JFS: Food Chemistry and Toxicology

Effects of Microbial Transglutaminase on the Wheat of and Croissant Dough J.A. GERRARD, S.E. FAYLE, P.A. BROWN, K.H. SUTTON, L. SIMMONS, AND I. RASIAH

ABSTRACT: Transglutaminase is a crosslinking enzyme that is finding increasing use in foods, yet the molecular Food Chemistry and Toxicology changes responsible for its effects are not fully understood. Proteins were extracted from bread and croissant doughs that had been treated with transglutaminase and compared to those from control doughs by size exclusion high performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. Transglutaminase increased the amount of extracted in the fraction in both bread and croissant doughs. In croissant doughs, a corresponding decrease in the protein extracted in the albumin to globulin fraction was seen. In each case, crosslinking of the high molecular weight glutenins was observed. The possible role of each of these changes on the functional properties of baked products is discussed. Key Words: transglutaminase, dough protein, flour improver

Introduction understanding of the action of transglutaminase on wheat RANSGLUTAMINASE (PROTEIN-GLUTAMINE ␥-GLUTAMYL- proteins may add insights into the mechanism of the coeliac Ttransferase, EC 2.3.2.13) catalyzes acyl-transfer reactions, response. Recent reports in the medical literature have im- introducing covalent crosslinks in proteins (Nonaka and oth- plicated gut tissue transglutaminase in the reaction of coeliac ers 1989). Crosslinks are formed between lysine residues and sufferers on exposure to wheat protein (Marsh 1997). The in- glutamine residues producing an ⑀-(␥-Glu)-Lys bond, with- testinal inflammation in coeliac disease is caused by expo- out reducing the nutritional value of the lysine residue (Seg- sure to wheat gliadin in the diet and has recently been asso- uro and others 1996). Transglutaminase may also catalyze ciated with an increase in the level of mucosal activity of the incorporation of amine groups into proteins via the transglutaminase (Mowat 1998). It is thought that trans- amide moiety of a glutamine residue and, in the absence of glutaminase may be involved in modulating the reactivity of available amines, hydrolyze a glutamine residue to a gliadin specific T cells by specific hydrolysis of the glutamine glutamate residue (Nielson 1995) (Figure 1). residues (Vandewal and others 1998). These observations are Transglutaminase is finding increasing use in a wide vari- entirely consistent with earlier reports (Watanabe and others ety of foods (Nielson 1995; Motoki and Seguro 1998), particu- 1994) that suggested that treatment of with trans- larly since large quantities of microbial transglutaminase be- glutaminase reduces its allergenicity. Thus, in addition to im- came commercially available (Zhu 1995). It is generally assumed that the effects of transglutaminase in foods are due to its crosslinking activity. However, the direct experi- mental evidence for this supposition is limited (Christensen and others 1996; Babiker and others 1996; Yildirim and Hetti- arachchy 1997; Yasunaga and others 1998) and the possible role of non-crosslinking activity has not been widely consid- ered. The exact nature of the effects of transglutaminase on different food proteins needs to be determined in order to understand the action of the enzyme and relate the molecu- lar changes to macroscopic changes in the final product. Furthermore, these studies need to be carried out in the food itself, since test-tube results do not necessarily translate successfully into the food-processing situation. We have recently reported the beneficial effects of trans- glutaminase in the breadmaking process (Gerrard and others 1998) and in pastry and croissants (Gerrard and others 2000). In this paper we report the results of an investigation into the effects of transglutaminase on wheat proteins in dough. Pre- vious studies have reported that wheat and high mo- lecular weight glutenins were a substrate for transglutami- nase (Alexandre and others 1993; Larre and others 2000), but did not extend the study to food systems. Figure 1—The 3 reactions catalyzed by microbial trans- In addition to furthering our understanding of the rela- glutaminase: protein crosslinking, incorporation of a free tionship between dough protein structure and function, an amine and hydrolysis of a glutamine residue

