Characteristics of oil–water emulsions stabilised by an industrial α-lactalbumin concentrate,cross-linked before and after emulsification,by a microbial transglutaminase

Industrial α-lactalbumin concentrate, cross-linked with a microbial transglutaminase, showed lower dilational surface viscosity at a planer oil–water interface than a non-cross-linked α-lactalbumin concentrate. Properties of emulsions containing cross-linked α-lactalbumin were influenced by the sequence of cross-linking and emulsification. Emulsions stabilised by α-lactalbumin concentrate (even without crosslinking) were generally unstable. While cross-linking before emulsification decreased the stability further, the emulsion stability was improved when cross-linking was carried out after emulsification. Results from the sodium dodecyl sulphate (SDS) gel electrophoresis of adsorbed protein suggested that, irrespective of the sequence of cross-linking and emulsification, the adsorbed protein was polymerised too extensively to be resolvable on the gel matrix. Results from the reverse-phase HPLC suggested that the amount of adsorbed protein in emulsions containing protein cross-linking before emulsification was lower than that containing cross-linking after emulsification.     

Cross-linking of β-casein by Trichoderma reesei tyrosinase and Streptoverticillium mobaraense transglutaminase followed by SEC–MALLS

Enzymatic modification of proteins, in order to produce functional materials such as hydrogels, adhesives and films via cross-linked networks or scaffolds of proteins, is a constantly evolving technology to create tailored micro- and nanostructured materials for food, cosmetic, and medical applications. For the successful utilization of oxidoreductases or transferases such as tyrosinases and transglutaminases, respectively, it is crucial to understand the action of these enzymes on protein substrates. In this study, cross-linking of the milk protein β-casein by Trichoderma reesei tyrosinase (TrTyr) was studied using size-exclusion chromatography (SEC) equipped with multi-angle light scattering (MALLS) and ultraviolet/visible (UV/Vis) detectors in order to determine the molecular mass (MM), radius of gyration (R_G) and degree of polymerization (DP) of the reaction products. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect early polymerization states. The widely used Streptoverticillium mobaraense transglutaminase (Tgase) was used for comparison to tyrosinase from T. reesei. The results showed that cross-linking of β-casein by these two different types of enzymes resulted in the formation of polymerized reaction products with MM ranging from 500 to 1700 kg mol⁻¹ depending on the enzyme dosage and incubation time. The DP varied from 21 to 71, respectively. In the case of TrTyr the polymerized reaction products were slightly colored, and formation of the covalent cross-linking of β-casein could be monitored by UV/Vis as a function of incubation time.     

Skin gelatin from bigeye snapper and brownstripe red snapper: Chemical compositions and effect of microbial transglutaminase on gel properties

Fish skin gelatin was extracted from the skin of bigeye snapper (Priacanthus macracanthus) and brownstripe red snapper (Lutjanus vitta) with yields of 6.5% and 9.4% on the basis of wet weight, respectively. Both skin gelatins having high protein but low fat content contained high hydroxyproline content (75.0 and 71.5 mg/g gelatin powder). The bloom strength of gelatin gel from brownstripe red snapper skin gelatin (218.6 g) was greater than that of bigeye snapper skin gelatin (105.7 g) (P<0.05). The addition of microbial transglutaminase (MTGase) at concentrations up to 0.005% and 0.01% (w/v) increased the bloom strength of gelatin gel from bigeye snapper and brownstripe red snapper, respectively (P<0.05). However, the bloom strength of skin gelatin gel from both fish species decreased with further increase in MTGase concentration. SDS-PAGE of gelatin gel added with MTGase showed the decrease in band intensity of protein components, especially, β- and γ- components, suggesting the cross-linking of these components induced by MTGase. Microstructure studies revealed that denser and finer structure was observed with the addition of MTGase.     

