Effects of transglutaminase treatment on the thermal properties of soy protein isolates

The effects of covalent cross-linking of microbial transglutaminase (MTGase) on the thermal properties of soy protein isolates (SPI), including the thermal denaturation and glass transition were investigated by conventional and modulated differential scanning calorimetry (DSC). The MTGase treatment significantly increased the thermal denaturation temperatures (including the on-set temperature of denaturation, T_m and the peak temperature of denaturation, T_d) of glycinin and β-conglycinin components of SPI (P ⩽ 0.05), and the thermal pretreatment of SPI further increased the extent of this improvement. The MTGase treatment also improved the ability of SPI to resist the urea-induced denaturation. Modulated DSC analysis showed that there were two glass transition temperatures (T_g) in the reversible heat flow signals of native SPI (about 5% moisture content), approximately corresponding to 45 and 180 °C, respectively. These T_g values of SPI were significantly decreased by the MTGase treatment (at 37 °C for more than 2 h) (P ⩽ 0.05). The improvement in the hydration ability of protein and the formation of high molecular biopolymers may account for the changes of thermal properties of soy proteins caused by the MTGase cross-linking.     

Assessing the rate of solid-phase crystallization for lactose: The effect of the difference between material and glass-transition temperatures

Assessment of the rate of solid-phase crystallization for lactose with recent experimental data is relevant for modelling and predicting the extent of crystallinity that can be expected during the drying of solutions using spray dryers. There is some evidence that the rates rise sharply when there are differences of around 30 K between the material and the glass-transition temperatures. There is also strong evidence, for any particular material, that this rate is related to the (positive) difference between the material and the glass-transition temperatures (T − T_g), as suggested by the WLF equation, and that the rate decreases significantly below the glass-transition temperature.     

Stickiness curves of high fat dairy powders using the particle gun

High fat (>42%) dairy powders are inherently sticky due to their high levels of liquid surface fat. Incorrect operating conditions when spray drying these powders can rapidly lead to blockages. The particle gun was used to characterise the stickiness curves of high fat cream and cheese powders. Stickiness was shown to increase with increasing temperature to a maximum at 50 °C after which it decreased until no stickiness was observed above 68 °C. A dramatic increase in stickiness for the powders was found when the relative humidity of the air was increased past a certain critical point for each temperature. This was attributed to the lactose component of the powder exceeding its glass transition temperature by a critical amount. Best estimates of the (T−T_g)_crit. values for White Cheese Powder, Low Fat Cream Powder and High Fat Cream Powder were 28, 37 and 38 °C, respectively.     

Effect of preheating and other process parameters on whey protein reactions during skim milk powder manufacture

Skim milk powder was manufactured in a milk powder plant using different preheating temperatures, concentrate heating temperatures and spray drying temperatures. Varying the preheating conditions from 70 °C for 52 s to 120 °C for 52 s had a marked effect on the denaturation of $\beta$-lactoglobulin A, $\beta$-lactoglobulin B, $\alpha$-lactalbumin, bovine serum albumin (BSA), and immunoglobulin G. In contrast, varying concentrate heating temperature (65–74 °C) and inlet/outlet air dryer temperature (200/101 °C–160/89 °C) had a minimal effect on whey protein denaturation. Most of the whey protein denaturation and association with the casein micelle occurred in the preheating section of the powder plant. Aggregation of β-lactoglobulin (β-lg) and BSA predominantly involved disulphide bonds. Although, greater than 90% of the β-lg and BSA was denatured after preheating at 120 °C for 52 s, the extent of association with the casein micelle was lower, 50% for β-lg and 75% for BSA.     

Stickiness of skim milk powder using the particle gun technique

The particle gun method for investigating the initiation of stickiness of dairy powders was examined using skim milk powder (SMP). The point at which significant stickiness occurred was at a constant temperature above the glass transition temperature (T_g) of amorphous lactose regardless of the temperature and relative humidity (RH) conditions of the air in the particle gun. This point has been called (T–T_g)_critical. The initial water activity of SMP did not significantly affect (T–T_g)_critical, confirming that stickiness is a surface phenomenon. Changing the particle feed rate did not effect (T–T_g)_critical or the rate at which the powder accumulated on the target plate. The errors in measuring the value of (T–T_g)_critical and the rate of stickiness development [slope of the deposition line versus (T–T_g)] for SMP were found to be 33.6 ± 0.8 °C and 3.1 ± 0.7% deposition °C^?1, respectively, after standardisation of the ambient air conditions around the powder feeder and the initial water activity of the SMP.     

