Thermal modifications of structure and codenaturation of α‐lactalbumin and β‐lactoglobulin induce changes of solubility and susceptibility to proteases

Study of heat denaturation of major whey proteins (β‐lactoglobulin or α‐lactalbumin) either in separated purified forms, or in forms present in fresh industrial whey or in recomposed mixture respecting whey proportions, indicated significant differences in their denaturation depending on pH, temperature of heating, presence or absence of other co‐denaturation partner, and of existence of a previous thermal pretreatment (industrial whey). α‐Lactalbumin, usually resistant to tryptic hydrolysis, aggregated after heating at ⪈85°C. After its denaturation, α‐lactalbumin was susceptible to tryptic hydrolysis probably because of exposure of its previously hidden tryptic cleavage sites (Lys‐X and Arg‐X bonds). Heating over 85°C of β‐lactoglobulin increased its aggregation and exposure of its peptic cleavage sites. The co‐denaturation of α‐lactalbumin with β‐lactoglobulin increased their aggregation and resulted in complete exposure of β‐lactoglobulin peptic cleavage sites and partial unveiling of α‐lactalbumin tryptic cleavage sites. The exposure of α‐lactalbumin tryptic cleavage sites was slightly enhanced when the α‐lactalbumin/β‐lactoglobulin mixture was heated at pH 7.5. Co‐denaturation of fresh whey by heating at 95°C and pH 4.5 and above produced aggregates stabilized mostly by covalent disulfide bonds easily reduced by β‐mercaptoethanol. The aggregates stabilized by covalent bonds other than disulfide arose from a same thermal treatment but performed at pH 3.5. Thermal treatment of whey at pH 7.5 considerably enhanced tryptic and peptic hydrolysis of both major proteins.     

Temperature-controlled gelation of casein concentrates enabled by the utilisation of acid whey permeate as a diafiltration medium in microfiltration

In this study, ultrafiltration permeate obtained from acid whey (AWP) was applied as a diafiltration (DF) medium during skim milk microfiltration. The thereby induced acidification of differently concentrated retentates was assessed regarding suitability to generate gels from micellar casein concentrates further downstream. While aggregation was avoided during cold DF, subsequent heating induced controlled spontaneous formation of gels with concentration- and pH-dependent firmness ranging from 26 to 1420 Pa. By employing its typical compounds, AWP as a rather problematic dairy side stream could thus be utilised for the production of calcium-rich acidic casein gels providing sensory characteristics of cultured dairy products.     

The effectiveness of butylated hydroxyanisole and α‐tocopherol in inhibiting oxidant‐induced chemical and structural changes of whey protein

The objective of the study was to investigate the role of butylated hydroxyanisole (BHA) and α‐tocopherol in protecting whey protein isolate (WPI) from oxidative modification. The results showed that oxidation increased protein carbonyls and decreased total sulfhydryls, and led to higher dityrosine and surface hydrophobicity (P < 0.05) than nonoxidised WPI. The presence of BHA and α‐tocopherol significantly reduced (P < 0.05) the extent of WPI oxidation, thus limiting the oxidation‐induced protein aggregates and structural changes. Therefore, BHA and α‐tocopherol may be used as potential antioxidants in WPI and WPI‐containing foods.     

Effect of temperature and pore size on the fractionation of fresh and reconstituted buttermilk by microfiltration

