Celiac disease occurs in the small intestine due

Celiac disease occurs in the small intestine due to the ingestion of gluten and triggers an immune response by T-cells resulting in tissue remodelling and malnutrition [6]. Earlier studies by many researchers have shown that the prolamins of gluten are the toxic agent for celiac disease, since they cannot be digested appropriately in the small intestine of humans because of their specific amino pituitary adenylate cyclase-activating peptide composition. The amount of consumed gluten is therefore crucial to celiac patients [1], [2], [4]. Gluten is rich in proline which as a unique cyclic side chain structure and imparts exceptional conformational rigidity. This distinctive structural form plays an important role in the physiological condition of different diseases by protecting immunogenic peptides from proteolytic degradation [7], [8], [9], [10]. Therefore proline is present in peptides causing various diseases such as Parkinson’s disease, depression, celiac sprue, annoroxia, bulimia nervosa as well as affecting blood pressure regulation. Even foods not containing whole wheat, barley, or rye often contain small amounts of prolamins from contaminations of cereals and can possibly induce allergy [11]. Gluten from these sources can be rendered safe, if it is degraded by prolyl endopeptidases into non-allergenic peptides and a number of enzyme preparations purporting to do this have come onto the market recently. The prolyl endopeptidase (PEP), EC 3. 4. 21. 26 is also known as proline specific endoprotease and belongs to the serine protease family, has the ability to cleave peptides at internal proline residues [12]. PEP is structurally and functionally closely related to the dipeptidyl peptidase IV (EC 3.4.14.5), oligo peptidase B (EC 3.4.21.83) and acyl-aminoacyl peptidase (EC 3.4.19.1) sub-families are members of the S9 peptidase family. As such, this enzyme class has been extensively investigated for potential pharmaceutical use to degrade gluten or to treat celiacs and for other therapeutic use [13], [14]. A number of studies have examined degradation of wheat gluten and to some extent barley gluten and components by prolyl endopeptidase initially extracted from Flavobacterium meningosepticum [15] and other microbes, such as Xanthomonas sp. [16], Aermonas hydrophilic[17], Sphingoonas capsulate [18], Halobacterium halobium S9 [19], Lactobacillus helveticus[20], Myxococcus xanthus[21], Asperillus niger[22], [23], [24] and Aspergillus oryzae[25]. However, these have shown ability to break down toxic gluten peptides only under in vitro conditions. In a very recent study Janssen et al. [6] a number of dietary supplements containing different types of gluten degrading enzymes were tested for their ability to degrade immunogenic gliadin peptides from wheat. It was found that none of these enzyme preparations could degrade the allergenic peptides, except for the Aspergillus niger prolyl endopeptidase (AN-PEP) produced by DSM in Holland. In another recent article by Walter et al. [26], it was reported that the PEP from A. niger was again found to be the only enzyme capable of delivering a gluten free starch from wheat (gliadin) and barley (hordein) and had activity 690000 times greater than from bran extracts. Stenman et al. [27] also found that enzymes from germinating barley could degrade rye secalin, eliminating toxic reactions, but few, if any studies on microbial derived enzymatic degradation of secalin, or avenin appear to have been reported. Moreover, all the enzymes discussed above are limited by the pH and temperature range in which they are active, which makes them less suitable for the majority of industrial food processes, where high temperatures are used. The mashing process of beer brewing (e.g. 50–80°C) is a crucial step for preparing the wort for the yeast fermentation. The mashing process takes approximately 2h and includes a series of rests at increasing temperature (typically ca. 54°C, 64°C and 78°C) at which the various enzymes have optimal activity; which includes α-amylase, β-amylase, proteases, cellulases and β-glucanases. Subsequently there is a boiling step for 60min at 100°C before the wort is cooled and fermentation is initiated by yeast starter culture [28]. Beer brewing practices thus already involve protein degradation to produce fermentable amino acids for yeast growth, and it is not unthinkable to completely degrade gluten protein from beer during the mashing process itself. In fact, different methods of degrading and removing gluten have been developed, including the use of different proteases as well as microbial transglutaminase. However, no heat stable gluten degrading enzyme has been reported, so current efforts focus on gluten degradation during fermentation, maturation or clarification, where temperatures are typically in the sub-zero to 20°C range. It has been reported that gluten concentration in the final beer can be reduced by use of prolyl endopeptidase during the fermentation part of the brewing process [29]. In addition, it has been demonstrated that use of acid proline-specific endoprotease from A. niger in low level in bottled beer prevents chill-haze formation (often caused by gluten) and had almost no effect on the beer [23]. However, gluten degradation during wort preparation would be advantageous, since it would result in more fermentable amino acids in the crucial initial stages of yeast growth.