Behaviour of homogenized fat globules during the spray drying of whole milk

The changes in milk fat globule size and fat globule surface proteins of both low-preheated and high-preheated concentrated milks, which were homogenized at low or high pressure prior to spray drying using a disc atomization drier, were examined. The average fat globule size (d₃₂) of the spray-dried milk powders was smaller than that of the corresponding concentrates, but a small proportion of very large globules (4–80 μm) was also formed during spray drying. As a consequence, total surface protein (mg protein g⁻¹ fat) increased due to the adsorption of casein micelles at the fat globule surface during spray drying. Confocal micrographs of the powders showed some apparent spreading of the fat on the surface of the powder particles, particularly when the concentrates were homogenized at low pressure. These results indicate disruption of the milk fat globules during spray drying, which consequently causes changes in the fat globule surface protein layer.     

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.     

Inactivation of Lactobacillus plantarum WCFS1 during spray drying and storage assessed with complementary viability determination methods

Survival of Lactobacillus plantarum WCFS1 spray-dried and stored under different conditions was investigated using complementary methods. One method involved a cell membrane integrity viability-based determination, the other assessed cell growth behavior in a liquid medium by means of detection time or by conventional plating. Survival decreased below 95% when spray drying was carried out at higher outlet spray drying temperatures (T_out > 70 °C). However, the membrane integrity method provided higher residual viability values compared to the detection time and conventional plating. This suggests that loss of viability may be due to a combination of damage to intracellular components and cell membrane. Also during storage viability based on growth behavior declined faster and was more temperature dependent compared to the viability as determined by the membrane integrity method. Also here additional damage to intracellular components is expected responsible to loss of viability. Major conclusion is that one should not only rely on a cell-membrane integrity based method to assess survival during spray drying and storage of bacteria. Previous studies that did so most probably underestimated viability as critical damage to intracellular components was not assessed.     

Binding of retinyl acetate to whey proteins or phosphocasein micelles: Impact of pressure-processing on protein structural changes and ligand embedding

A whey protein isolate (WPI) and native phosphocaseins (PC) at pH 6.5–6.6 were processed with retinyl acetate (RAC) using pressure-assisted technological tools to improve RAC embedding through processing-induced protein structural changes. To this end, protein-RAC dispersions were submitted to ultra-high pressure homogenisation (UHPH) at 300 MPa and an initial fluid temperature (T_in) of 14 °C or 24 °C, or isostatic high-pressure at 300 MPa and 14 °C or 34 °C for 15 min. A short-time thermal treatment (STTT, 73 °C for 4 s) able to generate WPI aggregates was assessed for comparison. Processing effects were investigated in terms of protein particle sizes and molecular weights (M_w). M_w calculated using protein size determination obtained from light scattering measurements were in agreement with the known values. The amounts of RAC retained in WPI particles (unfolded and/or aggregated proteins) or in PC assemblies were quantitated after protein precipitation by ammonium sulphate. A 2.3–3.7 nmol RAC was carried per mg of pressure-denatured whey proteins, significantly less than after STTT (6.3 nmol RAC per mg of heat-denatured whey proteins) indicating that RAC embedding varied according to the technological tool, pressure or temperature. A 3.8–5.4 nmol RAC was carried per mg of PC assemblies through pressure-induced dissociation/reassociation of PC micelles. Combined pressure and mild temperature increased RAC embedding in PC assemblies.     