782 JOURNAL OF FOOD SCIENCE—Vol. 66, No. 6, 2001 © 2001 Institute of Food Technologists Dough Proteins as affected by Microbial Transglutaminase . . . proving the functional properties of wheat-based products, Protein extraction transglutaminase may also have potential in the production Protein extraction was based on a modification of the of wheat-based foods appropriate for coeliac sufferers. methods of Hay and Sutton (1990) and of Batey and others The objectives of this study were to extract proteins from (1991). One-hundred-mg samples from each freeze-dried doughs that had been treated with transglutaminase and dough powder were weighed into 1.5-mL Eppendorf tubes. compare them to those that had not. Subsequent analysis of Protein concentrations were determined using the method the proteins allows us to deduce the action of the enzyme of Bradford (1976). within the dough itself. Albumins and globulins. Albumins and globulins were extracted by adding dilute saline solution (0.4 mL, 2% (w/v) Materials and Methods sodium chloride) to the 100-mg samples, with vortex mixing LL EXPERIMENTS WERE CARRIED OUT IN DUPLICATE OR TRIP- every 5 min for 30 min. After centrifugation (10,000 g, 5 min) Alicate to ensure reproducibility. the clarified supernatant was removed to a new Eppendorf tube and stored at –10 ЊC prior to analysis. Materials Gliadins. Gliadins were extracted by resuspending the re- Unless otherwise stated, all materials were obtained from sidual pellet from the above extraction in 70% (v/v) aqueous Sigma Chemical Co. (Sydney, NSW, Australia). Microbial trans- ethanol (0.4 mL) for 30 min with vortex mixing every 5 min. glutaminase was obtained from Amcor Trading Pty. Ltd (Mel- Following centrifugation (10,000 g, 5 min), the clarified super- bourne, Australia). Flours were obtained from a local mill. natant was removed and stored at –10 ЊC prior to analysis. SDS-soluble glutenin. Sodium dodecyl sulfate (SDS-) sol- Dough preparation uble glutenin was extracted by resuspending the residual pel- Bread and croissant doughs were prepared as described let in a SDS-buffer (0.4 mL, 0.5% (w/v) SDS, 0.05M phos- previously (Gerrard and others 1998, 2000). For both bread phate, pH 6.9) for 30 min with vortex mixing every 5 min. and croissants, control doughs were compared to those con- After centrifugation (10000 g, 5 min), the clarified superna- Food Chemistry and Toxicology taining 5000 ppm microbial transglutaminase. Doughs were tant was removed and stored at –10 ЊC prior to analysis. prooved for 30 min, flash frozen in liquid nitrogen, freeze- SDS-insoluble glutenin. SDS-insoluble glutenin was ex- dried to dryness, then ground to a fine flour using a mortar tracted by resuspending the residual pellet in the same SDS- and pestle, before protein extraction. buffer as for the soluble glutenin (0.4 mL, 0.5% (w/v) SDS, 0.05 M phosphate, pH 6.9) and sonicating the mixture at 30W Fat extraction from croissant dough for 15 s using a Branston sonic disrupter. The sample was To remove the fat from the croissant doughs we used the vortexed then clarified by centrifugation (10000 g, 5 min). method of Daniels and others (1967); samples were freeze- The clarified supernatant was removed and stored at -10 ЊC dried and ground using a mortar and pestle. A portion (5 g) prior to analysis. of each sample was weighed into an extraction thimble, which was then plugged with a cotton wool, and placed in a Analysis by SDS-PAGE labeled glass extractor. Boiling chips and n-hexane (250 mL) SDS polyacrylamide gel electrophoresis (SDS-PAGE) was were added to round bottom flasks in a fumehood, and carried out under reducing conditions according to the placed on a series of heating elements of a standard Soxhlet method of Dunn (1989) using a 3.5% stacking gel and a apparatus. After approximately 23 cycles, the water and 12.2% resolving gel. Samples were electrophoresed at a heating elements were turned off. After cooling, the appara- constant current of 30 mA until the bromophenol blue dye, tus was dismantled and the thimbles placed into labeled plas- contained in the 2 × treatment buffer, had reached the bot- tic bags. The bags were placed under reduced pressure to re- tom of the gel. This took approximately 5.5 h. Protein visu- move hexane from the samples. The samples were then alization was afforded by staining with Coomassie Brilliant placed into labeled containers, ready for protein extraction. Blue (Dunn 1989).

