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Final Report Summary - PROST! (Development of low-calorie beverages with prebiotic properties from cheap and abundant feedstocks)

This 4-year Marie Curie CIG fellowship was devoted to the development of a technology platform for the production of low-calorie, value-added beverages that have beneficial health effects. The objective of “PrOSt!” project is the valorization of cheap and abundant by-products of food processing industries, such as molasses and whey.

The proposed technology includes two key processing steps. In the first step, disaccharides (such as sucrose and lactose) originating from the raw materials are partially converted into valuable oligosaccharides (such as fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS)). The resulting products, FOS and GOS, are considered to be calorie-free, physiologically beneficial food ingredients with favorable technological properties such as low viscosity, good sweetness profile, and good pH and temperature stability. In the second processing step, the mixture of mono-, di-, and oligosaccharides is further processed in a selective fermentation step with appropriate microorganisms which are capable of utilizing mono- and disaccharides but lack the power to hydrolyze OS to a high extent. Thus, a prebiotic function can be added to the resulting mildly sweet product due to the presence of remaining oligosaccharides (OS).

With this goal in view, several processing steps of the proposed technology were studied in depth within the frame of the PrOSt! project.

A specific objective of the project was to investigate the applicability of enzyme membrane reactors (EMR) utilizing free enzymes for the bioconversion of disaccharides into prebiotic oligosaccharides. The EMR technology enables a continuous biotransformation by coupling a reaction with a separation process. It promises an innovative solution for process intensification by enabling enzyme re-use and enhancing overall productivity. In line with our objectives, we have constructed a semi-pilot scale EMR capable of producing 50 litres/day of OS from dimers as substrate, and tested its performance on both FOS and GOS systems. It was found that conventional batch reactors outperform EMR in term of conversion, but EMR compares favorably regarding productivity. This insight was obtained by analyzing both filtration and catalytic performance of the EMR. The filtration performance was investigated in terms of flux characteristics, fouling tendency and cleaning efficacy. We analyzed the major governing filtration parameters as well as critical conditions and process limitations, and interpreted the experimental data by mathematical models of ultrafiltration. Regarding the catalytic performance, we determined the relation of product composition to various operational parameters such as residence time and enzyme load. We also identified kinetic parameters from a series of progress curves and modelled the static and dynamic catalytic behavior of the system. Our results reveal that EMR represents a simple and convenient tool for partially converting disaccharides into oligosaccharides. The on-site industrial implementation of this technology might be attractive for food m¬anufacturers aiming at utilizing a value-added sweetener mixture with prebiotic properties.

Prebiotics represent one of the fastest-growing segments of the ingredient market. The growth of this industrial sector is mainly driven by the increasing health awareness of consumers. One of the key challenges in the production of OS is the real-time monitoring of the bio-manufacturing process. This is particularly important in continuous processes, such as EMR technology, that require a strict supervision in order to ensure a controlled and safe plant operation. A specific objective of the project was to find robust analytical techniques that allow the quantitative, on-line analysis of the relative amount of OS to di- and monosaccharides in the process solutions. In industrial practice, the chemical analysis of saccharides composition in OS manufacturing is done by HPLC. It is a sensitive and reproducible method, however, off-line, time-costly, laborious, and requires expensive instrumentation and technically trained personal. Being an off-line technique, there is a considerable long delay between sampling from the production line at the facility and receiving information on the oligo-/di-/mono-saccharides composition from the analytics lab. There is a need for analysis methods that could be applied in quality control of OS production. We have addressed this problem by investigating the applicability of several rapid techniques. Our proof-of-concept investigations pointed UV spectroscopy as the method with the highest potential for the quantitative analysis of saccharides composition. Our efforts lead to the development of robust spectroscopic techniques coupled with chemometrics tools that allow the real-time monitoring of the bioconversion during the manufacturing process and the detection of possible failures and disturbances in the EMR operation.

The ERC grant allowed us to investigate various techniques for valorization of whey. We proposed a multi-product process that is based on the fractionation of whey constituents and the subsequent enzymatic conversion of lactose into GOS. We have investigated a multi-step process in which partially demineralized whey is first concentrated and diafiltered by ultraltration (UF/DF) to obtain whey protein isolates, then lactose is concentrated by nanofiltration. We also performed a systematic study to investigate the hydrolytic and transgalactosylation activity of beta-galactosidases on whey-derived substrates. In addition, we have devoted a special attention to the synchronization of the individual process steps and provided component balances and relevant processing parameters required for flow-sheeting design and scale-up calculations. The data collected and the approaches proposed to the synchronization of the individual operations is useful for the design of whey valorization processes and may find potential applications in whey management practice.

