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DSM Nutrition Cluster
0
DSM Enzyme Innovation
New tools for constructing dairy bioactives
William King
October 20, 2010
DSM Nutrition Cluster
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
DSM Nutrition Cluster
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
DSM Nutrition Cluster
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
DSM Nutrition Cluster
4The DSM Nutrition Cluster – major product areas
DSM NutritionDSM Nutritional Products
DSM Food Specialties
• 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)
DSM Nutrition Cluster
5The DSM Nutrition Cluster – technology platforms
DSM NutritionDSM Nutritional Products
DSM Food Specialties
• 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
DSM Nutrition Cluster
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
DSM Nutrition Cluster
7
DSM Nutrition Cluster
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
DSM Nutrition Cluster
9DEC consists of 2 parts…
DSM Enzyme Collection (DEC)
Enzyme dataEnzyme samples
DSM Nutrition Cluster
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
DSM Nutrition Cluster
11Radical Innovation in the Nutrition Cluster
Diabetes
Protease toolboxMaxiPro, TPAP,
DPAP, …
Joint Health
I-OmegaTensGuardPep2Balance
PeptoPro
InsuVital
InfantPro
I-FlexFoodSafetyCeliac
disease
Cheeseripening
DSM Nutrition Cluster
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
DSM Nutrition Cluster
Novel peptidases for cheese & dairy products
Confidential
DSM Nutrition Cluster
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)
DSM Nutrition Cluster
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
DSM Nutrition Cluster
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
DSM Nutrition Cluster
Enzymatic bleaching of dairy products
Confidential
DSM Nutrition Cluster
18Application: Control of products color
Mozzarella, Feta and Ricotta cheeses made from cow milk
Whipped creamWhey
Confidential
DSM Nutrition Cluster
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
DSM Nutrition Cluster
Novel texturizing tools for dairy protein applications
Enzymatic cross linking of dairy proteins
DSM Nutrition Cluster
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)
DSM Nutrition Cluster
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
DSM Nutrition Cluster
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
DSM Nutrition Cluster
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
DSM Nutrition Cluster
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
DSM Nutrition Cluster
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
DSM Nutrition Cluster
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
DSM Nutrition Cluster
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
DSM Nutrition Cluster
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.
DSM Nutrition Cluster
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
DSM Nutrition Cluster
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
DSM Nutrition Cluster
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.
DSM Nutrition Cluster
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
DSM Nutrition Cluster
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.
DSM Nutrition Cluster
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.
DSM Nutrition Cluster
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.
DSM Nutrition Cluster
37Ingredients for food, nutrition, and health
The End
The Start