Protein quality and identification of the storage protein subunits of tofu and null soybean genotypes,using amino acid analysis,one- and two-dimensional gel electrophoresis,and tandem mass spectrometry

The protein quality of 11 null and 2 tofu soybean genotypes were determined from their total protein content, their amino acid composition, and their glycinin and β-conglycinin contents. There were highly significant differences (P < 0.001) in their total storage proteins, and amino acid contents. Total protein among these genotypes ranged from 33 to 37%, with arginine being the third highest amino acid (7.4–10.9 g/100 g protein) followed by glutamic and aspartic acids. Methionine accounted for only 1.6–2.4 g/ 100 g of protein. All genotypes contained a good balance of essential amino acids (EAA₉), ranging from 43.5 to 47.3% of the total protein, limited only in methionine and possibly threonine and valine. Two-dimensional gel electrophoretic (2-DE) reference maps, using narrow range immobilized pH gradient (IPG) strips, revealed unique differences in the proteome, and subunit expression of glycinin and β-conglycinin, among these null genotypes, which can then be correlated with their protein quality. Out of a total of 111 basic (pH 6–11), and 223 acidic (pH 4–7) protein spots separated by 2-DE, 41 soybean storage protein spots were excised, and identified by liquid chromatography on-line with electro spray LCQ DecaXP tandem quadrupole time-of-flight mass spectrometry (LC/MS/MS). These methods will enable accurate evaluation of protein quality in soybeans, based on their protein digestibility-corrected amino acid score, assessment of the genetic variability of soybean genotypes, and serve as very effective tools for assisting plant breeders in their selection of high quality soybean varieties.     

Effect of transglutaminase on rheological properties and microstructure of chemically acidified sodium caseinate gels

The mechanical properties and microstructure of 2.7% and 4.5% sodium caseinate gels chemically acidified by glucono-δ-lactone (GDL) and cross-linked by microbial transglutaminase (TG) were studied. The acidification was performed at different temperatures. According to SDS–PAGE TG clearly caused polymerisation of caseinate irrespective of the treatment temperature (4–50 °C), The cross-linking of the proteins was more extensive at temperatures 22–50 °C. Low amplitude viscoelastic measurements showed that 4.5% caseinate gels acidified at 50 °C were formed much faster than gels acidified at 22 °C. TG only slightly increased the time of gelling. Control gels prepared without TG at temperatures of 4, 22, 37 and 50 °C were mechanically weak. Examination of the control gels with a confocal laser scanning microscope showed that gels formed at 37 and 50 °C were coarse and porous with large cavities between particle aggregates, whereas those formed at 22 °C were much more homogeneous. The TG-treated and acidified sodium caseinate dispersions formed firm gels, indicating cross-linking of casein proteins. Interestingly, the strongest gels were formed at 22 and 37 °C. TG treatment improved the homogeneity of the gel structure at temperatures of 37 and 50 °C. The hardness of TG-treated gels acidified at 4 °C increased during 1 week of storage.     

Structures and some properties of soluble protein complexes formed by the heating of reconstituted skim milk powder

The effects of heat on reconstituted skim milk powder (RSMP) at a temperature of 90 °C have been determined, with respect to the formation of soluble protein complexes. Heating at the natural pH of the RSMP forms soluble complexes in the milk serum, which can be separated by size exclusion chromatography. The diameters of the particles were found to be up to 50 nm, as shown by light scattering and by scanning electron microscopy. The formation of the soluble complexes was strongly affected by the treatment of the RSMP before heating. Treatment of the milk with small amounts of glutaraldehyde, to fix the casein micellar structure, severely inhibited the formation of the soluble complexes. The pH at which the milk was heated (in the range 6.5–7.2) also had an effect on the amounts and the sizes of the complexes, as determined by size exclusion chromatography. The amount of soluble complex present in the RSMP affected the strength of the acid gels produced from the different milks, as was shown by exchanging the sera of milks treated differently with glutaraldehyde, and by following the gelation of milks heated at different pH values. Increased amounts of soluble complexes gave stronger acid gels. However, the relationship appeared to break down if milk was heated at pH 7.2, where the soluble material appeared to change and the acid gel was much weaker.     

