Gelation properties of salt-extracted pea protein isolate catalyzed by microbial transglutaminase cross-linking

Gelation is a fundamental functional characteristic of plant proteins. In this paper, a salt-extracted pea protein isolate (PPI) was mixed with microbial transglutaminase (MTG) to produce gels and the gelation properties were studied. When the MTG level increased, the magnitude of both the G′ and G″ moduli also increased, which means the gel strength increased. A second order polynomial equation was used to describe the relationships between the G′, G″ modulus and TG level. It was found that with increased heating and cooling rate at the same MTG level, G′ and G″ tended to decrease, resulting in a weaker gel. This was attributed to the rearrangement time of pea protein molecules; slower heating and cooling rates enabled protein molecules to have more time to rearrange and therefore form a stronger gel. At the same MTG level, higher pea protein concentration resulted in higher G′ and G″ values and a power law relationship was found between G′ and pea protein concentration or G″ and pea protein concentration. Frequency sweep data of PPI show that the MTG treatment resulted in higher G′ values and lower tan delta values, indicative of a stronger more elastic gel. The minimum gelation concentration was found to be 3% (w/v) with 10 U MTG treatment, lower than 5.5% required when no MTG was present. When compared to PPI and soy protein isolate (SPI) with and without 10 U MTG treatment, the gel strength of PPI with MTG was stronger than that of SPI with MTG treatment, whereas the opposite was true without the MTG treatment. SDS-PAGE showed that at the same pea protein concentration, higher MTG level induced more cross-linking as fainter bands were seen on the gel and there was a shift in the relative intensities of the bands in the molecular weight range of 35–100 kDa.     

大豆分离蛋白与甜菜果胶静电复合过程的研究

利用葡萄糖酸内酯(GDL)诱导大豆分离蛋白(SPI)与甜菜果胶(SBP)形成静电复合物。在不同SPI/SBP混合比例下,通过动态光散射和紫外可见分光光度计对pH酸化过程中的散射光强和浊度进行测定,跟踪复合物的生成及结构演变。实验结果表明,SPI与SBP可生成具有不同结构和不同稳定性的分子复合物,包括分子内可溶复合物、分子间可溶复合物以及分子间不可溶复合物。不同类型复合物的形成取决于溶液体系的pH和不同SPI/SBP混合比例。该研究系统地建立了SPI与SBP静电复合物的相图。      

Rheological properties of whey protein isolate stabilized emulsions with pectin and guar gum

Dynamic oscillatory and steady-shear rheological tests were carried out to evaluate the rheological properties of whey protein isolate (WPI) stabilized emulsions with and without hydrocolloids (pectin and guar gum) at pH 7.0. Viscosity and also consistency index of emulsions increased with hydrocolloid concentration. At γ = 20 s⁻¹, the value of viscosity of the emulsion with 0.5% (w/v) pectin was about fivefold higher than that of the emulsion without pectin. Flow curves were analyzed using power law model through a fitting procedure. Flow behaviour index of all emulsions except for containing 0.5% (w/v) guar gum was approximately in the range of 0.9–1.0, which corresponds to near-Newtonian behaviour. The shear thinning behaviour of emulsions containing 0.5% (w/w) guar gum was confirmed by flow behaviour index, n, of 0.396. Both storage (G′) and loss modulus (G″) increased with an increase in frequency. Emulsions behaved like a liquid with G″ > G′ at lower frequencies; and like an elastic solid with G′ > G″ at higher frequencies. Effect of guar gum was more pronounced on dynamic properties. Phase angle values decreased from 89 to <10° with increasing frequency and indicated the viscoelasticity of WPI-stabilized emulsions with and without pectin/guar gum.     

A novel improvement in whey protein isolate emulsion stability: Generation of an enzymatically cross-linked beet pectin layer using horseradish peroxidase

Extensive research has indicated that the electrostatic attraction between polysaccharides and proteins on the oil-water interface can improve the stability of emulsions. However, this electrostatic effect will be weakened or even eliminated as the solution pH or ionic strength of emulsions change, resulting in the shedding of the polysaccharide layer. We prepared primary oil-in-water emulsions at pH 7.0 using whey protein isolate (WPI) as an emulsifier and then beet pectin was added to form secondary emulsions. After the pH of emulsions was adjusted to 4.0 to promote electrostatic attraction between the beet pectin molecules and the protein-coated droplets, horseradish peroxidase was added to generate a cross-linked beet pectin coating. Results show that stable emulsions coated with WPI and cross-linked beet pectin interfaces could be formed. The sensitivity of the emulsions to the environmental stresses of pH changes, ions addition, thermal processing and freezing was also characterized in this work. Our results support the view that cross-linked beet pectin improves the stability of emulsions and is superior to simple deposition on the surface of lipid droplets. The interfacial engineering technology used in this study could be used to create food emulsions with improved stability to environmental stresses.     

