Prebiotics for functional food and bioactive cosmetics

Produced in intensified enzymatic processes

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About Project

Prebiotics for functional food and bioactive cosmetics produced in intensified enzymatic processes
PrIntPrEnzy

Rapid growth of market demand and extended applications of prebiotics brought challenges for biochemical industry, such as requirements to increase production capacity, decrease costs and to develop processes for novel prebiotic compounds.

General goal of PrIntPrEnzy project is to give contribution in solving these issues by designing novel intensified enzyme-based processes for the production of skin and gut prebiotics. Our ambition is to widen scope of current research activities at the FTM beyond established gut prebiotics and to give scientific impetus to rising and challenging research within field of emerging prebiotics by our expertise in biochemical engineering, enzymatic synthesis of bioactive compounds and process intensification.

About

PrIntPrEnzy will provide significant scientific impact through new optimization-based method for rapid bio-process development and intensification and significant contribution to elucidation of the structure-activity correlation for emerging skin and gut prebiotics. Additionally, development of prototypes of prebiotic-containing food and cosmetic products are expected in final stage of the project, hence PrIntPrEnzy will also provide tangible economic impact. Finally, the fact that inexpensive food industry waste will be used as raw materials indicates environmental impact and contribution to circular economy.

NOVEL ENZYME-BASED PROCESSES
FOR PRODUCTION OF PREBIOTICS

PrIntPrEnzy Logo White

Objectives

The main objective of the project PrIntPrEnzy is to design novel enzyme-based processes for the production of high added-value compounds – skin and gut prebiotics. The novelty of our project lies in contribution within three fields:

Novel enzymatic processes for the production of emerging prebiotics

Process intensification for production of both established and emerging prebiotics

Utilization of food industry wastes as raw materials for a significantly more lucrative purpose

Objectives-PrIntPrEnzy

State of the art. Decades ago it has been confirmed that human health is highly dependent on microbial communities which inhabit human organism, particularly the gut. In recent times, prebiotics, compounds that selectively stimulate growth of good microbiota, have been recognized as attractive products.

Prebiotic-related research is propulsive, especially since 2017, when redefined by The International Scientific Association for Probiotics and Prebiotics (ISAPP) as: “a substrate that is selectively utilized by host microorganisms conferring a health benefit”. This widely accepted new definition goes beyond effects on only the gut microbiota, and opened whole new areas of application including skin prebiotics, compounds with beneficial effect on health through interaction with skin microbiota. Analysis of current state of the art reveals that products containing “established” gut prebiotics, those with prebiotic activity confirmed in studies on humans, can be found in portfolio of several companies within food industry. Nevertheless, “emerging” prebiotics, compounds with prebiotic activity not yet confirmed in clinical trials, still present large scientific and technological challenge due to their structural diversity arising from heterogeneity of their macromolecular precursors and complex compositions of natural sources, as well as necessity of developing adequate methods for evaluation of their prebiotic potential.

Motivation for project PrIntPrEnzy is to widen scope of current research activities at the FTM beyond “established” gut prebiotics to the development of novel processes for “emerging” gut or skin prebiotics. Our firm opinion is that expertise in biochemical engineering and process intensification can give important impetus to scientific knowledge on emerging prebiotics and development of novel prebiotic technologies.

Concept and Methodology

PrIntPrEnzy project is an extension of previous, particularly industry-oriented projects delivered by members of Enzyme-derived Prebiotics Group (EDPG) focused on in situ enzymatic synthesis of established prebiotics (FOS and GOS) for their incorporation in food and feed. Ambition is to expand research area by applying bioengineering expertise for development of novel emerging prebiotics and applying expertise of Process modeling and intensification group (PMIG) of the Department of Chemical Engineering to intensify novel enzymatic processes.