Effects of lubrication and sample dimensions on compression property of fish–meat gels

Effects of lubrication and sample dimension on compression property of fish–meat gel were investigated using the modified Mooney–Rivlin equation. Cylindrical fish–meat gels were cut into 26 mm in diameter and 10, 20 and 30 mm in heights, respectively. Lubrication of samples led to the lower stress–strain curve. Stress at fracture was dependent on sample dimension in non-lubricated test. In lubricated compression, the dependant was reduced and the agreement of stress–strain data for all samples was observed at the low strain region. The similar results were observed in comparing compression properties of non-lubricated and lubricated fish–meat gels according to the modified Mooney–Rivlin equation. In order to reduce friction problems in compression test of fish–meat gels, the experiment setting can be suggested as follows: for the samples with small dimensions, the surface of gel sample has to be lubricated, or gel samples have to be made with large dimensions.     

Effect of chemical phosphorylation on solubility of buffalo milk proteins

Buffalo milk proteins (casein, co-precipitate or whey protein concentrate) were phosphorylated with phosphorus oxychloride (POCl₃) at three different pH values (5.0, 7.0 and 9.0). The solubilities of phosphorylated milk proteins were examined over the pH range 3.0–9.0 in water and ionic (0.1 m NaCl or 10–70 mm Ca²⁺) systems. The solubilities of buffalo milk proteins decreased at pH 3.0, while there was an increase in the solubilities of casein and co-precipitate near their isoelectric points upon phosphorylation. Solubilities of these phosphorylated milk proteins were pH dependent in 0.1 m NaCl but there was a decrease in their solubilities with increase in calcium ion concentration. This alteration could be due to the shifting of isoionic points of phosphorylated buffalo milk proteins towards acidic pH.     

Stability of casein micelle subjected to reversible CO2 acidification: Impact of holding time and chilled storage

Casein micelle stability and reactivity were assessed on milk subjected to reversible acidification by carbonation. Pressurised CO₂ was injected at 4 °C, leading to controlled acidification from 6.63±0.02 to a target pH (5.5 or 5.2). After holding the pressurised milk under these conditions for 15 or 60 min, the pressure was released and the milk pH returned to its initial value under stirring and vacuum degassing. Upon CO₂ treatment, calcium and protein partition, zeta potential and size of casein micelles were restored directly after neutralisation. The rheological properties of the gel obtained by acid coagulation of CO₂-treated milk did not change as a result of carbonation. Micelle hydration increased after neutralisation and during storage. Milk buffering capacity in the pH range of 4.5–5.5 decreased after neutralisation of milk acidified by carbonation, but increased during chilled storage of this milk. Holding time of carbonated milk at low pH was found to have no impact on the physicochemical characteristics of casein micelles and the rheological properties of the gel obtained by acid coagulation of this milk.     

Gel-Forming Characteristics of Milk Proteins. 1. Effect of Heat Treatment

Skim milk with or without preheating (60 to 80°C for 30 min) were acid coagulated at 60 to 80°C for 1 h with glucono-delta-lactone. Preheating below 70°C has no effect on gel firmness and water-holding capacity. When coagulated below 70°C, the gels were weak and had low water-holding capacity. When coagulated at 80°C, the gels were solid and had high water-holding capacity. Gels prepared from skim milks preheated to above 80°C had a different quality: when coagulated at less than 70°C, gel firmness increased slightly, and when coagulated at 80°C, gel firmness decreased sharply. Change in the accessibility of sulfhydryl groups in milk protein caused by heating, was also measured using Ellman's reagent. Changes in the gel-forming property of milk protein, caused by the heat treatment, were closely related to increase in available sulfhydryl groups in milk proteins, and also were related to heat denaturation of whey protein or the formation of β-lactoglobulin/κ-casein complex.     