Fabrication and characterization of TiO2/whey protein isolate nanocomposite film

Composite films of whey protein isolate and TiO₂ are formed through three simultaneous processes, i.e., the self-assembly of protein–protein, TiO₂–TiO₂, and the association of protein–TiO₂. All the processes could be controlled by adjusting TiO₂ concentration in the blended system. The self-assembly of protein–protein molecules constituted the main network of the composite film. A low TiO₂ concentration (<0.25%) dispersed the TiO₂ nanoparticles in the protein matrix, reinforced the association of protein–TiO₂, reduced the ability of UVC absorption, and promoted the fluorescence and tensile strength of the composite films. In contrast, a high TiO₂ concentration (>0.25%) enhanced the self-assembly of TiO₂–TiO₂ nanoparticles, brought fluorescent quenching, and produced a decline of the tensile strength and water vapor permeability. The transmittance of the visible, UVA, and UVB lights showed a first order exponential decay relative to the TiO₂ concentration.     

Peptide–peptide and protein–peptide interactions in mixtures of whey protein isolate and whey protein isolate hydrolysates

The extent of aggregation in whey protein isolate (WPI) hydrolysates induced by Bacillus licheniformis protease was quantified as a function of degree of hydrolysis (DH), temperature and ionic strength. The capacity of the hydrolysates to aggregate added intact protein was also studied. The amount of aggregated material and the size of the aggregated peptides were measured by nitrogen content and size exclusion chromatography, respectively. Aggregation increased with DH up to the practical end point of hydrolysis (DH 6.8%). The aggregates formed under the various conditions studied consisted of peptides with masses ranging from 1.4 to 7.5 kDa. The hydrolysates were also able to aggregate added WPI. The additional amount of aggregated material increased with increasing DH. Peptides involved in peptide–peptide interactions were also involved in protein–peptide interactions. It is hypothesized that hydrophobic interactions dominated peptide–peptide interactions, while protein–peptide interactions depended on the balance between hydrophobic attraction and electrostatic repulsion.     

Formation and characterization of noncovalent ternary complexes based on whey protein concentrate,high methoxyl pectin,and phenolic acid

Protein-polysaccharide-polyphenol noncovalent ternary complexes possess unique physicochemical, structural, and functional properties. In the present study, ternary complexes based on whey protein concentrate (WPC; 2%, wt/vol) and high methoxyl pectin (HMP; 0.5%, wt/vol) complexes and 0.2 to 0.6% (wt/vol) chlorogenic acid (CA) or rosmarinic acid (RA) were formed and characterized at 3 pH values (4, 4.5, and 5). The pH conditions were decided according to phase diagram of WPC and HMP during acidification. Fluorescence quenching experiments indicated that WPC-HMP complexes bound RA stronger than CA and the binding constant increased with increasing pH for both phenolic acids. Particle size of ternary complexes decreased and absolute ζ-potential increased with pH values changing from 4 to 5, and RA influenced the particle size of WPC-HMP complexes greater than CA. The CA and RA in ternary complexes showed good stability against UV light with pH order of pH 5 > pH 4.5 > pH 4. Fourier-transform infrared spectroscopy spectra indicated the involvement of hydrogen bonding between WPC-HMP and CA or RA. Antibacterial tests showed that ternary complexes had good antibacterial activity against Staphylococcus aureus and Escherichia coli at concentrations of 6.2 mg/mL and the ability increased with decreasing pH values. All ternary complexes possessed strong scavenging radical capacities with median inhibitory concentration (IC₅₀) values ranging from 2.71 ± 0.05 to 6.20 ± 0.41 μg/mL. Antioxidative ability of ternary complexes increased as pH went up and WPC-HMP-RA showed significantly higher antioxidative property compared with WPC-HMP-CA. Data may provide useful information for rational design of ternary complexes and applications of the formed complexes in food matrices such as beverages and emulsions.     