Dual-enzymatic synthesis of lactulose in organic-aqueous two-phase media

Lactulose has been successfully synthesized by dual-enzymatic method in organic-aqueous two-phase media using lactose and fructose as the raw materials. Cyclohexane–buffer system C₆H₁₂:buffer = 95:5 (v/v) was employed as the organic-aqueous media for the reaction. The dual-enzymatic system was consisted of immobilized lactase (IL) and immobilized glucose isomerase (IGI). Immobilized lactase was prepared by cross-linking the free lactase into Fe₃O₄-chitosan magnetic microspheres. The main enzymatic reaction parameters were investigated, including reaction temperature (T), pH value and reaction time (t). Under the optimum reaction conditions, i.e., lactose 0.8 g mL^?1, fructose 0.1 g mL^?1, IL 0.1 g mL^?1, IGI 0.05 g mL^?1, T = 30 °C, pH = 8.0 and t = 2 h, the obtained highest lactulose yield was approximately 151 g L^?1 and the corresponding productivity was 75.5 g L^?1 h^?1. Experimental results indicated that the organic-aqueous media can significantly promoted the transglycosidation activity of lactase and therefore improve the lactulose yield. The possible reaction mechanism of the synthesis of lactulose using IL and IGI in two-phase system was also proposed.     

Heat-Induced Protein Changes in Milk Processed by Vat and Continuous Heating Systems

This study investigated and compared the effects of heating system and residence times on the physicochemical properties and interactions of casein and whey proteins in heated milk. Milk was processed by vat heating (85°C for 10 to 40 min), HTST heating (98°C for .5 to 1.87 min), UHT heating (140°C for 2 to 8 s), cooled, and fractionated into casein and whey by isoelectric precipitation.β-Lactoglobulin A and B variants were partially denatured by HTST and UHT heating and totally denatured by vat heating. Increasing residence time caused significant (P<.01) increases in denaturation of both β-lactoglobulin variants in UHT and HTST heating systems and of α-lactalbumin in the vat heating system. Surface hydrophobicity and sulfhydryl content were negatively correlated with whey protein denaturation. Sodium dodecyl sulfate electrophoresis of the casein fraction of heated milk indicated the presence of a high molecular weight component that would not enter the gel. Addition of 2-mercaptoethanol to heated casein samples dissociated this component, with the concurrent appearance of β-lactoglobulin and α-lactalbumin bands. In HTST and UHT heating systems, ratio of β-lactoglobulin to κ-casein increased linearly from the complex with increasing residence time.     

In vitro digestion of foods using pH-stat and the INFOGEST protocol: Impact of matrix structure on digestion kinetics of macronutrients,proteins and lipids

It is currently admitted that food structure can facilitate or delay the release of nutrients during digestion and their absorption by the human body. The aim of this study is to propose an in vitro method able to assess the behavior of lipo-proteinic matrices with different structures during digestion. Two model matrices of exactly the same compositions (10% oil, 15% whey proteins, w/w) were designed: i) A liquid emulsion (LE) made of small fat droplets (1 μm) dispersed in a liquid continuous phase containing native whey proteins, ii) a solid emulsion (SE) made of a continuous whey protein gel entrapping large oil droplets (20 μm). The two matrices were digested through an in vitro gastro-intestinal protocol based on the INFOGEST guidelines using pH-stat to monitor the enzymatic hydrolyses of both proteins and lipids. By further digesting lipid-free matrices in the same conditions, the contributions of the proteolytic and lipolytic reactions were evaluated. Significant differences were observed between matrices at both short and long digestion times. The initial rates of both proteolysis and lipolysis were slower for SE than LE because of the gel state of the continuous phase. At the end of the experiments, SE led to a smaller extent of lipolysis (DH_{lip_SE} = 51% < DH_{lip_LE} = 81%) but a greater extent of proteolysis (DH_{prot_SE} = 80% > DH_{prot_LE} = 52%) because of the higher sensitivity to digestion of denatured whey proteins. These results highlight the impact of matrix structure on enzyme accessibility, and show that the proposed method is suitable to monitor the digestion of complex food matrices.     

Thermal properties of globulin from rice (Oryza sativa) seeds

The thermal properties of rice (Oryza sativa) seed globulin were studied by differential scanning calorimetry (DSC) under the influence of various medium conditions. The denaturation temperatures (T_d) of crude and purified rice globulin were 97.6 and 98.5 °C, respectively. Increasing salt concentration enhanced thermal stability of rice globulin. The effect of chaotropic anions on DSC characteristics of rice globulin followed the order of the lyotropic series indicating progressive decreases in both T_d and enthalpy (ΔH). Extreme pHs and protein perturbants (sodium dodecyl sulfate at above 20 mM, dithiothreitol, urea and ethylene glycol) caused marked protein denaturation and loss of thermal stability. In contrast, N-ethylmaleimide and SDS at below 20 mM exerted a stabilizing effect on rice globulin. Pre-heat treatments led to partial unfolding of rice globulin, and the level of denaturation increased progressively with increasing heating time.     

