Transglutaminase: effect on rheological properties, microstructure and permeability of set style acid skim milk gel

Transglutaminase: effect on rheological properties, microstructure and permeability of set style acid skim milk gel

Food Hydrocolloids - Vol. 11 no . 3 pp. 287-292, 1997 Transglutaminase: effect on rheological properties, microstructure and permeability of set st...

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Food Hydrocolloids

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Vol. 11 no . 3 pp. 287-292, 1997

Transglutaminase: effect on rheological properties, microstructure and permeability of set style acid skim milk gel M.Frergemand and K.B.Qvist Department of Dairy and Food Science, Dairy Section, The Royal Veterinary and Agricultural University, Rolighedsvej 305, DK-1958 Frederiksberg C, Denmark

Abstract Reconstituted skim milk was incubated with microbial transglutaminase to allow enzymatic cross-linking of the milk proteins. Subsequently the cross-linked skim milk was acidified by glucono-o-lactone. Three methods were applied to study the nature of the gels. A Bohlin VOR rheometer was used to follow the development of rheological properties during acidification; gel permeability was determined by measuring liquid flow through the gel; and the microstructure of the gels was investigated by confocal laser scanning microscopy (CLSM). It was found that skim milk incubated with transglutaminase formed a 4- to 6-Jold stiffer gel with a 2- to S-fold lower permeability on acidification than gels without transglutaminase. Images from CLSM revealed that skim milk gels with transglutaminase have a finer protein network with thin strands between the particles than the untreated acid skim milk gels, which were found to consist of coarser particles.

Introduction Several types of milk protein gels have traditionally been manufactured by acidification or protease action . These gel networks are considered stabilized mainly by weak non-covalent interactions. Introduction of new covalent bonds in milk protein gels may be expected to produce gels different from the traditional milk protein gels in structure and hence functionality. For food purposes most chemical modifications would not be desired because of possible toxicity or unwanted side reactions, and enzymatic modification would be preferred. Acid milk gels, e.g. yoghurt, are commercially important milk gels. Two essential parameters for consumer quality acceptance are consistency and low whey drainage (1). These parameters may change by introduction of new covalent bonds in the gel. Transglutaminase (EC 2.3.2.13, R-glutaminyl-peptide: amine y-glutamyltransferase) is an enzyme capable of forming inter- or intramolecular cross-links in many proteins. The enzyme catalyzes an acyl transfer reaction between y-carboxamide groups of peptide-bound glutamine residues as acyl donors and primary amines as acceptors. When the s-amino group of peptide-bound lysine acts as the acyl acceptor, the s-(y-glutamyl)lysine cross-link is formed (2,3). Transglutaminases are widely distributed in nature.

ICi Oxford University Press

Because of the important physiological role of transglutaminase in catalyzing the clotting of fibrin in blood coagulation (2), mammalian transglutaminases are well characterized, whereas the purpose of transglutaminases in other systems is not so well understood. Even though transglutaminase has been extensively studied for cross-linking activity in several food proteins, e.g. soybean protein (4,5), caseins (6-8) and whey proteins (8-10), its actual application in food models or food products has not received much attention. Several authors have stated that casein is a good substrate among food proteins for transglutaminase-catalyzed cross-linking. Traore and Meunier (II) were able to polymerize caseins by human placenta FXIIIa in the absence of a reductant, whereas for the polymerization of whey proteins a reductant was found to be required (8). Nonaka et al. (12) reported polymerization of o.sl-casein by microbial transglutaminase in the absence of a reductant and polymerization of albumins (bovine serum albumin , human serum albumin and conalbumin) only in the presence of dithiothreitol. Kurth and Rogers (13) found that, among soybean protein, gluten and casein, casein was the most effective substrate for trans glutaminase (bovine plasma)catalyzed cross-linking to myosin.

