Periodic Reporting for period 2 - FORMILK (Innovative technology for the detection of enzyme activity in milk) Reporting period: 2018-01-01 to 2019-12-31 Summary of the context and overall objectives of the project "The project ""Innovative technology for the detection of enzyme activity in milk"" (FORMILK) is focused on the development of novel methods of detection of activity of enzymes, such as plasmin and lactase, important for milk industry and for lactose-free diet. The objectives of the project are as follows: 1. Development of the method of detection the activity of milk enzymes plasmin and lactase in a volume based on high-resolution ultrasonic spectroscopy (HR-US), 2. Development of the assay of detection activity of plasmin at surfaces using electrochemical and acoustic methods, 3. Transfer of technology, 4. Organization of workshops and summer schools, 5. Project management. Project is important for the society due to several reasons. First it brings new technologies to the milk industry for improving quality of milk and milk products by analyzing protease activity such as plasmin. Second it improves technology of preparation of lactose-free milk products. Third it accelerates the collaboration between academic institutions and Industry. Fourth it is focused on training of early stage and experienced researchers in various novel analytical methods through secondments to highly ranking laboratories in Europe, Canada and USA as well as through participation on well focused workshops and international schools." Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far For the first time we applied high resolution ultrasonic spectroscopy (HR-US) for detection activity of plasmin and trypsin in a buffer at various conditions, such as enzyme concentration, temperature and pH. The assay was optimised and validate for detection plasmin in milk applicable for using in dairy laboratories. The cleavage of β-casein by these enzymes has been determined also by spectroscopic methods as well as by standard Enzyme Linked Immunosorbent Assay (ELISA). We also found optimal conditions for assay for preparation lactose-free milk by study of the activity of lactase at various conditions. Substantial progress was achieved in development acoustics biosensor based on β-casein layers for detection of the activity of proteases plasmin and trypsin. For the first time we used the electromagnetic piezoelectric acoustic sensor (EMPAS) for detection plasmin and trypsin cleavage with exceptionally high sensitivity of protease detection at pico molar level, which is best sensitivity reported so far. The acoustic sensor has been successfully validated in real milk samples. The protocol for detection plasmin in milk using thickness shear mode acoustic method (TSM) has been developed and validated in milk samples. The assay can be used for detection enzyme activity in dairy laboratories. The protocol has been compared with ELISA method and revealed similar sensitivity. However, in contrast with ELISA that determines only plasmin concentration, TSM provide information about plasmin activity. AFM topography of the array of short peptide substrate as well as casein layers allowed detection of the substrate topography following exposure to proteases such as plasmin and chymotrypsin. AFM method has been shown as a very effective tool for identification of bacteria in milk and milk products. The databases of potential end-users have been prepared. The miniature potentiostat, software and electrode holder has been constructed and validated. It has been demonstrated to the potential end-users Five international workshops and three international summer schools have been organized by FORMILK consortium. In all events several PhD students and young researchers from 10 countries participated, which allows them to obtain new knowledge in application of novel physical methods in analysis of protease activity in a volume and at surfaces. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Detection of protease activity is critical for assuring quality and safety of dairy products. Especially important is determination of plasmin activity. During the lactation period, typical plasmin concentrations fall in the 1–8 nM range. To prevent plasmin from cleaving β-casein, the main milk protein, causing the negative changes in milk, it is necessary to employ a proper heating technique, commonly ultra-high temperature (UHT) treatment, which can effectively inactivate plasmin. However, if the technique is not used properly, residual plasmin activity could remain and as the plasmin inhibitors are the least temperature-stable, and this activity will be enhanced by no regulated activation of plasmin. Traditional techniques of detection of residual plasmin activity are either not sensitive enough or too expensive to be employed for frequent use in dairy plasmin detection. Therefore one of important objective of FORMILK project is focused on development effective assay of plasmin detection in milk using novel technique based on high-resolution ultrasonic spectroscopy (HR-US) as well as on electrochemical and acoustics methods allowing detection of plasmin at surfaces. As a result of the effort of the project partners we were able to develop novel assay based on HR-US and thickness shear mode acoustic methods (TSM) for detection plasmin activity in milk. Both assays were validated in milk samples and detailed protocols applicable for dairy laboratories were prepared. For application of electrochemical methods of detection protease activity at surfaces we developed miniature potentiostat, software and electrode holder. The potentiostat was validated and its sensitivity compared with commercial instruments. It has been demonstrated to the potential end-users. Another important enzyme for milk industry is lactase that is responsible for cleaving of disaccharide lactose, into monosaccharides (glucose and galactose). Approximately 75% of the world population is affected by deficiency of enzyme lactase. Deficiency of lactase causes elevated concentration of lactose in the colon, where it suffers fermentation by resident bacteria. This can lead to medical disorders, termed lactose intolerance, after consumption of dairy products and therefore, requires milks and other dairy product with reduced level of lactose. The latter could be manufactured from the lactose free milk (obtained by hydrolysis of lactose). Another, low cost approach is an addition of lactase (β-galactosidase) in a form of liquid formulation (droplets) to milks (and other milk based products) prior their consumption, followed by incubation period. The composition of the formulation and the incubation time shall provide a reduction of the concentration of lactose in the milk to an acceptable level. It is, however necessary to find optimal conditions for incubation of lactase in a milk, which is another important objective of FORMILK. We were able to find these optimal conditions. Thus, the FORMILK project has substantial impact on the society in respect of improving food quality and safety. FORMILK is highly innovative project. It combines excellent science in nanotechnology and novel experimental techniques with transfer of technology to a milk industry. This also involved extensive collaboration between academic institutions and industrial partners. Involvement of young researchers from both sectors gives them possibility for better understanding what academy can bring into industry and what industry needs from academy. Innovative technologies that can academy bring into industry as well as better knowledge on the working conditions in academy and industry can affect also decision of young researchers on their further carrier. scheme-1.jpg