Physicochemical basis for cosolvent modulation of β-lactoglobulin functionality: Interfacial tension study

The impact of neutral cosolvents on the thermal stability of globular proteins in aqueous solutions depends on the nature of the cosolvent, e.g., sorbitol causes a pronounced increase in the thermal denaturation temperature (T_m) of β-lactoglobulin (β-lg), whereas glycerol does not. When a protein unfolds there is a change in the exposed surface area and in the type of molecular interactions that occur at the protein–solvent–cosolvent interface. These changes contribute to the free energy change associated with protein denaturation and depend on cosolvent type. In this study we measured the equilibrium interfacial tensions of aqueous glycerol (0–70% w/w) and sorbitol (0–55% w/w) solutions as a function of temperature to provide insights into the role of the interfacial energy on the thermal stability of β-lg. There was a slight increase in interfacial tension with increasing sorbitol concentration, indicating its preferential exclusion from the oil–water interface. On the other hand, there was an appreciable decrease in interfacial tension with increasing glycerol concentration, indicating its preferential accumulation at the oil–water interface. These changes were largely independent of the measurement temperature (30–80 °C). Our results suggest that sorbitol increases the T_m of β-lg mainly through a steric exclusion effect, whereas glycerol has little effect on T_m because the steric exclusion effect is counter-balanced by a differential interaction effect.     

Development and application of an optical biosensor immunoassay for α-lactalbumin in bovine milk

An automated, label-free biosensor-based immunoassay for α-lactalbumin in bovine milk utilising surface plasmon resonance (SPR) detection is described. α-Lactalbumin content was estimated from the specific interaction with an anti-bovine α-lactalbumin antibody immobilised on the sensor surface in a direct-binding assay format, although an alternative inhibition assay format is also described. Samples were prepared for analysis by direct dilution into buffer. Ligand selection and analysis conditions are defined, and non-specific binding considerations evaluated. Performance parameters include a working range of 10–1000 ng mL^?1, a method detection limit of 0.12 mg mL^?1 in milk, an overall instrumental reproducibility relative standard deviation (RSD_R) of 5.71%, a mean inter-assay RSD_R of 7.61% for an infant formula control sample and a surface stability of approximately 500 cycles. Accuracy was confirmed by comparison against an independent liquid chromatographic method. The technique was applied to the measurement of the α-lactalbumin content of consumer milk, colostrum, whey protein concentrates and infant formulae, the temporal change during early bovine lactation and a preliminary study of thermal denaturation.     

Cocoa brew inhibits in vitro α-glucosidase activity: The role of polyphenols and high molecular weight compounds

Cocoa brew showed a dose-dependent inhibitory activity against α-glucosidase (IC₅₀ 7.87 mg/mL). The cocoa brew was fractionated by ultrafiltration in a low molecular weight fraction (LMW < 10 kDa) and two melanoidin-rich fractions called high molecular weight (HMW > 30 kDa) and intermediate molecular weight (IMW 10–30 kDa) fractions. All fractions tested caused some inhibition with IMW that was the most active (IC₅₀ 2.37 mg/mL). LMW fraction was separated with Sephadex LH-20 in an unbound (containing monomeric and dimeric catechins) and a bound fraction. All the inhibitory activity was recovered in the unbound fraction. All the phenolic compounds identified with HPLC showed α-glucosidase inhibitory activity. IMW and HMW fractions were fractionated by ethanol precipitation. The fractions from IMW precipitated with 75 and 25% ethanol were found to contain power inhibitors of α-glucosidase activity (IC₅₀ 0.87 and 1.01 mg/mL, respectively). In the HMW sample, the fractions precipitated with 50 and 75% ethanol were found to be active against α-glucosidase activity. Most of the inhibitory activity against α-glucosidase of cocoa brew was due to the LMW fraction (56%) whereas IMW and HMW contributed for about 47% of the inhibitory activity. This study suggests that different components of cocoa may influence α-glucosidase activity.     

