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

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 10

th 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

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 [email protected]

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

mailto:[email protected]

ENVIRONMENT30%

INFRASTRUCTURES33%

KBBE31%

IDEAS-ERC3%

SME2011 1%

EURATOM-FISSION1%

INCO 1%

European projects of Tuscia University

(2009-2013)

Global market for food and beverage enzymes

The Food and beverage enzyme industry global market revenue was

$1.2bn in 2012 and is estimated to reach $2.3bn by 2018

0

2

4

6

8

10

12

0

1

2

3

2010 2012 2018

G

row

th (

%)

bn

$

Global food and beverage enzyme market: 2010-2018

Billion $ Growth %

2009 2010 2011

Turnover (€ billion) 929 953 1,017

Added value (€ billion) 193 203 206

Number of employees (million) 4.20 4.25 4.25

Europe’s food and everage industry

Global beverage industry: 2008-2017

0

1

2

3

4

5

6

7

0

500

1000

1500

2000

2500

2008 2012 2017

G

row

th (

%)

bn

$

Billion $ Growth %

.

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

2012 2017

Revenue ($ billion) 36 40

0 1 2 3 4 5 6 7 8

Dairy products

Ready-made meals

Soft drinks

Savory frozen products

Biscuits

Meat, delicatessen, poultry

Appetizer grocery products

Chocolate products

Cheeses2011

2013

The most innovative food sectors in Europe, 2011-2013 (% of total European food innovation)

EU consumer expectations a ording to XTC Trends Tree™

Food innovation trends in Europe: 2009 - 2013

0 5 10 15 20 25 30 35

Variety of sense

Sophistification

Easy to handle

Natural

Medical

Fun

Slimness

Time saving

Exoticism

Nomadism

Energy. Well-being

Vegetal

ecology

Solidarity

Cosmetics

2013 2009

2013 2009

2013 2009

2013 2009

2013 2009

2013 2009

2013 2009

2013 2009

2013 2009

2013 2009

2013 2009

2013 2009

2013 2009

2013 2009

%

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)

Dr Ilaria Benucci

Department for Innovation in Biological, Agro-food and Forest systems

(DIBAF)




Enzymes for beverage clarification

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

Protein haze

Pectic substances (Pectin-protein colloidal complexes)

Chill haze

Starch haze

Immediate turbidity

Age-related haze (during storage)

Cloud particles in beverages

Conventional treatments for beverages

stabilization

• Bentonite

• Silica sol

• Tannic Acid

• Gelatin

• Isinglass

• Polyvinylpolypyrrolidone

(PVPP)

Fining agents

addition

• Filter aids (diatomaceous; sludge

frame; kieselguhr)

Filtration

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)

• High capital costs for regeneration (PVPP)

Fining agents

addition

• Energy consumption;

• Harmful; require

special handling and

disposal procedures (filter aids)

Filtration

• Increase reaction specificity;

• Preserve product quality without altering

organoleptic properties;

• Reduce environmental impact (save raw materials,

energy, chemicals, water).

Enzymes: Advantages

• 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.

Enzymes: Drawbacks

Cloud particle prevention with enzymes

• Increase reaction specificity;

• Preserve product quality without altering

organoleptic properties;

• Reduce environmental impact (save raw materials,

energy, chemicals, water).

Enzymes: Advantages

• 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.

Enzymes: Drawbacks

Sustainability

of conventional and enzymatic treatments

0 20 40 60 80 100

Greenhouse gas emissions

Consumption of energy

Chemical inputs

Waste streams

Conventional treatments Free enzyme Immobilized enzyme

Dr Ilaria Benucci

Department for Innovation in Biological, Agro-food and Forest systems (DIBAF)




Thank you for your attention

mailto:[email protected]

© Fraunhofer IAP

Enzyme immobilization

Dr. Alexandra Latnikova,

Fraunhofer institute for applied polymer research

Potsdam, Germany

© 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

© 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.

© Fraunhofer IAP

Application of immobilized enzymes: continuous process

Potential advantages: Easier reactor operation and control

Easier product recovery and purification

Wider choice of reactors

© Fraunhofer IAP

Design of robust industrial catalysts

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!