ENZYME-DERIVED
PREBIOTICS GROUP

Emerging gut prebiotics, pectic- and xylo- oligosaccharides (POS and XOS), obtained by partial enzymatic hydrolysis of pectin and xylan will be in focus of Food prebiotics subgroup (FPS). PrIntPrEnzy project is targeting substrates rich in pectin and xylan which are produced in large quantities as by-products of the Serbian food industry – rapeseed and sunflower meal. General idea is to use simultaneous extraction and enzymatic hydrolysis (with pectinases and xylanases) of targeted polysaccharide components, instead of the two-step process of polysaccharide extraction and subsequent hydrolysis. This approach will enable the enhanced utilization of raw materials by producing high-added-value products. Bioseparation techniques that include the application of different membrane separation techniques (ultrafiltration and nanofiltration) and HPLC methods (ion exchange chromatography and gel filtration chromatography) will enable fractionation of obtained compounds and their prebiotic activity will be evaluated in fermentation studies in anaerobic conditions with mixed microbial cultures comprising representatives of beneficial and pathogenic gut microbiota to mimic conditions in the human gut. Structural analysis of these gut prebiotics will be enabled by applying several chromatographic separation methods with mass spectrometry analysis of separated products (GC-MS, HPAEC-PAD-MS/MS and/or HILIC-LC-MS/MS) in order to elucidate structure-function relationship. Selected fractions of the highest prebiotic activity will be incorporated in adequate matrices for application (fruit preparations or cream products) and their functional and sensory properties will be tested. 

As skin prebiotics, certain compounds of plant extracts, such as sugar alcohols and polyphenols will be obtained and evaluated by Skin prebiotics subgroup (SPS).

 Additionally, strategy of enzymatic functionalization of extracted biomolecules, with the target of enhancing prebiotic activity will be used. This strategy is based on the transformation of plant extracts’ polyphenols by glycosylation with transglycosylases, oligomerization with laccases and esterification by lipases. Regarding skin prebiotics, substrates rich in sugar alcohols and polyphenols, namely wild berries and local medicinal herbs (lemon balm, rose hip, pine needles, comfrey, blueberries, black currant, raspberries and blackberries), which are widespread in Serbia, will be chosen for the project.

Fig-4-Scheme-of-enzymatic-production
*available from Applied Microbiology
and Biotechnology

 Enzymatically assisted extractions will be utilized to enable faster extraction and higher yields of skin prebiotics. Due to the complex structure of plant cell walls several enzymes, such as cellulases, pectinases, hemicellulases and laccases will be used for disruption of complex plant cell structure and alleviation of the extraction process. Obviously, overlapping of enzymes utilized for production of gut and skin prebiotics provides added value to our concept with promising goal of fractionating one substrate into several bioactive products. RP-HPLC with UV/VIS or RID detection and RP-HPLC-MS/MS methods will be used for their separation, quantification and structural analyses. Activities of obtained skin prebiotics will be tested in fermentation studies (in anaerobic/microaerophilic conditions) by monitoring the growth of mixed culture comprising skin residents. The most promising products will be further evaluated by 3D human skin models performed by our partners from Radboud University, within TwinPrebioEnz project. Selected skin prebiotics will be incorporated in dermo-cosmetic formulations and their skin compatibility and transdermal delivery will be evaluated.

Process Modeling and
Intensification Group

In first stage of PrIntPrEnzy, intensification of enzymatic processes for production of “established” prebiotics will be in focus. One of the ways used to improve the performance of these processes is switching to continuous operation via equipment miniaturization accompanied by effective mixing.  We will explore and apply a systematic model-based approach, novel approach in which mathematical modeling is used to design the optimal reactors and other equipment and select appropriate process conditions. The methodology is developed by PMIG and was derived and applied to different chemical processes, within several scientific projects. Further, this  methodology will be expand on by introducing prototype design and 3D printing the experimental validation step, as well as second, more detailed 3D computational fluid dynamics (CFD/DEM) models. Within PrIntPrEnzy we will apply the described methodology for process development of established prebiotics (GOS and FOS) in oscillatory baffled reactors and spiral reactors (i.e. helicoidal tube).

