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Functional properties of enzymatically hydrolysed wheat gluten

S.K. Bhail1,3 and L. Day2,3

1RMIT, 124 La Trobe Street, Melbourne, VIC 3000, Australia
Food Science Australia, 671 Sneydes Road, Werribee, VIC 3030, Australia
Value-Added Wheat CRC, North Ryde, NSW 1670, Australia


Wheat gluten is a major co-product of the wheat-starch industry. However, lack of ionic character of gluten proteins limits its solubility in pH neutral solvents, restricting functional properties and consequently its utilisation in the food industry. The aim of this project was to investigate the effect of the two newer commercial enzymes of complex nature, Flavourzyme and Protamex®, on the chemical and physical properties of wheat gluten after hydrolysis reaction, with the goal to develop modified gluten having different physical and functional properties.

Materials and methods

Vital wheat gluten (VWG) was obtained from an Australian commercial producer. The protein content of the gluten was 73.5%. The enzymes used were food grade commercial enzymes Flavourzyme 500L and Protamex (Novozymes A/S, Denmark). Other chemicals were analytical grade from Sigma-Aldrich (Sigma-Aldrich Pty. Ltd, Sydney).

Preparation of gluten hydrolysates

Wheat gluten (20 g) was dispersed slowly into the preheated water (100 mL) under constant agitation. Enzyme (0.5 mL or 0.5 g) was added to the gluten dispersion. The reaction was carried out for 30 min and the enzyme was inactivated afterwards in accordance with the enzyme manufacturer’s protocol. The hydrolysate was then centrifuged at 11,000 g for 10 min. The supernatant was removed and the pellet was re-suspended in distilled water (approx. 100 mL) and centrifuged. The combined supernatant and the remaining pellet were freeze-dried.

Determination of protein content and protein solubility

The nitrogen content of the hydrolysed gluten products, the dried supernatant and pellet, was determined according to the AACC method 46-30. Protein solubility was determined by preparing a 10% (w/v, dry powder base) protein solution in water. The solution was centrifuged at 15,300 g for 5 minutes to remove insoluble materials. The protein solution was then dried for the determination of the nitrogen content. The soluble protein was calculated using the factor 5.7.

Gel filtration liquid chromatography

The molecular weight distribution of peptides in gluten hydrolysates (supernatant) was analysed using a Liquid Chromatography system equipped with a gel filtration Superdex 200 10/300GL column (Amersham Biosciences Pty Ltd., Castle Hill, NSW). The elution buffer was 100mM Tris, 50mM NaCl, 0.02% NaN3, at pH 7.0. Protein standards with a range of molecular weights were used for the calibration of the column.

Emulsification capacity

Emulsification capacity was determined according to the method of Vuillemard et al. (1990). Protein solutions of the supernatant fractions after the enzyme hydrolysis and the original VWG were prepared at concentrations of 0.025%, 0.075%, 0.1% and 0.15% (w/v) (protein base).

Results and discussion

Upon hydration, the polypeptides of VWG interact to form a viscoelastic mass. The resultant product is characteristically insoluble in solutions of neutral pH, which consequently limits its application in the food industry. This property is attributed to the non-polar moieties of gluten proteins which exhibit a hydrophobic effect. In addition, the relatively large molecular size of the gluten proteins, in particular the glutenin proteins, decreases the number of solvent molecules surrounding the proteins (Singh and MacRitchie, 2001). Thus in order to diversify the application of gluten proteins as a high-protein food additive, it is essential to increase the solubility of the proteins over a wide range of pH (Ahmedna et al, 1999). Potentially, this might be achieved by structural modification of gluten proteins. Chemical or enzymic hydrolysis has been used to improve the functional properties of wheat gluten (Batey, 1985; Mimouni et al, 1994; Drago & González, 2000). Recently, enzymic modification of corn and soy proteins using Flavourzyme and Protamex have been reported to show greater improvement of the emulsifying and foaming properties of these plant proteins (Kim et al, 2004; Surówka et al, 2004). However, there are no specific reports for the hydrolysis of wheat gluten using Flavourzyme or Protamex.

