StrAtegie agronomiche, tecnologiche e microbiologiche per Controllare e mitigare il Rischio di formazione di Acrilammide nella pasta e nel pane di frumento duro
ProjectThe project focuses on the crucial factors that increase the risk of exposure of consumers of cereal-based foods—particularly durum wheat pasta and bread—to acrylamide. Acrylamide is a toxin with well-established carcinogenic potential, which develops during high-temperature processing. This issue has never been studied in pasta, whereas many studies have been conducted on soft wheat bread. In Italy, especially in the central-southern regions, the production and consumption of durum wheat bread are widespread.
In pasta production, the spread of high- and very-high-temperature drying cycles has undoubtedly increased the productivity of pasta factories; however, it has also increased the risk of formation of potentially harmful substances, including acrylamide. This project proposal aims to provide answers based on scientific results concerning the effects of agronomic, microbiological, and technological techniques on the formation of this compound.
It is known that during the drying phase, more than 50% of the total water contained in pasta is removed, and the moisture content reaches values of 12–13%. These moisture conditions, equivalent to a water activity of 0.8, in the presence of reducing sugars and free asparagine, are recognized as optimal conditions for the development of the Maillard reaction and the subsequent formation of acrylamide. The same occurs in bread, where baking is certainly the most critical phase for acrylamide formation, in the presence of the aforementioned precursors.
In wheat, free amino acids generally account for less than 5% of the total nitrogen present in the kernels; moreover, nitrogen availability is positively correlated with the total content of free amino acids, including asparagine. In cereals, mineral fertilization also plays a fundamental role in determining protein quality and quantity, particularly nitrogen availability and the phenological stage at which fertilization is carried out. Indeed, the nitrogen fertilizations most effective in increasing protein content are those performed post-anthesis.
Among the factors involved in the increase of free asparagine, a particularly severe sulfur deficiency may cause an increase of up to 30-fold compared with a situation in which sulfur is not deficient. Under these conditions, free asparagine may account for up to 50% of the total pool of free amino acids.
Therefore, in this project proposal, which is structured into six WPs, we have planned agronomic fertilization trials based on sulfur and nitrogen in two locations, using two durum wheat varieties among the most widely cultivated and used for pasta- and bread-making. The agronomic trials will be conducted in two environments with different pedoclimatic conditions, at the two farms of OU1 CREA and OU2 UNITUS, both highly specialized in conducting agronomic trials on cereals. Specifically, trials will be carried out in Viterbo at the Nello Lupori Teaching and Experimental Farm of the University of Tuscia, and in Foggia at the Manfredini experimental farm of CREA-CI.
The field experiment will use an experimental design with the following treatments: two durum wheat varieties; two doses of nitrogen fertilizer, 80 kg/ha and 110 kg/ha; two doses of sulfur fertilizer, 30 kg/ha and 50 kg/ha; and three replicates. The agronomic technique applied with regard to intervention timing will be the one normally adopted in central Italy for durum wheat production.
During the crop vegetative cycle, physiological parameters will be recorded in order to understand the plant’s resource-use efficiency. Before sowing and after harvesting, soil samples will be collected to determine the main fertility parameters. The assessment of biotic stress affecting the crop as a function of the experimental treatments will be carried out in the field through epidemiological and etiological analysis of the main durum wheat diseases, with particular reference to Fusarium head blight and black point of the ear.
The grain obtained from the agronomic trials will be analyzed for free asparagine content at the laboratories of the CREA-AN Centre, which has consolidated experience in food research. Analysis of the main functional groups—“fingerprint” analysis—will be performed on the grain samples obtained from the agronomic trials using Fourier-Transform Infrared Spectroscopy with Attenuated Total Reflectance, FTIR-ATR. FTIR-ATR is an example of an innovative, rapid, non-destructive, green technique, requiring minimal sample preparation and minimal solvent use, which allows the “fingerprint” of a product to be identified.
Free asparagine content will also be measured using LC-MS, Liquid Chromatography–Mass Spectrometry, which will provide a preliminary indication of correct fertilization management in terms of both quantity and timing of application. At the same time, the samples showing the most contrasting free asparagine contents will be forwarded to the subsequent technological transformation phases by CREA-CI and CREA-IT.
After conditioning, the durum wheat samples will be milled using a roller mill, in order to obtain semolina and milling by-products, namely bran and fine bran. These by-products will be micronized and used in mixtures with semolina to obtain reconstituted wholemeal flours. The subsamples derived from the different milling techniques will be analyzed to determine free asparagine content, using LC-MS by CREA-AN, and the results will indicate which milling technique is most suitable for limiting the presence of free asparagine.