In first stage of PrIntPrEnzy, intensification of enzymatic processes for production of “established” prebiotics will be in focus. One of the ways used to improve the performance of these processes is switching to continuous operation via equipment miniaturization accompanied by effective mixing.  We will explore and apply a systematic model-based approach, novel approach in which mathematical modeling is used to design the optimal reactors and other equipment and select appropriate process conditions. The methodology is developed by PMIG and was derived and applied to different chemical processes, within several scientific projects. Further, this  methodology will be expand on by introducing prototype design and 3D printing the experimental validation step, as well as second, more detailed 3D computational fluid dynamics (CFD/DEM) models. Within PrIntPrEnzy we will apply the described methodology for process development of established prebiotics (GOS and FOS) in oscillatory baffled reactors and spiral reactors (i.e. helicoidal tube).

EDPG

ENZYME-DERIVED
PREBIOTICS GROUP

Emerging gut prebiotics, pectic- and xylo- oligosaccharides (POS and XOS), obtained by partial enzymatic hydrolysis of pectin and xylan will be in focus of Food prebiotics subgroup (FPS). PrIntPrEnzy project is targeting substrates rich in pectin and xylan which are produced in large quantities as by-products of the Serbian food industry – rapeseed and sunflower meal. General idea is to use simultaneous extraction and enzymatic hydrolysis (with pectinases and xylanases) of targeted polysaccharide components, instead of the two-step process of polysaccharide extraction and subsequent hydrolysis. This approach will enable the enhanced utilization of raw materials by producing high-added-value products. Bioseparation techniques that include the application of different membrane separation techniques (ultrafiltration and nanofiltration) and HPLC methods (ion exchange chromatography and gel filtration chromatography) will enable fractionation of obtained compounds and their prebiotic activity will be evaluated in fermentation studies in anaerobic conditions with mixed microbial cultures comprising representatives of beneficial and pathogenic gut microbiota to mimic conditions in the human gut. Structural analysis of these gut prebiotics will be enabled by applying several chromatographic separation methods with mass spectrometry analysis of separated products (GC-MS, HPAEC-PAD-MS/MS and/or HILIC-LC-MS/MS) in order to elucidate structure-function relationship. Selected fractions of the highest prebiotic activity will be incorporated in adequate matrices for application (fruit preparations or cream products) and their functional and sensory properties will be tested. 

As skin prebiotics, certain compounds of plant extracts, such as sugar alcohols and polyphenols will be obtained and evaluated by Skin prebiotics subgroup (SPS).

 Additionally, strategy of enzymatic functionalization of extracted biomolecules, with the target of enhancing prebiotic activity will be used. This strategy is based on the transformation of plant extracts’ polyphenols by glycosylation with transglycosylases, oligomerization with laccases and esterification by lipases. Regarding skin prebiotics, substrates rich in sugar alcohols and polyphenols, namely wild berries and local medicinal herbs (lemon balm, rose hip, pine needles, comfrey, blueberries, black currant, raspberries and blackberries), which are widespread in Serbia, will be chosen for the project.

Fig-4-Scheme-of-enzymatic-production

 Enzymatically assisted extractions will be utilized to enable faster extraction and higher yields of skin prebiotics. Due to the complex structure of plant cell walls several enzymes, such as cellulases, pectinases, hemicellulases and laccases will be used for disruption of complex plant cell structure and alleviation of the extraction process. Obviously, overlapping of enzymes utilized for production of gut and skin prebiotics provides added value to our concept with promising goal of fractionating one substrate into several bioactive products. RP-HPLC with UV/VIS or RID detection and RP-HPLC-MS/MS methods will be used for their separation, quantification and structural analyses. Activities of obtained skin prebiotics will be tested in fermentation studies (in anaerobic/microaerophilic conditions) by monitoring the growth of mixed culture comprising skin residents. The most promising products will be further evaluated by 3D human skin models performed by our partners from Radboud University, within TwinPrebioEnz project. Selected skin prebiotics will be incorporated in dermo-cosmetic formulations and their skin compatibility and transdermal delivery will be evaluated.

