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Tuscia University and Veltha ivzw, with the support of Lazio Region representative office in Brussels, are pleased to invite you at the:

Workshop on Innovation in beverage industries: towards more sustainable enzymatic processes 10th DECEMBER 2014 - h. 14:00 BRUSSELS - Rond Point Schuman, 14 -(Lazio Region Office)-8th Floor Beverage industry needs specific, robust and cost effective enzyme-based catalyst systems that can be directly implemented into the industrially relevant beverage production and clarification processes. The development of immobilized systems over the free enzyme systems could led to several advantages, including multiple reusability, as well as an easy separation from the reaction mixture to avoid the permanence of residues which could affect food matrices stability. Implementation of these new solutions should result in novel, techno-economically viable large-scale bioprocess, which will become a sustainable alternative to the traditional ways of beverage treatment, ensuring the competitiveness of European industries in the knowledge-based economy in Europe and world-wide. The main themes of the discussion are: - Application of enzymes in food and beverage industry - Sustainable clarifying process in beverage industry: issues & solutions The workshop is organised as an discussion table where, after some brief presentations of the speakers, the participants may exchange their points of view and/or questions. Programme 14,00 - Welcome coffee and registration 14,30 - Welcome speech - MEP Silvia Costa – European Parliament 14,40 - Introduction to the two themes - Marco Esti - Tuscia University 14,50-Speakers:  Tuscia University - (Ilaria Benucci)  Fraunhofer Institute for Applied Polymer Research IAP - (Alexandra Latnikova)  European Commission - DG Research and Innovation –Advanced Manufacturing Systems and Biotechnologies (Carmine Marzano)  Slovak University of Technology - (Martin Rebros)  University Politehnica Bucharest - (Michaela Dina Stanescu)  CLEA Technologies B.V. - (Pieter Koning)  Eaton Technologies GmbH - (Ilona Schneider) 16,45 - Round table (Moderator: Carlo Polidori- Veltha ivzw) 18,00 End of the Workshop

-

List of attendees: Association of Manufacturers and Formulators of Enzyme Products (AMFEP) European Technology Platform Food for Life Anheuser-Busch InBev nv/sa Juice Products Association LentiKat´s a.s. Vitalgrana Pomegranate S.L.


Workshop on Innovation in beverage industries: towards more sustainable enzymatic processes

10/12/2014 Lazio Region Representation in Brussels Rond Point Schuman, 14


10th DECEMBER 2014 – BRUSSELS Rond Point Schuman, 14 -(Lazio Region Office)

Innovation in beverage industries: towards more sustainable enzymatic processes Prof. Marco Esti Department for Innovation in Biological, Agro-food and Forest systems (DIBAF) University of Tuscia Via S. Camillo de Lellis 01100 Viterbo - ITALY +39 0761 357426 esti@unitus.it


European projects of Tuscia University (2009-2013)

KBBE 31%

INFRASTRUCTURES 33%

ENVIRONMENT 30%

IDEAS-ERC 3%

SME2011 1% EURATOM-FISSION 1% INCO 1%


Global market for food and beverage enzymes Global food and beverage enzyme market: 2010-2018 Billion $

Growth % 12

bn $

10 8

2

6 4

1

Growth (%)

3

2 0

0

2010

2012

2018

The Food and beverage enzyme industry global market revenue was $1.2bn in 2012 and is estimated to reach $2.3bn by 2018


Europe’s food and everage industry

Turnover (€ billion) Added value (€ billion) Number of employees (million)

2009

2010

2011

929 193 4.20

953 203 4.25

1,017 206 4.25


Global beverage industry: 2008-2017

Growth %

7 6

bn $

2000

5 1500

4

1000

3 2

500

1

0

0

2008

2012

2017

Growth (%)

Billion $

2500


.

Europe accounts for about 40% of the global beverage industry, expected to rise to 916.5bn liters by the end of 2017


European juice market

Revenue ($ billion)

2012

2017

36

40


The most innovative food sectors in Europe, 2011-2013 (% of total European food innovation) Dairy products Ready-made meals Soft drinks

Savory frozen products Biscuits Meat, delicatessen, poultry

Appetizer grocery products Chocolate products 2011

Cheeses

2013

0

1

2

3

4

5

6

7

8


EU consumer expectations a ording to XTC Trends Tree™


Food innovation trends in Europe: 2009 - 2013 Variety of sense

2013

2009 2013

Sophistification

2009 2013

Easy to handle

2009 2013

Natural

2009

2013 2009

Medical 2013 2009

Fun Slimness

2013 2009

Time saving

2013 2009

Exoticism Nomadism

2013 2009

Energy. Well-being

2013 2009

Vegetal

2013 2009

ecology

2013 2009 2013 2009

Solidarity

2013 2009

Cosmetics 0

5

10

15

%

20

25

30

35


Conclusions The two industrial sectors of food enzymes and beverages have great innovation potential and considerable economic growth potential Impact of food and beverages on the senses is the most important factor in terms of costumer value expectations.


10th DECEMBER 2014 – BRUSSELS Rond Point Schuman, 14 -(Lazio Region Office)

Enzymes for beverage clarification

Dr Ilaria Benucci Department for Innovation in Biological, Agro-food and Forest systems (DIBAF) University of Tuscia Via S. Camillo de Lellis 01100 Viterbo - ITALY +39 0761 357426 ilaria.be@unitus.it


Haze development in beverages

Haze is the result of light scattering by colloidal or large particles suspended in a beverage.


