Effect of Transglutaminase, Citrate Buffer, and Temperature on a Soft Wheat Flour Dough System Marcela P. Bagagli,1,2 Sahar Jazaeri,3 Jayne E. Bock,3 Koushik Seetharaman,4 and Helia H. Sato1 ABSTRACT
Cereal Chem. 91(5):460–465
Transglutaminase (TGase) can improve the functional characteristics of proteins by introducing covalent bonds inter- or intrachains. Temperature and pH interfere with the protein structure and the catalytic activity of enzymes. Because these three factors can act synergistically, TGase, citrate buffer, and temperature were evaluated for their effects on the rheological and chemical changes in low-protein wheat flour dough. Dough strength, measured by microextension test, significantly increased with increasing levels of TGase (8 U/g of protein), with changes in pH of the citrate buffer
(pH 6.5), and by the effect of interaction between these factors. The same trend was observed in the size-exclusion HPLC measurements, indicating that these two parameters have the effect of increasing gluten protein aggregation. Temperature had a significant effect on dough extension, measured by microextension test. The changes in secondary structure of gluten protein were investigated by FTIR second-derivative spectra (amide I region, 1,600–1,700 cm–1) and showed an increase in β-sheet structures initiated by TGase, citrate buffer pH, and their interaction.
Wheat is one of the most important crops in the world, and its flour has the ability to form dough with unique viscoelastic properties that are specifically related to the gluten network (Shewry et al 2001). The proportion of gliadins and glutenins, the main fractions of gluten proteins, in the gluten network influences the rheological behavior of dough and consequently the breadmaking performance of flour (Delcour et al 2012). During dough mixing, mechanical energy promotes the breakage and formation of covalent bonds (e.g., disulfide bonds) and noncovalent bonds (e.g., hydrophobic interactions), thereby changing the conformational structure of proteins. The chemical bonds between and within gluten proteins are strongly related to dough strength and viscoelastic properties (Singh and MacRitchie 2001; Delcour et al 2012; Sivam et al 2013). Gluten structure can be changed by several additives, including enzymes that are generally recognized as safe for human consumption. As a consequence, the breadmaking and rheological properties of the dough are affected (Steffolani et al 2010). Transglutaminase (TGase, E.C. 2.3.2.13) enhances functional properties of food products such as elasticity and water-holding capacity (Zhang et al 2009). TGase catalyzes the acyl-transfer reaction between a γ-carboxyamide group of the peptide bond of a glutamine residue and the ε-amino group of a lysine, resulting in an isopeptide bond, ε-(γ-glutamyl)-lysine, resistant to protease action or mechanical damage (Zhang et al 2009). Gliadins and glutenins are reported as substrates for TGase, even though the amount of lysine in these protein fractions is relatively low (Bauer et al 2003a; Autio et al 2005). The TGase-mediated isopeptide bond was reported to change the characteristics of cereal flour doughs, especially those with low protein contents such as soft wheat flours and gluten-free formulations, thereby improving the breadmaking properties of the dough (Medina-Rodríguez et al 2009; Renzetti et al 2012). The enzyme has also been used to improve the characteristics of baked products made with a mixture of wheat flour and other cereal flours (e.g., soybean and oats), increasing the nutritional value and improving the technological functionality of these products (Bonet et al 2006; Ribotta et al 2010).
The extent of TGase treatment is dependent on the amount of glutamine and lysine residues on the surface of proteins and accessible to the enzyme. The exposure of TGase substrates in proteins can be obtained by increasing the ionic strength, thermal treatments, or the addition of denaturants. Wang et al (2007) observed that the thermal treatment of gluten proteins before TGase treatment increased the number of lysine and glutamine residues on the surface of gluten and improved the gelation behavior and gel properties of TGase-treated gluten. The pH and ionic strength of citrate buffer and temperature of mixing are supposed to interfere with the gluten protein network (Tanaka et al 1967; Bloksma and Nieman 1975; Farahnaky and Hill 2007) and the catalytic site of TGase (Kashiwagi et al 2002; Macedo et al 2010). The aim of this study was to evaluate the effect of the amount of TGase, citrate buffer pH, temperature, and their interactions on soft wheat dough rheology and biochemical characteristics.