PMIG

Process Modeling and
Intensification Group

In first stage of PrIntPrEnzy, intensification of enzymatic processes for production of “established” prebiotics will be in focus. One of the ways used to improve the performance of these processes is switching to continuous operation via equipment miniaturization accompanied by effective mixing.  We will explore and apply a systematic model-based approach, novel approach in which mathematical modeling is used to design the optimal reactors and other equipment and select appropriate process conditions. The methodology is developed by PMIG and was derived and applied to different chemical processes, within several scientific projects. Further, this  methodology will be expand on by introducing prototype design and 3D printing the experimental validation step, as well as second, more detailed 3D computational fluid dynamics (CFD/DEM) models. Within PrIntPrEnzy we will apply the described methodology for process development of established prebiotics (GOS and FOS) in oscillatory baffled reactors and spiral reactors (i.e. helicoidal tube)

In first stage of PrIntPrEnzy, intensification of enzymatic processes for production of “established” prebiotics will be in focus. One of the ways used to improve the performance of these processes is switching to continuous operation via equipment miniaturization accompanied by effective mixing.  We will explore and apply a systematic model-based approach, novel approach in which mathematical modeling is used to design the optimal reactors and other equipment and select appropriate process conditions. The methodology is developed by PMIG and was derived and applied to different chemical processes, within several scientific projects. Further, this  methodology will be expand on by introducing prototype design and 3D printing the experimental validation step, as well as second, more detailed 3D computational fluid dynamics (CFD/DEM) models. Within PrIntPrEnzy we will apply the described methodology for process development of established prebiotics (GOS and FOS) in oscillatory baffled reactors and spiral reactors (i.e. helicoidal tube).

PrIntPrEnzy Logo White

Project Impact

Impact within partner institutions. It is expected that the PrIntPrEnzy project, by assembling scientists at different career stages and with complementary research backgrounds, will significantly contribute to the expansion of current research scope and accelerate the transfer of scientific results to industrial processes for the production of novel prebiotic-based products.  PrIntPrEnzy project will have a significant impact on the career development of early-career scientists, because it will provide an excellent opportunity to extend their research beyond current scientific areas.

Project-Impact-PrIntEnzy

Technological and economic impact. Project PrIntPrEnzy is designed to increase exploitation capacity of indigenous herbs of Serbia and the utilization of waste materials from the food industry using environmentally acceptable enzymatic technologies and digital tools for process intensification. Hence, project topic is in congruence with the Economic and Investment Plan for the Western Balkans, especially its mission to spur the twin green and digital transition of Western Balkan economies.

Societal impact. Scientific results and technologies obtained within PrIntPrEnzy will be promoted in social media and public media providing significant contribution to public awareness about health benefits of prebiotics. Special attention will be given to reach public groups with the highest potential interest – consumers of functional food and users of bioactive cosmetics, consumers preferring sustainable products from natural sources, nutritionists and young population in general. The use of food industry wastes as raw material for production of food with added value will increase public awareness of importance and usefulness of circular economy concept in region of Western Balkan.

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Prebiotics for functional food and bioactive cosmetics Produced in intensified enzymatic processes

About us

Research will be performed at Department of Biochemical Engineering and Biotechnology and Department of Chemical Engineering of Faculty of Technology and Metallurgy (FTM), by researchers of the FTM and Innovation Center of FTM. Due to our cooperation with EU scientific research organizations through TwinPrebioEnz project within Horizon Europe, important contribution to research within PrIntPrEnzy will be given by our colleagues from Maastricht University (Maastricht, Netherlands), Spanish National Research Council (Madrid, Spain) and Radboud University Medical Center (Nijmegen, Netherlands) as part of our joint research.