Haze-Active (HA) molecules in beverages HA Proteins Proline-rich proteins

HA Phenolic compounds Low molecular weight flavans (mainly dimers)

Acidic polysaccharides Pectic substances

Starch


Time course of haze development in beverages Age-related haze (during storage)

Immediate turbidity Protein haze

Pectic substances (Pectin-protein colloidal complexes)

Starch haze

Chill haze


Time course of haze development in beverages Age-related haze (during storage)

Immediate turbidity Protein haze

Pectic substances (Pectin-protein colloidal complexes)

Starch haze

Chill haze


Cloud particles in beverages


Conventional treatments for beverages stabilization • Bentonite • Silica sol • Tannic Acid • Gelatin • Isinglass • Polyvinylpolypyrrolidone (PVPP)

Fining agents addition

Filtration • Filter aids (diatomaceous; sludge frame; kieselguhr)


Drawbacks of conventional treatments • Non-specific treatments • Overall reduction in sensory quality of beverages • High labour input and associated costs • Disposal of spent fining agents constitutes a non-negligible source of waste • Release of contaminant elements (bentonite)

Filtration • Energy consumption; • Harmful; require special handling and disposal procedures (filter aids)

• High capital costs for regeneration (PVPP)

Fining agents addition


Enzymes: Advantages • Increase reaction specificity; • Preserve product quality without altering organoleptic properties; • Reduce environmental impact (save raw materials, energy, chemicals, water).

Enzymes: Drawbacks • High cost; • Low stability; • After each cycle of operation the enzymes can not be recovered for further use; • They are inevitably present in the final product.


Cloud particle prevention with enzymes


Enzymes: Advantages • Increase reaction specificity; • Preserve product quality without altering organoleptic properties; • Reduce environmental impact (save raw materials, energy, chemicals, water).

Enzymes: Drawbacks • High cost; • Low stability; • After each cycle of operation the enzymes can not be recovered for further use; • They are inevitably present in the final product.


Sustainability of conventional and enzymatic treatments Conventional treatments

Free enzyme

Immobilized enzyme

Waste streams

Chemical inputs

Consumption of energy

Greenhouse gas emissions 0

20

40

60

80

100


Thank you for your attention Dr Ilaria Benucci Department for Innovation in Biological, Agro-food and Forest systems (DIBAF) University of Tuscia Via S. Camillo de Lellis 01100 Viterbo - ITALY +39 0761 357426 ilaria.be@unitus.it


Enzyme immobilization Dr. Alexandra Latnikova, Fraunhofer institute for applied polymer research Potsdam, Germany

Š Fraunhofer IAP


What has to be considered about enzymes? Enzymes are relatively expensive We would like to use them multiple times. Therefore, we need an easy way to recover them from the reactive mixture and be able to re-use them

Enzymes are sensitive to the processing conditions Therefore, we would like to stabilize them

Their presence in the final product can cause intolerance by consumers Therefore, we need an easy way to remove them from the product after they did their job

Possible solution: enzyme immobilization

Š Fraunhofer IAP


Enzyme immobilization strategies Depending on the enzyme type, some methods will improve the performance, while some can even worsen it. Within one immobilization strategy, the performance will depend on the material used, preparation conditions etc.

Š Fraunhofer IAP


Application of immobilized enzymes: continuous process

Potential advantages: Easier reactor operation and control Easier product recovery and purification Wider choice of reactors

Š Fraunhofer IAP


Design of robust industrial catalysts

The enzyme type - immobilization strategy combination is specific for each application The major problem in enzyme immobilization is not how to immobilize enzymes, but how to design the performance of the immobilized enzyme at will. Unfortunately, the approaches currently used to design robust industrial immobilized enzymes are, without exception, labelled as irrational , because they often result from screening of several immobilized enzymes and are not designed. As a result, many industrial processes might be operating under suboptimum conditions.

In order to make it rational, the dialog between the fundamental research and industry is needed!

Š Fraunhofer IAP


Size

Carrier-based approach

The right set of criteria, specific for each application, has to be fulfilled Therefore: one has to be able to vary all this parameters simultaneously in a controlled manner

Surface chemistry

Porosity

Material

All parameters are interconnected

Š Fraunhofer IAP

Expertise of Fraunhofer IAP


Expertize of “Microencapsulation and particle applications” group at Fraunhofer IAP Available particle size: 100 nm – 1 mm

Possible particle architectures: Compact, core-shell, multicompartment, porous

Available materials: Conventional: polyethylene, polypropylene etc. Synthetic: melamine-(form)aldehyde, polyurethane, polyamide, polystyrenes, polyacrylates, polyacrylonitrile, copolymers of those etc. Bio-based/friendly: polylactide (stereocomplexes of), modified starch, modified cellulose, lignin-based polymers etc.

Surface chemistry: -

— SO3 —OSO3—OH —COOH —SH —NH2 —NR3+ —CONH2 -- CONHR -- etc.

© Fraunhofer IAP

More than 25 years of experience


Organization of Fraunhofer IAP

Š Fraunhofer IAP


Fraunhofer Institutes in Germany

 67 institutes and research units  more than 23,000 staff  €2 billion annual research budget totaling. Of this sum, more than 1.7 billion euros is generated through contract research  2/3 of this sum is generated through contract research on behalf of industry and publicly funded research projects  1/3 is contributed by the German federal governments in the form of base funding

© Fraunhofer IAP


Fraunhofer worldwide Glasgow

Dublin Southampton Wrocław Brussels Vienna Paris Budapest Bolzano Graz

Vancouver Londo

San José

Gothenburg

Boston East n Cambridg Lansing Plymouth Storrs e Maryland Newark

Porto Thessaloniki

Beijing

Seoul

Sendai Tokyo

Jerusalem Cairo Dubai Bangalore

Ampang Singapore Jakarta

Salvador Campinas São Paulo Santiago de Chile

     

Subsidiary Center Project Center ICON / Strategic Cooperation Representative / Marketing Office Senior Advisor