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Functional properties of proteins cross-linked by transglutaminase have been investigated by only a few authors. Most of the work in this area has been by Motoki and co-workers, who extensively studied the modification of us\-casein by transglutaminase-catalyzed cross-linking (5,14,15). These studies showed the ability of (guinea pig liver) transglutaminase to induce increased viscosity and gelation of usl-casein solutions and clearly demonstrated the covalent nature of such gels. Nio et al. (5) demonstrated the superiority of u s\-casein as a protein substrate over soybean lIS and 7S globulins. In this study us\-casein solutions formed gels at concentrations of 3% w/w whereas both the soybean globulins required 8% w/w protein for gel formation. Gel-forming ability was confirmed in this study by a breaking strength> O. Recently, a few studies more directly related to food have been published on rheological properties of sodium caseinate and skim milk gels (16), and several other food protein gels (7) formed by transglutaminase-catalyzed cross-linking. Nonaka et al. (16) reported formation of gels from sodium caseinate and reconstituted skim milk solutions by crosslinking with a microbial transglutaminase, Both systems were able to form self-supporting ge1s-even though skim milk gels apparently were only formed at quite high solid levels (-43 %). Self-supporting gels were not formed in the absence of transglutaminase, These studies show that transglutaminase has a potential use in providing texture for food protein systems. Whereas most of the studies on transglutaminasecatalyzed cross-linking of food proteins so far have been concerned with the formation of novel gel foods, this paper is a study of the effect of trans glutaminase-catalyzed cross-linking of caseins in a model of an existing food product, namely a set style acid skim milk gel.

Materials and methods Enzyme

A microbial transglutaminase purified from Streptomyces Iydicus was provided by Novo Nordisk A/S (Bagsvserd, Denmark). The enzyme has a molecular weight of 37 kDa and pH optimum of 7.5 (T.Mathiesen, personal communication). Transglutaminase activity was determined by the hydroxamate procedure with CBZ-L-glutaminyl-glycine as substrate (17). The enzyme activity unit was defined as one unit (U) causing the formation of I umol/dm? of hydroxamic acid per min at pH 6.0 and 37°C. The specific activity of the enzyme preparation was 8.5 U/mg. Skim milk

Low-heat skim milk powder containing 34% protein was obtained from MD Akafa AlS (Svenstrup, Denmark). Skim milk was prepared by dissolving 11% (w/w) low-heat skim milk powder in distilled water at room temperature. The milk was mixed using a magnetic stirrer for 30 min and stored at

5°C for 20 h to allow the proteins to hydrate. Preparation of cross-linked skim milk

Reconstituted skim milk was incubated with microbial transglutaminase at an enzyme/milk protein ratio of 0.4% (w/w) at 41°C for 60 min to allow cross-linking of the milk proteins. Measurement of ammonia

Ammonia was measured by flow injection analysis (FIA) using an FIAstar Analyzer equipped with a Chemifold type V and an FIAstar 5023 Spectrometer (Tecator, Hoganass, Sweden). In this instrument, the sample is injected in a carrier stream which is mixed with an NaOH stream whereby ammonia is released as gas from the sample. The gas passes a gas diffusion membrane and is then mixed with an indicator stream. The change in absorbency at 590 nm of this indicator solution is proportional to the concentration of ammonia in the sample (18). Cal ibration was performed on 25 times diluted milk samples with added NH 4Cl (1-7 mg/I NH 4) . The samples for ammonia determination in milk were prepared as follows: reconstituted skim milk was incubated with the microbial transglutaminase. At various intervals samples were withdrawn and diluted 25 times in water at BO°e. The diluted samples were held at BO°C for 5 min in order to inactivate the enzyme. Control samples were prepared by incubating with enzyme solution that had been denatured at BO°C for 5 min. Samples were stored at - 18°C until analyzed. FIA was then carried out on the thawed samples at room temperature according to the method described above. 80S-PAGE

For confirmation of intermolecular cross-linking, the transglutaminase-treated skim milk was analyzed by SDS-PAGE. Samples were prepared by diluting skim milk 10 or 20 times with SDS buffer (0.01 mol/dm ' Tris-HCI, pH B.O, 2.5% SDS, 0.01% bromophenol blue). Samples with denatured enzyme were prepared by heating skim milk immediately after enzyme addition at 80°C for 5 min in order to check the heat inactivation of the enzyme. The separation of samples was performed on a Phastgel Homogeneous 12.5% gel using Phastsystem" (Pharmacia, Allered, Denmark). Densitometry was performed by analyzing scanned images of the gels using Quantimet 500+ (Leica, Glostrup, Denmark). Rheological measurements