Influence of ultrasound and enzymatic cross-linking on freeze-thaw stability and release properties of whey protein isolate hydrogel

This study investigated the effect of ultrasound and enzymatic cross-linking on the freeze-thaw (FT) stability and release properties of whey protein isolate hydrogels. We evaluated the FT stability by the changes in the microstructure, riboflavin retention, syneresis, water holding capacity (WHC), and texture of gels subjected to 3 FT cycles. High-intensity ultrasound (HUS) and transglutaminase (TGase)-mediated cross-linking improved the FT stability of whey protein isolate hydrogels loaded with riboflavin (WPISAR), as demonstrated by a more uniform and denser porous structure, significantly higher riboflavin retention, WHC, and textural properties, and lower syneresis after 3 FT cycles than those of untreated hydrogels. Furthermore, HUS- and TGase-mediated cross-linking decreased protein erosion and swelling ratio of WPISAR in simulated gastrointestinal fluids (SGIF) and reduced the riboflavin release rate in SGIF both with and without the addition of digestive enzymes. After 3 FT cycles, faster riboflavin release occurred due to a more porous structure induced by ice crystal formation compared with their unfrozen counterparts as detected by confocal laser scanning microscopy. High-intensity ultrasound- and TGase-mediated cross-linking alleviated the FT-induced faster riboflavin release rate in SGIF. High-intensity ultrasound- and TGase-treated gel samples showed that both diffusion and network erosion were responsible for riboflavin release regardless of FT. These results suggest that HUS- and TGase-mediated cross-linking improved the FT stability of WPISAR with a high riboflavin retention, and might be a good candidate as a controlled-release vehicle for riboflavin delivery to overcome undesired FT processing.     

微生物转谷氨酰胺酶催化乳清蛋白聚合研究

采用SDS－PAGE分析，研究了不同条件下微生物转谷氨酰胺酶（MTGase）催化乳清蛋白（WPI）聚合。结果显示，MTGase可催化乳清蛋白的β－乳球蛋白（β-LG）和α－乳清蛋白（α－LA）聚合，形成低聚物或生物聚合物，其中β-LG更易受MTGase的催化，当TGase酶浓度一定时（0.5U/mL),TGase催化WPI聚合的最佳底物质量分数范围为2％－4％，对WPI进行加热预处理，同时添加还原剂，可明显提高MTGase对WPI的催化活性，MTGase催化WIP的最适PH值范围为6．5－7．5，当WPI经预热处理（85℃，15min），同时添加20mmol/L的DTT，TGase催化WPI聚合12h，可使质量分数为92％的β-LG和质量分数为75％的α－LA聚合。     

Enzyme catalyzed oxidative gelation of sugar beet pectin: Kinetics and rheology

Sugar beet pectin (SBP) is a marginally utilized co-processing product from sugar production from sugar beets. In this study, the kinetics of oxidative gelation of SBP, taking place via enzyme catalyzed cross-linking of ferulic acid moieties (FA), was studied using small angle oscillatory measurements. The rates of gelation, catalyzed by horseradish peroxidase (HRP) (EC 1.11.1.7) and laccase (EC 1.10.3.2), respectively, were determined by measuring the slope of the increase of the elastic modulus (G′) with time at various enzyme dosages (0.125–2.0 U mL⁻¹). When evaluated at equal enzyme activity dosage levels, the two enzymes produced different gelation kinetics and the resulting gels had different rheological properties: HRP (with addition of H₂O₂) catalyzed a fast rate of gelation compared to laccase (no H₂O₂ addition), but laccase catalysis produced stronger gels (higher G′). The main effects and interactions between different factors on the gelation rates and gel properties were examined in response surface designs in which enzyme dosage (0.125–2.0 U mL⁻¹ for HRP; 0.125–10 U mL⁻¹ for laccase), substrate concentration (1.0–4.0%), temperature (25–55 °C), pH (3.5–5.5), and H₂O₂ (0.1–1.0 mM) (for HRP only) were varied. Gelation rates increased with temperature, substrate concentration, and enzyme dosage; for laccase catalyzed SBP gelation the gel strengths correlated positively with increased gelation rate, whereas no such correlation could be established for HRP catalyzed gelation and at the elevated gelation rates (>100 Pa min⁻¹) gels produced using laccase were stronger (higher G′) than HRP catalyzed gels at similar rates of gelation. Chemical analysis confirmed the formation of ferulic acid dehydrodimers (diFAs) by both enzymes supporting that the gelation was a result of oxidative cross-linking of FAs.     