© Fraunhofer IAP

Stellenbosch

Sydney


Joseph von Fraunhofer

Discovery of the “Fraunhofer lines” in the solar spectrum

New methods for processing lenses

Director and partner in a glassworks

© Fraunhofer IAP

The FraunhoferGesellschaft

Researcher

Inventor

Entrepreneur

Research and development on behalf of industry and state

mp3 music format, white LED, highresolution thermal camera

Research volume: approx. €2 billion annually


Thank you for your attention

Š Fraunhofer IAP


The story of success

sweet

Table sugar intermediate

much sweeter

US, 1950s: Sugar is produced by extraction from sugarcane and sugar beets A considerable part of table sugar is used in liquid inverted form, e.g. hydrolyzed into GLU and FRU form Liquid sugar markets is an enticing target for the corn syrup and glucose manufacturers The problem: glucose is less sweet than fructose Therefore, a process that would transform glucose into fructose is urgently needed This transformation takes place at basic conditions, but: too many by-products --> color and off flavors Therefore, it does not have industrial success 1957, USA: Marshall and Kooi: xylose isomerase, which transforms D-glucose into D-fructose 1960, USA: a process of GLU-FRU transformation using the enzyme isolated from Pseudomonas hydrophilia 1966, Japan: Takasaki described an industrial process of GLU-FRU using the soluble glucose isomerase 1967, USA: Clinton Corn Processing Company produces first commercial high fructose corn syrup (HFCS), contains 15% FRU 1968, USA: Clinton introduces 42% fructose syrup prepared with immobilized enzyme 1969, Japan: A process described, in which enzyme-containing cells were reused either in batch reactors or columns 1972, USA: continuous system using an immobilized enzyme was put into operation by Clinton

Š Fraunhofer IAP


The result: Nowadays, HFCS corn syrup is produced from starch and comprises 3 enzyme-mediated stages: 1. liquefaction of starch (by Îą-amylase) 2. saccharification (by glucoamylase)

3. glucose-fructose transformation The product, 55% enriched fructose syrup is obtained at a price 10-20 % lower than that of sucrose (based on sweetening power)

Š Fraunhofer IAP


Biotechnology in Horizon 2020 Carmine Marzano Directorate "Key Enabling Technologies" DG Research and Innovation Workshop on Innovation in beverage industries: towards more sustainable enzymatic processes Brussels 10th Dec 2014 Policy Research and Innovation


Outline: -

The KETs and Biotechnology Biotechnology in Horizon 2020 Biotechnology KET calls 2014-2015 Status WP 2016-17 Conclusion

2 Policy Research and Innovation


Industrial mastering and deployment of Key Enabling Technologies (KETs) • Nanotechnologies

What are KETs? • Six strategic technologies • Driving competitiveness and growth opportunities

• Contributions to solving societal challenges • Knowledge- and Capitalintensive

• Cut across many sectors

• Advanced Materials • Micro- and nanoelectronics • Photonics • Biotechnology • Advanced Manufacturing European KET Strategy: • EC Communications (2009)512 & (2012)341 • KET High-level Group 3

Policy Research and Innovation


Horizon 2020 •

Priority 1: Excellent Science

Priority 2: Industrial Leadership Leadership in enabling and industrial technologies (LEIT) (i) ICT including micro- and nano-electronics and photonics (ii) Nanotechnologies (iii) Advanced Materials This part of the Work Programme (iv) Biotechnology (v) Advanced Manufacturing & Processing (vi) Space Access to risk finance Leveraging private finance and venture capital for R&I Innovation in SMEs Fostering all forms of innovation in all types of SMEs

Priority 3: Societal Challenges Policy Research and Innovation

4


FP7 KBBE ~ € 650 mio Industrial biotechnology

Environmental biotechnology

Horizon 2020 > € 1500 mio Pillar 1: LEIT Biotechnology •Biotechnology-based industrial processes, including environmental and health biotechnology • Boosting cutting-edge biotechnologies as future innovation drivers

Emerging trends in biotechnology

• Innovative and competitive platform technologies NEW

Novel sources of biomass and bioproducts

Marine and fresh-water biotechnology

Pillar 2: Societal challenge •Food security, sustainable agriculture, marine and maritime research and the bioeconomy •Bio-Based Industries JTI

Biorefinery

•Marine biotechnology research


• Biotechnology-based industrial processes driving competitiveness and sustainability 

Aim: Maintain European leadership in industrial and environmental biotechnology.  Biocatalysts and bio-processing: bioprospecting, optimization of biocatalysts and biocatalytic process design

 Novel and improved microorganisms: understanding gene expression in the light of stress conditions; understanding mixed cultures and the dynamics of microbial communities  Bioreactors and downstream processing: address possible bottlenecks in downstream processing and facilitate the transfer of new developments into operational environments  Environmental biotechnology: focussing on the development and practical use of know-how in terms of bioprospecting; biodetection of 6 environmental pollution; bioremediation


• Cutting-edge biotechnologies as future innovation drivers 

Aim: Assuring that the European industry stays at the forefront of innovation, also in the medium and long term  Synthetic biology: creation of minimal cells, design of robust and sustainable bio-molecular circuits and pathways, biosafety and bioethics  Systems biology: investigating the operations of biological systems in order to optimize industrial applications of biotechnology  Bioinformatics: provision of powerful tools to store, retrieve and analyse biological data, in support of modelling and process design

 Nano-biotechnology: combining both fields of research for application development in a wide range of sectors, biosafety and bioethics  Biotechnology in ICT: provision of ICT-devices incorporating biological materials 7


• Innovative and competitive platform technologies 

Aim: Develop platform biotechnologies for a wide range of sectors to support European industrial leadership.  Platform technologies: Development of platform technologies and their integration across applications in support of the commercial use of biotechnology

 Industrial applications of 'omics': Bringing closer to the market 'omics'-derived industrial applications, including prototyping and demonstration

8


Calls 2014

Biotechnologies

• Cutting-edge biotechnologies as future innovation drivers BIOTEC 1: Synthetic Biology – construction of organisms for new products and processes RIA