Higher proportion of the soluble fraction – the supernatant by weight, was produced using Protamex in comparison with Flavourzyme. The protein content in the supernatant fraction was also slightly high when Protamex was used (Table 1). This indicates that Protamex may have better specificity for gluten proteins than Flavourzyme. All the protein in a 10% solution of the supernatant fraction was soluble, while the remaining proteins in the pellet had very low solubility.

Table 1. The effects of Flavourzyme and Protamex on the solubility of gluten.




Enzyme used

Proportion of product (%)

Protein Content (%)

Protein solubility (%)

Proportion of product (%)

Protein Content (%)

Protein Solubility (%)

















Chromatographic analysis of enzyme hydrolysed wheat gluten showed that both enzymes were able to produce soluble peptides with a wide range of molecular weights (Figure 1). Although the size distribution of the peptides was similar between the two enzymes used (Table 2), the relative amounts of each peptides were likely to be different as showed by the relative sizes of the peaks. Flavourzyme is more effective than Protamex in hydrolysing gluten protein into small peptides, most noticeably the smallest peptide peak 5.

The molecular sizes of the peptides presented in the soluble portion of the hydrolysed gluten were between approx. 48,000 to 1,000 g/mol. This is comparably lower than the native gluten proteins; up to 80,000 for gliadins and greater than 500,000 for glutenin polymers.

Figure 1. Chromatograms of the soluble proteins hydrolysed by (a) Flavourzyme, and (b) Protamex. The molecular weights of the peaks were summarised in Table 2.

Table 2. Molecular weight distrubition of the solubilised gluten proteins determined by the gel filtration chromatography.


Molecular weight of the solubilised gluten protein(g/mol)

Peak no.






Enzyme used















The method for the determination of emulsification capacity measures the total oil emulsified by the dispersed protein in water prior to the point of emulsion inversion, i.e., the maximum amount of oil that can be retained in an oil-in-water phase by protein. The results were expressed as the amount of oil being emulsified by per milligram of protein (Figure 2). When protein concentration increased, the total amount of oil emulsified by the same volume of protein solution increased, the emulsification capacity of the enzyme hydrolysed gluten expressed as g oil/mg protein decreased. The trend was similar to that of milk protein, and was caused by the destabilization of the emulsion due to close packing of the oil globules which occurs when the volume of the oil phase reaches 65-85% of the total emulsion volume (Vuillemard et al, 1990). Therefore the results above the protein concentrations of 0.075% should not be related to the true emulsification capacity of the protein as the amount of oil used has exceeded 65% of the total volume.

The differences in the emulsification capacities of the enzyme hydrolysed soluble proteins and the vital wheat gluten were observed at 0.025% (w/v, protein base) (P=0.001). Enzyme hydrolysis led to a clear increase in the emulsifying properties of gluten proteins. Flavourzyme produced soluble gluten protein hydrolysate with an emulsification capacity of 6.1±0.6%, while Protemax produced soluble hydrolysate with the greatest emulsification capacity of 8.1±0.4%. The results showed an increased surface activity at the oil-water interface presumably provided by the exposure of more hydrophilic residues through changing in protein conformations and sizes caused by the enzyme hydrolysis reactions.

Figure 2. Comparison of the maximum emulsification capacities of enzyme hydrolysed gluten with the vital wheat gluten at various protein concentrations.


Wheat gluten hydrolysed with complex enzymes, in particular Protamex, demonstrated enhanced total protein content, content of soluble proteins and improved emulsification capacity at 0.025% (w/v) protein concentration.


This research was funded by the Value Added Wheat CRC summer vacation studentship to Ms S. Bhail. The authors would also like to thank Dr. R. Williams (Food Science Australia, Werribee) for setting up the HPLC analyses and the emulsification capacity determination systems.


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Drago, S.R. and González, R.J. (2000). Innov. Food Sci. & Emerging Technol. 1(4), 269-273.

Mimouni, B.; Raymond, J.; Merle-Desnoyers, A. M.; Azanza J. L. and Ducastaing, A. (1994). J. Cereal Sci. 20 (2) 153-165.

Kim, J. M.; Whang, J. H. and Suh, H. J. (2004). Eur. Food Res. Technol. 218(2), 133-138.

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