Stabilization of oil-in-water emulsions by enzyme catalyzed oxidative gelation of sugar beet pectin

Enzyme catalyzed oxidative cross-linking of feruloyl groups can promote gelation of sugar beet pectin (SBP). It is uncertain how the enzyme kinetics of this cross-linking reaction are affected in emulsion systems and whether the gelation affects emulsion stability. In this study, SBP (2.5% w/v) was mixed into an oil-in-water emulsion system (4.4% w/w oil, 0.22% w/w whey protein, pH 4.5). Two separate, identically composed, emulsion systems were prepared by different methods of preparation. The emulsions prepared separately and subsequently mixed with SBP (referred as Mix A) produced significantly larger average particle sizes than the emulsions in which the SBP was homogenized into the emulsion system during emulsion preparation (referred as Mix B). Mix B type emulsions were stable. Enzyme catalyzed oxidative gelation of SBP helped stabilize the emulsions in Mix A. The kinetics of the enzyme catalyzed oxidative gelation of SBP was evaluated by small angle oscillatory measurements for horseradish peroxidase (HRP) (EC 1.11.1.7) and laccase (EC 1.10.3.2) catalysis, respectively. HRP catalyzed gelation rates, determined from the slopes of the increase of elastic modulus (G′) with time, were higher (P < 0.05) than the corresponding laccase catalyzed rates, but the final G′ values were higher for laccase catalyzed gels, regardless of the presence of emulsions or type of emulsion preparation (Mix A or Mix B). For both enzymes, rates of gelation in Mix A were higher (P < 0.05) than in Mix B, and higher stress was needed to break the gels in Mix A than in Mix B at similar enzyme dosage levels. These differences may be related to a lower availability of the feruloyl groups for cross-linking when the SBP was homogenized into the emulsion system during preparation.     

Preparation and characterization of whey protein isolate films reinforced with porous silica coated titania nanoparticles

Whey protein isolate (WPI) films embedded with TiO₂@@SiO₂ (porous silica (SiO₂) coated titania (TiO₂)) nanoparticles for improved mechanical properties were prepared by solution casting. A WPI solution of 1.5 wt% TiO₂@@SiO₂ nanoparticles was subjected to sonication at amplitudes of 0, 16, 80 and 160 μm prior to casting in order to improve the film forming properties of protein and to obtain a uniform distribution of nanoparticles in the WPI films. The physical and mechanical properties of the films were determined by dynamic mechanical analysis (DMA), thermogravimetric analysis (TGA), and tensile testing. Water vapor permeability (WVP) measurements revealed that the water vapor transmission rates are slightly influenced by sonication conditions and nanoparticle loading. The DMA results showed that, at high sonication levels, addition of nanoparticles prevented protein agglomeration. The thermal stability of the materials revealed the presence of 3–4 degradation stages in oxidizing the protein films. The addition of nanoparticles strengthens the WPI film, as evidenced by tensile stress analysis. Sonication improved nanoparticle distribution in film matrix; such films can potentially become effective packaging materials to enhance food quality and safety.     

Hydrolysis of whey protein isolate using subcritical water

Hydrolyzed whey protein isolate (WPI) is used in the food industry for protein enrichment and modification of functional properties. The purpose of the study was to determine the feasibility of subcritical water hydrolysis (SWH) on WPI and to determine the temperature and reaction time effects on the degree of hydrolysis (DH) and the production of peptides and free amino acids (AAs). Effects of temperature (150 to 320 °C) and time (0 to 20 min) were initially studied with a central composite rotatable design followed by a completely randomized factorial design with temperature (250 and 300 °C) and time (0 to 50 min) as factors. SWH was conducted in an electrically heated, 100-mL batch, high pressure vessel. The DH was determined by a spectrophotometric method after derivatization. The peptide molecular weights (MWs) were analyzed by gel electrophoresis and mass spectrometry, and AAs were quantified by high-performance liquid chromotography. An interaction of temperature and time significantly affected the DH and AA concentration. As the DH increased, the accumulation of lower MW peptides also increased following SWH (and above 10% DH, the majority of peptides were <1000 Da). Hydrolysis at 300 °C for 40 min generated the highest total AA concentration, especially of lysine (8.894 mg/g WPI). Therefore, WPI was successfully hydrolyzed by subcritical water, and with adjustment of treatment parameters there is reasonable control of the end-products.     

乳清分离蛋白成膜工艺的研究

以乳清分离蛋白(WPI)为主料,通过添加甘油(增塑剂)和半胱氨酸(还原剂),制备乳清分离蛋白膜.同时,对其制备工艺与性能进行了详细分析与测定,从而确定了成膜最佳工艺为:乳清分离蛋白含量为8%,增塑剂添加量为4%.还原剂的添加量为0.6mmol/L.在此工艺条件下测定乳清分离蛋白膜性能:厚度0.101±0.013mm,透明度0.055±0.005.抗拉强度1165.2±20.8g.断裂伸长率70.06%±1.62%,透H2O性17.13±0.63g/m2·h,透O2性3.60±0.08g/m2·d,透CO2性445.56±5.26g/m2·d.