1 Department
of Food Science, University of Campinas, Rua Monteiro Lobato, 80, P.O. Box 6121, CEP 13083-862, Campinas-SP, Brazil. author. Phone: +55 1935212175. E-mail:
[email protected] 3 Department of Food Science, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada. 4 Deceased; formerly Department of Food Science, University of Guelph, Guelph, ON, Canada. 2 Corresponding
http://dx.doi.org/10.1094 / CCHEM-09-13-0176-R © 2014 AACC International, Inc.
460
CEREAL CHEMISTRY
MATERIALS AND METHODS Flour. Commercial soft wheat flour (Everlite Farine enriched, Kraft Canada, Mississauga, ON, Canada) was used for all experiments. The protein content of the flour (7.44%; rate of nitrogen, 5.7) was determined with an automated Dumas protein analysis system (LECO Instruments, Mississauga, ON, Canada). Ash content of the flour (0.66%) was determined according to AACC International Approved Method 08-01.01, and moisture (12.3%) was measured with a halogen moisture analyzer (MB35, Ohaus, Parsippany, NJ, U.S.A.). Enzyme. A commercial TGase with a corn starch carrier was purchased from Kinry Food Ingredients (Shanghai, China). The TGase activity was tested based on the colorimetric hydroxamate assay with the substrate N-carbobenzoxy-L-glutaminyl-glycine according to the method of Macedo et al (2010). One unit of TGase activity was defined as the amount of enzyme needed to produce one micromole of hydroxamic acid per minute at 37°C. A calibration curve was prepared with L-glutamic acid γ-monohydroxamate. The optimum pH and temperature of TGase were 6.5 and 37°C, respectively. Dough Preparation. The flour was mixed in a Brabender farinograph (C. W. Brabender, Hackensack, NJ, U.S.A.) with TGase and citrate buffer (0.05M) in a 50 g bowl for 6 min at 63 rpm. Temperature, TGase amount, and citrate buffer amount varied as indicated in Table I. The total amount of buffer added to the flour was fixed at 53% of the flour weight. This value was based on the water absorption to reach 500 BU for the same flour mixed in the farinograph with distilled water at 30°C and 63 rpm. After mix-
ing, the dough was allowed to rest for 30 min inside the bowl (constant temperature) and was then divided into two pieces: one was used for texture analysis, and the other was freeze-dried for further analysis. Dough Rheology. Dough uniaxial extensibility was analyzed with a TA.XT Plus texture analyzer (Stable Micro Systems [SMS], Godalming, U.K.) and the SMS/Kieffer rig (5 kg load cell). Dough was placed in a Teflon strip form, pressed, and allowed to relax for 40 min at 25°C. The strips were placed on the platform and extended at a speed of 3.3 mm/s (Ribotta et al 2010). Seven measurements were taken for each dough batch. Resistance to extension (Rm, maximum force), maximum extensibility (Em, extension to break), and the area under the curve (work input) were calculated with Texture Exponent 32 software (SMS). Soluble Proteins (Size-Exclusion HPLC). Freeze-dried dough was crushed and milled with particle size standardized at 0.5 mm. The soluble proteins were extracted by adding 1 mL of 2% SDS phosphate buffer (0.05M, pH 6.8) to 20 mg of each sample and subjecting samples to shaking at 25°C for 1 h. Samples were centrifuged at 10,000 rpm for 5 min at room temperature. The supernatants were filtered through a 0.45 μm filter. Chromatography was performed with a BioSep-SEC-s4000 size-exclusion analytical column (300 × 7.8 mm, Phenomenex, Torrance, CA, U.S.A.) coupled in an HPLC system (LC 10A, Shimadzu, Kyoto, Japan) with a UV-visible detector (SPD 10A, Shimadzu). Proteins were eluted isocratically by a 50% (v/v) acetonitrile–water solution (0.05% trifluoroacetic acid) at a flow rate of 1 mL/min with a run time of 30 min (Medina-Rodríguez et al 2009). The absorbance was recorded at 214 nm. The total area under the chromatogram was used to evaluate the extractability of dough proteins in the presence of 2% SDS. SDS-PAGE. SDS-PAGE was carried out under reducing conditions with a 10% polyacrylamide gel and Coomassie blue staining. For each sample, 11 mg of freeze-dried dough was dissolved in 400 μL of SDS sample buffer (50 mM dithiothreitol), heated at