The objective of this research was to evaluate the effect of temperature (7, 25, and 50 degrees C) and pore size (0.1, 0.8, and 1.4 micro m) on the separation of proteins and lipids (neutral lipids and phospholipids) during microfiltration (MF) of fresh or reconstituted buttermilk. Buttermilk was subjected to MF using a pilot-scale unit mounted with ceramic membranes. The MF runs were carried out in a uniform transmembrane pressure (UTP) mode. Changes in processing temperature had no significant impact on protein transmission, whereas increasing temperature reduced both lipid and phospholipid transmission. A maximum concentration factor (CF) for lipids was reached at 25 degrees C, as protein CF remained essentially unaffected by temperature. The use of the smaller pore size (0.1 microm) resulted in low lipid (10%) and protein (approximately 20%) transmission. Larger pore sizes (0.8 and 1.4 microm) resulted in higher levels of protein, lipid, and phospholipid transmission (>50%), but gave high permeation fluxes. Transmission of both proteins and lipids was markedly different when using fresh buttermilk as opposed to reconstituted buttermilk. This study showed that MF temperature, pore size, and buttermilk type influence fractionation but that MF alone cannot achieve optimal separation of lipids and proteins for the production of novel ingredients from buttermilk.     

Influence of whey peptides on the surface activity of κ‐casein and β‐lactoglobulin A

Whey protein hydrolysate (WPH) was fractionated by reverse‐phase chromatography to obtain fractions of varying surface‐hydrophobicities. A model oil–water interface (MI) was pre‐coated with the WPH or fractions thereof. Contact angle (θ) of sessile drops of κ‐casein (κ‐CN) or β‐lactoglobulin A (β‐LGA) were measured on the MI. Pre‐coating of MI with un‐fractionated WPH decreased θ, that is, increased surface activity, of both κ‐CN (35–8.3°) and β‐LGA (38–21.3°). Conversely, pre‐coating of MI with the fractions significantly increased θ of both proteins as a function of hydrophobicity. Data provide insight into variability of whey protein functionality in food applications.     

Impact of pH on the interactions between whey and egg white proteins as assessed by the foamability of their mixtures

The impact of protein–protein interactions on foaming properties of mixtures consisting of egg white proteins (EWP) and whey proteins (WP) with total protein content of 60 g/L was examined at pH 5, 7 and 9. The ratio between EWP and WP in the mixtures was varied between 67:33, 50:50 and 33:67 (in %; w/w). The ionic strength was adjusted to that of milk (I = 176 mM). The foamability of the protein products was characterized by the foam capacity, stability and firmness. In addition, the hydrophobicity in the protein solutions was assessed as a measure for the physical behaviour and ability of proteins to adsorb at the air–water interface.The individual egg white proteins and whey proteins each showed the best foaming properties at pH 5 and pH 9, respectively. At pH 9 a synergism was observed in the capacity and stability of the foams from EWP/WP-mixtures. This effect appears to be caused by the electrostatic interactions between egg white and whey proteins which occur in the bulk solution after the pH adjustment prior to the foaming. In contrast, at pH 5 no positive influence of foaming the components in a mixture as well as no indication of intermolecular interactions was found. At pH value near the pI of ovalbumin the protein interactions occur when the proteins have adsorbed at the air–water interface. The protein systems foamed at pH 7 showed intermediate foamability compared to the values obtained at alkaline and acidic pH.     

Impacts of glucosamine/oligochitosan glycation and cross‐linking by transglutaminase on the structure and in vitro antigenicity of whey proteins

With the fixed and selected conditions, whey protein concentrate (WPC) was glucosamine‐/oligochitosan‐glycated and cross‐linked by transglutaminase, resulting in glucosamine conjugation of 4.18–5.88 g/kg. Electrophoretic analysis showed cross‐linking and glycation of whey proteins, while circular dichroism analysis indicated that the two reactions contributed less ordered secondary structure to the two products. Enzyme‐linked immunosorbent assay results showed that the two products lost 64–95% antigenic responses of WPC, and oligochitosan was more powerful than glucosamine to reduce the responses. Rocket immuno‐electrophoretic analysis also evidenced antigenicity loss. This applied treatment is efficient to modify the structure and to decrease the allergenicity of whey proteins.     