Figure 2—The effect of transglutaminase on the percent- Figure 3—The effect of transglutaminase on the percent- age of wheat proteins extracted in the albumin and globu- age of wheat proteins extracted in the albumin and globu- lin, gliadin, SDS-soluble glutenin, and SDS-insoluble glute- lin, gliadin, SDS-soluble glutenin, and SDS-insoluble glute- nin fractions of bread dough nin fractions of croissant dough

Vol. 66, No. 6, 2001—JOURNAL OF FOOD SCIENCE 783 Results and Discussion Results and L) were injected onto the column, which was column, onto the injected L) were ␮ The mixing of flour and water causes some of the wheat The mixing of flour proteins to combine to form a mass known as gluten. Gluten proteins to combine held together by a range of chemi- consists of many proteins in- Waals der Van bonds, hydrogen cal interactions including interactions and disulfide bonds teractions, hydrophobic the unique viscoelastic properties of glu- (Belton 1999). It is in- the functional properties of a dough, ten that account for which in turn determines and elasticity, cluding its strength product (Kauffman and others 1986). the quality of the final fi- to improve been shown has previously Transglutaminase the further strengthening by presumably nal product quality, mechanism of action and the precise However, gluten mass. by the en- whether specific dough proteins are crosslinked Wheat proteins in dough Wheat proteins in Samples (20 Samples were elut- and proteins at ambient temperature, maintained = A:B for 35 of 50:50 at a solvent composition ed isocratically were Eluted components rate of 0.5 ml/min. min at a flow 210 nm. detected at —Vol. 66, No. 6, 2001 66, No. —Vol. m filter, rapid sparging with helium sparging rapid m filter, software operating on a personal operating software ␮ 32 7.8 mm) with a matching guard column (75 7.8 mm) with a matching ϫ 7.8 mm). Solvents used were (A) water containing 7.8 mm). Solvents JOURNAL OF FOOD SCIENCE ϫ C overnight to ensure complete reduction of disulfide reduction of to ensure complete C overnight Њ Samples were mixed with an equal volume of a solution equal volume of mixed with an Samples were HPLC Waters was carried analysis out using a SE-HPLC m polyvinylidenefluoride membrane syringe-filter prior to syringe-filter membrane m polyvinylidenefluoride mm containing 4% SDS and 2% dithiothreoitol and incubated at and 4% SDS and 2% dithiothreoitol containing 20 were was 6.9. Samples final pH of the samples bonds. The through a 5- min and filtered at 10000 g for 10 centrifuged ␮ high performance liquid chroma- analysis by size exclusion tography (SE-HPLC). 2690 injector/solvent Alliance Waters a system consisting of detec- 490 UV/visible Waters delivery/control system and a using controlled and data recorded were Instruments tor. the Waters Millennium 784 Figure 4—SE-HPLC traces of bread dough proteins: (a) the albumin and globulin fraction from control dough; (b) the Figure 4—SE-HPLC traces of bread dough proteins: (a) the albumin and gliadin fraction from control dough; (d) the albumin and globulin fraction from transglutaminase-treated dough; (c) the gliadin fraction from transglutaminase-treated dough. Dough Proteins as affected by Microbial Transglutaminase . . . Transglutaminase Microbial by affected as Proteins Dough by SE-HPLC Analysis (100 mL/min for 10 min), and constant slow bubbling of heli- (100 mL/min for 10 min), and constant (10 mL/min). um into capped, vented solvent reservoirs 0.1% (v/v) trifluoroacetic acid (TFA)0.1% (v/v) trifluoroacetic and (B) acetonitrile con- vacuum by was achieved Deaeration TFA. taining 0.1% (v/v) through a 0.22- filtration computer. The column used was a Phenomenex BioSep SEC- was a Phenomenex BioSep The column used computer. 4000S (300 mm

