Enzimi nei prodotti dolci da forno: individuazione, quantificazione e studio degli effetti su impasti e prodotti a lievitazione naturale

Breadmaking quality of wheat flour is largely determined by the quality and the quantity of its gluten proteins. The quality of gluten is also dependent on different factors such as wheat variety and growing conditions. In this context, the treatment of flours with functional additives must be considered. Chemical improvers have been used for decades in breadmaking as a way of adjusting the variation in flour properties and baking conditions. Nowadays, the baking industry is deeply involved in research for alternatives to chemical compounds because of their potential hazards. The enzymatic treatment of wheat flours is an interesting alternatives to improve functional properties of flours and, in consequence, to generate changes in the structure of the dough. The intentional inclusion of enzymes in bread formulas dates back to more than one century. Today, a wide range of enzymes produced especially for breadmaking is available for bakers and a variety of aims may be pursued by enzyme addition. During dough mixing, wheat flour is hydrated and the gluten proteins are transformed into a continuous cohesive viscoelastic gluten protein network. Starch is relatively inert during dough mixing where plays a role as a “filler” that contributes to increase dough viscoelasticity but has a critical influence during the baking process when it gelatinizes, and during subsequent storage, when retrogradation accounts for the major part of bread staling. Talking about enzymes involved in gluten modification, different enzymes affect different protein fraction depending on their particular mechanism. The type of protein being cross-linked appears to be highly correlated with the character of qualitative changes in the final product. For this reason, association of different gluten modifying enzymes could be an excellent option to improve overall quality of backed products. To avoid an excessive increase in dough tenacity the treatment with gluten degrading enzymes is also proposed. Besides the gluten network, other secondary minor compounds of flour such as arabinoxylans and pentosans can be modified and the combined use of these enzymes could induce synergistic effect on dough behaviour or product quality. Amylolytic enzymes have been also used as active contributors towards fresh bread quality and anti-staling behaviour during storage. Bacterial or fungal enzymes are being used in bakery for years, but the increased number of new available enzymes is not followed by an increase in the knowledge of their activity mechanism. The purpose of this PhD project is to improve the knowledge on the specific action of the major enzymes used in the bakery, predicting the effect that they will have in the final product. For this reason this project is a collaboration between the university and an industry involved in bakery. The work can be divided in two separated parts deeply connected one each other. In the first part we search for a fast and effective method to determine the enzymatic components located in the enzymatic mixtures used in the production of final products and their concentrations. For this purpose three mixes (called A, B and C) were tested for a quali-quantitative analysis of the enzymes present in them. All the samples were analyzed with a zymogram for the detection of enzymatic activity on electrophoretic native gels. To check for the presence or the absence of the most commonly used enzymes in bakery, we optimized the zymogram’s protocols for seven different type of enzymes: fungal and bacterial amylases, xylanases, proteases, transglutaminases, glucose-oxidases and laccase. The 3 mixes examined with all the six zymogram techniques have shown the presence of one isoform of amylase and one isoform of xylanase in mixes A and B, whereas the third mix is composed only of amylase but obtained from two different organisms. The zymograms have also shown the absence of protease, transglutaminase, glucose-oxidase and laccase in all the mixes. With specific enzymatic activity essays it’s possible to find out the percentage of amylase and xylanase present in the tested sample on the total amount of protein. The enzymes are then characterized creating a curve of enzyme activity related to variation of temperature and pH to realize how the activity could change in different working condition. To validate this part of the work, the three mixes used in the production of backed food were recreated and two final backed products are obtained: one containing the commercial blend and one containing the enzymes in the calculated concentrations. They were compared in a triangular test by a panel of experts people and the results shown that there’s no difference between the two product indicating that the commercial mix and the recreated from the calculation one are comparable in the effects leading in the final products. With these techniques has been also possible to provide a constant monitoration, throughout a whole year, of the flours that are used in the productive area. This was particularly important to find out that there isn’t exogenous amylase addition by the flour producer. The second part analyze modifications in the dough and moreover in the characteristics of the final product, due to the additions of different types of enzymes and combinations of them. This analysis will help to better understand the action mechanism of previously described enzymes and then to realize previously the effect that they can have on dough and on the final product. The final goal is to be able to identify an ideal enzymatic composition for every desired characteristic of a new product. Tested enzymes can be grouped in two categories: those increasing the formation of protein bonds (transglutaminase, glucose oxidase, and laccase) and those that destroy different type of bonds (amylase, protease and xylanase). Rheological tests are done with different technique that give different information about the strength of a flour, the characteristic of its gluten protein and its starch: amylograph, farinograph and estensograph. Than simple breads are prepared and the samples are tested after 15, 30, 60, 90, and 120 days for the shelf-life analysis. After these storing periods, a consolidated analytical methods of texture analysis, the Texture Profile analysis (TPA), was applied to the products. Some physical parameters like hardness, cohesiveness, elasticity, adhesiveness and chewiness were obtained by an instrument called “Texture Analyzer”. In this project has been possible to evaluate not only the effect of every enzyme in the dough and into the final product, but also the different effects of an enzyme in combination with six other different ones, considering both the rheological behaviour and the shelf life profile. To analyze the huge amount of data, a dedicated software has been created. With this program is possible to collect all the data, analyze them easily with the filtering tool and understand them quickly with the generation of different graph related to all the different analyzed features. It has been very useful to compare two samples containing only one enzyme with the sample containing both of them. In fact two enzymes can have not only a synergistic effect, but also a competitive and an antagonistic effect. Now it’s possible to understand how they relate each others and what type of effect they have, alone or combined, in the dough and in the final product. Moreover the mentioned software is also necessarily for the purpose of creating enzymatic mixes ad hoc for every new product: filtering for the particulars feature or features desired in the final product is possible to go back at the enzyme or enzymes that leads to that particular conformation.