Rheological,gelling and emulsifying properties of a glycosylated and cross‐linked caseinate generated by transglutaminase

The impacts of oligochitosan glycosylation and cross‐linking on some properties of a commercial caseinate were investigated in this study. The glycosylated and cross‐linked caseinate with glucosamine content of 4.74 g kg^?1 protein was generated by transglutaminase (TGase) and oligochitosan at pH 7.5 and 37 °C, with fixed substrate molar ratio of 1:3 (acyl donor to glucosamine acceptor), caseinate content of 50 g L^?1, TGase of 10 kU kg^?1 protein and reaction time of 3 h, respectively. In comparison with the caseinate, the glycosylated and cross‐linked caseinate had decreased reactable amino groups (0.58 vs. 0.51 mol kg^?1 protein), higher apparent viscosity, decreased emulsifying activity index (about 14.5%) and statistically unchanged emulsion stability index (92.6 vs. 90.5%). Based on the mechanical spectra of the acid‐induced gels, the glycosylated and cross‐linked caseinate showed shorter gelation time (95 vs. 200 or 220 min) than the caseinate or cross‐linked caseinate. The gels prepared from the glycosylated and cross‐linked caseinate also had enhanced hardness, springiness and cohesiveness. The results indicated that TGase‐mediated oligochitosan glycosylation and cross‐linking has the potential to obtain new protein ingredients.     

A study on the preparation and some functional properties of a cross‐linked casein–gelatin composite by a microbial transglutaminase

A microbial transglutaminase (TGase) was used in this work as biocatalyst to prepare a cross‐linked casein–gelatin composite, a modified protein product with 4‐hydroxyproline about 41 mg g^?1 peptides. Some cross‐linking conditions such as total protein content, the ratio of caseinate to gelatin and original pH were fixed at 5% (w/v), 4:1 (w/w) and 7.5, respectively. Other suitable conditions selected by single factor trials were TGase addition of 20 U g^?1 peptides, reaction temperature of 45 °C and reaction time of 4 h. Peptide profiles from SDS‐PAGE analysis showed the composite was peptide polymers. Compared to that of the original caseinate or the cross‐linked caseinate, the dispersion of the composite exhibited a markedly enhanced apparent viscosity, storage modulus and viscous modulus. Meanwhile, the composite also showed a better water holding capacity, unchanged oil binding capacity and lower enzymatic digestibility in vitro, conferring its applicability as a new protein ingredient.     

Effect of phosphatase/transglutaminase treatment on molar mass distribution and techno-functional properties of sodium caseinate

Sodium caseinate was enzymatically dephosphorylated by alkaline or acid phosphatase prior to incubation with microbial transglutaminase. It was demonstrated that a higher degree of protein cross-linking by transglutaminase was achieved in dephosphorylated sodium caseinate than in non-dephosphorylated sodium caseinate. During transglutaminase treatment, about 70% protein polymers >200.000 g/mol were produced from untreated sodium caseinate, but about 90% protein polymers >200.000 g/mol from dephosphorylated sodium caseinate. Phosphatase/transglutaminase-treated sodium caseinate exhibited techno-functional properties similar to transglutaminase-treated sodium caseinate, but performed improved interfacial stabilisation behaviour as well as higher viscosity.     

Zinc‐loaded whey protein nanoparticles prepared by enzymatic cross‐linking and desolvation

Zinc‐loaded whey protein nanoparticles were prepared by enzymatic cross‐linking whey protein followed by ethanol desolvation. Whey protein isolate (WPI) was denatured by heating (80 °C for 15 min) at pH 7.0 and then cross‐linked by transglutaminase at 50 °C for 4 h while stirring. Transglutaminase was inactivated by heating at 90 °C for 10 min, and then, ZnSO₄·7H₂O (1–10 mm ) was added. Zinc‐loaded whey protein nanoparticles were formed by adding ethanol at one to five times the volume of the protein solution at pH 9.0. The desolvated solutions were diluted by adding distilled water at ratio of 1:100 (w/v) immediately after desolvation. Dynamic light scattering (DLS) data showed that the particle size of zinc‐loaded whey protein nanoparticles increased with the amount of zinc and the volume of ethanol. Scanning electron microscopy micrographs revealed an almost spherical morphology for zinc‐loaded whey protein nanoparticles. The zinc loading efficiency was determined ranging from 76.7% to 99.2%. In vitro test data showed that the zinc release rate was low in simulated gastric fluid but high in simulated intestinal fluid. The results indicated that enzymatic cross‐linked whey protein nanoparticles may be used as a good vehicle to deliver zinc as a supplement.     