Polymerization of β-lactoglobulin and bovine serum albumin at oil-water interfaces in emulsions by transglutaminase

Polymerization of β-lactoglobulin and bovine serum albumin at the oil—water interfaces in n-tetradecane-in-water emulsions induced by the transglutaminase reaction was studied. The emulsions were incubated with transglutaminase for various times, and adsorbed and unadsorbed protein fractions at the oil—water interfaces were analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis. While only monomers were detected in the unadsorbed fractions, polymers were observed in the adsorbed fractions of the both proteins. The sizes and amounts of the polymers increased with incubation time. The incubation with transglutaminase caused much flocculation of the emulsion stabilized by β-lactoglobulin. An increase in viscosity was also observed with the flocculation. The flocculation was probably initiated by the formation of ε-(γ-glutamyl)-lysyl isopeptide bonds between β-lactoglobulin molecules adsorbed on different oil droplets. In the case of the emulsion stabilized by bovine serum albumin, however, the flocculation and the increase in viscosity occurred to only limited extents by the transglutaminase reaction. This suggests that ε-(γ-glutamyl)-lysyl isopeptide bonds induced by the transglutaminase reaction were formed only between neighboring molecules of bovine serum albumin on the same droplet.     

Influence of other whey proteins on the heat-induced aggregation of α-lactalbumin

The thermally induced aggregation of α-lactalbumin in solution and in the presence of serum protein-free casein micelles has been studied using gel permeation chromatography. No aggregation of α-lactalbumin was detected after heating at 90°C for 24 min. However, addition of β-lactoglobulin or serum albumin to the serum protein-free casein micelles + α-lactalbumin system caused aggregation of the α-lactalbumin, the rate and extent of this aggregation being dependent upon the concentration of free sulphydryl groups present in the other whey protein. It is therefore the sulphydryl group which is important and which appears to function by inducing cleavage of intramolecular disulphide bonds in the α-lactalbumin. This leads to the formation of intermolecular disulphide bridges and hence to aggregation of the α-lactalbumin.     

Evaluation of microbial transglutaminase and ribose cross-linked soy protein isolate-based microcapsules containing fish oil

Soy protein isolate (SPI)-based microcapsules containing fish oil were prepared using a modified coacervation method followed by cross-linking treatments. The procedure yielded 95–98% microcapsules containing 0.5–0.6 g fish oil/g capsule with a volume mean diameter ranged from ~ 260 to ~ 280 μm. Four types of microcapsules produced were SPI with sucrose (MC-C/S), SPI with ribose (MC-C/R), SPI with sucrose and microbial transglutaminase (MC-MTG/S) and SPI with ribose and MTGase (MC-MTG/R). Protein cross-linking due to ε-(γ-glutamyl)lysine bonds and “Maillard cross-linking” were evidenced in the SDS-PAGE profiles of MC-C/R, MC-MTG/S and MC-MTG/R. Even though the microcapsules prepared with cross-linking treatments had significantly (P < 0.05) lower protein solubility as compared to that of the control, the results of fish oil release in pepsin solution at 37 °C indicated that the core release of all microcapsules prepared with ribose (MC-C/R and MC-MTG/R) was significantly (P < 0.05) lower than other microcapsules. During storage, microcapsules prepared with ribose had longer shelf life as compared to other microcapsules. This may be due to the release of antioxidative Maillard reaction products during heating and storage and a slower rate of gas permeability through the capsules.

Industrial relevance

The use of protein-based wall materials in the food industry for sensitive ingredients is limited because proteins are generally unstable with heating and damaged by organic solvents and the cross-linking agent is usually harmful. Therefore a novel method of combining two familiar cross-linking agents such as the microbial transglutaminase and ribose can convert SPI microcapsules into a stable form. The application of SPI in industry would be increased.     

Enzymatic cross-linking of ewe's milk proteins by transglutaminase

Enzymatic cross-linking of ewe's milk proteins in the presence of transglutaminase was studied and the extent of cross-linking was analysed by capillary gel electrophoresis. Up to now, no publications are available that study the relative susceptibility of individual ewe's milk proteins. Transglutaminase has been demonstrated to induce cross-linking of the ewe's milk proteins. Moreover, a heat treatment of the milk before the reaction with transglutaminase enhanced the susceptibility of the individual ewe's milk proteins towards the cross-linking reaction. The specificity of transglutaminase has been shown to vary with the type of ewe's milk proteins (α_s2-casein, α_s1-casein, α_s0-casein, κ-casein, β-casein A¹, β-casein A², α-lactalbumin and β-lactoglubulin). From our findings, the reactivity for ovine α-caseins was reduced with respect to that of ovine κ-casein and ovine β-caseins. An optimisation strategy based on desirability functions together with experimental design has been used to optimise the preheating conditions (temperature and time) of ovine milk that maximised the cross-linking reactions catalysed by transglutaminase.     

