dsm nutrition cluster · 2013-09-29 · dsm nutrition cluster. tensguard – a source of...

DSM Nutrition Cluster

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DSM Enzyme Innovation

New tools for constructing dairy bioactives

William King

October 20, 2010


1

Evolution

Life Science Products

Coal

Fertilizers

Petrochemicals

Performance Materials

Bioterials / Biologics

Chemical engineering

Polymer technology

Fine chemicals

Material science

Mechanical engineering

Biotechnology

Technological competences1902 1930 19701950 1990 2000 2010

classical modern

DSM - Over a century of successful transformations


2Who is DSM?

Industrial ChemicalsPerformance MaterialsPharmaNutrition

DSM Managing Board

DSM Innovation Center Corporate Staff

Shared Competences & Business Support

DSM Nutritional Products

DSM Food Specialties

DSM Resins

DSM Engineering Plastics -DSM Dyneema

DSM Elastomers

DSM Agro

DSM Fibre Intermediates

DSM Pharmaceutical Products

DSM Anti-InfectivesDSM Energy


3DSM, the Life Sciences and Materials Sciences company

• Some 22,700 employees• Net sales 2009: € 7,7 billion• Innovation driven• Some 300 new patents filed in

2009• Strong, solid balance sheet • A+ status for 3P reporting• Leader in Dow Jones Sustainability

Index 2009• FTSE4 Good Index Series• DSM best practice in Product

Carbon Footprint (PCF) Germany

28%

11%

29%

15%17%

Nutrition PerformanceMaterials

Base Chemicals& Materials

Pharma

PolymerIntermediates


4The DSM Nutrition Cluster – major product areas

DSM NutritionDSM Nutritional Products


• Vitamins and carotenoids; bio-active plant extracts (eg, resveratrol)

• Dairy ingredients: starter cultures, coagulants, preservation ingredients

• Food processing enzymes (baking, fruit processing, brewing, wine making)

• Food safety solutions (pathogens, allergens, impurities, acrylamides)

• Yeast derived products (food flavors, salt replacement, fermentation)

• PUFA’s (ARA, DHA) for use in infant nutrition and functional food

• Fuctional food ingredients (functional peptides, fortification aids, etc)


5The DSM Nutrition Cluster – technology platforms

DSM NutritionDSM Nutritional Products


• Fermentation – fungal expression, bacterial expression, yeasts, dairy cultures

• Molecular biology – genomics, enzyme development, directed evolution

• Extraction and Purification – enzymes, functional ingredients, flavors, colors

• Chemical synthesis – organic intermediates, vitamins, supplements, colors

• Formulation – encapsulation, bioavailability, nano-emulsions, drying

• Anti-infectives – antibiotics, anti-fungals, natural antimicrobials

• Clinical studies – design, execution, biomarker development, publications


6DSM has access to a unique enzyme toolbox

• DSM determined the first complete DNA sequence of the food-grade fungus Aspergillus niger

• Genome contains a large number of interesting genes for the development of new enzymes, e.g.:

• The genome of Aspergillus niger contains 13,000 genes• About 200 genes encode for proteases• About 60 of these are secreted proteases (= protein hydrolase = peptidase)

• These are well-defined, purified, specific, single enzymes, and offer exciting possibilities for new food processes, digestive aids, food safety applications, and bioactive protein hydrolysates and peptides

• DSM holds a strong patent position on proprietary enzymes, production processes, and selected applications


7


8Proteases in Aspergillus niger

Proteolysis (total) about 198 genes 100%

Cytosolic, nuclear degradation 84 42%

Proteasome 36 18%

Lysosomal degradation 14 7%

Vacuolar degradation 20 10%

Other proteolytic proteins 44 22%

Overall 198 proteases identified (intra- and extracellular)

-Endo-proteases-Amino peptidases -Carboxypeptidases


9DEC consists of 2 parts…

DSM Enzyme Collection (DEC)

Enzyme dataEnzyme samples


Bio-informatics Search Bio-diversity Search

Design & run high throughput screens

Genetic optimization, Cloning, and Expression

Looking for enzymes in complex matrices?

Looking for enzymes for synthetic applications?