• Biotechnology-based industrial processes driving competitiveness and sustainability BIOTEC 3: Widening industrial application of enzymatic processes IA

BIOTEC 4: Downstream processes unlocking biotechnological transformations IA BIOTEC 5: SME-boosting biotechnology-based industrial processes driving competitiveness and sustainability SME-Instrument (70% funding) Policy Research and Innovation


Results 2014

Biotechnologies

• Total call budget € 47.9 million • 26 proposals in second stage evaluation with a total requested EU budget of € 223.515 million • 5 proposals on main list, currently in grant preparation (EU budget € 37.6 million) • Possibility to fund 2 additional proposals • Areas covered by the successful proposals include: • Recycling of plastic waste for production of PHAs • Vaccine chassis for livestock industry • New vaccine/biomolecules purification technologies (DSP, 2 projects) • Biocatalysts for higher alcohols

10 Policy Research and Innovation


Calls 2015

Biotechnologies

• Cutting-edge biotechnologies as future innovation drivers BIOTEC 2: New bioinformatics approaches in service of biotechnology, RIA

• Biotechnology-based industrial processes driving competitiveness and sustainability BIOTEC 5: SME-boosting biotechnology based industrial processes driving competitiveness and sustainability, SME Instrument (70%)

• Innovative and competitive platform technologies BIOTEC 6: Metagenomics as innovation driver, RIA Policy Research and Innovation


Calls 2015 • BIOTEC 2 and 6 (2 stage evaluation) • Deadlines: • First stage 26/03/2015 • Second stage 08/09/2015 • Total Budget: 28.84 Million Euro

12 Policy Research and Innovation


Status WP 2016-17 • Currently we are in the process of finalising the scoping paper on which the 2016-17 WP will be designed upon • The work programme 2016-17 will be prepared during the first half of 2015, aiming to publish it in July 2015 (tentative deadline)

13 Policy Research and Innovation


Conclusion • Biotechnology relevant activities are covered substantially in H2020 (SC2, BBI JTI, BIOTEC KET)

• The Biotechnology KET will support activities to strenghten the competitiveness of the European Biotechnology Industry (including SMEs) • Bio-catalysis and industrially relevant activities will continue to be supported by the KET in H2020 (one topic already in 2014) • The scoping paper is being finalised, and the WP 2016-2017 might be available already in July 2015

14

Policy Research and Innovation


HORIZON 2020 Thank you for your attention! Find out more: http://ec.europa.eu/programmes/horizon2020/


Immobilized biocatalysis

Rebros Martin

Institute of Biotechnology and Food Science Faculty of Chemical and Food Technology Slovak University of Technology, e-mail: martin.rebros@stuba.sk

1


Immobilization PVA gel Advantages:

- cheap - non-toxic - non-biodegradable - good mechanical stability - no side effect - excellent long-term stability 2


LentiKats® technology

Stloukal, R., Rosenberg, M., Rebroš, M.: PCT - WO2007104268, www.lentikats.eu

3


LentiKats® technology Suitable for: • Enzymes (so far 5 hydrolases)

• Bacteria (10 natural producers, 2 recombinant)

• Yeast (Saccharomyces sp.)

4


Immobilization of enzymes α-L-rhamnosidase

α-L-rhamnosidase Rutin

Isoquercitrin

Re o i a t α-L-rhamnosidase of Aspergillus terreus immobilization in polyvinylalcohol hydrogel and its application in rutin derhamnosylation Rebroš M.,Pilniková A., Ši číková D., Weignerová L., Stloukal R., Kře V., and Rosenberg M., Biocatalysis and Biotransformation, accepted 5


Immobilization of enzymes Îą-L-rhamnosidase

Îą-L-rhamnosidase Rutin

Wine aroma release

Isoquercitrin

6


Background and skills Laboratories of applied biocatalysis

• immobilization cells/enzymes

• biocatalysis/biotransformations • fermentation technology • process development

• scale up

7


Background and skills Optimalization bioreactors

8


Background and skills Scale up of fermentation up to 400 l

9


Background and skills Downstream equipment

10


Immobilized biocatalysis Rebros Martin

Thank you for your attention! Institute of Biotechnology and Food Science Faculty of Chemical and Food Technology Slovak University of Technology, e-mail: martin.rebros@stuba.sk

11


LACCASE A VERSATILE ENZYME AND ITS APPLICATIONS IN FOOD AND TEXTILE DOMAINS Michaela Dina Stănescu U iversity „POLITEHNICA” Bucharest, „C.D. Ne itzescu” Depart e t of Orga ic Chemistry,Polizu Str. 1, Bucharest, Romania


Sustainable development • A development that meets the needs of the present, without compromising the ability of the future generations to meet their own needs. A development that provides economic, social, and environmental benefits in the long term, having regards to the needs of living of future generations • The concept was introduced in 1987, by the World Commission on Environment and Development in the report Our Common Future , Gro Harlem Brundtland known also as Bruntland Report.


Sustainable development • New approaches concerning the evaluation of products and technologies: – – – – –

product life cycle; ecological footprint; blue print; sustainability; greenness, etc.

• Most resource-efficient and environmentally friendly products have to be marketed.


Bio-based economy Fostering a bio-based economy leads to: • A lower carbon economy and sustainable primary production —reduction of CO2 emissions, resource and land-use efficiency;

• Building competitive bio-industries— potential for value creation through cascading use of biomass and reuse of waste materials; E C Bio-based economy in Europe: state of play and future potential 2011


Bio-based economy • A resilient and sustainable food chain —contribution to global food security, new agricultural practices to avoid competition between food and non-food use of biomass, and improved animal health and welfare;

• Developing the European science base and stimulating high-skilled jobs as well as research and innovation excellence in Europe. E C Bio-based economy in Europe: state of play and future potential 2011


LACCASE • Classification: Oxydase (EC 1.10.3.2) – metallo-enzyme (copper) • Sources: plants (from the latex of Japanese laquer tree), bacteria and fungi; • Substrates (unspecific): usually aromatic compounds having – OH or -NH2; • Optimum pH values: 2.6-8.0; • Optimum temperature: 45-55 0C


LACCASE (structure) • Apo enzyme: protein with ~ 500 aminoacids in a 3 βbarrel domains + oligocarbohydrates • Reaction centre : 4 copper ions 330 nm

600 nm


LACCASE (catalysis)


LACCASE (catalysis) • Advantages: – Diminish of the spent energy for the substrate oxidation; – Water as side product – Biodegradability; – Reactions in water.