On solid-like rheological behaviors of globular protein solutions

Dynamic viscoelastic and steady flow properties of β-lactoglobulin, bovine serum albumin, ovalbumin, and α-lactalbumin aqueous solutions were investigated at 20°C. When a sinusoidal strain in the linear viscoelastic region was applied, the solutions of the globular proteins except for α-lactalbumin showed typical solid-like rheological behavior: the storage modulus G′ was always larger than the loss modulus G″ in the entire frequency range examined (0.1–100 rad/s). Under a steady shear flow, strong shear thinning behavior was observed with increasing shear rate from 0.001 to 800 s⁻¹, for the globular proteins except for α-lactalbumin. The values of the steady shear viscosity η were lower than those of the dynamic shear viscosity η* at a comparable time scale of observation, violating the Cox–Merz rule, and thus suggesting that a solid-like structure in a globular protein solution was susceptible to a steady shear strain. During isothermal gelation of the protein colloids at 70°C, no crossover between G′ and G″ was observed so that the gelation point was judged by an abrupt increase in the modulus or a sudden decrease in tanδ.     

Competitive adsorption of α-lactalbumin in the molten globule state

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

Application and sensory evaluation of enzymatically texturised vegetable proteins in food models

Using simplified model systems, the effects of salts and oil on enzymatic texturisation of protein isolates from soy (Glycine max (L.) Merr.; SPI), pea (Pisum sativum L.; PPI) and sweet lupine (Lupinus albus L.; LPI) were evaluated. In aqueous systems, protein cross-linking by microbial transglutaminase (MTG) was significantly improved when NaCl (1–2 g hg⁻¹) was added, but respective doses of CaCl₂ reduced gel strengths. As shown by emulsion model systems of PPI and SPI with oil/protein ratios of 1 and 2 g g⁻¹, emulsification of corn oil into aqueous protein suspensions prior to enzymatic cross-linking enhanced gel formation depending on the emulsification technique. The impact of NaCl and oil varied among the protein isolates as to obtainable maximum gel strengths and optimum doses of these ingredients. The applicability of MTG to leguminous proteins in complex plant foodstuffs was finally deduced from their performance in complex food models of the liquid (thickened soup), foamed (mousse) and solid (sausage-like substitute) type, respectively. The sensory characteristics of the latter were evaluated by trained panellists relative to their milk-, gelatin- and meat-based counterparts. Texture was appealing in the foamed and solid food models, but the liquid soup model suffered from unfavourable grittiness. Without masking their beany off-flavour, the food models containing leguminous proteins deviated from the reference products. On the whole, MTG-catalysed cross-linking rendered the leguminous proteins suitable for the food applications in terms of visual appearance, texture and colour. Especially, the gels representing mousse-type foams and cuttable sausage-like vegetarian substitutes were very promising.     

Rheological characterizations of texturized whey protein concentrate-based powders produced by reactive supercritical fluid extrusion

A powder blend comprising (by weight) 94% whey protein concentrate (WPC80), 6% pre-gelatinized corn starch, 0.6% CaCl₂, and 0.6% NaCl was texturized using a supercritical fluid extrusion (SCFX) process. The blend was extruded at 90 °C in a pH range of 2.89 to 8.16 with 1% (db) supercritical carbon dioxide (SC-CO₂) and 60% moisture content. The texturized WPC-based (TWPC) samples were dried, grounded into powder, reconstituted in water, and evaluated using a range of rheological studies. Most TWPC samples exhibited shear thinning behavior and their mechanical spectra were typical of weak gel characteristics. The TWPC produced under extremely acidic condition of pH 2.89 with SC-CO₂ yielded the highest η^* (10,049 Pa s) and G′ (9,885 Pa) compared to the unprocessed WPC (η^* = 0.083 Pa s and G’ = 0.036 Pa). The SCFX process rendered WPC into a product with cold-setting gel characteristics that may be suitable for use as a food texturizer over a wide range of temperatures.     