Mining the DSM Enzyme Collection

http://dsm.starbrand.com/scripts/dsm.exe?ctx=21c063&view=1&object=BasketImage:004cc2e0

http://dsm.starbrand.com/scripts/dsm.exe?ctx=21bfc3&view=1&object=BasketImage:004ca873

http://dsm.starbrand.com/scripts/dsm.exe?ctx=21bfc3&view=1&object=BasketImage:004cc2ee


11Radical Innovation in the Nutrition Cluster

Diabetes

Protease toolboxMaxiPro, TPAP,

DPAP, …

Joint Health

I-OmegaTensGuardPep2Balance

PeptoPro

InsuVital

InfantPro

I-FlexFoodSafetyCeliac

disease

Cheeseripening


12

Val Pro

Leu

Pro

Lys

Arg

Gly

GluVal

Met

Gly

ValSer

LysVal

Lys

Glu AlaMet

Ala

Pro

Leu

Gln

PheProArg

Lys

Enzymes offer exciting possibilities for the development of novel flavor & texture profiles in dairy products

• Improving taste / removing bitter off-flavours

Endoproteasescleave peptide bonds in the inner regions of a peptide chain, to release oligopeptides

Exoproteasescleave residues from the ends of a peptide chain, to release free amino acids

Di-/tri-peptidasescleave after every 2nd/3rd amino acid, to release di- and tripeptides

Many different functionalities• Endo, Exo and tripeptidases• Broad versus narrow specificity• Differences in preference• ……

Protein

Enzyme toolbox


Novel peptidases for cheese & dairy products

Confidential


14Accelerzyme® CPG is a Carboxypeptidase

LEF

G RI

VD E

PA

K L CS

F

V

VK

D

G

NH2

COOH

Carboxy-peptidase (Accelerzyme CPG)

Endo-protease (Accelerzyme NP)


15

Ripening accelerator

Accelerzyme® assisted ripening

€15/ month/ tonne of cheese

Accelerzyme® CPG

•Reduction cheese maturation time thereby reducing storage time and costs

• Eliminates bitter peptides in cheese, protein hydrolysatesprocessed cheese, EMC

•Softening soft cheeses (Brie, Camembert)

• Speed-up blue fungus growth in Blue Cheese

• Increased flavor development fermented meat

50%Storage Time Reduction


16Enzymes offer exciting possibilities for the development of novel flavor & texture profiles in dairy products

• Improving taste / removing bitter off-flavours• Improving yields, color, and gelation properties

Enzyme toolboxβ-carotene


Enzymatic bleaching of dairy products

Confidential


18Application: Control of products color

Mozzarella, Feta and Ricotta cheeses made from cow milk

Whipped creamWhey

Confidential


19Carotenoid degrading fungal enzymes

• Cyathus palidus X

• Ischnoderma benzoinum X

• Marasmius scordonius (MsP) !

• Trametes versicolor X

• Lepista irina X

Screening on β-carotene agar plates

Light halo around Marasmius scrodonius growing colony

Confidential


Novel texturizing tools for dairy protein applications

Enzymatic cross linking of dairy proteins


21Use of Lysyl oxidase

0.8% milkpowderc.i.u. 2.5 €cent / liter

< 0.5% milkpowderc.i.u. < 1.5 €cent / liter

LOX costs 10%0.25 €centValue-based Pricing will be applied

7 € /ton profit

- Reduction of milkprotein (whey) up to 50% giving same (or higher) viscosity in final yoghurt

- Treatment of milk protein prior to adding in yoghurt process- Adding milk protein to milk, LOX treatment, pasteurization, cultures yoghurt

- Reduction of caloric value of yoghurt-Other applications (IceCream, Soy-based applications)


22Enzymes offer exciting possibilities for the development of novel flavor & texture profiles in dairy products

• Improving taste / removing bitter off-flavours• Improving yields, color, and gelation properties• Improving food digestibility and safety

LactoseIntolerance

Gluten Intolerance

Celiac Disease

IBS


2323AN-PEP : an enzyme to degrade gluten epitopes

Toxic gluten protein Non-Toxic gluten peptides

Aspergillus niger - active at low (gastric) pH

- cleaves peptides after a proline residue

prolyl endoprotease

Immune response in celiac patients

Immune response in celiac patients


24

2 4 6 8 10 120

20406080

100120

FM-POP

AN-PEP

pH

Enzy

mat

ic a

ctiv

ity [%

]An-PEP optimized for stability to stomach conditions

NT 0' 15' 30' 60'0

20406080

100120

Enzy

mat

ic a

ctiv

ity [%

]