95°C for 4 min, and centrifuged at 10,000 rpm for 5 min. Supernatants (10 μL) were loaded into the gel and electrophoresed at 120 V (Laemmli 1970). The gel was scanned with a Gel Doc EZ imager (Bio-Rad Laboratories, Hercules, CA, U.S.A.). Infrared Spectroscopy. Fresh dough was carefully removed from the mixer after 30 min of rest and was placed on a clean, dry diamond crystal with a deuterated L-alanine doped triglycine sulfate detector. Infrared spectra (100 scans) were recorded with a resolution of 2 cm–1 with an FTIR spectrophotometer (IRPrestige21, Shimadzu) at room temperature in the region 600–4,000 cm–1. Spectra were normalized and baseline corrected before spectral analysis. Spectral analysis was conducted with OPUS 7.0 software (Bruker Optics, Billerica, MA, U.S.A.). Mixtures of H2O and D2O were used as standards according to the method of Bock and Damodaran (2012) for subtraction of H2O in the amide I region. Second-derivate spectra were calculated with a five-point Savitzky–Golay function to reveal structural changes in gluten protein structure in the amide I region (1,600–1,700 cm–1). The area of the second-derivative curve associated with each secondary structure was calculated by integration and divided by the total area of the curve to predict the secondary structure content. The α-helix structures were assigned from 1,653 to 1,658 cm–1 of the spectra; the β-sheet structures were assigned from 1,620 to 1,643 cm–1 of the spectra; the β-turn structures were assigned from 1,659 to 1,673 cm–1 of the spectra; and the random structures were assigned from 1,644 to 1,652 cm–1 of the spectra. Factorial Design. A 23 factorial design with four genuine replicates at the center point was used to evaluate the effect of TGase, citrate buffer pH, and temperature on dough development, rheological properties, and biochemical properties. The effects (the change in the response as we move from the low to the high level of the variables) were calculated and evaluated with Statistica 8.0 software (StatSoft, Tulsa, OK, U.S.A.). The higher the modulus of effect, the higher the change promoted by the variables on the parameters. All results were evaluated at a 90% confidence level and R2 > 0.60. Table I shows the values for each factor in the 12 experiments of the factorial design.
TABLE I Factorial Design for the Evaluation of Effect of Transglutaminase, pH, and Temperature on Soft Wheat Dough Experiment
Enzyme (U/g of protein)
Citrate Buffer pH
Temperature (°C)
2 8 2 8 2 8 2 8 5 5 5 5
4.5 4.5 6.5 6.5 4.5 4.5 6.5 6.5 5.5 5.5 5.5 5.5
30 30 30 30 37 37 37 37 33.5 33.5 33.5 33.5
1 2 3 4 5 6 7 8 9 10 11 12
RESULTS Effect of TGase, Citrate Buffer pH, and Temperature on Dough Rheology. The effects of TGase, citrate buffer pH, and temperature on maximum resistance (Rm), maximum extensibility (Em), and work input (A) are presented in Table II, and the extensigrams are shown in Figure 1. TGase and pH showed a positive and significant effect on Rm, and the interaction between these parameters was significant at a 90% confidence level. Temperature had no significant effect on maximum resistance. The addition of citrate buffer (50 mM, pH 6.5) to the dough increased the resistance by a factor of 2 in comparison to dough made with distilled water. Tanaka et al (1967) observed a reduction of Rm with increasing pH, adjusted by acetic acid and the addition of salt. In this study, the addition
TABLE II Effect of Transglutaminase Amount, Buffer pH, and Temperature on Soft Wheat Dough Protein: Resistance to Extension (Rm), Maximum Extensibility (Em) and Work Input (A) Rm (g) Factor Mean Enzyme (U/g of protein) Buffer pH Temperature (°C) Enzyme × buffer pH Enzyme × temperature Buffer pH × temperature R2
Em (mm)
A (g × mm)
Effect
P Value
Effect
P Value
Effect
P Value
14.99 6.06 11.71 –0.07 2.61 0.95 –0.29 0.98