The effect of modifying the distribution of sucralose and quinine on bitterness suppression in model gels

This study investigated the effect of simultaneously manipulating the spatial distribution of a sweet masking agent and bitter tastant within gelatine–agar gels on bitterness suppression, using sucralose and quinine. Sixty subjects participated in a series of paired comparison tests comparing different gel designs containing both sucralose and quinine, against a homogeneous control gel of identical overall sucralose (0.3 mM) and quinine content (0.3 mM). Twenty subjects also determined the bitterness reduction achieved in the homogeneous control gel by the addition of 0.3 mM sucralose to 0.3 mM quinine. Separating quinine and sucralose into different portions of the gel had no influence on bitterness suppression. Inhomogeneously distributing the quinine and sucralose into the same parts of the gel reduced the effectiveness of bitterness suppression. Inhomogeneously distributing the sucralose, while maintaining a homogeneous distribution of quinine, had no influence on bitterness suppression. Although an inhomogeneous distribution of sucralose increased sweetness perception and the addition of sucralose (0.3 mM) was found to substantially reduce bitterness in the homogeneous control, bitterness suppression was not enhanced when distributing the masking agent (sucralose) inhomogeneously.     

High-pressure treatment effects on proteins in soy milk

Effects of high-pressure treatment on the modifications of soy protein in soy milk were studied using various analytical techniques. Blue shifts of λ_max could be observed in the fluorescence spectra. Spectrofluorimetry revealed that the soy protein exhibited more hydrophobic regions after high-pressure treatment. Electrophoretic analysis showed the change of soy protein clearly and indicated that soy proteins were dissociated by high pressure into subunits, some of which associated to aggregate and became insoluble. High-pressure denaturation occurred at 300 MPa for β-conglycinin (7S) and at 400 MPa for glycinin (11S) in soy milk. High pressure-induced tofu gels could be formed that had gel strength and a cross-linked network microstructure. This provided a new way to process soy milk for making tofu gels.     

Modifying the microstructure of low-fat yoghurt by microfluidisation of milk at different pressures to enhance rheological and sensory properties

The effects of microfluidisation of milk at different pressures, prior to heat treatment, on structural and sensory properties of low-fat stirred yoghurt, were investigated. Low-fat yoghurts prepared from microfluidised milk were compared with low-fat (1.5%) and full-fat (3.5%) control yoghurts made with homogenised (20/5 MPa) milk. The microstructure of low-fat yoghurts prepared with microfluidised milk consisted of smaller and more uniform fat globules, well incorporated into more interconnected fat-protein gel networks, compared with those of control yoghurts. This modification in microstructure caused significant changes in gel particle size, sensory profile and rheological behaviour. Microfluidisation increased the gel particle size, gel strength and viscosity; marked beneficial effects were found at higher pressures (50–150 MPa). Microfluidising milk at 50–150 MPa increased the gel strength by 171–195% and viscosity by 98–103%, creating low-fat yoghurts with creaminess and desirable texture properties similar to, or better than, full-fat conventional yoghurt.     

Effect of soy protein substitution for sodium caseinate on the transglutaminate-induced cold and thermal gelation of myofibrillar protein

The influence of soy protein isolate (SPI) substitution for sodium caseinate (SC) on the properties of cold-set (4 °C) and heat-induced gels of pork myofibrillar protein (MP) incubated with microbial transglutaminase (TG) was investigated. The strength of cold-set MP–SC gels (formed in 0.45 M, NaCl, 50 mM phosphate buffer, pH 6.25) increased with time of TG incubation, but those gels with more than 66% SPI substituted for SC had a >26% reduced strength (P < 0.05). Upon cooking, both incubated and non-incubated protein sols were quickly transformed into highly elastic gels, showing up to 6000 Pa in storage modulus (G′) at the final temperature (72 °C). However, no differences (P < 0.05) in G′ were observed between heated samples with SPI and SC. Myosin heavy chain, casein and soy proteins gradually disappeared with TG incubation, contributing to MP gel network formation. Both cold-set and heat-induced gels had a compact protein matrix, attributable to protein cross-linking by TG.     