AN-PEP / pH 2 / pepsin

FM-POP / pH 2

FM-POP / pH 2 / pepsin

Stepniak D et al. Am J Physiol Gastrointest Liver Physiol, 2006; 291: G621-9


25

Val Pro

Leu

Pro

Lys

Arg

Gly

GluVal

Met

Gly

ValSer

LysVal

Lys

Glu AlaMet

Ala

Pro

Leu

Gln

PheProArg

Lys

Enzymes offer exciting possibilities for the development of novel flavor & texture profiles in dairy products

• Improving taste / removing bitter off-flavours• Improving yields, color, and gelation properties• Improving digestibility and food safety• Development of new bioactive peptides

Endoproteasescleave peptide bonds in the inner regions of a peptide chain, to release oligopeptides

Exoproteasescleave residues from the ends of a peptide chain, to release free amino acids

Di-/tri-peptidasescleave after every 2nd/3rd amino acid, to release di- and tripeptides

Many different functionalities• Endo, Exo and tripeptidases• Broad versus narrow specificity• Differences in preference• ……

Protein

Enzyme toolbox


26Release of bioactive peptides using novel DSM enzymes

• Milk proteins are known to contain many biologically active peptides & glycopeptides

• Bioactive peptides are inactive in their native protein carrier – they are released through bacterial or enzymatic action

• Both human and bacterial proteases may be active in releasing bio-actives from milk and colostrum

• Bioactive peptides are being discovered by genomic characterization, supported by the International Milk Genomics Consortium

Pro

Leu

Leu

Pro

Gly

Pro Phe

Met

Gly

ValSer

AsnIle

Pro

Pro LeuMet

Ala

Pro

Val

Val

Val

Pro

Gln

LysGlu

Arg Lys

Glu

ArgBioactivepeptides

Protein

Releasing the bioactive peptides in proteins


27Example of functional peptides in β-casein

Ser

Lys

Val Leu Pro Val Pro Gln Lys Ala Val Pro Tyr Pro Gln Arg Asp Met Pro Ile Gln Ala

LeuLeuTyrGlnGluProValLeuGlyProValArgGlyProPheProIleIleVal

IleThrArgIleAsnLysLysIleGluLysPheGlnSerPGluGluGlnGlnGlnThrGlu

Asp

Glu Leu Gln Asp Lys Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro

ProPro IleAsnSerLeuProPro ProProVal Val Val Thr ThrGln Leu Ile Asn Gln

Pro

Pro ProPhe Leu Gln Glu Val Met Gly Val Ser Lys Val Lys Glu Ala Met Ala Pro Lys

SerPArg Glu Leu Glu Glu Leu Asn Val Pro Gly Glu Ile Val Glu SerP Leu SerP SerP Glu Glu

His

Phe

Gly

Ser

Antihypertensive caseinophosphopeptide

Immunopeptide β casomorphine 7

β casomorphine 11Antihypertensive

Antihypertensive

β -casokinin 7

Immunopeptide


28PeptoPro beverages for sports recovery

• Muscle glycogen resynthesis is one of the most important factors which determine the time needed to recover from exercise, and eventually energy and performance levels.

• With regular sport drinks, it takes about 20 hours to restore energy levels

• Athletes need to recover within 12 hours in order to be able to perform the next day

• PeptoPro sports accelerates uptake of the glucose in the muscle cells and improves muscle glycogen resynthesis


29

N C = O

O-

C

O

N C

O

CH CH2 CH

NH2

CH3

CH3

N C = O

O-

C

O

N C

O

CH CH

NH2CH3

C2H5

N C = O

O-

C

O

N C

O

CH CH

NH2

CH3

CH3

IPP IC50 = 2 μM

VPP IC50 = 9 μM

LPP IC50 = 10 μM

Selected peptides behave as ACE inhibitors

+X

HZn++

Angiotensin I converting enzymeThese dairy peptides are:1) resistant to digestion2) small enough to pass the

intestinal barrier, and3) with strong ACE inhibitory

properties.


30TensGuard – a source of Lactotripeptides

• Casein contains high levels of ACE inhibitor peptides.

• These peptides are largely degraded by stomach enzymes before reaching the intestinal tract

• Controlled digestion of casein with DSM endo-proteases releases the tri-peptides without loss of function.