In addition, the use of FTIR-ATR will be tested in order to develop predictive models for free asparagine, using as reference data those obtained with conventional methods, namely LC-MS. A very important aspect that will also be considered in this project is the determination of the technological quality of the flours. This activity will be carried out by CREA-CI and CREA-IT, which have consolidated experience in these activities.
The technological quality indicators considered will include: protein content, hectoliter weight, thousand-kernel weight, gluten index and quantity, color index, alveographic indices W and P/L, mixographic and farinographic indices, and amylase activity.
The project also includes the development of a microbiological approach for reducing free asparagine levels in flours. The activities to be carried out at the UNIMORE laboratories, OU3, will focus on identifying microbial strains belonging to the group of lactic acid bacteria, LAB, capable of metabolizing the free asparagine present in flours.
Subsequently, at the technology laboratories of CREA-CI and CREA-IT, the most suitable stage of the milling and transformation process for applying the selected bacterial culture will be identified, with the aim of achieving a significant reduction in asparagine. In particular, it is expected that the levels of this amino acid may be especially high in the aleurone layer and in wholemeal products. It is therefore hypothesized that LAB could be used during the wheat conditioning phase, before milling, when water is added to the grain and it is left to rest before grinding.
Moreover, in the bread-making process, anti-asparagine LAB will be tested through bread-making trials in order to evaluate the effect of the strain during dough leavening under the three planned conditions: baker’s yeast, sourdough, and sourdough plus microbial starter; and with different baking temperatures and times, with one decreasing as the other increases. All working conditions and parameters will be optimized and standardized, including fermentation time and temperature and inoculum concentration.
The research work will continue with pasta-making and bread-making tests on the flours with the highest free asparagine content, in order to quantify the extent of the thermal effect on acrylamide formation in pasta, both dry and cooked, and in bread. The pasta-making and bread-making trials will be carried out using the experimental pilot plants available at the CREA-CI Centres in Foggia and Acireale and at CREA-IT, while analyses of acrylamide content, using LC-MS, will be performed by the CREA-AN Centre.
In addition, aspects related to product texture, sensory analysis, and antioxidant activity will be evaluated by CREA-CI and CREA-IT. Coding systems, LanguaL™ and FoodEx2, will be applied to the products obtained from the different production stages—grain, flours, pasta, and bread—according to standardized and harmonized procedures at European and international level by CREA-AN.
Coding is a tool that makes it possible to discriminate among different products along the agri-food chain on the basis of specific characteristics related, for example, to environment, production, processing technology, and other factors. The sharing, traceability, and dissemination of data for the protection of all consumers will make it possible to understand and promote the main characteristics of the products under study.
In pasta production, the spread of high- and very-high-temperature drying cycles has undoubtedly increased the productivity of pasta factories; however, it has also increased the risk of formation of potentially harmful substances, including acrylamide. This project proposal aims to provide answers based on scientific results concerning the effects of agronomic, microbiological, and technological techniques on the formation of this compound.
It is known that during the drying phase, more than 50% of the total water contained in pasta is removed, and the moisture content reaches values of 12–13%. These moisture conditions, equivalent to a water activity of 0.8, in the presence of reducing sugars and free asparagine, are recognized as optimal conditions for the development of the Maillard reaction and the subsequent formation of acrylamide. The same occurs in bread, where baking is certainly the most critical phase for acrylamide formation, in the presence of the aforementioned precursors.
In wheat, free amino acids generally account for less than 5% of the total nitrogen present in the kernels; moreover, nitrogen availability is positively correlated with the total content of free amino acids, including asparagine. In cereals, mineral fertilization also plays a fundamental role in determining protein quality and quantity, particularly nitrogen availability and the phenological stage at which fertilization is carried out. Indeed, the nitrogen fertilizations most effective in increasing protein content are those performed post-anthesis.
Among the factors involved in the increase of free asparagine, a particularly severe sulfur deficiency may cause an increase of up to 30-fold compared with a situation in which sulfur is not deficient. Under these conditions, free asparagine may account for up to 50% of the total pool of free amino acids.
Therefore, in this project proposal, which is structured into six WPs, we have planned agronomic fertilization trials based on sulfur and nitrogen in two locations, using two durum wheat varieties among the most widely cultivated and used for pasta- and bread-making. The agronomic trials will be conducted in two environments with different pedoclimatic conditions, at the two farms of OU1 CREA and OU2 UNITUS, both highly specialized in conducting agronomic trials on cereals. Specifically, trials will be carried out in Viterbo at the Nello Lupori Teaching and Experimental Farm of the University of Tuscia, and in Foggia at the Manfredini experimental farm of CREA-CI.