The quality of set‐style yoghurt responsible to partial lactose hydrolysis followed by protein cross‐linking of the skimmed milk

Skimmed milk used for set‐style yoghurt production was treated with lactase at 0.1 g/kg for 30 min to give partial lactose hydrolysis and then treated with horseradish peroxidase and glucose oxidase at 200 and 6 kU/kg protein to result in protein cross‐linking. Two treatments conferred higher apparent viscosity on the milk, but led to the yoghurt prepared from it with insignificantly different chemical compositions to the counterparts (P > 0.05). The prepared yoghurt also showed decreased syneresis (about 17.7%), higher apparent viscosity and viscoelastic modulus, firmer texture and finer microstructure. This ternary enzyme system is a potential approach to improving the quality of set‐style yoghurt.     

Structure and Property Changes of Transglutaminase-Induced Modification of Sodium Caseinate in the Presence of Oligochitosan of 5 kDa

Sodium caseinate was modified by transglutaminase-catalyzed reaction in the presence of oligochitosan of 5 kDa at two different levels, aiming to generate two glycated and crosslinked caseinate products (GC-caseinate I and II) and to clarify their respective structure and property changes. Electrophoretic analysis with aid of protein and saccharide staining confirmed that both GC-caseinate I and II were glycated and crosslinked protein products, while high-performance liquid chromatrography analysis demonstrated that both GC-caseinate I and II contained glucosamine (12.8 and 30.8 g/kg protein, respectively). In comparison with sodium caseinate, GC-caseinate I and II also contained less reactable –NH₂ groups (0.50 and 0.52 versus 0.62 mol/kg protein), more –OH groups in their molecules, but they had more ordered secondary structure than sodium caseinate. In addition, GC-caseinate I and II showed higher water-binding capacity, larger hydrodynamic radius (173.9 and 168.2 versus 145.3 nm), and larger negative zeta-potential (–32.9 and –30.9 versus –27.6 mV) in aqueous dispersions, but they exhibited lower thermal stability (namely, greater mass loss) at temperature higher than 286ºC. Oligochitosan-glycated sodium caseinate at lower and higher extents was observed to be unfavorable and favorable to the in vitro digestibility of GC-caseinate I and II, respectively. It is concluded that application of transglutaminase and the oligochitosan can modify structure and property of sodium caseinate to generate new protein ingredients with good hydration and colloidal stability.     

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

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

Effect of waxy rice starch on textural and microstructural properties of microwave‐puffed cheese chips

Protein‐based expanded snacks sometimes do not have homogeneous structures. However, starches are widely used in expanded food snacks because of their good puffing characteristics. We aimed to use waxy rice starch to partially substitute for sodium caseinate in caseinate‐based and caseinate plus soya protein (double‐protein) microwave‐puffed imitation cheese snacks to improve their textural, microstructural and sensory properties. The single‐protein chips without waxy rice starch and the double‐protein chips with a low degree of expansion were not well appreciated, whereas the chips with up to 8 g of waxy rice starch per 100 g of product were scored highly by the panellists. Our results demonstrated that waxy rice starch can improve the expansion, homogeneity, hardness, crispness and colour of imitation cheeses.     