Glucono-d-lactone (GDL) at a concentration of 2% (w/w) was used to slowlyacidify the cross-linked skim milk at 41"C, Immediately after GDL addition, 2.5 ml of the reaction mixture was transferred to a coaxial cylinder measuring system (CI4) of a Bohlin VOR or a Bohlin CS rheometer (both Bohlin Rheology, Lund, Sweden), where the acidification took place. The surface of the milk was covered

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Effect of transglutaminase on acid skim milk gel

with a few drops of low-viscous oil to prevent drying of the sample. Oscillation measurement was performed at 1 Hz and a strain of 0.002. This was found to be well within the linear viscoelastic region of the formed gels. The amount of GOL was chosen to give a final pH of 4.45. On completion of the acidification a frequency sweep from 0.0015 to 9 Hz (strain 0.002) was performed in the Bohlin VOR monitoring the elastic modulus (G), the viscous modulus (G'), the complex modulus [G*, defined as I G*l = V(G2 + iG'2)] and the loss angle [0, defined as tan(o)G'IG] . A yield stress test was performed in the Bohlin CS rheometer. This test applies a ramped stress to the sample, monitors the induced strain and from that calculates an apparent viscosity. As the material starts to flow, the apparent viscosity will change rapidly from an increasing to a decreasing value, and at this maximum the yield stress is determined. This test thus provides an apparent yield value at the chosen time scale of the experiment. The yield stress test was performed under the following conditions: the stress ramp was applied linearly from I to 1388 Pa in 50 steps in 120 s. All measurements were at 41°C.

prepared in special object glasses with a polished cavity -10 mm in diameter and 0.7 mm deep. The milk protein was stained with 0.001% (w/w) Rhodamine B (Merck) prior to cross-linking. Cross-linking was performed as already described. Immediately after GDL was added, some of the reaction mixture was transferred to the object glass. It was then covered and sealed with nail polish to prevent evaporation. The object glass sample was incubated at 41°C and the acidification followed in a parallel sample. At pH 4.45 the object glass was transferred to 5°C, where it was stored until examination on the following day. The gels were examined by CLSM consisting of a Leica TCS 40 confocal laser scanner with a Leitz DM RB/E* microscope (Leica, Glostrup, Denmark). CLSM enables optical sectioning and depth viewing of samples (20). Images were recorded with oil-immersion optics (N.A. = 1.4). Care was taken to focus at a suitable distance below the cover glass at which there seemed to be an increased concentration of protein particles.

Permeability measurements

Typical frequency sweeps from small deformation oscillatory experiments are shown in Figure 1. The complex modulus of the transglutaminase treated gel was up to six times higher than that of the gel without transglutaminase (Fig. la). As the complex modulus describes the total stiffness of the gel, arising from both the viscous and the elastic response (20), this result demonstrates that the acid milk gel becomes stiffer as new covalent bonds are introduced by transgl utaminase-catalyzed cross-linking. The formation of e-(y-glutamyl)lysine cross-links was suggested by following the release of ammonia from the transglutaminase-catalyzed reaction (2). Results from FIA (not shown) demonstrated that the release of ammonia had reached a plateau value corresponding to -2 mol NHJmol casein after 60 min of reaction. Care should be taken in

Permeability was determined by measuring liquid flow through the gel according to Roefs (19). GOL was added to cross-linked skim milk and then acidification to pH 4.45 was performed in glass tubes at 41°C. The tubes were then cooled to 5°C and placed in the measuring vat of a permeability apparatus (purchased from Wageningen Agricultural University, The Netherlands). The measuring vat containing whey was kept at 5°C and the liquid flux was measured and permeability coefficients (B) calculated as described by Roefs (19). Microscopy Gels for confocal laser scanning microscopy (CLSM) were

