Production of Invert Sugar using Lentikats Biotechnology

Content Our aim What is Lentikats Biocatalyst Production of Lentikats Biocatalyst Benefits of Lentikats Biotechnology Lentikats Biocatalyst in Food Industry Our guarantees Our partners Contacts 2

LentiKat’s a.s. in Food Industry LentiKat’s a.s. follows up on nine year of research and development of Lentikats Biotechnology. Our production know-how for commercial use of our Biocatalyst and for its industrial applications is patented internationally. Our main focus is in intensification of production processes that leads to: higher process yields enhanced process stability, Increased revenue, Reduction of environmental footprint. Biotechnologies are changing the world…

Lentikats Biotechnology Unique shape of the matrix and original immobilisation procedure enables to encapsulate free enzymes or microorganisms (bacteria, yeasts, fungi) inside a hydrogel porous carrier from polyvinyl alcohol (PVA). The advantages of PVA matrix are: excellent physical and mechanical characteristics that result in a long-term mechanical stability hardly biodegradable no toxic effects Lentikats Biocatalysts enable easy separation of enzymes or microorganisms from the bulk media and therefore can be used repeatedly. With biotechnologies to a better future… 4 4

How does the Lentikats Biocatalyst work? A maximum surface in combination with the minimum volume of Lentikats Biocatalyst results in a substantial reduction of diffusion limitations associated with the transfer of substrate into the matrix and the product out of the matrix. 5 5

Why polyvinylalcohol? Biotechnologies are changing the world… It is an inexpensive material Encapsulation takes place under optimal, cell-sparing conditions It allows a production of hydrogel with excellent mechanical properties It does not have any side effects on fermentation It offers high capacity for the cell loading The process is stable and easy to control It is a non-toxic material Biotechnologies are changing the world… 6

How is Lentikats Biocatalyst produced?

Lentikats Biocatalysts Biocatalyst Types for Use in Lentikats Encapsulation: Free Cells dead cells viable, non growing cells viable and growing cells Free or Immobilised Enzymes. Lentikats Biotechnology allows to immobilise any type of enzyme The size of Lentikats Biocatalyst pores varies depending on the size and diameter of the Biocatalyst due to: PVA additive type (size of macromolecule) additive concentration biomass concentration

Lentikats Biocatalysts Manufacturing Plant

Laboratory Units for Encapsulation For low-capacity production of encapsulates (especially for research purposes), a laboratory device is available

What are the benefits? Matrix containing high concentration of biomass Higher yields (in comparison to conventional technologies using free biomass) Higher reaction rates Reduction of reaction tanks’ volume Operational costs savings (electricity, heat) Possibility of repeated usage Operational costs savings Enhanced resistance of modified and immobilised biomass Higher process stability Easy separation of biomass from reaction media Reduced costs for downstream processes (separation) Lower production of waste and by-products Environmentally friendly disposal 11

..and also… Shortening of the reaction times Much higher yields in comparison to conventional technologies (up to 5 – 15 % higher) Repetitive use of the Biocatalyst, easy separation from media (sieve separator) Higher productivity of the Biocatalyst Lower risk of contamination during the production process High stability and easy control of production process Biotechnologies are changing the world… 12 12

Technological set-up         - inlet of reaction media  - filling of Lentikats Biocatalyst  - medium with Lentikats Biocatalyst  - stirrer  - sieve separator  - outlet of separated media  - outlet of Lentikats Biocatalyst

Lentikats and food industry We currently offer solutions for the following processes: production of lactose free milk Lentikats Biocatalyst with β-galaktosidase activity production of invert sugar Lentikats Biocatalyst with invertase activity whey processing Lentikats Biocatalyst with β-galaktosidase activity for: production of D-galaktose from whey lactose (pharmaceutical quality) production of galacto-oligosaccharides from whey lactose production of special syrups Lentikats Biocatalyst with invertase activity for production of glucose-fructose syrups Lentikats Biocatalyst with immobilised glucoamylase for production of glucose from sugar and starch-based materials or glucose syrups from maltotriose or maltose syrup

Lentikats and food industry Following solutions are currently under development and testing: production of organic acids Lentikats Biocatalyst with immobilised bacteria Bacillus coagulans for production of lactic acid from lactose, glucose and sucrose Lentikats Biocatalyst with immobilised filamentous fungi Aspergillus niger for production of gluconic acid from sucrose and glucose Lentikats Biocatalyst with immobilised filamentous fungi Aspergillus terreus for production of itaconic acid from sucrose and glucose Biotechnologies are changing the world…

Invert Sugar in Food Industry Sucrose source – sugar beat, sugar cane Mixture of glucose and fructose (sucrose hydrolysis) Invert sugar as a product: composition 33% sucrose, 35% glucose and 32% fructose » composition 4% sucrose, 51% glucose and 45% fructose invert sugar is more expensive than syrup » invert sugar is sweeter and contains also other monosaccharides apart from sucrose large variety of applications: kids food, pastry, soups and sauces, candy and sweets, fruit drinks, dairy products.