Lactotripeptides (mg/g protein) DoseIPP LPP VPP Total BPPeq (g/day)

AmealPeptide 1,8 1,1 1,4 4.3 3,2 >1.5

TensGuard 21,0 2,2 nd 23.2 22,3 0,2-0,5

DSM developed clean flavor formulations with high levels of Lactotripeptides

Pro

Leu

Leu

Pro

Gly

Pro Phe

Met

Gly

ValSer

AsnIle

Pro

Pro LeuMet

Ala

Pro

Val

Val

Val

Pro

Gln

LysGlu

Arg Lys

Glu

ArgBioactivepeptides

Enzyme


31Tensguard bioavailability and absorption

• Human volunteers ingested a yogurt drink enriched with IPP and VPP. Blood plasma samples were collected and analyzed for IPP and VPP

• The presence of IPP, but not VPP, was demonstrated in measurable amounts in the circulation

• Tensguard contains high levels of IPP, the most effective of the ACE peptides

Ile-P

ro-P

ro (p

mol

/L)

Time (min)0 30 60 90 120

200

400

600

800

1000Lactotripeptide enriched drinkPlacebo drink

Foltz et al (2007), Journal of Nutrition 137, 953-9


32Balancing of blood sugar peaks

15%-30% reduction

Breakfast Lunch Dinner

Blo

od s

uger

leve

ls

Healthy people

Diabetes

Diabetes taking InsuVital

= Insulin response

• InsuVital restores the impaired insulin function in type 2 diabetes

• It works by helping your body release insulin right at the start of a meal, when you need it the most. Better meal time glucose control lowers your overall level of blood sugar

• InsuVital works on complementary to medication. It is not intended to replace medication, but is a nutritional intervention to lower glucose peaks.


33

• InsuVital is a casein hydrolysate that lowers postprandial glucose peaks by improving the impaired insulin secretion in type 2 diabetes

• An art-13 dossier to substantiate health claims has been submitted

promotes healthy blood sugar levelslowers postprandial blood sugar to healthy levelshelps to regulate blood sugar peaks after a meal

• Safe; extensive history of safe use

• DSM has own IP to claim blood glucose regulation by protein hydrolysates

• InsuVital can be applied in a wide variety of products within the functional food or medical nutrition range

• Current observed/expected customer and consumer interest is high


34Where we are today . . .

• DSM’s large gene bank and high throughput screening are potent capabilities for the creation of new food grade enzymes.

• New enzymes lead to innovation in food processing, functionality, and safety• New enzymes can lead to new bio-active ingredients for nutrition, metabolic

health, and for treatment of specific chronic conditions. • It has been shown that bioactive peptides can survive digestion and be

absorbed into the blood stream – dosages can be measured and controlled. • Taste, purification, and bioavailability issues can be overcome with novel

enzymes, refining, chromatography and formulation. • Bioactive peptides from milk demonstrate clinically significant effects – a

starting point that may lead to targeted nutrition and medical foods.


35Peering into the future . . .

• Conserved sequences in mammalian milk suggest a range of new functional possibilities for nutrition & health – satiety, immune function, etc.

• Genomic characterization of mammalian and human milk is starting to identify new bio-actives embedded in casein and whey protein fractions.

• New analytical tools are discovering bio-active components in other milk fractions, eg, phospholipids, oligosaccharides, lipoproteins, sugars, etc.

• Enzymes will be essential tools to releasing this potential for commercial applications.

• DSM supports Open Innovation by partnering with dairy processors, DMI, Dairy Centers, and universities.


36What we need from the research world . . .

• Ongoing genomic characterization and annotation of key mammalian and human milk genes, especially those coding for novel enzymes and proteins.

• Better phylogenetic understanding of high value enzymes and proteins – how and why have key components changed during mammalian evolution.

• Genomic and functional characterization of conserved milk components, eg, conserved peptides embedded in milk protein.

• Isolation and characterization of potential bioactive components• Effects of potential bioactives on gene expression (in vitro)• Linkage of bioactives to specific nutrition, development, or health effects.• Effect of potential bioactives on the microbiome. • Bio-availability and gastro-intestinal uptake of potential bioactives. • Clinical evalution and validation of selected bio-actives.

• DSM supports the development and commercialization of new functional ingredients for food processing, nutrition, and health.

• The best place to find these ingredients is in nature’s most evolved food.


37Ingredients for food, nutrition, and health

The End

The Start

dsm nutrition cluster · 2013-09-29 · dsm nutrition cluster. tensguard – a source of...

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