• Problems: - Unstable; - Reaction conditions imposed by the enzyme (pH, temp.).


LACCASE (immobilization) Method

Advantages

Disadvantages

Physical methods Adsorption

Easy to perform

Easy loss of enzyme

Entrapment

Enhanced enzyme stability

Enzyme loss in time

Chemical methods Chemical bond with the carrier

Enhanced enzyme stability

May change enzyme parameters

Cross linking

Enhanced enzyme stability

May reduce enzyme activity due to modified conformation


The carrier • Matrix cryogel polymer:

1)High-molecular gel precursors; 2) Solvent; 3) Low-molecular gel precursors or solutes; 4) Crystals of frozen solvent; 5) Unfrozen liquid micro-phase; 6) Polymeric framework of the cryogel; 7) Đœacro-pores; 8) Solvent.


The carrier • Cryogel properties: – macroporous texture; – free of monomers and oligomers (reduced toxicity); – good mechanical properties; – easy available; – reasonable costs, etc.


The carrier • Cryogel type PVA


LACCASE (immobilization) • Covalent bond immobilization of Laccase

M.D. Stanescu, M. Fogorasi, B. L. Shaskolskiy, S.Gavrilas, V. I. Lozinsky, Appl Biochem Biotechnol, 2010,160, 1947–1954


Immobilized LACCASE (commercial) Content of the Commercial laccase 0.57 mg/g


Immobilized LACCASE (commercial) • Stability of the free enzyme (1) and immobilized enzyme (2) 2 1

M.D. Stanescu, M. Fogorasi, B. L. Shaskolskiy, S.Gavrilas, V. I. Lozinsky, Appl Biochem Biotechnol, 2010,160, 1947–1954


LACCASE (activity) • 2,2’-azinobis-(3-etylbenzothiazoline-6-sulfonate), (ABTS) - λmax (420 = 3.6·104 M-1cm-1)


Commercial LACCASE (activity) Catalyst

Free enzyme

Immobilized enzyme

pH

Specific activity (µmol min-1 g-1)

3.8 4.7 5.8 3.8 4.7 5.8

1301.2 1017.1 965.0 196.5 268.4 76.7

M.D. Stanescu, M. Fogorasi, B. L. Shaskolskiy, S.Gavrilas, V. I. Lozinsky, Appl Biochem Biotechnol, 2010,160, 1947–1954


Biosynthesized LACCASE Biosynthesis T. Pubescens Innoculum

Centrifugation Diafiltration Chromatography a) Ionic - Agarose–OCH2-O-(CH-OH)-CH2O-(CH-OH)-CH2O-N+(CH3)3 b) Gel – Superdex (Agarose + Dextran) Protein content: 12.51 mg/mL


LACCASE (immobilization) • Progress of the immobilization process by following the protein content in enzyme solution

• Biocatalyst: content -5.2 mg /g , activity - 22250 µmol/min g M. D. Stanescu, S. Gavrilas, R. Ludwig, D. Haltrich, V. I. Lozinsky, Eur Food Res Technol, 2012, 234, 655–662


APPLICATIONS IN FOOD DOMAIN Polyhydroxyphenol oxidation catalyzed by laccase


LACCASE (commercial) • Polyhydroxyphenols:

• catechol (1); quercitine (2), catechin (3), chlorogenic acid (4) and caffeic acid (5) S. Gavrilaş, F. Dumitru, M. D. Stănescu, U.P.B. Sci. Bull., Series B, 2012, 74 (4), 4-10.


LACCASE (commercial) • Catechin (3) oxidation: dimer of 3

S. Gavrilaş, F. Dumitru, M. D. Stănescu, U.P.B. Sci. Bull., Series B, 2012, 74 (4), 4-10.


Covalently immobilized LACCASE • Polyhydroxyphenols:

catechol (I), chlorogenic acid (II), caffeic acid (III), catechin (IV) M. D. Stanescu, S. Gavrilas, R. Ludwig, D. Haltrich, V. I. Lozinsky, Eur Food Res Technol, 2012, 234, 655–662


Covalently immobilized LACCASE (biosynthesis) • Oxidation kinetics of the compounds I-IV

M. D. Stanescu, S. Gavrilas, R. Ludwig, D. Haltrich, V. I. Lozinsky, Eur Food Res Technol, 2012, 234, 655–662


Covalently immobilized LACCASE (biosynthesis Variation of phenol content in: apple juice (AJ), untreated(UAJ), treated with free laccase (FLAJ) or immobilized laccase (ILAJ) a ) Mean value of a triplicate b) Expressed as CE—catechol equivalent

c) Expressed as QE—quercitine equivalent

M. D. Stanescu, S. Gavrilas, R. Ludwig, D. Haltrich, V. I. Lozinsky, Eur Food Res Technol, 2012, 234, 655–662


Covalently immobilized LACCASE (biosynthesis • Solid deposits in 10 mL of AJ samples taken after 2 h and stored 24 h at 5 °C, from: a) untreated apple juice (UAJ); b) free laccase-treated apple juice (FLAJ); c) immobilized laccase-treated apple juice (ILAJ)

M. D. Stanescu, S. Gavrilas, R. Ludwig, D. Haltrich, V. I. Lozinsky, Eur Food Res Technol, 2012, 234, 655–662


CONCLUSIONS • New biocatalysts have been obtained by covalently immobilizing commercial and laboratory-prepared laccases. • The biocatalyst has higher stability compared with the free enzyme. • The PVA-cryogel carrier is macro-porous, allowing the access of voluminous phenolic compounds to the reaction center. • The kinetic parameters of polyphenols oxidation revealed the influence of the hydrophobicity and electrochemical potential of the substrates on the reaction progress.