Invert sugar in Distilleries Sucrose source – sugar beat, sugar cane Mixture of glucose and fructose molasses – benefits of sucrose hydrolysis a longer shelf-life » possibility of a year-round operation for molasses distilleries a lower water binding capacity – possibility to a achieve higher thickness prior to storage (80%) » lower demands for storage space and transportation

Lentikats Biotechnology and production of invert sugar Lentikats Biocatalyst with invertase activity = immobilised saccharase (β-D-fructofuranosidase) saccharase source – Baker’s yeast (Candida utilis) invertase activity 600 - 1000 g sucrose/kg LB/hr optimum temperature 30 - 35°C optimum pH 4.5 – 5.0 operational stability 600 – 1000 hodin 18 18

Lentikats Biotechnology and production of invert sugar The active compound is immobilised invertase for sucrose hydrolysis to glucose and fructose. - Standard activity assay: batch test standard sucrose solution with the concentration 100 g/l temperature 30°C pH 4.5 (10 mM acetate buffer) conversion time 50 min conversion rate 100% 19

Case studies

1) Hydrolysis of sucrose solution Invert sugar production Input parameters sucrose concentration 450 g/l pH = 4.5 temperature = 30°C batch operation mode solution volume: 2 222 m3 Lentikats Biocatalyst activity 1000 g SUC/kg LB/hr stability 900 hr amount 1 ton Outlet concentration: sucrose 10 g/l glucose 220 g/l fructose 220 g/l volume 2 222 m3 Invert sugar production amount 1 000 000 kg 14 EUR/m3 solution 0.03 EUR/kg invert sugar 21

2) Hydrolysis of sucrose solution Ethanol production Input parameters sucrose concentration 450 g/l pH = 4.5 temperature = 30°C batch operation mode Solution volume: 2222 m3 Lentikats Biocatalyst activity 1000 g SUC/kg LB/hr stability 1000 hr amount 1 ton Output concentration: sucrose 10 g/l glucose 220 g/l fructose 220 g/l volume 2 222 m3 Ethanol production volume 615 333 litres a.a. 14 EUR/m3 solution 0.05 EUR/litre a.a. 22

3) Molasses distillery 4 EUR/m3 molasses 0.05 EUR/litre a.a. Ethanol production Input parameters sucrose concentration 120 g/l pH = 4.5 temperature = 30°C continuous operation mode molasses volume: 8 333 m3 Lentikats Biocatalyst activity 1000 g SUC/kg LB/hr stability 1000 hr amount 1 ton Output concentration: sucrose 30 g/l glucose 45 g/l fructose 45 g/l volume 8 333 m3 Ethanol production volume 629 167 litres a.a. 4 EUR/m3 molasses 0.05 EUR/litre a.a. 23

Summary – application of 1 ton of LB Sucrose solution – 450 g/l sucrose Volume of solution processed [m3] Ethanol produced [l a.a.] Price [EUR/m3 SUC solution] [Kč/litr a.a.] 1 ton LB 2222 615 333 14 0.05 Molasses – 120 g/l sucrose Volume of molasses processed [m3] Ethanol produced [l a.a.] Price [EUR/m3 molasses] [Kč/litr a.a.] 1 ton LB 8333 629 167 4 0.05

Investment and Investment Return Guarantees You can easily verify our great results by testing our Biocatalyst in your or other facilities with the material of your choice. Sample of Lentikats Biocatalyst free of charge Our company provides assistance and full technical support. Our company offers a long-term cooperation to companies, whose main concerns are reduced investment costs and maximal process profitability. Biotechnologies change the world… 25

Investment and Investment Return Guarantees We guarantee the following: output parameters operational stability of Lentikats Biocatalyst and its activity and productivity

Our partners in the field of industrial applications Fermenta Biotech, DIL Complex, Ghodbunder Road, Thane (West) - 400 610, Maharashtra, India www.fermentabiotech.com Protean, Pod Lesem 9, 373 16 Dobra Voda u Ceskych Budejovic, Czech Republic www.protean.cz Pharmix s.r.o., Na Sadkach 2798, 767 01 Kromeriz, Czech Republic www.pharmix.cz 27 27

Our partners in the field of research and development Fermenta Biotech, DIL Complex, Ghodbunder Road, Thane (West) - 400 610, Maharashtra, India www.fermentabiotech.com Institute of Microbiology, Academy of Science of the Czech Republic Vídeňská 1083, 142 20 Prague 4, Krč, Czech Republic www.biomed.cas.cz STU Bratislava, Fakulta chemické a potravinářské technologie, Ústav biotechnologie a potravinářství, Radlinského 9, 81237 Bratislava, Slovakia www.stuba.sk VŠCHT Praha, Ústav kvasné chemie a bioinženýrství, Technická 5, 166 28 Praguea 6 www.vscht.cz University of Alberta, Edmonton Alberta, T6G 2J9 Canada www.ualberta.ca Masaryk University, National Centre for Biomolecular Research, Kamenice 5, 625 00 Brno-Bohunice www.ncbr.chemi.muni.cz 28 28

Thank you for attention Laboratory: INBIT LentiKat‘s a.s. Kamenice 34 625 00 Brno Czech Republic Tel.: + 420 511 205 288 Tel.: + 420 511 205 287 Company Headquarters: Office Building ARGO LentiKat‘s a.s. Evropska 846/176a 160 00 Prague 6 Czech Republic Tel.: + 420 255 710 680 Fax: + 420 255 710 699 E-mail: info@lentikats.eu Production plant: Pod Vinicí 83 471 27 Stráž pod Ralskem Tel.: + 420 487 851 393 Fax: + 420 487 851 399 E-mail: info@lentikats.eu 29 29