Food Chemistry and Toxicology Dough Proteins as affected by Microbial Transglutaminase . . . zyme remain undetermined. SDS PAGE analysis Dough proteins have been classified into 4 groups, ac- As had been the case with SE-HPLC, no major differences cording to their solubility properties. The 1st group, the al- were observed when the SDS-PAGE results for bread doughs bumins and globulins, constitute approximately 15% of and croissant doughs were compared. Figure 5 and 6 show a wheat flour protein and are generally regarded as non- comparison of each of the 4 fractions of proteins extracted dough forming proteins. Gliadins are monomeric proteins from croissant dough, with and without transglutaminase. of relatively low molecular weight (approximately 30 to 50 In the albumin fraction (Figure 5, lanes 1 and 2), aggregat- kDa) and are alcohol soluble. The larger glutenins consist of ed protein that is too large to enter the gel (labeled A) ap- polymers of high molecular weight, and can be classified as pears only in the transglutaminase-treated dough. Some of either soluble or insoluble in SDS solutions (Bushuk 1985). the proteins in the control dough appear unchanged by the addition of transglutaminase, while others—the protein la- Extraction and analysis methods beled B, running slightly behind the 55-kDa marker, and Protein extraction was based on the classification described those in the area labeled C, of low molecular weight—appear above and was carried out using a modification of the methods to have been crosslinked, as judged by their diminished in- of Hay and Sutton (1990) and of Batey and others (1991). In or- tensity in the transglutaminase-treated dough. The gliadins der to simplify the analysis by SE-HPLC and PAGE, all fractions do not appear to have been crosslinked by transglutaminase, were reduced with dithiothreitol prior to analysis. in contrast to the observations of Alexandre and others Bread and croissant doughs were prepared by previously (1993) on the purified proteins. described methods (Gerrard and others 1998, 2000). In each Figure 6 also shows an increase in aggregated material (la- case, some of the dough was baked to confirm the previously beled P) at the top of the gel in the proteins from the trans- reported differences in functional properties (data not glutaminase-treated doughs. In addition, there is evidence shown). To investigate the molecular effects of transglutami- for specific crosslinking of the proteins in the molecular nase in dough, a parallel study was carried out on bread and weight range 60 to 120 kDa (labeled Q, R, S, and T). These Food Chemistry and Toxicology croissant doughs to establish whether the enzyme had the proteins correspond to the high molecular weight (HMW) same effect on the proteins of each, or whether differences in glutenin subunits, proteins that have previously been associ- formulation and processing of the dough affected the enzyme activity. The main difference in formulation between bread and croissant doughs is the presence of fat in the latter. Since fat interfered with both chosen analyses (PAGE, SE-HPLC), it was removed by Soxhlet extraction prior to analysis.

SE-HPLC analysis In the 1st instance, the effect of transglutaminase on the overall quantity of the 4 fractions of wheat proteins was ex- amined. Figures 2 and 3 show the effect of transglutaminase on bread dough and croissant dough proteins. In each case, the percentage of extractable protein ap- pearing in the gliadin fraction increases. In the croissant dough, the addition of transglutaminase reduces the albumin and globulin fraction. The effect on the glutenins is less pro- nounced, although there is a drop in the level of SDS-insolu- ble proteins in the bread dough. One explanation for the im- provement in quality of the croissants might, therefore, be the removal of the non-dough forming albumins and globu- lins and the formation of dough forming proteins, especially those extracting with the gliadins. The difference in extract- able proteins in each fraction may be a function of the dif- ferent formulation and processing conditions, and warrants further investigation. Detailed examination of the SE-HPLC traces for the bread and croissant doughs suggested that transglutaminase was having a very similar effect in each case, but that the effect on the albumins and globulins was more marked in the croissant case. Inspection of the albumin and globulin and gliadin fractions suggested that the presence of transglutami- nase might affect specific albumin proteins. Figure 4 shows individual traces for the bread dough samples, showing high- er molecular weight peaks on addition of transglutaminase, with the most dramatic change highlighted. While the SE-HPLC method was well suited to the quanti- Figure 5—PAGE analysis of croissant doughs. Lanes are, fication of individual fractions of wheat proteins, it was not from left to right: (1) albumins and globulins from control the method of choice for analysis of specific proteins that dough; (2) albumins and globulins from transglutaminase- may be affected by the addition of transglutaminase. Our at- treated dough; (3) gliadins from control dough; (4) gliadins tention, therefore, turned to analysis by SDS-PAGE. from transglutaminase-treated dough; and (5) molecular weight markers.