Gelation properties of salt soluble meat protein and soluble wheat protein mixtures

Salt soluble meat proteins (SSMP) and commercially available soluble wheat proteins (SWP) were characterised by SDS-polyacrylamide gel electrophoresis, differential scanning calorimetry (DSC) and small and large deformation testing. DSC scans indicated transitions similar to those of native actomyosin for the salt soluble meat extract whereas SWP did not indicate any transitions between 20 and 120 °C. Small deformation tests on SWP indicated a G′/G″ crossover gelation temperature of 90 °C and weak gels as judged by frequency sweeps. In contrast, SSMP gelled at 40 °C and formed strong gels on heating to 90 °C. However, on autoclaving at 120 °C, 20% SWP in distilled water produced strong elastic gels with little syneresis, compared with the more brittle gels produced with 20% (w/w) SSMP as indicated by large deformation testing. Mixtures of the two proteins in the ratio SSMP/SWP (15:5) gave strong elastic gels similar to the SWP gels. Even the presence of very small amounts of SWP in the mixture, e.g. SSMP/SWP 20:1 trebled the elastic modulus compared with a SSMP gel and reduced syneresis. This was probably due to the close association of SWP with actomyosin strands as viewed by transmission electron microscopy. However, increased levels of SWP in the mixture, for example SSMP/SWP 10:10 ratio, resulted in the separation of the two protein phases as shown by phase contrast microscopy, and consequently led to lower G′ values in the mixed gels. The addition of 20 mM chloride salts showed that potassium reduced the shear modulus, sodium had no effect and calcium enhanced the shear modulus for SWP gels formed at 120 °C. In contrast, SSMP gels were stronger in the presence of potassium, followed by sodium and calcium.     

Texture and structure of pressure-shift-frozen agar gel with high visco-elasticity

To determine the effects of sucrose and high-pressure-freezing, two kinds of agar gel were compared; A gel with high visco-elasticity and B gel, an ordinary dessert gel. Both agar gels with 0, 5, 10 or 20% sucrose were frozen at 0.1–686 MPa and −20 °C. They were frozen during pressurization, and exothermic peaks were detected at 0.1, 100, 600 and 686 MPa and −20 °C (freezing). However, at 200 MPa, they did not freeze but froze with released pressure (pressure-shift-freezing). Thus, the amount of syneresis from gel pressure-shift-frozen at 200 MPa was smaller than that from gel frozen at other pressures. Also, amount of syneresis from A was smaller than B. In addition, compared to control gels, the appearance of 0% sucrose–agar gels frozen at 0.1, 100, 600 and 686 MPa differed greatly due to syneresis and a volumetric shrinkage of the gel. It was apparent that the rupture stress of the gels decreased, strain and size of ice crystals increased and quality declined. Conversely, due to quick freezing, the texture and structure of both A and B pressure-shift-frozen at 200 MPa were better than the other pressure-treated gels and gels frozen in freezers (−20, −30 or −80 °C) at atmospheric pressure. Consequently, pressure-shift-freezing was more effective. However, texture, structure and syneresis of A were somewhat better than that of B. It was found that the addition of sucrose to the gel was effective in improving the quality of frozen agar gels.     

Small and large deformation rheology and microstructure of κ-carrageenan gels containing commercial milk protein products

The rheological behaviour of commercial milk protein/κ-carrageenan mixtures in aqueous solutions was studied at neutral pH. Four milk protein ingredients; skim milk powder, milk protein concentrate, sodium caseinate, and whey protein isolate were considered. As seen by confocal laser microscopy, mixtures of κ-carrageenan with skim milk powder, milk protein concentrate, and sodium caseinate showed phase separation, but no phase separation was observed in mixtures containing whey protein isolate. For κ-carrageenan concentrations up to 0.5 wt%, the viscosity of the mixtures at low shear rates increased markedly in the case of skim milk powder and milk protein concentrate addition, but did not change by the addition of sodium caseinate or whey protein isolate. For κ-carrageenan concentrations from 1 to 2.5 wt%, small and large deformation rheological measurements, performed on the milk protein/κ-carrageenan gels, showed that skim milk powder, milk protein concentrate or sodium caseinate markedly improved the strength of the resulting gels, but whey protein isolate had no effect on the gel stength.     