The enzyme type - immobilization strategy combination is specific for each application

© Fraunhofer IAP

Carrier-based approach S

ize

Ma

teria

l P

oro

sity

Su

rface

che

mis

try

All parameters are interconnected 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

Expertise of Fraunhofer IAP

© 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.

More than 25 years of experience

© Fraunhofer IAP

Organization of Fraunhofer IAP

© 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

Dubai

Bangalore

Jakarta

Beijing Seoul

Tokyo

Cairo

Ampang

Santiago de Chile

Singapore

Brussels

Porto

Vienna

Bolzano Graz Budapest

Wrocław

Gothenburg

Thessaloniki

Sydney

Salvador

Sendai

Paris

São Paulo Campinas

Jerusalem

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

Stellenbosch

Boston

Plymouth

East Lansing San José

Newark Maryland

Cambridge

London

Vancouver

Storrs

Glasgow

Southampton Dublin

© Fraunhofer IAP

Joseph von Fraunhofer

Director and partner in

a glassworks

Discovery of the

“Fraunhofer lines” in the solar spectrum

New methods for

processing lenses

Research volume:

approx. €2 billion

annually

Research and

development on behalf

of industry and state

mp3 music format,

white LED, high-

resolution thermal camera

Researcher

Inventor

Entrepreneur

The Fraunhofer-Gesellschaft

http://www.fraunhofer.de/fhg/Images/jvf_brosch_tcm5-5806.pdf

© Fraunhofer IAP

Thank you for your attention

© Fraunhofer IAP

The story of success

Table sugar intermediate

sweet 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

© Fraunhofer IAP

The result: Nowadays, HFCS corn syrup is produced from starch and comprises 3 enzyme-mediated stages:

1. liquefaction of starch (by α-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)

Policy Research and Innovation

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


Outline:

- The KETs and Biotechnology

- Biotechnology in Horizon 2020

- Biotechnology KET calls 2014-2015

- Status WP 2016-17

- Conclusion

2


Industrial mastering and deployment of Key Enabling Technologies (KETs)

What are KETs?

• Six strategic technologies

• Driving competitiveness and growth opportunities

• Contributions to solving societal challenges

• Knowledge- and Capital- intensive

• Cut across many sectors

• Nanotechnologies

• Advanced Materials

• Micro- and nano- electronics

• Photonics

• Biotechnology

• Advanced Manufacturing

European KET Strategy:

• EC Communications

(2009)512 & (2012)341

• KET High-level Group

3


Leadership in enabling and industrial technologies (LEIT)

(i) ICT including micro- and nano-electronics and photonics

(ii) Nanotechnologies

(iii) Advanced Materials

(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

Horizon 2020

• Priority 2: Industrial Leadership

• Priority 1: Excellent Science

• Priority 3: Societal Challenges

This part of the Work Programme

4

FP7 KBBE ~ € 650 mio

Novel sources of biomass and bioproducts

Marine and fresh-water biotechnology

Biorefinery

Pillar 2: Societal challenge

•Food security, sustainable agriculture, marine and maritime research and the bioeconomy

•Bio-Based Industries JTI

•Marine biotechnology research

Environmental biotechnology

Emerging trends in biotechnology

Industrial biotechnology

Pillar 1: LEIT Biotechnology

•Biotechnology-based industrial processes, including environmental and health biotechnology

• Boosting cutting-edge biotechnologies as future innovation drivers

• Innovative and competitive platform technologies

NEW

Horizon 2020 > € 1500 mio

• Biotechnology-based industrial processes driving competitiveness and sustainability

6

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 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


8

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



Calls 2014 Biotechnologies


BIOTEC 1: Synthetic Biology – construction of organisms for new products and processes RIA

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)


• 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

Results 2014 Biotechnologies



Calls 2015 Biotechnologies


BIOTEC 2: New bioinformatics approaches in service of biotechnology, RIA

BIOTEC 5: SME-boosting biotechnology based industrial processes

driving competitiveness and sustainability, SME Instrument (70%)

BIOTEC 6: Metagenomics as innovation driver, RIA



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


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


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

HORIZON 2020

Thank you for your attention!