Cross‐linking of bovine and caprine caseins by microbial transglutaminase and their use as microencapsulating agents for n‐3 fatty acids

Bovine and caprine caseins were cross‐linked with microbial transglutaminase (mTG). The mTG‐cross‐linked bovine or caprine casein dispersion, mixed with 14.5% maltodextrin (DE = 40), was used to prepare emulsions with 10.5% algae oil. Oxidative stability of emulsions was evaluated by peroxide values (PVs) and anisidine values. Adding liposoluble rosemary extract rich in carnosic acid and δ‐tocopherol lowered the formation of hydroperoxides and their subsequent decomposition products in emulsions. Emulsions stabilised with liposoluble rosemary extract rich in carnosic acid and δ‐tocopherol were spray‐dried at 180/95 °C. Algae oil microencapsulated with mTG‐cross‐linked bovine casein reduced PV by ≈ 34%, while the algae oil microencapsulated with mTG‐cross‐linked caprine casein with low levels of α_s1‐casein reduced PV by ≈ 42% at 4 weeks of storage at 30 °C. The investigation suggests that liposoluble rosemary extract rich in carnosic acid and δ‐tocopherol effectively protected algae oil during the coating process with mTG‐cross‐linked bovine and caprine caseins. The above results clearly indicated that the choice of milk caseins (bovine vs. caprine) cross‐linked with mTG impacts the oxidative stability of spray‐dried algae oil emulsions (microcapsules) enriched with n‐3 fatty acids.     

Characterisation and quantification of the reaction(s) catalysed by transglutaminase using the o‐phthaldialdehyde reagent

The potential application of the o‐phthaldialdehyde (OPA) reagent for quantification of the type and extent of the reaction(s) catalysed by transglutaminase (TGase) during incubation with sodium caseinate (NaCN) was investigated. Initial studies were performed to ensure that NH₃, a by‐product of TGase activity, could be determined with the OPA reagent in trichloroacetic acid (TCA) supernatants of NaCN solutions. The detectable concentration of exogenously added NH₃ (at NH₃ concentrations > 10 mM ) was found to decrease during extended incubation at 23, 37 and 50°C and at either pH 7.0 or 8.0 in 4% w/v NaCN solutions, even when taking into account the evaporation of water from the sample. The recovery of NH₃ from 12% w/v TCA supernatants of NaCN solutions spiked with 5 mM NH₃ at 23°C and pH 7.0 was found to be 88%. The release of NH₃ and the decrease in ε‐amino groups on incubating NaCN with TGase was subsequently quantified using the OPA reagent. Incubation of NaCN (4% w/v) with TGase at 23°C resulted in progressive increases and decreases, respectively, in NH₃ and ε‐amino group concentration with increased incubation time. These changes were dependent on TGase : NaCN. It was estimated that approximately 20% of the available Lys residues in NaCN were involved in TGase‐catalysed cross‐links. However, the observed decrease in ε‐amino group concentration was higher than expected. This may be due to concealment of noncross‐linked amino groups in polymerised NaCN, making them unavailable for reaction with the OPA reagent.     

Casein gelation under simultaneous action of transglutaminase and glucono‐δ‐lactone

Casein solutions (5% w/v) were treated with microbial transglutaminase (MTG) and glucono‐δ‐lactone (GDL) under varying conditions in order to obtain gels. Storage modulus (G ′) and gelation time of the gels were measured by oscillation rheometry, while protein cross‐linking was determined by gel permeation chromatography. The addition of only GDL to milk resulted in very weak gels, while MTG on its own was not able to create gel networks. Simultaneous action of both ingredients led to gels, the firmness of which was linearly related to the added amount of MTG, but passed through a maximum with rising GDL concentrations. Using chromatographical analysis, increasing G ′ values were interrelated with the formation of MTG‐induced oligomers. The gelation time was directly proportional to the GDL concentration but not influenced by the addition of MTG within the studied range of concentration.     

