CDH OxyPlus

Cellobiose dehydrogenase OxyPlus (with increased oxygen affinity) was kindly provided by Ms.

M. Preims. from the Institute of Food Biotechnology. The cross-linking reaction was carried out in a 2% AX standard solution, containing 15 mM D-Glucose, at 25°C and pH 6.0. Approximately 5.9 U CDH OxyPlus were present per gram of the final sample (standard solution plus enzyme solution).

Figure 37 Rheological characterization of CDH OxyPlus

Table 43 CDH Oxyplus, initial and final value of viscosity, loss and shear modulus

Time (s) G`(Pa) G''(Pa) η *(Pas)

5 0.0442 0.5282 0.0840

5000 0.0385 0.5540 0.0880

CDH OxyPlus did not show a clear cross-linking effect, but a slight rise of G’ at the very beginning of the measurement could be observed. Further investigations would be necessary to verify this assumption and should include application of higher enzymatic activity as well as the use of higher concentrated enzyme solutions.

6 Conclusions and Outlook

The expression of the enzymes PnGOx and Cgl1, based on their DNA sequence published in the respective paper, did not give the expected results for PnGOx (2.0 U/mL vs. 615 U/mL of Pichia supernatant by Gao Z. et al. (2012)) and no activity at all for Cgl1. Also the addition of amino acids by site-directed mutagenesis to the published PnGOx sequence, that must have been added to the PnGOx sequence accidentally, when cloned into the vector pPIC9 by Gao Z.

et al. (2012), did not lead to the desired high expression yield. PnGOx moreover did not show any AX crosslinking effect in preliminary rheological tests when high amounts of enzyme were used. As a consequence, Cgl1 could not be characterized in terms of its cross-linking ability and PnGOx was not produced on a large scale for subsequent baking trials.

During rheological characterization, 5.9 U of CDH OxyPlus per gram of the final test sample did not show a clear crosslinking effect. Therefore further rheological investigations should be made and higher activities applied, as in case of a positive result its field of application could be expanded. Further experiments with CDH OxyPlus are suggested to be done using a more concentrated enzyme solution, as otherwise the AX solution would be strongly diluted with enzyme solution.

BliLac showed a pronounced crosslinking effect even when comparably low activities were applied. It might also be interesting to produce the BliLac double mutant in large quantities for baking trials to verify whether it works in the more complex matrix equally well as in the standard rheological test. To make BliLac also applicable for large-scale production in food industry, the experiments of Koschorrek et al. (2009) could be continued in a way to increase the expression yield of the double mutant and its activity with ferulic acid even further.

2.3 U of PcPOx as well as 34.8 U of BaLac were required to show a pronounced cross-linking effect. Similarly to BliLac, one could use genetic engineering methods on BaLac in order to find a mutant with increased activity with ferulic acid. Prior to this, the development of a standard assay for laccases and their activity with ferulic acid would be necessary; alternatively one could stick to measuring the relative oxidation of numerous phenolic acids by laccase with HPLC, like stated by Koschorrek et al. (2009).

It has to be kept in mind that all produced oxidases are subsequently not applied in a simple environment like the arabinoxylan standard, but in a dough with many other components, where the enzyme’s mobility is much more limited and pH and matrix constitution vary throughout time.

Small bacterial laccases will certainly have an advantage, it is however difficult to predict which of the produced oxidases will perform best. Nevertheless, results from rheological tests show first signs for this new gluten network replacement approach to be successful. An interesting

option for subsequent baking tests would be to not only apply one single enzyme per trial, but also the combination of laccase and pyranose 2-oxidase, as those enzymes differ in their cross- linking mechanisms and substrates and might have a synergistic effect. In addition, as pH will drop during the sourdough fermentation, the pH at the beginning of the process is closer to the pH optimum of pyranose 2-oxidase and in the end closer to that of laccase. For now, however, no statement can be made whether the enzymes will be inhibited in their activity by other components in the dough matrix that were not considered in the preliminary rheological test.

BaLac is definitely not inhibited by chloride ions, which allows a first positive prognosis for its cross-linking ability even in the presence of sodium chloride in dough.

In the course of this work BaLac and PcPOx were successfully produced in high quantities – 71722 U and 11742 U, respectively - for further baking trials, in which their cross-linking ability in dough will be investigated. Still, laccase and PcPOx that were produced within this project are not safe for consumption: GRAS is an acronym for “Generally Recognized As Safe”, and is granted to a food additive after approval by the FDA. PcPOx does not have GRAS status since it was produced in E. coli, which belongs to the group of enterobacteria. Laccase was produced from Pichia pastoris, which has GRAS status, but anyway its induction with methanol is not appropriate for the production of food products. An approach for a continuation of this project would be, for example, the attempt to express PcPOx and BliLac in food grade hosts and BaLac under control of a constitutive promoter, so they can be added as technical aids to gluten-free bread that can actually be consumed. In food grade hosts the main focus is put on the absence of antibiotic resistance markers or markers that require heavy metals for selection, and on using alternative selection markers instead. (Peterbauer et al., 2011) Food grade expression would be for instance possible in lactobacilli like L. plantarum, which have the additional benefit that they could also be used directly in sourdough as a part of the mixed starter culture of yeast and lactobacilli. So far the absence of gluten in gluten-free bread was tried to be compensated by the addition of additives, such as hydrocolloids (Lazaridou et al., 2007); these did not give fully satisfying viscoelastic dough properties and partially poor quality profiles (Thompson, 2009).

Moreover, the addition of such additives has to be declared, which gives food a rather negative image as there is currently an increasing trend for consumption of natural foods without additives. The use of food grade enzymes would give food products a more natural image as according to EU Regulation No 1169/2011, art 20 (b) their use does not have to be declared if they are used as processing aids.

Apart from AX crosslinking there is another possibility to get enzymes involved in the production of gluten-free bread: endo-xylanases may be used for solubilization of water-insoluble arabinoxylan and lead to improved extraction of WEAX from raw material like rye bran

(Figueroa-Espinoza et al., 2004). For research purposes one could try to express and apply xylanases from literature, which are not yet commercially available, as alternatives to the predominantly used pentosanase derived from T. lanuginosus (Pentopan®, Novozymes).

In any case, in this work various enzymes were proven beneficial for the production of sensorically and texturally appealing gluten-free bread. As discussed previously, there are many ways to continue this part of the project and to further explore the world of arabinoxylan cross- linking enzymes.

7 Appendix