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

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

Enhancement of transglutaminase-induced protein cross-linking by preheat treatment of cows’ milk: A statistical approach

Optimization of heat treated milk towards protein cross-linking induced by transglutaminase was carried out. Capillary electrophoresis was employed to study the extent of cross-linking under different preheating temperatures (70–90 °C) and times (15–60 min). The experiments were arranged according to a central composite statistical design (3²+centre points). Response surface methodology was used to assess factor interactions and empirical models regarding relative peak area (%) of individual protein (α_s2-casein, α_s1-casein, α_s0-casein, κ-casein, β-casein A¹, β-casein A², α-lactalbumin and β-lactoglobulin) and total α_s-caseins, total β-caseins and whey proteins (sum of α-lactalbumin and β-lactoglobulin). Multi-response optimization was also performed on the total α_s-caseins, total β-caseins, κ-casein and whey proteins data set of the factorial design. The desirability function was the statistical tool employed in this multi-optimization step. The optimum preheating conditions that maximized the cross-linking reactions catalyzed by transglutaminase were achieved within 60 min at 84.5 °C.     

Influence of complexing carboxymethylcellulose on the thermostability and gelation of α-lactalbumin and β-lactoglobulin

Thermostability and gelation of the main proteins of whey, α-lactalbumin (α-lac) and β-lactoglobulin (β-lg) recovered by selective complexation with carboxymethylcellulose (CMC) was studied to evaluate its functionality in food systems. Their behavior was compared to the non-complexed proteins. Both complexes showed a maximum stability at pH 4, that is close to the pH of obtention of β-lg/CMC coacervate (pH 4) and α-lac/CMC coacervate (pH 3.2). Protein complexation increased the thermostability of β-lg by approximately 6–8 °C and that of α-lac by approximately 26 °C due to immobilization of protein molecules in a complex, mainly by electrostatic interactions and because of different amounts of bound polysaccharide. The denaturation enthalpy of complexed proteins markedly decreased as compared to free proteins. Storage modulus (G′) and loss modulus (G″) were recorded to reflect the structure development during heating β-lg/CMC and α-lac/CMC complexes at different pH values. β-lg/CMC complex at 20 wt% was a viscoelastic liquid at pH values within 2 and 8 but upon heating turned to a particulate viscoelastic gel. However, α-lac/CMC complex formed before heating opaque, large visible white particulate aggregates that sticked together to give a solid viscoelastic structure that was not further modified by thermal processing.     

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.     

Heat-induced aggregation of β-lactoglobulin A and B with α-lactalbumin

β-Lactoglobulin A and β-lactoglobulin B were heated at 75°C in the absence and presence of α-lactalbumin, and the aggregation products were characterized by size exclusion chromatography in combination with multi-angle laser light scattering and electrophoretic techniques. α-Lactalbumin did not form aggregates when heated alone, but in admixture with β-lactoglobulin it was incorporated into both the disulphide-bonded and the hydrophobically associated aggregates as well as forming α-lactalbumin dimers and other oligomers. The presence of α-lactalbumin diminished the proportion of smaller aggregates and increased the number of very large aggregates within both variant protein mixtures. In the presence of α-lactalbumin, β-lactoglobulin A was converted into a series of disulphide-bonded and the hydrophobically associated aggregates more slowly, but with a greater proportion of hydrophobically associated aggregates, than β-lactoglobulin B. These patterns are similar to that when β-lactoglobulin A or B are heated on their own. These and other results indicate that the mechanism of aggregation of α-lactalbumin/β-lactoglobulin mixtures is governed by β-lactoglobulin.     