Heat processed whey-protein food emulsions and growth of shear-induced cracks during cooling

When high fat (40 g oil /100 g) food dressings emulsified by whey protein were heat-filled (80°C) into plastic-bottles, large visible cracks developed in the dressings after cooling. The occurrence of cracks was dependent on the length of the heat treatment, pH and protein concentration. Heating (2 h at 80°C), low pH (pH 3) and high protein concentrations (1.5 g/100 g) increased the number of cracks. Dressings with cracks were more viscous and had a broader particle size distribution than dressings devoid of cracks. During heating at 80°C the complex modulus (G¹) measured at low deformation increased sharply, signifying the formation of a three-dimensional network as a result of aggregation of whey protein bridging the fat-droplets. Confocal laser scanning microscopy revealed differences in microstructure dependent on cooling rate and pH. At slow cooling and pH 3 the network structure was inhomogeneous with large voids, while at pH 4, closer to the isoelectrical point, the structure was more compact aggregated and homogeneous. The gels at pH 3 were strain sensitive and seemed more prone to localized fracture. A strong (r=0.86) relationship between a visible quality defect called cracks and the gel properties of the emulsion was found. Avoiding cracks called for a strict control of protein concentration, pH and holding time at higher temperatures.     

The effect of pH on the gelling behaviour of canola and soy protein isolates

The present study investigates the gelation mechanisms of a canola protein isolate (CPI) as a function of a pH (3.0–9.0), and compares it to that of a commercial soy protein isolate (SPI). A rheological investigation found that CPI was non-gelling at pH 3.0, and then formed a gel with increasing strength as pH was raised from pH 5.0 to 9.0. In contrast, the commercial SPI ingredient was found to be non-gelling at pH 9.0, but formed the strongest networks at pH 5.0 near its isoelectric point (pI = 4.6). Denaturation temperature as determined by differential scanning calorimetry were found to occur at ~ 78 °C for CPI at pH 5.0, then shifted to higher temperatures (~ 87 °C) at pH 7.0/9.0, whereas detection of SPI denaturation could not be obtained due to instrument sensitivity. Gelling temperatures were similar for both CPI and SPI (~ 82–86 °C) at all pHs, with the exception of SPI at pH 5.0 (~ 46 °C). Overall CPI networks were stronger than SPI, since the latter had weaker inter- and intramolecular junction zones. Confocal laser scanning microscopy images indicated that CPI gels became denser with lower lacunarity values as pH increased from 3.0 to 9.0. Moreover, the fractal dimension of CPI gels was found to increase from ~ 1.5-1.6 to ~ 1.8 as pH increased from 5.0/7.0 to 9.0, respectively suggesting diffusion-limited cluster-cluster aggregation. Images of SPI networks were not concurrent with fractal analysis under the conditions examined. Despite CPI having excellent gelling properties that are comparable to SPI, its need for alkaline pH conditions will limit its applicability in foods.     

A proteomic approach to detect lactosylation and other chemical changes in stored milk protein concentrate

Milk proteins undergo chemical changes such as lactosylation, deamidation and protein cross-linking during processing and storage of milk products. A proteomic technique combining two-dimensional gel electrophoresis and mass spectrometry was used to investigate chemical modifications to proteins, in milk protein concentrate (MPC80), during storage. Lactosylation, deamidation and protein cross-linking were observed on 2-DE gels. They were storage temperature-, humidity- and time-dependent. Lactosylated whey proteins were well separated on 2-DE in vertical stacks of spots. The masses of the spots varied by multiples of 324, indicating the attachment of lactose to lysine residues in the proteins. The trypsin-digested spots of α-lactalbumin were analysed by MALDI-TOF mass spectrometry, which indicated multiple lactosylation sites. The lactose adducts on gels were quantified by image analysis, allowing development of adducts over time to be monitored. The results show that proteomics can be used for the detection and quantification of chemical modifications to proteins in stored MPC80.     