CONCLUSIONS • The immobilized laccase proved to be a good catalyst for stabilizing the phenol content of the apple juice by fastening oxidative processes and leading to more soluble products (less polymers). • Despite its lower activity, the catalyst is appropriate for the application due to the following considerations: – easy separation, – possibility of reuse, – improved quality of juice (clarity, colour, smell, etc.).


APPLICATIONS IN TEXTILE DOMAIN Degradation of textile dyes


The carrier • N,N-dimethylacrylamide (DMAAm), • allyl glycidyl ether (AGE) , • N,N’-methylene-bis acrylamide (MBAAm), • N,N,N’,N’-tetramethylethylene diamine (TMEDA) • iminodiacetic acid (IDA) Properties: • Macroporous; • High mechanical resistance.


LACCASE (immobilization) • Coordinative bond immobilization of commercial Laccase Roglyr Lite 1540 :

• Biocatalyst : content: 0.30 mg protein/mL, 24,6 µmol/min mL M.D. Stanescu , A.Sanislav R.V. Ivanov, A.Hirtopeanu, V. I. Lozinsky, Appl Biochem Biotechnol, 2011, 165, 1789–1798.


Coordinatively immobilized LACCASE (commercial) 窶「 Studied compounds: Acid Blue 62 (2) and bromaminic acid (1), :

A. Sanislav, F. Dumitru, M. D. Stトハescu, U.P.B. Sci. Bull., Series B, 2013, 75( 4), 91-100.


Coordinatively immobilized LACCASE (commercial • Decolorization of anthraquinone compounds: 1 (a) and 2 (b), with free (FL) and immobilized laccase (IL):

IL FL

IL FL M.D. Stanescu , A.Sanislav R.V. Ivanov, A.Hirtopeanu, V. I. Lozinsky, Appl Biochem Biotechnol, 2011, 165, 1789–1798.


Coordinatively immobilized LACCASE (commercial • Kinetic constants of oxidation reaction of 1 and 2, catalyzed by free and immobilized laccase

M.D. Stanescu , A.Sanislav R.V. Ivanov, A.Hirtopeanu, V. I. Lozinsky, Appl Biochem Biotechnol, 2011, 165, 1789–1798.


CONCLUSIONS • The coordinatively immobilized laccase proved to be a good catalyst for amino-anthraquinone compounds. • Despite its lower activity, the catalyst is appropriate for the application due to the following considerations: – easy separation, – possibility of reuse, – less polluting procedure.


APPLICATIONS IN TEXTILE DOMAIN Improving hemp quality


Mediators • Mediators improve the efficiency of the oxidation process being a electron shuttles extending the range of substrates:


LACCASE (commercial Hemp treatment:


LACCASE • Lignin oxidation


LACCASE (commercial) • Laccase treatment with or without US (FT-IR Vertex 70 )

US

No US


LACCASE (commercial) • Cristallinity after treatment initial


CONCLUSIONS • Laccase is an ecological solution for lignin elimination from hemp. • Laccase treatment improve the hemp fibre quality. • Use of mediators enhance the efficiency of treatment. • Use of US leads to an advanced degradation of lignin.


GENERAL CONCLUSIONS • Laccase is a nonspecific oxidase accepting as substrate: – Polyphenols, – Dyes, – Lignin. • Immobilization improve laccase treatments due to: – easy separation of the catalyst, – gentle process with less polymers, – possibility of reuse, – less polluting procedure.


Financial support • European Science Foundation for a STMS at Boku University through COST 928 Programme Control and Exploitation of Enzymes for Added-Value Food Products”

• Romanian Academy (grant 1R-2008), and the Russian Foundation for Basic Research (Project # 07-03-91682_RA_a).


Thank you for attention! • POZE


CLEA Technologies

Cross-linked Enzyme Aggregates in food applications

10/12/2014

Brussels

mCLEAs in Food

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Company Overview •

CLEA Technologies B.V.

Independent SME since 2002

Situated in Delft, The Netherlands

Currently 15 employees

Making enzymatic processes work. Sustainable & profitable

Proprietary enzyme immobilization technology

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mCLEAs in Food

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CLEA Technologies as partner •

Specialist on Biocatalysis – –

Roots in Delft University, Biocatalysis department Founder: Professor Roger Sheldon

Technology and service provider to a.o. – – –

pharma and specialty chemical manufacturers cosmetic ingredients, flavors and fragrances oil and fat, starch, biofuel (1st & 2nd generation)

What we do for our customers: – – – –

enzyme screening route development process optimization enzyme immobilization

Working for major players in each market 10/12/2014

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mCLEAs in Food

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Cross-linked Enzyme Aggregates

X-linking + Copolymerization

Precipitation

• “i ple a d roadly appli a le • Cost-effective (no carrier, no need for pure enzyme) • Short time-to-market (low development costs) • Scalable protocols

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mCLEAs in Food

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Magnetic CLEAs Synthesis of mCLEA 1. Synthesis of ferromagnetic nanoparticles 2. Functionalisation of the nanoparticles 3. CLEAtion: cross-linking the enzyme- and the nanoparticles

Characteristics

10/12/2014

Magnetic separation

Magnetic strength can be adjusted

No change in CLEA activity

Every CLEA can be made into mCLEA

Brussels

mCLEAs in Food

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(m)CLEA Properties • Very high enzyme loading – A tivity per kg o para le to ’free e zy es’

• Particle size typically 5-50 mm

• Mechanically robust – High resistance to shear, flexible

• Excellent operational and storage stability – Temperature, pH, organic solvents, long shelf life, hypoallergenic

• (Almost) all enzymes can be made into a mCLEA • Dedicated CLEA development within weeks • Short term to market 10/12/2014

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mCLEAs in Food

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mCLEA Properties in action • Covalent bond: No leaching of enzyme – No contamination of the product reaction system

• Easy product / enzyme separation – by permanent magnet, >99% separation efficacy. Low investment. – reduced down stream processing – applicable in multi-solid systems (fermentation, crystallization)

• Recyclable – depending on enzyme and process, > 50 cycles possible. 10 as target.

