Elsevier

Journal of Cereal Science

Volume 60, Issue 2, September 2014, Pages 440-447
Journal of Cereal Science

Targeted proteomics-based analysis of technical enzymes from fungal origin in baked products

https://doi.org/10.1016/j.jcs.2014.04.007Get rights and content

Highlights

  • The paper addresses the detection issues of technical enzymes in bakery.

  • Possibilities of targeted proteomics-based analysis of food proteins.

  • An alternative to antibody recognition for specific peptides is put forward.

  • Method can be adapted for other protein based supplementary ingredients.

Abstract

The application of technical enzymes is a potential tool in modulating the dough and baking quality of cereal products. No endogenous amylases (α- and β-forms) are present in mature wheat grains; they may be synthesized or activated during germination. Hence, microbial α-amylases are added to the dough, being resistant to the endogenous α-amylase/trypsin inhibitors. Here, we report on the initial identification of two technical enzymes from a commercial sample based on an in-gel tryptic digestion coupled with MALDI-MS analysis. The primary component of the protein fraction with 51.3 kDa was α-amylase from Aspergillus species. A second major protein with 24.8 kDa was identified as endo-1,4-xylanase from Thermomyces lanuginosus. In the following experimental work up, a targeted proteomics approach utilizing the combination of specific proteolytic digestion of the added amylase and xylanase in wheat flour, dough or baked products, solid phase extraction of released peptides and their detection using LC-MS/MS was optimized. The targeted (MRM) MS/MS peptide signals showed that the peptide “ALSSALHER” (MW = 983) originating from amylase and “GWNPGLNAR” (MW = 983) from xylanase can be used to identify the corresponding technical enzymes added. Consequently, locally available baked products were tested and found to contain these enzymes as supplementary ingredients.

Introduction

In order to achieve product specific quality requirements and to meet the demands of high production turnovers, the food industry has been observed to augment the application of technical enzymes. One particularly interesting and important field of such utilization has crystallized in the baking process, where it has been shown to improve some essential functional properties such as the formation of bread crust, bread volume and to enhance its taste properties (Huber, 2006).

One of the best known technical approaches applied for some years to improve the bread quality is the use of α-amylase. The amylases of potential commercial importance used to degrade starch granules are those that split α-1,4 bonds in these structures. Many amylases are produced from animals or microorganisms, although plant substrates constitute substantial sources as well. A variety of amylases, mostly from microbial, mammalian and cereal sources are well characterized. α-amylases commercially available and used extensively in processing food technology are mostly from fungal origin (e.g. from cultures of Aspergillus species) (Sahnoun et al., 2012). The advantages of such systems are enhanced yeast growth reflected in increased dough volume, an improved bread structure and texture (firmness of breads) and darkened crust coloration (Cauvain and Young, 2007, Kim et al., 2006). A further substantial improvement of bread quality indicators such as volume, porosity and elasticity were also obtained by combining fungal α-amylase and glucose oxidase (Stoica et al., 2010).

Synergistic effects with other polysaccharide degrading enzymes (e.g. xylanase), in breadmaking systems have also been reported (Butt et al., 2008, Caballero et al., 2007, Hemalatha et al., 2010). Xylanases are hydrolytic enzymes, which internally cleave the β-1,4 chains of cereal xylans, often referred to as arabinoxylans (Han et al., 2013, Paes et al., 2012). According to their protein sequence homology, most xylanases are classified into the glycoside hydrolase (GH) family, differing in their substrate specificity (Paes et al., 2012). Their use in excess is to overcome the high endogenous inhibitory effect of xylanase inhibitors (TAXI- Triticum aestivum xylanase inhibitor, XIP- xylanase inhibitor protein and TLXI-thaumatin-like xylanase inhibitor) from wheat (Butt et al., 2008, Paes et al., 2012). Xylanases have been reported to improve the bread volume, crumb structure and reduce stickiness (Butt et al., 2008, Hilhorst et al., 1999, Jiang et al., 2005). At optimum levels, the use of the enzymes yields significant improvement of bread shelf life and reduces bread staling (Caballero et al., 2007).

Under consideration of consumer protection issues and the up-coming discussion with regard to the allergenic potential of technical enzymes used in bakeries to improve the bread quality (Sander et al., 2000, Smith, 2004, Smith et al., 1997), the final products thus produced underlie strict regulative labelling requirements (EG/1332/2008). Currently, no enzyme labelling in baked goods is being practised. Accordingly, it is necessary to scrutinize if the additive applied, including enzymes applied in the food industry, can be detected in changed or unchanged form in the end product (EG/1169/2011). Therefore, analytical methods which could address such detection and labelling issues of technical enzymes are required. Some immune assay based methods to measure e.g. α-amylase have been reported (Lillienberg et al., 2000). Considering the complexity of the matrix present during food processing and the specific antibodies not always being available, alternative methods should be proposed.

Therefore, the main objective of this study was to investigate the possibility of the application of proteomics-based analysis tools in establishing a detection method for technical enzymes of fungal origins. The study was confined to two enzyme representatives using electrophoresis coupled with in-gel tryptic digestion and MALDI-MS analysis. This was followed by a targeted proteomics approach using the combination of specific proteolytic digestion, solid phase extraction of the peptides and the identification of the enzymes in wheat dough by their specific peptides using LC-MS/MS.

Section snippets

Materials

Wheat flour type 405 was obtained from the local supermarket (moisture, ca. 7%; protein, ca. 11%; ash, ca. 0.4%; Carbohydrates, ca. 80%). Commercially available enzyme preparations (Fungamyl Super AX, Novozymes Switzerland AG, Dittingen Switzerland) were employed. Local baked products tested: 1 = marble cake, 2 = chocolate cake, 3 = multi-grain bread, 4 = lemon cake, 5 = multi-grain roll (Crustigette), 6 = protein bun, 7 = Crustini roll, 8 = wheat-rye bread. Bovine serum albumin (BSA), p

Preliminary characterisation

Fungamyl Super AX is a commercially available Novozymes blend of two enzymes: Pentopan 500 (GH11 xylanase) and Fungamyl (alpha-amylase). It is generally applied for special dough conditioning. The preparation is not completely soluble in water and the solubility as determined by the Bradford method was 12.5 μg/mg Fungamyl Super AX sample. The preparation is more soluble in SDS solution and the solubility can be increased in 1% SDS-solution amounting to 210 μg/mg (Lowry protocol). Therefore, the

Concluding remarks

Generally, such technical enzymes have been recommended to improve or enhance some essential functional properties such as the dough formation, bread crust and bread volume in bakery products. An application of a higher amount of technical enzyme would lead to a deterioration of these quality parameters. The lowest amount applied in this study was 0.5 g Fungamyl®/kg flour amounting to 500 ppm on weight basis. Since we found that only ca. 10% of the sample Fungamyl Super AX can be attributed

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