The field experiment will use an experimental design with the following treatments: two durum wheat varieties; two doses of nitrogen fertilizer, 80 kg/ha and 110 kg/ha; two doses of sulfur fertilizer, 30 kg/ha and 50 kg/ha; and three replicates. The agronomic technique applied with regard to intervention timing will be the one normally adopted in central Italy for durum wheat production.
During the crop vegetative cycle, physiological parameters will be recorded in order to understand the plant’s resource-use efficiency. Before sowing and after harvesting, soil samples will be collected to determine the main fertility parameters. The assessment of biotic stress affecting the crop as a function of the experimental treatments will be carried out in the field through epidemiological and etiological analysis of the main durum wheat diseases, with particular reference to Fusarium head blight and black point of the ear.
The grain obtained from the agronomic trials will be analyzed for free asparagine content at the laboratories of the CREA-AN Centre, which has consolidated experience in food research. Analysis of the main functional groups—“fingerprint” analysis—will be performed on the grain samples obtained from the agronomic trials using Fourier-Transform Infrared Spectroscopy with Attenuated Total Reflectance, FTIR-ATR. FTIR-ATR is an example of an innovative, rapid, non-destructive, green technique, requiring minimal sample preparation and minimal solvent use, which allows the “fingerprint” of a product to be identified.
Free asparagine content will also be measured using LC-MS, Liquid Chromatography–Mass Spectrometry, which will provide a preliminary indication of correct fertilization management in terms of both quantity and timing of application. At the same time, the samples showing the most contrasting free asparagine contents will be forwarded to the subsequent technological transformation phases by CREA-CI and CREA-IT.
After conditioning, the durum wheat samples will be milled using a roller mill, in order to obtain semolina and milling by-products, namely bran and fine bran. These by-products will be micronized and used in mixtures with semolina to obtain reconstituted wholemeal flours. The subsamples derived from the different milling techniques will be analyzed to determine free asparagine content, using LC-MS by CREA-AN, and the results will indicate which milling technique is most suitable for limiting the presence of free asparagine.
In addition, the use of FTIR-ATR will be tested in order to develop predictive models for free asparagine, using as reference data those obtained with conventional methods, namely LC-MS. A very important aspect that will also be considered in this project is the determination of the technological quality of the flours. This activity will be carried out by CREA-CI and CREA-IT, which have consolidated experience in these activities.
The technological quality indicators considered will include: protein content, hectoliter weight, thousand-kernel weight, gluten index and quantity, color index, alveographic indices W and P/L, mixographic and farinographic indices, and amylase activity.
The project also includes the development of a microbiological approach for reducing free asparagine levels in flours. The activities to be carried out at the UNIMORE laboratories, OU3, will focus on identifying microbial strains belonging to the group of lactic acid bacteria, LAB, capable of metabolizing the free asparagine present in flours.
Subsequently, at the technology laboratories of CREA-CI and CREA-IT, the most suitable stage of the milling and transformation process for applying the selected bacterial culture will be identified, with the aim of achieving a significant reduction in asparagine. In particular, it is expected that the levels of this amino acid may be especially high in the aleurone layer and in wholemeal products. It is therefore hypothesized that LAB could be used during the wheat conditioning phase, before milling, when water is added to the grain and it is left to rest before grinding.
Moreover, in the bread-making process, anti-asparagine LAB will be tested through bread-making trials in order to evaluate the effect of the strain during dough leavening under the three planned conditions: baker’s yeast, sourdough, and sourdough plus microbial starter; and with different baking temperatures and times, with one decreasing as the other increases. All working conditions and parameters will be optimized and standardized, including fermentation time and temperature and inoculum concentration.
The research work will continue with pasta-making and bread-making tests on the flours with the highest free asparagine content, in order to quantify the extent of the thermal effect on acrylamide formation in pasta, both dry and cooked, and in bread. The pasta-making and bread-making trials will be carried out using the experimental pilot plants available at the CREA-CI Centres in Foggia and Acireale and at CREA-IT, while analyses of acrylamide content, using LC-MS, will be performed by the CREA-AN Centre.
In addition, aspects related to product texture, sensory analysis, and antioxidant activity will be evaluated by CREA-CI and CREA-IT. Coding systems, LanguaL™ and FoodEx2, will be applied to the products obtained from the different production stages—grain, flours, pasta, and bread—according to standardized and harmonized procedures at European and international level by CREA-AN.
Coding is a tool that makes it possible to discriminate among different products along the agri-food chain on the basis of specific characteristics related, for example, to environment, production, processing technology, and other factors. The sharing, traceability, and dissemination of data for the protection of all consumers will make it possible to understand and promote the main characteristics of the products under study.