• No Carrier: – highly cost effective

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mCLEAs in Food

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Separation. Lab scale • Using € 1 permanent magnet • Separation: 1 minute movie stills

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mCLEAs in Food

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Separation: 10 – 1000 liter scale •

Using magnetic rod(s) in flow tube.

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mCLEAs in Food

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Separation: large scale •

Wet drum separation

– – – –

Proven technology Robust Low cost Standard mining equipment

10/12/2014

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mCLEAs in Food

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in beverage clarification • Fruit Juices – Just liquid or with solids? – With (high amounts of) solids: mCLEA only option

• Issues – Viscosity reduces movement between immobilized enzymes and substrate – On bead, as with large molecules: only enzymes on surface will be used – Smaller particles in favor: higher specific surface – Higher risk of attrition: strong bead or (m)CLEA

• Application – No solids present

->

bead:

– Solids present

->

mCLEA: magnetic separation

10/12/2014

use column or settling

Brussels

mCLEAs in Food

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Food Status Immobilized Enzymes • In general: from food additive to processing aid – Enzyme should still be food approved – In our vision: removal of immobilized enzymes is always advantageous – Removal of immobilized enzymes to check using activity assay? Leakage?

• Food status ingredients – Carrier Based: work with food approved (ion exchange) resins – cross-linked: an ingredient asks for attention: glutaraldehyde

• Food status glutaraldehyde – EU: Flavour Ingredient FL-nr 05.149 – FDA 173.357 Materials used as fixing agents in the immobilization of enzyme preparations. – Standard procedure immobilizing enzymes for (HF)CS production

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mCLEAs in Food

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EU Direct and Indirect Legislation • EU direct legislation: – EC 1331/2008 – –

regulations on food additives, enzymes and flavourings EFSA approval guidelines EFSA example on new enzyme http://www.efsa.europa.eu/en/efsajournal/doc/3778.pdf

– – – – – –

EC 1332/2008 EC 1333/2008 EC 1334/2008 EC 1935/2004 EC 1130/2011 ’

regulation on food enzymes regulation on food additives regulations on flavourings, Annex 1: substances Food contact materials framework Union list of approved food additives Food Contact Material Dbase

• EU indirect: German legislation – Use of glutaraldehyde in artificial sausage coatings

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mCLEAs in Food

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EU legal situation interpretation • • • •

The 1331-1333/2008 describe the use of enzymes in food, either as food enzyme or as food additive The application of enzymes to immobilize, remove and re-use restricts the legal position to processing aids (EC 1332-1333/2008) Alternatively, it may (also) be judged as Food Contact Material, like ion exchange resins (EC 1935/2004, EC 1130/2011) but EC 1130/2011 and DBase do mention but not cover enzyme carriers by any means. Ergo:

To the best of our knowledge, there is no EU legal base for any immobilized enzyme for use in food contact yet, only member state regulations. •

Ho e er, the EF“A e a ple o a e e z e e tio s o a i ert carrier , therefor judging the safety as enzyme solely http://www.efsa.europa.eu/en/efsajournal/doc/3778.pdf

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mCLEAs in Food

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Wrap Up • CLEAs – – – –

powerful and costs effective enzyme immobilization method no leaching much higher activity / ml than any carrier based enzyme can be hard to separate

• mCLEA – slightly more expensive than a CLEA – easy to separate at high efficacy at very limited investment – applicable in multi-solid systems (fermentation, crystallization)

• Legal status

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mCLEAs in Food

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CLEA Technologies B.V. Pieter Koning, Business Development

p.koning@clea.nl

Delftechpark 34 2628 XH Delft The Netherlands +31 15 76 00 300 www.cleatechnologies.com

Let us show you that it is not only better to use enzymes but also how to use enzymes better. 10/12/2014

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mCLEAs in Food

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Eaton’s Filtration Division

Begerow Product line – Life Science Beverage Treatment Products

© 2010 Eaton Corporation. All rights reserved.


Begerow Product line – Beverage Treatment Products Biotechnology (Yeast, Enzymes) Filter Aids

Beer Stabilization

Fining

Stabilization Beverage Treatment Product

Š 2009 Eaton Corporation. All rights reserved.

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Eaton’s Filtration Division

Wine Segment

© 2010 Eaton Corporation. All rights reserved.


Concept total solution provider in Wine Processing flow

Products we offer

Grape processing - Pressing - Fining

SIHA Gelatine, SIHA Activated Carbon, SIHA Lime

Alcoholic Fermentation

SIHA/SIHAFERM (Yeast), SIHA PROFERM (Yeast Nutrients)

Malolactic Fermentation

SIHALACT Malolactic bacteria,

SIHA Enzymes

SIHA bacteria nutrients

Clarification

Fining

SIHA Gelatine, SIHA Bentonite, SIHA Gum arabic ,… SIHA Active Carbon BECOGUR (Diatomite), BECOLITE (Perlite), BECOCEL (Cellulose)

Pre - filtration

End - filtration

Bottling

BECO + BECOPAD DFS, Modules BECO Filter Cartridges (membrane, depth) BECO Integra (Plate & Frame filters), BECO StepFlow, Housings

© 2009 Eaton Corporation. All rights reserved.