Vol. 66, No. 6, 2001—JOURNAL OF FOOD SCIENCE 785 - ␥ -( ⑀ , independent transglutaminase 2+ References derived from micro-organisms. Agric. Biol. Chem. 53:2619-2623. derived from micro-organisms. Agric. Biol. Chem. quality of British HMW glutenin subunit composition and the breadmaking grown wheat varieties. J. Sci. Food Agric. 40:51-65. available source for lysine glutamyl)lysine moiety in crosslinked casein is an for rats. J. Nutrition 126:2557-2562. 1998. Cutting edge – selective deamidation by tissue transglutaminase strongly 161:1585-1588. J. Immunology reactivity. T-cell enhances gliadin specific Bio- Biotech. Biosci. flour. of wheat proteins for production of hypoallergenic chem. 58:388-390. on addition and Chum Salmon Pollack Walleye and two-step heated gel from of microbial transglutaminase. Bulletin of the Japanese Society of Scientific Fisheries 64:702-709. bean 11S globulin with proteins using transglutaminase. J. Food Sci. 62:270- 275. of its production and application in food processing. Applied Microbiology and Biotechnology 44:277-282. Wheat gamma-gliadin as a substrate for bovine plasma factor XIII. J. Ag. Food bovine plasma factor XIII. as a substrate for Wheat gamma-gliadin Chem. 41:2008-2214. transglutaminase for improvement of the functional tein digests by microbial 29:627-634. properties. Food Res. Int. the study of wheat flour proteins: an improved chro- liquid chromatography in Cereal Chemistry 68:207-209. matographic procedure. 107. utilising the principle of protein dye binding. gram quanitities of protein 72:248-254. Analytical Biochemistry 30:447-451. World Foods Cereal J. cross-linking sites in bovine caseins. isation of potential transglutaminase Ag. Food Chem. 44:1943-1947. dveloped doughs. Chem. Ind. 17:955-956. lipid binding in mechanically Press. Univ at Oxford IRL Press York: New approach. cation methods: A practical p 21-27. transglutaminase. J. properties and crumb strength as affected by microbial Food Sci. 63:472-475. J. Food Sci. and croissant volume as affected by microbial transglutaminase. 65:312-314. NZ J. Crop Hort. Sci. 18:49- wheat / durum wheat and rye cultivars by RP-HPLC. 54. World. Foods Cereal reactions. models and disulfide interchange structural 31:820-824. of gluten modified by 2000. Biochemical analysis and rheological properties transglutaminase. Cereal Chemistry 77:32-38. Nature Medicine 3:725-726. eliac disease. Gut. 43:599-600. 9:204-210. Technology Science and in Food Trends Biotechnology 9:119-156. inase – review of the literature and patents. Food Polymerisation of several proteins by Ca Payne PI, Nightingale MA, Krattiger AF, Holt LM. 1987. The relationship between The relationship LM. 1987. Holt MA, Krattiger AF, PI, Nightingale Payne The M. 1996. H, Motoki C, Sakamoto Kuraishi Y, K, Kumazawa Seguro F. G, Koning L, Papadopoulos S, Mearin Pena P, Vanveelen Y, Kooy Y, Vandewal enzymatic treatment S. 1994. Controlled Z, Arou Ikezawa T, M, Susiki Watanabe gel K. 1998. Change in quality of preheated Arai F, Nishioka Y, K, Abe Yasunaga Yidirim M, Hettiarachychy NS. 1997. Biopolymers produced by crosslinking soy- – a review transglutaminase J. 1995. Microbial Tramper A, J, Rinzema Bol Y, Zhu MS 19991108 This work was funded by the New Zealand Foundation for Research, Science, and Technol- Science, and for Research, Zealand Foundation the New was funded by This work of Plant Dept Healy, Ltd and Jackie Research Crop and Food Ross, thank Marcela We ogy. for technical assistance. Canterbury, of Univ Sciences, and Microbial Authors Fayle, Sutton, and Simmons are with the New Zealand Institute of Crop and Food Research Ltd., Christchurch, New Zealand. Author Gerrard Pri- of Canterbury, Univ. Sciences, Microbial and of Plant is with the Dept. vate Bag 4800, Christchurch, New Zealand. Authors Brown and Rasiah are with both organizations. Direct correspondence to author Gerrard (E-mail: [email protected]). Alexandre M-C, Popineau Y, Viroben G, Chiarello M, Lelion AP, Geugen J. 1993. M, Lelion AP, Viroben G, Chiarello Y, Popineau Alexandre M-C, of soy pro- Kato A. 1996. Polymerisation Khan MAS, Matsudomi N, Babiker EFE, high-performance of size-exclusion 1991. Use F. MacRitchie RB, IL, Gupta Batey Science 29:103- of Cereal Journal the elasticity of wheat gluten. On P.1999. Belton and sensitive method for the quantitation of micro- Bradford M. 1976. A rapid in dough and bread. and functionality 1985. Flour proteins : structure W. Bushuk LK. 1996. Local- TE, Rasmussen Petersen P, E, Hojrup Christensen BM, Sorenson of air on Coppock JBM 1967. Effect Russell Eggitt PW, Daniels NWR, Richmond JW, purifi- Protein S editors. Angel ELV, Harris planning. In MJ. 1989. Initial Dunn S. 1998. Dough M. Kavale Ross MP, AJ, Newberry Wilson SE, JA, Fayle Gerrard lift S. 2000. Pastry SE, Kavale M, Fayle AJ, Ross Wilson JA, NewberryGerrard MP, of New Zealand bread Hay RL, Sutton KH. 1990. Identification and discrimination 1986. Dough rheology: of the A review O. Fennema RC, Hoseney Kauffman SP, J. G, Desserme C, Lefebvre Deshayes Y, S, Popineau CS Denery-Papini Larre gluten and coeliac disease – food for thought. Transglutaminase, MN. 1997. Marsh autoimmunity in co- Mowat AMI. 1998. Dietary modifications: Food dependent and its use for food processing. Transglutaminase K. 1998. M, Seguro Motoki applications of transglutam- Nielson PM. 1995. Reactions and potential industrial A. 1989. K, Matsura H, Urneda M, Ando A, Motoki H, Okiyama Tanaka M, Nonaka cific molecular changes responsible for the dramatic effects dramatic for the responsible changes molecular cific and croissants. in bread of transglutaminase CROISSANT