Studies on the interaction of ‐epigallocatechin‐3‐gallate from green tea with bovine β‐lactoglobulin by spectroscopic methods and docking

The binding interaction between‐epigallocatechin‐3‐gallate (EGCG) and bovine β‐lactoglobulin (βLG) was thoroughly studied by fluorescence, circular dichroism (CD) and protein–ligand docking. Fluorescence data revealed that the fluorescence quenching of βLG by EGCG was the result of the formation of a complex of βLG–EGCG. The binding constants and thermodynamic parameters at two different temperatures and the binding force were determined. The binding interaction between EGCG and βLG was mainly hydrophobic and the complex was stabilised by hydrogen bonding. The results suggested that βLG in complex with EGCG changes its native conformation. Furthermore, preheat treatment (90 °C, 120 °C) and emulsifier (sucrose fatty acid ester) all boosted the binding constants (K_a) and the binding site values (n) of the βLG‐EGCG complex. This study provided important insight into the mechanism of binding interactions of green tea flavonoids with milk protein.     

Enhancement of the functional properties of whey protein by conjugation with maltodextrin under dry‐heating conditions

Conjugation of whey protein isolate (WPI) and maltodextrin (MD, dextrose equivalent of 6) was achieved by dry‐heating at an initial pH of 7.0, at 60 °C and 79% relative humidity, with WPI: MD6 ratio of 1:1, for up to 24 h. Conjugation was achieved with limited development of colour and advanced Maillard products on 24 h of heating. Conjugation increased the protein solubility at pH 4.5, by 7.1–8.5%, compared to the unheated and heated WPI controls. Conjugation of WPI with MD6 enhanced the stability and retention of clarity in protein solutions heated at 85 °C for 10 min with 50 mM added NaCl.     

Altering functional attributes of proteins through cross linking by transglutaminase – A case study with whey and seed proteins

The effect of cross linking of the major whey protein β-lactoglobulin (β-Lg) and major protein fractions (11S) of soybean (Glycinin) and sesame seed (α-globulin) with the microbial enzyme transglutaminase (EC 2.3.2.13) was studied. The formation of polymerized proteins was followed by poly acrylamide gel electrophoresis, gel filtration high pressure liquid chromatography (HPLC) and evaluation of functional properties. Cross linked proteins were less turbid on heating to higher temperature as compared to untreated samples and the temperature at which the protein turns turbid also increased in the treated samples. In case of β-Lg and α-globulin of sesame seed when the control showed turbidity at 60 °C, the enzyme treated sample indicated at 65 °C and higher. Similar results were obtained in the case of soybean also. The treated samples showed higher emulsifying activity when compared to the control. The control showed an emulsifying activity of 0.55 ± 0.02, and the treated sample showed an emulsifying activity of 0.72 ± 0.02 (optical density at 500 nm is taken as emulsifying activity). Foaming capacity did not improve significantly with the enzyme treatment. The complex formed was investigated by gel filtration chromatography. Nearly 30% of the proteins (11S protein fractions and β-Lg) formed the complex and increase in the concentration of the proteins or the enzyme did not show any increase in the complex formation. The changes in the fluorescence intensity indicated changes in the microenvironment of the chromophores induced by the enzymatic cross linking.     

Effect of heat shock protein 27 on the in vitro degradation of myofibrils by caspase‐3 and μ‐calpain

This study was designed to investigate the effect of heat shock protein 27 (HSP27) on the in vitro degradation of myofibrils induced by caspase‐3 or μ‐calpain. Myofibrillar proteins were prepared from at‐death beef muscles and incubated with caspase‐3 or μ‐calpain with and without HSP27, or with HSP27 alone, at 30 °C for 2 h, and protein degradation was assessed. Results showed that caspase‐3 promoted the degradation of titin, nebulin and troponin‐T, and μ‐calpain promoted the degradation of nebulin, desmin and troponin‐T, observed during normal PM ageing. Moreover, the addition of HSP27 restricted the degradation of troponin‐T in μ‐calpain‐ and caspase‐3‐treated myofilaments, and restricted the degradation of desmin in μ‐calpain‐treated myofilaments. Therefore, HSP27 may indirectly or directly interact with caspase‐3 and μ‐calpain, reducing their activity and mediating PM proteolysis of muscle proteins to affect meat tenderisation.     