Results and discussion

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Figure 2 (a) SDS-PAGE of skim milk incubated with transglutaminase for 60 min. Lane 1: skim milk, lOx diluted; lane 2: skim milk, 20x diluted; lane 3: skim milk + transglutaminase, IOx diluted; lane 4: skim milk + transglutaminase, 20x diluted; lane 5: skim milk + transglutaminase, heat-treated sample, lOx diluted; lane 6: skim milk + transglutaminase, heat-treated, 20x diluted; lane 7: skim milk, heat-treated, lOx diluted; lane 8: skim milk, heat-treated, 20x diluted. (b) Densitogram of lanes 1 (dotted line) and 3 (solid line). Peaks: a, end line drawn on the gel; b, a-lactalbumin; c, ~-lactoglobulin; d, asl-casein; e, ~-casein; f, bovine serum albumin; g, band formed by transglutaminase action; h, unknown; i, start of gel: polymers with molecular weight >250 kDa. (c) Densitogram of lanes 6 (solid line) and 8 (dotted line) showing the denaturation and aggregation of whey proteins and the lack of formation of oligomers by the heat-inactivated transglutaminase.

interpreting ammonia release as a measure of cross-linking, as the formed ammonia may derive from both primary amine incorporation and possible deamidation. These measurements do, however, demonstrate that only a small number of cross-links per casein molecule are needed to have a considerable effect on rheological properties. Formation of cross-linked products was confirmed by SDS-PAGE in Figure 2a. A new high molecular weight band was formed by transglutaminase action, which was validated by densiometry as shown in Figure 2b and c. This new band had a molecular weight of -75 kDa, as determined by molecular weight standards (gel not shown). The microbial transglutaminase has a molecular weight of 37 kDa (as determined by SDS-PAGE; not shown) and is not visible on this gel, as the enzyme concentration is only 0.4% of the milk protein concentration. In the samples with the heat-inactivated transglutaminase, the 75 kDa product is apparently not formed. The molecular weight of -75 kDa corresponds to a trimer of uSI-casein, and as the us l-casein band seems to decrease, this could well be the case. From a kinetic/statistical point of view cross-linking between casein micelles is much less probable than cross-linking within or at the surface of micelles, where proteins are constantly very close. Formation of uSI-casein oligomers within the casein micelles would most likely affect the disaggregation step in the structure formation of acid

milk gels, where uSI-casein apparently constitutes the backbone in the aggregate network as J3-casein and x-casein dissociate from the micelles (21). Disaggregation would probably be less pronounced, and this would eventually lead to stable aggregates. Also, formation of covalent casein oligomers would probably in itself give rise to stiffer particles and thereby a higher modulus in the final gel. The loss tangent is a measure of the character of the bonds forming the gel (22). As shown in Figure Ib, the loss angle of the acid skim milk gel was not altered significantly by transglutaminase treatment. At higher frequencies the loss angle tends to be slightly smaller for the transglutaminase cross-linked gel, but the difference is not significant in the results shown here. Bohlin et al. (23) found similar results for the addition of CaCl z before coagulation of milk with chymosin-Le. a higher G* but a constant loss angle as a function of CaCl z addition. For practical applications it could prove to be important that the viscoelastic character of the skim milk gel is not changed, indicating that the 'yoghurt'-like character of the product is maintained although gel stiffness is increased. The apparent yield stress determined by the stress ramp shows the same trend shown in the oscillatory experiment. Figure 3 shows that the transglutaminase-treated gel has a higher resistance to flow than the gel without enzyme. Large deformation properties are often considered to be better