Gel‐enhancing effect and protein cross‐ linking ability of tilapia sarcoplasmic proteins

BACKGROUND: Tilapia (Oreochromis niloticus) sarcoplasmic proteins contain substantial transglutaminase (TGase) activity. The enzyme catalyzes the protein cross‐linking reaction, resulting in a more elastic gel. The objective was to investigate the gel‐enhancing effect of sarcoplasmic proteins from tilapia as related to TGase activity. RESULTS: Total TGase activity of sarcoplasmic proteins concentrate (SpC) increased about 3.6‐fold after ultrafiltration using 30 kDa membrane, but specific activity remained unchanged, indicating minimal TGase purification by ultrafiltration. Addition of 1 mg mL^?1 SpC containing 40 units TGase activity induced cross‐linking of tilapia actomyosin, and the extent of cross‐linking increased with added level of SpC. Myosin heavy chain (MHC) and troponin were preferably cross‐linked by tilapia SpC, while actin and tropomyosin were not affected. Higher retention of MHC was observed concomitantly with greater content of cross‐linked protein when SpC was added to lizardfish surimi. Lizardfish surimi with 10 g kg^?1 SpC added and pre‐incubated at 37 °C for 1 h exhibited 91.6% and 26.7% increase in breaking force and deformation, respectively, when compared to the control. CONCLUSIONS: Residual TGase activity in SpC played an important role in catalyzing the protein cross‐linking and enhancing actomyosin gelation. SpC could be a potential ingredient for improving textural properties of fish protein gel. Copyright © 2007 Society of Chemical Industry     

Appetite‐Inducing Effects of Homoeriodictyol: Two Randomized,Cross‐Over Interventions

1 Scope

Anorexia of aging, characterized by a decrease in appetite and/or food intake, is a major risk factor of under‐nutrition and adverse health outcomes in elderly people. Recent in vitro evidence suggests homoeriodictyol (HED), a naturally occurring, bitter‐masking flavanone, as a promising agent to increase appetite and food intake.

2 Methods and results

In two cross‐over intervention trials, 30 mg NaHED, either solely (n = 10, Study I) or in combination with a 75 g glucose load (n = 17, study II) were administered to healthy adult subjects. Ratings of hunger were assessed at fasting and either 30 min (Study I) or 120 min (Study II) post intervention. Ad libitum energy intake from a standardized breakfast and plasma changes in hunger‐/satiety‐associated hormones PYY, GLP‐1, ghrelin and serotonin were determined after blood drawings. Effects were more pronounced when NaHED was administered in combination with 75 g glucose since ad libitum energy (+ 9.52 ± 4.60%) and protein (+ 7.08 ± 7.97%) intake as well as plasma ΔAUC ghrelin values increased in study II solely, whereas plasma serotonin concentrations decreased after both interventions.

3 Conclusions

NaHED demonstrated appetizing effects in healthy adults when administered with a glucose load. Long‐term intervention studies are warranted to verify these effects in compromised subjects.     

Evaluation of mechanical and water barrier properties of transglutaminase cross‐linked zein films incorporated with oleic acid

In this study, four concentrations of transglutaminase were used in zein films incorporated with four oleic acid concentrations, and subsequently, the mechanical and water barrier properties were evaluated. Enzyme concentration significantly affected mechanical and barrier properties of the films. Transglutaminase concentration at 1% improved tensile strength of control sample from 17.5 to 26.9 MPa while solubility decreased from 6.4% to 4.4%. Incorporation of oleic acid into transglutaminase‐treated zein films irrespective of enzyme concentration decreased water vapour permeability and solubility with the 1% transglutaminase‐treated zein films incorporated with 3% oleic acid registering the lowest permeability (0.37 g mm m^?2 h^?1 kPa^?1) and solubility (2.8%) values while elongation at break was not significantly improved. The use of transglutaminase at 1% concentration in cross‐linking zein films coupled with incorporation of controlled concentrations of oleic acid can be an effective approach in improving mechanical and water barrier properties of zein‐based films.     