Find out more: http://ec.europa.eu/programmes/horizon2020/

1

Rebros Martin

Institute of Biotechnology and Food Science

Faculty of Chemical and Food Technology

Slovak University of Technology,

e-mail: [email protected]

Immobilized biocatalysis

2

PVA gel

Advantages: - cheap

- non-toxic

- non-biodegradable

- good mechanical stability

- no side effect

- excellent long-term stability

Immobilization

3

LentiKats® technology

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

4

LentiKats® technology

• Enzymes (so far 5 hydrolases)

• Bacteria (10 natural producers, 2 recombinant)

• Yeast (Saccharomyces sp.)

Suitable for:

5

α-L-rhamnosidase

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

Rutin Isoquercitrin α-L-rhamnosidase

Immobilization of enzymes

6

Rutin Isoquercitrin α-L-rhamnosidase

Wine aroma release

α-L-rhamnosidase

Immobilization of enzymes

7

Background and skills

• immobilization cells/enzymes • biocatalysis/biotransformations • fermentation technology

• process development • scale up

Laboratories of applied biocatalysis

8


Optimalization bioreactors

9


Scale up of fermentation up to 400 l

10


Downstream equipment

11

Rebros Martin

Institute of Biotechnology and Food Science

Faculty of Chemical and Food Technology

Slovak University of Technology,

e-mail: [email protected]

Immobilized biocatalysis

Thank you for

your attention!

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 ,

known also as Bruntland Report. Gro Harlem Brundtland

http://upload.wikimedia.org/wikipedia/commons/3/36/Gro_Harlem_Brundtland.ogg




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 β-

barrel domains + oligocarbohydrates

• Reaction centre : 4 copper ions

600 nm

330 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) Мacro-pores; 8) Solvent.

• Cryogel properties:

– macroporous texture;

– free of monomers and oligomers (reduced

toxicity);

– good mechanical properties;

– easy available;

– reasonable costs, etc.

The carrier

The carrier

• Cryogel type PVA


• 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



LACCASE (activity)

• 2,2’-azinobis-(3-etylbenzothiazoline-6-sulfonate),

(ABTS) - λmax (420 = 3.6·104 M-1cm-1)

Commercial

LACCASE (activity) Catalyst pH Specific activity

(µmol min-1 g-1)

Free enzyme

3.8 1301.2

4.7 1017.1

5.8 965.0

Immobilized

enzyme

3.8 196.5

4.7 268.4

5.8 76.7



Biosynthesized LACCASE

T. Pubescens

Innoculum

Biosynthesis

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


• 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)



Covalently immobilized

LACCASE (biosynthesis)

• Oxidation kinetics of the compounds I-IV




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




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)



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.


• 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)

• Studied compounds: Acid Blue 62 (2) and

bromaminic acid (1),

:

A. Sanislav, F. Dumitru, M. D. Stănescu, U.P.B. Sci. Bull., Series B, 2013, 75( 4),

91-100.


LACCASE (commercial

• Decolorization of anthraquinone compounds: 1 (a)

and 2 (b), with free (FL) and immobilized laccase (IL):

IL

FL

IL

FL


Biotechnol, 2011, 165, 1789–1798.


LACCASE (commercial

• Kinetic constants of oxidation reaction of 1

and 2, catalyzed by free and immobilized

laccase


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,


– 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 treatment with or without US (FT-IR -

Vertex 70 )

US

No US


• 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,


– 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

10/12/2014 mCLEAs in Food slide | 1Brussels

CLEA Technologies

Cross-linked Enzyme Aggregates in food applications


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


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


Cross-linked Enzyme Aggregates

PrecipitationX-linking + Co-

polymerization

• “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


Magnetic CLEAsSynthesis of mCLEA

1. Synthesis of ferromagnetic nanoparticles

2. Functionalisation of the nanoparticles

3. CLEAtion: cross-linking the enzyme- and the nanoparticles

• Magnetic separation

• Magnetic strength can be adjusted

• No change in CLEA activity

• Every CLEA can be made into mCLEA

Characteristics


(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


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


Separation. Lab scale• Using € 1 permanent magnet

• Separation: 1 minute movie stills


Separation: 10 – 1000 liter scale

• Using magnetic rod(s) in flow tube.