Enzymatic properties of transglutaminase produced by a new strain of Bacillus circulans BL32 and its action over food proteins

The aim of this study was to physicochemically characterize transglutaminase (TGase) from Bacillus circulans BL32, a strain recently isolated from the Amazon basin region, for its application in food systems. The effects of pH and temperature on the enzyme activity were determined by Central Composite Rotatable Design (CCRD), with maximal TGase activities obtained for pH between 5.7 and 8.7 and temperatures of 25-45 °C. This microbial TGase showed to be remarkably stable: over 90% of its activity was retained after 120 min of incubation at 50 °C. The Ca²⁺ and Mg²⁺ cations enhanced enzyme activity and its thermal stability when in concentrations of up to 2 and 1 mol L⁻¹, respectively. Casein, isolated soy protein, and hydrolysed animal protein were treated with this TGase. The decrease in the amount of free amino groups, especially for casein, showed the cross-linking of protein catalysed by this enzyme, while the emulsifying properties of these proteins were improved with treatment. These results suggest that this microbial TGase has a good potential to be used in food and other industrial applications.     

Gelling of microbial transglutaminase cross-linked soy protein in the presence of ribose and sucrose

Soy protein isolate (SPI) was incubated with microbial transglutaminase (MTGase) enzyme for 5 (SPI/MTG(5)) or 24 (SPI/MTG(24)) h at 40 °C and the cross-linked SPI obtained was freeze-dried, and heated with 2% (w/v) ribose (R) for 2 h at 95 °C to produce combined-treated gels. Longer incubation period resulted in more compact and less swollen SPI particle shape when reconstituted with sugar solution. Thus, this MTGase treatment affected samples in terms of flow behaviour and gelling capacity. Rheological study showed different gelling profiles with the cross-linking treatments and combined cross-linked SPI gave a higher G′ value compared to single treated samples. These are due to the formation of additional ε-(γ-glutamyl)lysine bonds and “Maillard cross-links” within the SPI protein network during the MTGase incubation and heating in the presence of ribose (i.e. reducing sugar). Network/non-network protein analysis found that network protein increased with cross-linking treatment, which also resulted in different SDS–PAGE profiles. As in non-network protein fraction, A₄ subunit was suggested to become part of the network protein as a result of combined cross-linking.     

Enzymatic modification of protein preparation obtained from water-washed mechanically recovered poultry meat

Studies were conducted on a protein preparation obtained from washed mechanically recovered poultry meat (MRPM). The effect of addition of 3 g/kg microbial transglutaminase (MTG) to poultry meat protein was evaluated in terms of texture changes by dynamic mechanical analysis (DMA) and nuclear magnetic resonance (NMR) to determine water content in the preparation and its effect on protein. Samples with the addition of MTG were pre-incubated at 5–6 °C for 1.5, 3, 4.5, 6, 7.5, 9 and 24 h. The largest changes for both texture parameters and rheological properties were observed in the interval of approx. 4–7 h incubation. The protein preparation with the enzyme added had significantly higher values of the moduli of elasticity (G₁) and losses (G₂) in comparison to the control system. Samples with the addition of MTG also showed a higher water-binding capacity. From the NMR studies it was found that the greatest amount of water was bound by protein in the period of approx. 2.5–5 h incubation. After that time an increase was found in the amount of free water in the sample, which suggests that it was displaced from the system by stronger protein–protein bonds.     

Structural characterization of barley β-glucan extracted using a novel fractionation technique

A barley β-glucan concentrate prepared according to a novel technology was further purified and subjected to detailed structural characterization by NMR spectroscopy. β-Glucan was hydrolysed with β-glucan-4-glucanohydrolase (lichenase). Fractions of hydrolysate were collected using an HPLC-fraction collector. Intact β-glucan and the major fractions collected were subjected to MALDI-TOF–MS and NMR analyses. The two major oligosaccharides produced by lichenase hydrolysis of purified barley β-glucan were identified as β-d-Glc p-(1 → 4)- β-d-Glc p-(1 → 3)-β-d-Glc p and β-d-Glc p-(1 → 4)-β-d-Glc p-(1 → 4)-β-d-Glc p-(1 → 3)-β-d-Glc p based on ¹³C and ¹H NMR data. Spectrums were similar to those documented for barley β-glucan in the literature.