Functional properties of limited hydrolysed cross‐linked casein–gelatin composites

A casein–gelatin composite was prepared by cross‐linking of caseinate and bovine gelatin with a microbial transglutaminase, and the impact of limited proteolysis by trypsin on some functional properties of the composite was investigated in the present work. Two hydrolysed composites were prepared with degree of hydrolysis (DH) of 1 and 2% and analysed by SDS‐PAGE to reflect polypeptide profiles. Some functional properties of the two hydrolysed composites were evaluated, among which solubility, digestibility in vitro, surface hydrophobicity and emulsifying properties showed dependence on the DH. Limited proteolysis of the composite improved its solubility in pH 3–10, especially when the DH was 2%. Compared to the composite, the hydrolysed composites showed an increased emulsifying activity index (about 357–408%), emulsion stability index (about 23–28%) and digestibility in vitro (about 65–80% for pepsin, whereas 2–3% for pepsin–trypsin hydrolysis), together with a decreased surface hydrophobicity (about 55–62%) and oil absorption capacity (about 20–23%). The applied proteolysis also led to the aqueous dispersions of the two hydrolysed composites much lower apparent viscosity, storage and loss modulus.     

Improvement of microbial transglutaminase‐induced gelation of β‐conglycinin by conjugation with dextran

This work focused on the effect of glycosylation on the gelation ability of β‐conglycinin induced by microbial transglutaminase (MTGase). Rheological results indicated that the gels of β‐conglycinin‐dextran conjugate products exhibited higher G′ value (172.2 ± 8.6 Pa) compared with those of dry‐heated β‐conglycinin (75.2 ± 5.1 Pa), β‐conglycinin (53.3 ± 4.0 Pa) and β‐conglycinin‐dextran mixture (38.6 ± 2.6 Pa) after 4 h incubation with MTGase. The gels prepared from β‐conglycinin‐dextran conjugate products had higher hardness, fracturability, springiness and cohesiveness values determined by textural profile analysis. The turbidity of β‐conglycinin‐dextran conjugate products solution incubated with MTGase increased faster than those of the other three protein samples. The conjugated dextran in β‐conglycinin‐dextran conjugate products could inhibit extensive protein–protein interactions which might result in the formation of more ordered and stronger gel network structures during MTGase cross‐linking process. A compact and homogeneous gel networks in β‐conglycinin‐dextran conjugate products gels were also observed by scanning electron microscopy.     

Effect of a microbial calcium‐independent transglutaminase on functional properties of a partially purified cowpea (vigna unguiculata) globulin

A partially purified globulin from cowpea seeds was polymerised by calcium‐independent microbial transglutaminase. The level of free amino groups in the globulin decreased with increase in enzyme concentration, suggesting that the cross‐linking reaction involved participation of the amino residues. At transglutaminase concentrations of 6–18 μg ml⁻¹ emulsifying activity decreased with increasing enzyme concentration and degree of cross‐linking, while a similar trend was observed for the foaming property at 6–12 μg ml⁻¹ transglutaminase concentrations. The resultant emulsions and foams formed by the cross‐linked proteins were more stable than those formed by the untreated protein. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that of the two major polypeptides, the 55 kDa protein was more susceptible to transglutaminase action than the 50 kDa protein. Scanning electron microscopy revealed an amorphous structure for the control protein gel while a more defined and cross‐linked gel structure was observed for the protein treated with transglutaminase. The results suggest that protein products differing in functionalities can be obtained by controlling the degree of enzymatic protein cross‐linking. © 1999 Society of Chemical Industry     

Proximate composition,provitamin A retention,and shelf life of extruded orange‐fleshed sweet potato and bambara groundnut‐based snacks

Evaluating the effect of processing on nutrient content and shelf life is important when developing new products. Six formulations of orange‐fleshed sweet potato (OFSP) and bambara groundnut were extruded at a feed rate of 10.15 kg/hr, screw speed of 30 rpm and at 100 and 130 °C in first and second zones respectively. Proximate composition was determined using standard methods. Provitamin A was determined using high‐performance liquid chromatography. An untrained panel (n = 73) was used to determine consumer acceptability. Shelf life was predicted by using peroxide values. Concentration of OFSP or bambara groundnut significantly (p < .05) affected proximate composition of the snacks. Moisture (4.79–8.34 g/100 g), carbohydrates (55.53–78.99 g/100 g) and provitamin (0.54–17.33 mg/100 g) contents increased with increase in proportion of OFSP. Protein (4.08–15.03 g/100 g), fat (4.20–12.74 g/100 g), fiber (5.29–6.46 g/100 g), ash (0.09–4.80 g/100 g), and energy (366.13–396.9 kcal/100 g) increased with increasing proportion of bambara groundnut in the formulation. Extrusion significantly (p < .05) reduced provitamin A content from 0.90–20.73 to 0.54–17.33 mg/100 g. Presence of OFSP improved provitamin A retention and consumer acceptability. Predicted shelf life (ranged from 118 to 150 days at room temperature) was inversely proportional to the concentration of bambara groundnut.