Enzyme-derived
prebiotics Group

Dr-Dejan-Bezbradica

Dr Dejan Bezbradica

Full Professor
FTM
(EDPG Leader)

dbez@tmf.bg.ac.rs

Food Prebiotic
Subgroup (FPS)

Skin Prebiotic
Subgroup (SPS)

Dr-Milica-Simovic

Dr Milica Simović

Senior Research
Associate FTM

mcarevic@tmf.bg.ac.rs

Dr-Marija-Corovic

Dr Marija Ćorović

Senior Research
Associate FTM

mstojanovic@tmf.bg.ac.rs

Dr-Katarina-Banjanac

Dr Katarina Banjanac

Research Associate
ICFTM

kbanjanac@tmf.bg.ac.rs

Dr-Ana-Milivojevic

Dr Ana Milivojević

Research Associate
FTM

amilivojevic@tmf.bg.ac.rs

Milica-Veljkovic

M. Sc. Milica Veljković

Research Assistant
ICFTM

mveljkovic@tmf.bg.ac.rs

Dr-Rada-Pjanovic

Dr Rada Pjanović

Full Professor
FTM

rada@tmf.bg.ac.rs

Ana-Vukoicic

M. Sc. Ana Vukoičić

Junior Research
Assistant
ICFTM

avukoicic@tmf.bg.ac.rs

Anja-Petrov-Ivankovic

M. Sc. Anja Petrov Ivanković

Research Assistant
ICFTM

apetrov@tmf.bg.ac.rs

Process Modeling and
Intensification Group

Dr-Nikola-Nikacevic

Dr Nikola Nikačević

Full Professor
FTM
(PMIG Leader)

nikacevic@tmf.bg.ac.rs

Dr-Branislav-Todic

Dr Branislav Todić

Assistant Professor
FTM

btodic@tmf.bg.ac.rs

Dr-Radoslava-Pravilovic

Dr Radoslava Pravilović

Research Associate
FTM

rpravilovic@tmf.bg.ac.rs

Enzyme-derived
prebiotics Group

Dr-Dejan-Bezbradica

Dr Dejan Bezbradica

Full Professor
FTM
(EDPG Leader)

dbez@tmf.bg.ac.rs

Food Prebiotic
Subgroup (FPS)

Skin Prebiotic
Subgroup (SPS)

Dr-Milica-Simovic

Dr Milica Simović

Senior Research
Associate FTM

mcarevic@tmf.bg.ac.rs

Dr-Marija-Corovic

Dr Marija Ćorović

Senior Research
Associate FTM

mstojanovic@tmf.bg.ac.rs

Dr-Katarina-Banjanac

Dr Katarina Banjanac

Research Associate
ICFTM

kbanjanac@tmf.bg.ac.rs

Dr-Ana-Milivojevic

Dr Ana Milivojević

Research Associate
FTM

amilivojevic@tmf.bg.ac.rs

Milica-Veljkovic

M. Sc. Milica Veljković

Research Assistant
ICFTM

mveljkovic@tmf.bg.ac.rs

Dr-Rada-Pjanovic

Dr Rada Pjanović

Full Professor
FTM

rada@tmf.bg.ac.rs

Ana-Vukoicic

M. Sc. Ana Vukoičić

Junior Research
Assistant
ICFTM

avukoicic@tmf.bg.ac.rs

Anja-Petrov-Ivankovic

M. Sc. Anja Petrov Ivanković

Research Assistant
ICFTM

apetrov@tmf.bg.ac.rs

Process Modeling and
Intensification Group

Dr-Nikola-Nikacevic

Dr Nikola Nikačević

Full Professor
FTM
(PMIG Leader)

nikacevic@tmf.bg.ac.rs

Dr-Branislav-Todic

Dr Branislav Todić

Assistant Professor
FTM

btodic@tmf.bg.ac.rs

Dr-Radoslava-Pravilovic

Dr Radoslava Pravilović

Research Associate
FTM

rpravilovic@tmf.bg.ac.rs

Dissemination

- Publications

Rabab Salih, Katarina Banjanac, Ana Vukoičić, Jelena Gržetić, Ana Popović, Milica Veljković, Dejan Bezbradica, Aleksandar Marinković: Acrylic modified kraft lignin microspheres as novel support for immobilization of laccase from M. thermophila expressed in A. oryzae (Novozym® 51003) and application in degradation of anthraquinone textile dyes. Journal of Environmental Chemical Engineering, vol. 11, 109077, 2023

