymer particles were formed by heating (85 °C, 20 min) an aqueous solution containing a globular protein (β-lactoglobulin) and an anionic polysaccharide (beet pectin) above the thermal denaturation temperature (T_m) of the protein under pH conditions where the biopolymers formed electrostatic complexes (pH 5). The impact of two neutral cosolvents (glycerol and sorbitol) on the self-association of β-lactoglobulin and on the formation of β-lactoglobulin–pectin complexes was examined as a function of solution pH (3–7) and temperature (30–95 °C). Glycerol had little impact on the pH-induced self-association or aggregation of the biopolymers, but it did increase the thermal aggregation temperature (T_a) of the protein–polysaccharide complexes, which was attributed to its ability to increase aqueous phase viscosity. Sorbitol decreased the pH where insoluble protein–polysaccharide complexes were formed, and greatly increased their T_a, which was attributed to its ability to increase T_m, alter biopolymer–biopolymer interactions, and increase aqueous phase viscosity. This study shows that neutral cosolvents can be used to modulate the properties of biopolymer nanoparticles prepared by thermal treatment of protein–polysaccharide electrostatic complexes.     

Competitive adsorption of α-lactalbumin in the molten globule state

The molten globule state of α-lactalbumin is a partially denatured form with native-like secondary structure and disordered tertiary structure. Using circular dichroism measurements, it was demonstrated that the molten globule state was produced by decreasing the pH to 2.0 at 25°C or by removing bound Ca²⁺ by treatment with ethylenediamine—tetraacetic acid (EDTA) at pH 7.5 and 40°C. Tension measurements showed that α-lactalbumin in the molten globule state is more easily unfolded at liquid interfaces than is the native protein. Results of competitive adsorption experiments involving α-lactalbumin and β-lactoglobulin at the oil droplet surface in emulsions are consistent with preferential adsorption of α-lactalbumin during emulsification when it is in the molten globule state. In contrast to the difficulty of exchange between α-lactalbumin and β-lactoglobulin at the oil-water interface in emulsions at 25°C, it has been found that the two whey proteins are able partially to displace one another from the oil—water interface at 40°C. While native α-lactalbumin was found to be readily displaced from the oil—water interface by β-lactoglobulin at 40°C, it was found that α-lactalbumin in the molten globule state in the presence of EDTA at 40°C had itself the capacity for displacing β-lactoglobulin from the interface.     

α-Anomer-selective glucosylation of (+)-catechin by the crude enzyme, showing glucosyl transfer activity, of Xanthomonas campestris WU-9701

α-Anomer-selective glucosylation of (+)-catechin was carried out using the crude enzyme, showing α-glucose transferring activity, of Xanthomonas campestris WU-9701 with maltose as a glucosyl donor. When 60 mg of (+)-catechin and 50 mg of the enzyme (5.25 units as maltose hydrolysing activity) were incubated in 10 ml of 10 mM citrate-Na₂HPO₄ buffer (pH 6.5) containing 1.2 M maltose at 45°C, only one (+)-catechin glucoside was selectively obtained as a product. The (+)-catechin glucoside was identified as (+)-catechin 3′-O-α- -glucopyranoside (α-C-G) by ¹³C-NMR, ¹H-NMR and two-dimensional HMBC analysis. The reaction at 45°C for 36 h under the optimum conditions gave 12 mM α-C-G, 5.4 mg/ml in the reaction mixture, and the maximum molar conversion yield based on the amount of (+)-catechin supplied reached 57.1%. At 20°C, the solubility in pure water of α-C-G, of 450 mg/ml, was approximately 100 fold higher than that of (+)-catechin, of 4.6 mg/ml. Since α-C-G has no bitter taste and a slight sweet taste compared with (+)-catechin which has a very bitter taste, α-C-G may be a desirable additive for foods, particularly sweet foods.     

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.     

Thermal denaturation of mixtures of α-lactalbumin and β-lactoglobulin: a differential scanning calorimetric study

The thermal denaturation of α-lactalbumin (α-lac), β-lactoglobulin (β-lg) and a mixture of the two proteins in the presence of several sugars, sodium salts and at various pH values was studied by differential scanning calorimetry. The effects of N-ethylmaleimide (NEM), cysteine, urea and sodium dodecyl sulfate (SDS) were also investigated. The temperature of denaturation (T_d) of β-lg decreased from 71.9°C in the absence of α-lac to 69.1°C in its presence. In contrast, an increase of 2.5°C was observed in the T_d of apo-α-lac when heated in the presence of β-lg suggesting that α-lac was made more thermally stable in the presence of β-lg. Glucose and galactose had the greatest effect in stabilizing the proteins against thermal denaturation with the effect being greater for β-lg than for α-lac. A decrease in thermal stability of both proteins was observed in the presence of sodium bicarbonate; sodium ascorbate, however, had a stabilizing effect. Renaturation of α-lac was prevented in the presence of cysteine and NEM, but not in urea or SDS. Translucent gels were formed when the α-lac/β-lg mixtures were heated in the presence of all five sugars and in the presence of cysteine, urea and SDS but not in NEM. This suggests that disulfide–sulfhydryl interchange reactions may be primarily responsible for the gelation of α-lac/β-lg mixtures.     

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.