Practical applications

Vitamin A deficiency (VAD) is the leading cause of preventable blindness affecting many children and women of reproductive age globally. Provitamin A enriched foods can be developed from locally available provitamin A‐rich foods and their consumption promoted to help prevent VAD. In this study, recipes incorporating orange‐fleshed sweet potatoes and bambara groundnut were used to develop acceptable and shelf stable vitamin A enriched extruded snacks. The formulations and process used in this study can be adopted at commercial level to produce affordable snacks that can contribute towards reducing the burden of VAD. The results from this study can also be used in similar studies to develop nutrient enhanced extruded snacks.     

Composition and in vitro digestibility of raw versus cooked white‐ and colour‐flowered peas

Ten pea cultivars (four white‐flowered, Pisum sativum ssp. hortense, and six colour‐flowered, Pisum sativum ssp. arvense) grown in Latvia were analyzed and tested in in vitro experiments, as raw and cooked seeds. The colour‐flowered (CF) had a greater proportion of hulls and a higher acid detergent fibre (ADF) content than white‐flowered (WF) pea seeds (10.7 vs. 8.2% and 92.2 vs. 84.5 g/kg dry matter (DM), respectively). Three out of six CF varieties had a significantly greater amount of protein bound to neutral detergent fibre (NDF) than WF peas. The tannin content was higher in CF than in WF peas (8.46 vs. 0.37 g/kg DM). In vitro protein and amino acid digestibility was about 8% higher in WF than in CF varieties. Cooking decreased the tannin content in CF peas (8.46 vs. 5.51 g/kg DM) but had no effect on in vitro protein digestibility. Heat treatment reduced significantly trypsin inhibitor activity and amount of protein bound to NDF in CF and WF varieties (from 6.50 to 0.52 and from 6.54 to 0.46 trypsin inhibitor units (TIU)/mg DM; from 1.250 to 0.831 and 0.761 to 0.209 g N/100 g NDF, respectively). However, the protein bound to NDF content in pea DM increased in CF and decreased in WF varieties (from 1.525 to 2.145 and from 0.913 to 0.502 g N/kg DM, respectively). Cooking resulted in an increased NDF content over two times in both CF and WF pea seeds (from 122 to 259 and from 120 to 262 g/kg DM, respectively). The results suggest that colour‐flowered pea may be considered as an interesting dietary alternative to white‐flowered pea since cooking removes trypsin inhibitor activity (TIA), decreases tannins, and increases dietary fibre contents.     

Comparing the efficiency of different food‐grade emulsifiers to form and stabilise orange oil‐in‐water beverage emulsions: influence of emulsifier concentration and storage time

The aim of this study was to compare the efficiency of three different food‐grade emulsifiers to form and stabilise an orange oil‐in‐water emulsion. The emulsifier type and concentration had a profound effect on the initial particle size of the oil droplets with Tween 80 being the most effective in reducing the particle size (1% w/w, 1.88 ± 0.01 μm) followed by sodium caseinate (10% w/w, 2.14 ± 0.03 μm) and gum arabic (10% w/w, 4.10 ± 0.24 μm). The long‐term stability of the concentrated beverages was monitored using Turbiscan analysis. The Turbiscan stability indices after 4 weeks of storage followed the order: Tween 80 (1.70 ± 0.08) < gum arabic (4.83 ± 0.53) < sodium caseinate (6.20 ± 1.56). The protein emulsifier was more capable to control the oxidation process, and this was attributed to the excess amount of emulsifier present in the aqueous phase. This study provides useful insights into the formulation of flavour emulsions by the beverage industry.