Flavour profiles of low-fat comminuted sausages as affected by the addition of various sugars in combined with 0.1 m lysine

The study was performed to evaluate the flavour profiles of low-fat comminuted sausage (LFS) as affected by the addition of various concentrations of glucose, fructose and sucrose combined with 0.1  m lysine. Among thirty-five volatile compounds, the concentrations of furfural, 2-furan methanol, 2-methoxy phenol, 2-methoxy-4-methyl phenol, myristicine and pentadecanal occupied approximately 60% of the total concentration of volatile compounds identified in full- and low-fat smoked sausages. Phenols and hetero-compounds derived mainly from the smoking process were the predominant chemical groups. Nine volatile compounds were affected by fat content and the reduction of fat predominantly increased the headspace concentration of myristicin and pentadecanal. The headspace concentration of total volatile compounds detected in LFS control was significantly higher than those of LFS treatments with the various sugars and lysine. The headspace concentration of most volatile compounds decreased with the addition of various sugars. The results indicate that the addition of glucose, fructose and sucrose at the concentrations higher than 0.05  m in combination with 0.1  m lysine delays the release of some flavour compounds in LFS.     

Factors influencing the production of o/w emulsions stabilized by β-lactoglobulin–pectin membranes

A primary emulsion was prepared by homogenizing 10 wt% corn oil with 90 wt% aqueous β-lactoglobulin solution (0.5 wt% β-lg, pH 3 or 7) using a two-stage high-pressure valve homogenizer. This emulsion was mixed with aqueous pectin (citrus, 59% DE) stock solution (2 wt%, pH 3 or 7) and NaCl solution to yield secondary emulsions with 5 wt% corn oil, 0.225 wt% β-lactoglobulin, 0.2 wt% pectin and 0 or 100 mM NaCl. The final pH of the emulsions was then adjusted (3–8). Primary and secondary emulsions were ultrasonically treated (30 s, 20 kHz, 40% amplitude) to disrupt any flocculated droplets. Secondary emulsions were more stable than primary emulsions at intermediate pHs. Secondary emulsions prepared at pH 7 had smaller particle diameters (0.35 to 6 μm) than those prepared at pH 3 (0.42 to 18 μm) across the whole pH range studied, and also had smaller diameters than the primary emulsions (0.35 to 14 μm). Ultrasound treatment reduced the particle diameter of both primary and secondary emulsions and lowered the rate of creaming. The presence of NaCl screened the charges and thus the electrostatic interaction between biopolymer molecules and primary emulsion droplets. Secondary emulsions were more stable to the presence of 100 mM NaCl at low pHs (3–4) than primary emulsions. This study shows that stable emulsions can be prepared by engineering their interfacial membranes using the electrostatic interaction of natural biopolymers, especially at intermediate pHs where proteins normally fail to function.     

Interactions of polysaccharides with β-lactoglobulin spread monolayers at the air–water interface

In the present work we have studied the static (film structure and elasticity) and dynamic characteristics (surface dilatational properties) of β-lactoglobulin (βLG) monolayers spread at the air–water interface in the presence of polysaccharides in the aqueous phase, at 20 °C and at pH 7. The measurements were performed on a fully automated Wilhelmy-type film balance. As polysaccharides with interfacial activity we have used propylene glycol alginates (PGA). To evaluate the effect of the degree of PGA esterification and viscosity, different commercial samples were studied-kelcoloid O (KO), kelcoloid LVF (KLVF) and manucol ester (MAN). Xanthan gum (XG) and λ-carrageenan (λC) were studied as non-surface active polysaccharides. The results reveal a significant effect of surface active and non-surface active polysaccharides on static—when the polysaccharide was added in the subphase the π-A isotherms shifted to higher surface pressure values as the time increased-and dynamic—the presence of polysaccharide in the aqueous phase decreased the surface dilatational modulus of a pure β-lactoglobulin monolayer-characteristics of β-lactoglobulin monolayers. To explain the observed effects three phenomena were taken into account: (i) the ability of the polysaccharide to adsorb at the interface by it-self and to increase the surface pressure, (ii) the interfacial complexation of the polysaccharide with the adsorbed protein and (iii) the existence of a limited thermodynamic compatibility between the protein and polysaccharide, depending on the protein-polysaccharide system.     