• Wet drum separation– Proven technology

– Robust

– Low cost

– Standard mining equipment

Separation: large scale


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: use column or settling

– Solids present -> mCLEA: magnetic separation


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


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 regulation on food enzymes

– EC 1333/2008 regulation on food additives

– EC 1334/2008 regulations on flavourings, Annex 1: substances

– EC 1935/2004 Food contact materials framework

– EC 1130/2011 Union list of approved food additives

– ’ Food Contact Material Dbase

• EU indirect: German legislation

– Use of glutaraldehyde in artificial sausage coatings


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


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


CLEA Technologies B.V.

Pieter Koning, Business Development [email protected]

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.

http://www.cleatechnologies.com/

2 2 © 2009 Eaton Corporation. All rights reserved.

Begerow Product line – Beverage Treatment Products

Biotechnology (Yeast, Enzymes)

Filter Aids

Beer Stabilization

Fining

Stabilization

Beverage Treatment Product



Wine Segment


Concept total solution provider in Wine

Grape processing

- Pressing

- Fining

Alcoholic Fermentation

Malolactic Fermentation

Clarification

Fining

Bottling

SIHA Enzymes

SIHA/SIHAFERM (Yeast), SIHA

PROFERM (Yeast Nutrients)

SIHALACT Malolactic bacteria,

SIHA bacteria nutrients

Pre - filtration

End - filtration

SIHA Gelatine, SIHA Bentonite, SIHA

Gum arabic ,…

SIHA Active Carbon

BECOGUR (Diatomite), BECOLITE

(Perlite), BECOCEL (Cellulose)

BECO + BECOPAD DFS, Modules

BECO Filter Cartridges (membrane,

depth)

SIHA Gelatine, SIHA Activated

Carbon, SIHA Lime

BECO Integra (Plate & Frame filters),

BECO StepFlow, Housings

Processing flow Products we offer


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.


Market view EMEA –beverage treatment products

• Small, middle and big cooperative

wineries

• Sales resp. well educated and skilled

• Reputation and Image in the wine

industry

• Distribution network via distributor,

reunion of customer, direct sales

• „Consultant“ role • Product price: middle and high price

5%

28%

27%

25%

15%

European Wine Production by Country

2014, in M hl

Germany France

Italy Spain

ROE


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.)

Communication strategy to influence decision makers in the wine industry…



Juice Segment


Concept solution provider Fruit Juice

Fruit

-Mashing

Pressing

Juice treatment:

Starch / Pectin Finning

Storage Concentrate

or Storage Juice

Panzym, Panzym XXL

Panzym, Panzym XXL

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

Processing flow Products we offer

10 © 2012 Eaton Corporation. All rights reserved.

Process Apple/Pear

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

Enzyme addition during milling:

40-80 ml/t Panzym Yield Mash or

50-120 ml/t Panzym First Yield

> 30 min. or 1-2 h at 25 – 27 °C,

No stirring

Juice app.. 11,5 ° Bx

Pasteurisation

Centrifuge

Concentration

Direct concentrate with 45-65 °Bx

Storage < 4°C

Juice appr.11,5 ° Bx

Aroma - recovery

Degradation of pectin and starch

Finning and FIltration

Concentration

Juice- concentrate with

70-72° Bx storage <4°C

Apple/Pear

Mill

Enzymzation

Mash holding time

Press / Decanter pomace

Option:

Pomace enzymation

220 ml/t Panzym Second Yield


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


Market view EMEA – beverage treatment products

• 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

EU-28: Apple production

in 1.000 t 2011 2012 2013 2014 * ´13

* Prognose %

Poland 2.500 2.900 3.170 3.540 12

Italie 2.293 1.939 2.122 2.388 13

France 1.701 1.169 1.576 1.487 - 6

Germany 953 972 804 1.036 29

Hungaria 301 750 585 780 33

Spain 507 391 464 446 - 4

Netherland 418 281 314 350 11

Belgium 305 220 220 307 40

Rumania 412 351 367 285 - 22

Protugal 265 221 284 264 - 7

Greece 308 242 236 231 - 2

United Kingdom 226 162 204 225 10

Austria 199 157 155 188 21

Czech Republic 79 118 121 116 - 4

Slovenia 73 45 56 68 21

Danemark 20 18 23 24 4

Sum 10.560 9.936 10.701 11.735 10


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


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



Begerow Product line – Life Science Beverage Treatment Products

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

Documents

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