Influence of pH and NaCl on rheological properties of rennet-induced casein gels made from UF concentrated skim milk

Milk concentrates are used in the production of cast cheese. The effects of pH (5.19–6.21) and NaCl concentration (0, 1.75% and 3.50%, w/w) on the rheological and microstructural properties of rennet-induced casein gels made from ultra filtered skim milk (19.8%, w/w casein) were investigated. Low pH and high NaCl concentration reduced the development rate of the gel elasticity after coagulation of the ultra filtrated skim milk. Strain at fracture and stress at fracture from uniaxial compression of casein gels 48 h after coagulation showed maximum and minimum values at pH ∼5.8 and 5.29, respectively. Young's modulus from uniaxial compression of the same gels was almost constant between pH 5.52 and 6.21 but much lower at pH 5.28. Addition of NaCl resulted in increased Young's modulus in the interval pH 6.21–5.52. As pH decreased, the level of colloidal calcium phosphate decreased concomitantly, giving less cross-links in the casein network and partly explaining the changes in the rheological properties. Increased ionic strength by adjusting pH and addition of NaCl also influenced rheological results. The microstructure examined with confocal laser-scanning microscopy was unaffected by the changes in pH and concentrations of NaCl in the range studied as revealed by image analysis and calculations of two- and three-dimensional data from micrographs.     

Gelling properties of protein fractions and protein isolate extracted from Australian canola meal

Gelling properties of canola albumin and globulin fractions, and canola protein isolate (CPI) were examined in this study. The effects of pH and salt concentration on canola protein gelling properties were studied primarily by means of dynamic oscillatory rheology and gel texture analysis. The findings were supported by confocal laser scanning microscopy (CLSM) images of the gels, isoelectric point, and solubility measurement data. All canola proteins showed typical heat-set gel protein profiles. Gels formed at higher pH had better gelling properties including higher overall resistance to deformation (G*), higher gel elasticity (low tan δ ), higher fracture stress and firmness, and denser gel microstructure. Isoelectric points of canola proteins used in this study were in the range of pH 3.0–4.7 where low protein solubility was observed. The albumin fraction was able to form a very weak gel at pH 4, whereas the globulin fraction and CPI precipitated due to loss of protein surface charge. The effects of NaCl on gelling were protein sample dependent. The presence of NaCl negatively affected gelling properties of albumin and globulin fractions, with decreases in overall resistance to deformation (G*), and fracture stress and firmness, but positively affected CPI gels in the same aspects. The elasticity (tan δ) of all canola protein gels remained constant in the presence of NaCl. Frequency sweep analysis revealed that the albumin fraction and CPI formed weak gels, whereas the globulin fraction formed a strong gel. Strain sweep analysis further confirmed that the globulin fraction formed a stronger gel with a critical strain of at least 10%. This study demonstrates the high potential of canola proteins, particularly the globulin fraction, as a prospective gelling agent.     

Effect of polysaccharides with different ionic charge on the rheological,microstructural and textural properties of acid milk gels

The effects of addition of polysaccharides with different ionic charge on rheology, microstructure, texture and water holding capacity (WHC) of acid milk gels were studied and compared to that of gelatin addition. Similar to gelatin, starch (neutral) and xanthan gum (anionic) did not prevent milk gelation in the first 30 min of the acidification stage, even at high concentrations, and the typical casein network in acid milk gels could still be seen from electron micrographs; gelling and melting of these hydrocolloids were observed during the cooling and heating stages at specific concentrations. On the other hand, two neutral polysaccharides, guar gum (≥ 0.05%) and locust bean gum [LBG] (≥ 0.1%) inhibited milk gelation from the beginning of the acidification stage; the microstructure of the gel was modified greatly and no gelling/melting was observed during the cooling or heating stages. Another anionic polysaccharide, carrageenan, induced earlier milk gelation at low concentration (≤ 0.05%), but inhibited gelation entirely at high concentration (0.2%); inflections at ~ 27 °C and 21 °C were also observed during the cooling and heating stages at 0.05% concentration. The gel microstructure was not changed greatly, but showed smaller particle size at a carrageenan concentration of 0.05% than control sample. None of the polysaccharides showed as much improvement in WHC of the milk gels as gelatin did. Hence, xanthan and starch were found to be closer to gelatin in their effect on acid milk gels compared to guar gum, LBG and carrageenan.