Secondary structural studies of bovine caseins: temperature dependence of β-casein structure as analyzed by circular dichroism and FTIR spectroscopy and correlation with micellization

To obtain a molecular basis for the similarities and dissimilarities in the functional, chemical, and biochemical properties between β-casein and the other caseins, three-dimensional models have been presented. Secondary structural prediction algorithms and molecular modeling techniques were used to predict β-casein structure. The secondary structure of bovine β-casein was re-examined using Fourier transform infrared and circular dichroism spectroscopies to test these predictions. Both methods predict a range of secondary structures for β-casein (28–32% turns, 32–34% extended) at 25°C. These elements were highly stable from 5 to 70°C as viewed by circular dichroism. More flexible conformational elements, tentatively identified as loops, helix and short segments of polyproline II, were influenced by temperature, increasing with elevated temperatures. Another view is that as temperature decreases, these elements are lost (cold denaturation). Several distinct transitions were observed by circular dichroism at 10, 33 and 41°C, and another transition, extrapolated to occur at 78°C. Calculations from analytical ultracentrifugation indicate that the 10, 33 and 41°C transitions occur primarily in the monomeric form of the protein. As β-casein polymers are formed, and increase in size, the transitions at higher temperature may reflect changes in the more flexible conformational elements as they adjust to changes in surface charge during polymer formation. The transition at 10°C may represent an actual general conformational change or cold denaturation. Over the range of temperatures studied, the sheet and turn areas remain relatively constant, perhaps forming a supporting hydrophobic core for the monomers within the micelle-like polymer. This interpretation is in accord with the known properties of β-casein, and those predicted from molecular modeling.     

An improved method for the measurement of 3‐monochloropropanediol esters by matrix solid‐phase dispersion supported liquid–liquid extraction

3‐Monochloropropanediol (3‐MCPD) esters are contaminants produced from the high‐temperature processing of edible oils. The accurate measurement of 3‐MCPD using an easy‐to‐follow and reliable method that uses a readily available instrument is important. Here, we report an acid transesterification heptafluorobutyrylimidazole (HFBI) derivatisation method for the accurate measurement of 3‐MCPD esters in edible oils. We developed a dispersed matrix solid‐phase supported liquid–liquid extraction (DMSP‐SLE) system to remove impurities. Both the transesterification and DMSP‐SLE conditions were optimised. A good linear relationship was obtained within the range of 0.05–10 mg kg^?1 (R² ≥ 0.999) in both blank solvent and an oil sample. The limit of detection was 20.36 μg kg^?1. The average recovery of the 3‐MCPD esters spiked at 0.5, 1.0 and 2.0 mg kg^?1 into a blank oil matrix was in a range from 105.09 ± 2.77% to 120.16 ± 10.88%. The method we developed was further confirmed by performing detection on a Food Analysis Performance Assessment Scheme (FAPAS) sample.     

Desizing of untreated cotton fabric with the conventional and ultrasonic bath procedures by immobilized and native α‐amylase

In this work, starch‐sized, untreated 100% cotton plain woven fabric was treated with native and immobilized α‐amylase enzyme in two different system (conventional and ultrasonic bath procedures) and time (15 and 30 min). Weight loss, the amount of removed starch, tensile properties, contact angle, and scanning electron micrograph morphology were investigated. Sonification and treatment time enhance the desizing performance. Free enzyme, treatment time, and ultrasound brings about a significant increase in the starch‐size removal along with a decrease in the retained strength values, regardless of the used fabric. Immobilized enzyme can be use many times which enables to improve environment friendly pretreatment processes for the textile industry.