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Key technical elements in the wine industry – view from the beverage treatment products… •

Climate impact on flowering and ripening

Yeast – alcoholic fermentation: small, middle and large scale

Yeast nutrients

Enzymes – clarification and maceration of grapes, mash, juice, wine

Malolactic bacteria – metabolism malic acid to lactic acid + aroma

Fining, Stabilization & treatment of off-flavours: Gelatine, Bentonite

Filtration:

Pre-filtration with filter aids

Filtration with filter sheets, filter modules

Filtration with filter cartridges

We serve complete customer solutions in the wine industry.

© 2009 Eaton Corporation. All rights reserved.

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Market view EMEA –beverage treatment products European Wine Production by Country 2014, in M hl

5% 15%

Small, middle and big cooperative wineries

Sales resp. well educated and skilled

Germany

France

Reputation and Image in the wine industry

Italy

Spain

Distribution network via distributor, reunion of customer, direct sales

„Consultant“ role

Product price: middle and high price

28%

25% ROE

27%

© 2009 Eaton Corporation. All rights reserved.

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Communication strategy to influence decision makers in the wine industry… Competency / Expertise: • Sales group: skilled and experienced people  talk the customer language • Master, Bachelor – Thesis: Application and R&D products • Customer contact – Q1/Q2 : filtration and beverage treatment product Q3/Q4 : biotechnology and beverage treatment products •

Customer requirements and our answers: • • • • • • • •

lab services, specific product documentation technical articles, broschure recognition on trade shows @-newsletter customer training Enology symposium Germany, Austria Microscopy seminar: schools, customer Customer seminar: Germany, eastern european countries (crotia, slovenia, moldova, ect.)

© 2009 Eaton Corporation. All rights reserved.

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Eaton’s Filtration Division

Juice Segment

© 2010 Eaton Corporation. All rights reserved.


Concept solution provider Fruit Juice Processing flow Fruit

Products we offer Panzym, Panzym XXL

-Mashing Panzym, Panzym XXL

Pressing

Juice treatment: Starch / Pectin Finning

Pre - filtration End - filtration

Panzym, Panzym XXL, SIHA Bentonit, SIHA Bevasil, SIHA Gelatine, SIHA Activated Carbon SIHA Puranit UF

BECOGUR (Diatomite), BECOLITE (Perlite), BECOCEL (Cellulose)

BECO + BECOPAD DFS

Storage Concentrate or Storage Juice Š 2009 Eaton Corporation. All rights reserved.

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Process Apple/Pear Apple/Pear Mill Enzymzation

Option:

Enzyme addition during milling:

Pomace enzymation 220 ml/t Panzym Second Yield

pomace

Mash holding time

40-80 ml/t Panzym Yield Mash or

Press / Decanter

50-120 ml/t Panzym First Yield > 30 min. or 1-2 h at 25 – 27 °C, No stirring

Juice app.. 11,5 ° Bx

Juice appr.11,5 ° Bx

Pasteurisation

Aroma - recovery

Centrifuge

Degradation of pectin and starch

Concentration

Finning and FIltration

Direct concentrate with 45-65 °Bx

Concentration

Storage < 4°C

Juice- concentrate with

20-40 ml/1000l. Panzym XXL or 30-50 ml/1000l. Panzym Pro Clear 20-60 ml/1000l. Panzym HT 300 or Panzym AG XXL

70-72° Bx storage <4°C

© 2012 Eaton Corporation. All rights reserved.

10


Key technical elements in the fruit juice industry – view from the beverage treatment products… • climate impact on flowering and ripening • enzyme treatment mashing, clarification, stabilization • Beverage treatment (Stabilisation products): Gelatine & Bentonite & Active carbon • Filtration: • Filtration with filter sheets

© 2009 Eaton Corporation. All rights reserved.

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Market view EMEA – beverage treatment products EU-28: Apple production in 1.000 t 2011 Poland Italie France Germany Hungaria Spain Netherland Belgium Rumania Protugal Greece United Kingdom Austria Czech Republic Slovenia Danemark Sum

2.500 2.293 1.701 953 301 507 418 305 412 265 308 226 199 79 73 20 10.560

2012 2.900 1.939 1.169 972 750 391 281 220 351 221 242 162 157 118 45 18 9.936

2013 2014 * * Prognose 3.170 3.540 2.122 2.388 1.576 1.487 804 1.036 585 780 464 446 314 350 220 307 367 285 284 264 236 231 204 225 155 188 121 116 56 68 23 24 10.701 11.735

´13 %

-

-

-

-

12 13 6 29 33 4 11 40 22 7 2 10 21 4 21 4 10

High market share

Sales rep. well educated and skilled especially in poland

Increase demand of fruit processing, selling of fruit concentrate in new regions

„Consultant“ role, also for cider production

Stable AJC market

© 2009 Eaton Corporation. All rights reserved.

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Growth – PC Fruit juice – EMEA market • Product related with the partner companies: • Novozymes: production capabilities - surface and solid phase production • Panzym (traditional) Panzym XXL (GMM) • Panzym XXL and Panzym Yield Mash XXL (new product formulation)

• Industry trends, utilise these in our product development and communicate to our customer base

© 2009 Eaton Corporation. All rights reserved.

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PC Juice – EMEA market

Development of a new technology to stabilize cloudy beverages •

Pro`s: •

New Trends development with the SIHA, PANZYM and BECO products

Development of enzyme & extraction methods to get healthy juices

© 2009 Eaton Corporation. All rights reserved.

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Eaton’s Filtration Division

Begerow Product line – Life Science Beverage Treatment Products

© 2010 Eaton Corporation. All rights reserved.

Workshop on Innovation in beverage industries: towards more sustainable enzymatic processes  

Beverage industry needs specific, robust and cost effective enzyme-based catalyst systems that can be directly implemented into the industri...

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