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correlated to sensory properties than small deformation properties, which is why these are important to consider in investigations of food functionality. The small and large deformation studies in this paper show that transglutaminase treatment makes acid milk gels stifTer and more resistant to flow. Another important parameter in consumption qualities of milk gels is stability towards whey drainage. Susceptibility of acid gels to whey drainage can be reduced by creating a finer-meshed network that immobilizes a greater amount of water (24). Permeability is a measure of the gel network characteristics and depends mainly on the evenness of the distribution of the strands of particles, and thus on the size of pores in the network, of the gel (25). Permeability of milk gels depends on several factors and can be reduced by decreasing the acidification rate (by applying a lower acidification temperature), or increasing the protein (19) or fat content (25). A finer-meshed network can also be formed when heat treatment is applied to milk prior to acidification. This is due to the association of denatured ~-lactoglobulin with casein micelles (24). Figure 4 shows that the permeability coefficient of the acid skim milk gel is reduced by a factor of about two by transglutaminase-catalyzed cross-linking. This indicates that cross-linking produces a finer-meshed gel network structure, probably with better water (or whey) holding capacity. To probe whether the reduced permeability induced by cross-linking actually reflected an altered structure of the gel network, microscopy was applied. In Figure 5 typical images of the microstructure of the acid milk gels are shown. The images show that transglutaminase-catalyzed cross-linking yields an acid milk gel with a finer protein network with thinner protein particle strands between the aggregated particles than the untreated gel. The formation of a finer protein network leads to smaller pores in the network, which causes a lower permeability.

Figure 5 Images from confocal laser scanning microscopy (Cl, SM) of acid skim milk gels. Bars indicate 25 um. (a) Untreated skim milk gel; (b) transglutaminase-treated acid skim milk gel.

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The changed microstructure is most likely due to changes on the surface of the micelles caused by the cross-linking reaction, thus altering the interactions between micelles. The exact mechanism for this is yet to be determined. There seems to be a close quantitative correlation between microstructure and permeability data. The effect of transglutaminase-catalyzed cross-linking mirrors the results found by Harwalkar and Kalab (26) when increasing total milk solids. They found that increasing the solids content resulted in a denser microstructure and a lower whey drainage. Consequently, transglutaminase-induced crosslinking could possibly replace added milk solids in the production of set style acid milk gels. From an ethical and consumer acceptance point of view, it could be important that the enzyme loses its activity at pH 5 and is completely inactive at pH 4.3 (T.Mathiesen, personal communication), which is the approximate pH of the consumed product. Hence the microbial transglutaminase shows great promise for practical application as a structuring agent in the production of set style acid milk gels, with a potential to replace added milk solids or stabilizers currently used.

Acknowledgements The authors thank Novo Nordisk A/S for providing transglutaminase and the Department of Chemistry at the Royal Veterinary and Agricultural University for use of their FIA instrument.

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14. Nio,N., Motoki,M. and Takinami,K. (1986) Agric. Bio/. Chem., 50, 851-855. 15. Motoki,M., Nio,N. and Takanami,K. (1984) Agric. Bio/. Chem., 48, 1257-1261. 16. Nonaka,M., Sakamoto,H., Toigguchi,S., Kawajiri,H., Soeda,T. and Motoki,M. (1992) J. Food Sci., 57, 1214-1218. 17. Folk,IE. and Cole,P.W. (1966) J. Bio/. Chem., 241, 5518-5525. 18. Tecator (1992) Determination of Ammonia Nitrogen in Water by Flow Injection Analysis, Application Note ASN 50-02/92. 19. Roefs,S.P.F.M. (1985) Structure of Acid Casein Ge/s; a Study of Ge/s Formed After Acidification in the Co/d. Ph.D. thesis, Agricultural University, Wageningen, The Netherlands. 20. Heerje,I., van der Vlist.P, Blonk,IC.G. and Hendrickx, H.A.C.M. (1987) Food Microstruct., 6, 115-120. 21. Heertje,I., Visser,I and Smits,P. (1985) Food Microstruct., 4,267-277. 22. van Vliet,T., Roefs,S.P.F.M., Zoon,P. and Walstra,P. (1989) J. Dairy Res., 56,529-534. 23. Bohlin,L., Hegg,P.-o. and Ljusberg-Wahren,H. (1984) J. Dairy Sci., 67, 729-734. 24. Dannenberg,F. and Kessler,H.-G. (1988) Mi/chwissenschaft, 43, 632--635. 25. Walstra,P. and van Vliet,T. (1986) Neth. Milk Dairy J., 40, 241-259. 26. Harwalkar,Y.R. and Kalab,M. (1986) Food Microstruct., 5,287-294.

Received on September 18, 1995; accepted on March 7, 1996