Glass transitions of barley starch and protein in the endosperm and isolated from

When studying the glass-to-rubber transition inside natural materials, it is important to take into account not only the moisture content but also the moisture distribution over the components in the material. We measured the T_g of protein and starch isolated from barley at different moisture contents using differential scanning calorimetry (DSC) (heating rate 10 °C/min) and by thermo mechanical compression tests (TMCT) (heating rate 2 °C/min). The measurement of the T_g of partially crystalline materials, such as barley starch, is more difficult using TMCT because the mechanical effect of expansion of these materials is smaller. For both measurement sets the glass transition lines were modeled using the Gordon–Taylor equation. The lines were adapted for the differences in moisture content over the endosperm by using the sorption isotherms of isolated barley starch and protein and whole barley endosperm. The glass transition lines measured by TMCT were closer together than the ones measured by DSC.     

Determination of Whey Protein Denaturation in Heat-Processed Milks: Comparison of Three Methods

Fast protein liquid chromatography was used to determine the extent of whey protein denaturation in various heat-treated milk samples: Sordi-indirect UHT (145°C/3 s), Dasi-direct UHT (142°C/3 s), HTST (80°C/30 s), and batch (63°C/30 min). Results were compared with other published methods (differential scanning calorimetry, whey protein nitrogen index, and Kjeldahl nitrogen on salt fractions). Results of the differential scanning calorimetry method were too erratic to be used to quantify whey protein denaturation. The remaining methods (fast protein liquid chromatography, Kjeldahl nitrogen, and whey protein nitrogen index) gave reproducible results and the extent of denaturation (highest to lowest) was consistently predicted as Sordi > Dasi > HTST > batch. There was no difference between fast protein liquid chromatography and Kjeldahl nitrogen, but there was a significant difference between fast protein liquid chromatography and whey protein nitrogen index and between Kjeldahl nitrogen and whey protein nitrogen index. Fast protein liquid chromatography appears to be an effective method to determine whey protein denaturation in heat-treated milks.     

Flavor release from spray-dried amorphous matrix: Effect of lactose content and water plasticization

Glass-forming carbohydrates are widely used as matrix for encapsulation of nutrients and bioactive compounds. In this study, encapsulation systems with lactose/whey protein isolate (WPI) mixtures (4:1, 1:1, and 1:4), or WPI as wall materials and ethyl butyrate as core material were prepared by spray drying. The effects of lactose content and water plasticization on encapsulation efficiency and flavor release were investigated. Wall material consisting of lactose/WPI (4:1) mixture had significantly (P < 0.05) higher encapsulation efficiency. The flavor retention in powders did not have significant decrease with equilibration at 0.33 a_w, while it was dramatically decreased at 0.54 a_w and 0.65 a_w as a result of lactose crystallisation. Mechanical property study showed that the molecular mobility and free volume of encapsulation systems with higher lactose content increased more significantly with increasing water content, which accelerated the diffusion of flavor molecules. Those results may use in the assessment of protection and release characteristics of flavor components in formulated systems.     

α-Glucosidase and α-amylase inhibitory constituent of Carex baccans: Bio-assay guided isolation and quantification by validated RP-HPLC–DAD

Carex baccans is used extensively as food additive for its medicinal and nutritional properties. Its extract demonstrated significant inhibition of α-glucosidase and α-amylase with IC₅₀ 43.32 ± 1.22 and 562.18 ± 5.98 μg/mL, respectively. A bio-assay guided approach was employed to identify the active constituent(s). Fractionation and purification of the extract led to the isolation of a potent inhibitor, (+)-α-viniferin, and of weak inhibitors smiglasides A and B. (+)-α-Viniferin was quantified in the extract and fractions using HPLC and the method was validated for linearity, limit of detection, limit of quantification, precision, and accuracy. The calibration curve showed good linearity (r² > 0.999) in the range of 7.813–250 μg/mL. The identification of α-glucosidase and α-amylase inhibitory activity in C. baccans supports the possible use of the plant as functional food for the management of diabetes. The validated HPLC method for the study of plant extracts will be useful in future research.