The UF/DF process is an essential step in whey processing with the twin objectives of obtaining a purified lactose solution for further bioconversion and a concentrated protein solution as a valuable co-product. Motivated by our experimental investigations, we have also investigated this separation problem in depth from the economic point of view, and developed novel control strategies of optimal control to determine sustainable and economically optimal production. This has be done in a full generic way such that the research outcomes can be applied to any batch UF/DF process.

The current OS production route via biosynthesis has a significant weakness, namely the incomplete conversion. The enzymatic catalysis results in a mixture of carbohydrates consisting of OS, remaining disaccharides (i.e. non-reacting substrates), and by-product monosaccharides. Generally, yields over 50% are rarely exceeded, mostly due to by-product inhibition. Within the frame of the project, we have developed microbial processes for removal of monosaccharides (glucose and galactose) and disaccharides (lactose) from GOS-containing carbohydrate mixtures. We have carried out a systematic screening for appropriate microorganism among yeast and lactic acid bacteria strains. We have investigated two basic fermentation strategies for preparing calorie-reduced beverages with prebiotic functions from whey. The first concept uses the GOS-containing saccharides mixtures (i. e. the product stream of the enzymatic conversion step) as medium in the fermentation step. The second concept uses deproteinated (UF) permeate as medium in the fermentation step, and the GOS-containing saccharide mixture is used to enrich the fermentation broth at the end or in the last phase of the fermentation. We have performed experimental investigations on both systems and identified suitable strains. In addition, further fermentation studies helped us to reveal how the origin and amount of nitrogen and carbon source influences the microbial growth rate and the digestion of individual saccharide fractions.

A core results of the CIG is that the PI is now fully integrated within the Department of Food Engineering and expects to have a long term career within the Szent István University. An important activity related to the transfer of knowledge to the Host included the development of teaching material for a number of courses at both undergraduate and post-graduate level. The PI has introduced model-based engineering tools (such as numerical software packages and flowsheeting softwares) as educational aids into the courses coordinated by him. In addition, he successfully integrated research into classroom teaching, also by involving equipment and methodologies developed within the PrOSt! project in lab courses of Bioseparations and Unit Operations.

The PI has established his independent research group with long-term research perspectives at the Department of Food Engineering. His research is now supported by three full time doctoral students under his supervision (one co-supervised with Prof. Maráz at the Department of Microbiology and Biotechnology). He has acquired a large span of scientific competence necessary to deal with the interdisciplinary nature of the PrOSt! project, especially on process analytical technology and microbiology-related disciplines, by collaborating with the Department of Physics and Control, the Department of Biometrics and Agricultural Informatics, and the Department of Microbiology and Biotechnology at the host university.

The PI has also strengthened and expanded his international professional networks. The grant allowed him to continue his collaboration with the Prof. Czermak’s group (THM, Giessen, Germany) on oligosaccharides biosynthesis, with Prof. Fikar’s group (STU, Bratislava, Slovakia) and Dr. Paulen (TU Dortmund, Germany) on economic optimization of membrane processes and parameter estimation problems in biocatalytic reactions, and with Dr. Grachten (JKU, Austria) on artificial neural networks.

The scientific results obtained within the project have been disseminated in 2 book chapters, 5 papers published in peer-reviewed journals (further 5 papers are submitted and currently under revision), and 10+ contributions to international scientific conferences (including participation as invited lecturer (3), chairman (2) and member of scientific committee (1)). He supervised and co-supervised 10+ Bioengineering and Food Engineering MSc/BSc students. Notably, two of them joined the PrOSt! team as PhD students after receiving their academic degree. As a confirmation of our efforts in supporting young talents, one PrOSt! student was awarded by the Special Prize of the Hungarian Scientific Society for Food Industry, and two students received 1st Award from the National Scientific Student's Association for their theses under the PI’s supervision.

The PI has received a permanent position as an Associate Professor. His habilitation thesis has been reviewed by the Doctoral School of Food Science at the host university, and his defense before the academic committee is scheduled for winter semester in 2017. As a major step forward in research management, the PI has obtained the Bolyai Research Scholar by the Hungarian Academy of Sciences. This grant allows him the continuation of his research that builds upon the findings of the PrOSt! project. The grant supports distinguished young researchers to prepare their thesis for applying the degree of Doctor of Science at the Hungarian Academy of Sciences – a title that is currently a pre-requirement for access to a professorship at the host university. Overall, an important progress in the PI's career has been made with long term career perspectives, largely as a result of the accelerated start provided by the CIG.

Zoltán Kovács, Principal Investigator
Department of Food Engineering
Szent István University
Ménesi st 44, H-1118 Budapest, Hungary

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