AND

BREAD

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Conclusion TRANSGLUTAMINASE

OF

JOURNAL OF FOOD SCIENCE

dough has specific effects on the wheat proteins. The dough has specific DDITION

The presence of transglutaminase in a croissant dough re- The presence of transglutaminase 786 Figure 6—PAGE analysis of croissant doughs. Lanes are, analysis of croissant 6—PAGE Figure from left to right: (1) SDS-soluble glutenins from control dough; (2) SDS-soluble glutenins from transglutaminase- treated dough; (3) SDS- insoluble glutenins from control dough; (4) SDS- insoluble glutenins from transglutaminase- treated dough; (5) molecular weight markers. A Dough Proteins as affected by Microbial Transglutaminase . . . Transglutaminase Microbial by affected as Proteins Dough explanation Another (Payne 1987). quality with bread ated on the addi- of the products in quality for the improvement be that trans- therefore, may, tion of transglutaminase into HMW glutenin subunits can crosslink the glutaminase aggregates. useful protein more functionally specific effects are similar for bread and croissants doughs, specific effects are more marked in the case of the crois- although they seem formulation and processing, therefore, sants; differences in at a effect on the action of the enzyme appear to have some molecular level. al- amount of protein extracting in the sults in a diminished ex- fraction, and an increased amount bumin and globulin specific fraction. Additionally, in the gliadin tracting en- appear to be crosslinked by the HMW-glutenin subunits not dough forming, zyme. Since albumins and globulins are these small, it may be that transglutaminase is crosslinking that are benefi- soluble proteins, forming protein aggregates the beneficial effects of Alternatively, quality. cial to product to crosslinking of the transglutaminase may be attributed to be linked to the HMW-glutenin subunits, proteins known underway to experiments are Further flour. quality of bread and identify the spe- differentiate between these possibilities

Food Chemistry and Toxicology