Milica Veljković, Relja Stepanović, Katarina Banjanac, Marija Ćorović, Ana Milivojević, Milica Simović, Milan Milivojević, Dejan Bezbradica: Continuous production of fructo-oligosaccharides using selectively immobilized fructosyltransferase from Aspergillus aculeatus onto Purolite® A109, Journal of Industrial and Engineering Chemistry, vol. 117, p. 149-156, 2023

Radoslava Pravilović, Branislav Todić, Milica Simović, Katarina Banjanac, Dejan Bezbradica, Nikola Nikacević: Kinetic Model for Galacto-Oligosaccharide Synthesis, Industrial & Engineering Chemistry Research, vol. 61, p. 14189–14198, 2022

Milica Veljković, Milica Simović, Katarina Banjanac, Marija Ćorović, Ana Milivojević, Milan Milivojević, Dejan Bezbradica: Heterofunctional epoxy support development for immobilization of fructosyltransferase from Pectinex® Ultra SP-L: batch and continuous production of fructo-oligosaccharides, Reaction Chemistry & Engineering, vol. 7, p. 2518-2526, 2022

Anja Petrov, Marija Ćorović, Ana Milivojević, Milica Simović, Katarina Banjanac, Rada Pjanović, Dejan Bezbradica: Prebiotic effect of galacto-oligosaccharides on the skin microbiota and determination of their diffusion properties, International Journal of Cosmetic Science, vol. 44, p. 309-319, 2022

Ana Milivojević, Marija Ćorović, Milica Simović, Katarina Banjanac, Rada Pjanović, Dejan Bezbradica: Evaluation of In Vitro Skin Permeation of Enzymatically Synthesized Phloridzin Acetates from Emulsions and Liposomes Dispersed in Gel, European Journal of Lipid Science and Technology, Vol. 124, 2200073, 2022.

- Conferences

Milica Veljković, Milica Simović, Marija Ćorović, Ana Milivojević, Anja Petrov, Katarina Banjanac, Dejan Bezbradica, Selective Immobilization of Fructosyltransferase onto Glutaraldehyde Modified Support and Its Application in the Production of Fructo oligosaccharides, ICAPP 2022: XVI. International Conference on Advances in Probiotics and Prebiotics, Copenhagen, Denmark, July 19-20, 2022, Book of Abstracts, ISSN: 1307-6892.

Anja Petrov, Milica Veljković, Marija Ćorović, Ana Milivojević, Milica Simović, Katarina Banjanac, Dejan Bezbradica, Evaluation of Herbal Extracts for Their Potential Application as Skin Prebiotics, ICAPP 2022: XVI. International Conference on Advances in Probiotics and Prebiotics, Copenhagen, Denmark, July 19- 20, 2022, Book of Abstracts, ISSN: 1307-6892.

Milica Veljković, Milica Simović, Anja Petrov Ivanković, Ana Vukoičić, Katarina Banjanac, Dejan Bezbradica, In situ transformation of sucrose in maple syrup in order to produce fructo-oligosaccharide enriched product. 10th International Congress of Food Technologists, Biotechnologists and Nutritionists, Zagreb, Croatia, November 31 st – December 2 nd , 2022, Book of Abstracts, ISSN: 2975-4313.

Anja Petrov Ivanković, Milica Veljković, Ana Vukoičić, Ana Milivojević, Marija Ćorović, Rada Pjanović, Dejan Bezbradica, Enzyme-assisted extraction of various bioactive components from blackcurrant (Ribes nigrum). 10th International Congress of Food Technologists, Biotechnologists and Nutritionists, Zagreb, Croatia, November 31 st – December 2 nd , 2022, Book of Abstracts, ISSN: 2975-4313.

Media

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Workshop

on enzymatic production of prebiotics for food and cosmetics

Highlights from the Workshop

The PrIntPrEnzy and TwinPrebioEnz projects have successfully organized the Workshop on enzymatic production of prebiotics for food and cosmetics at the FTM, December 5 – 6, 2023.

WorkShop-on-enzymatic-products-of-prebiotics-for-food-and-cosmetics