Provides a multifunctional view of the drivers behind the development and application of alpha lactalbumin as a valuable dairy protein ingredient in nutritional foods

1. LACTALBUMIN
BIOACTIVES
LACTOFERRIN
PHOSPHOLIPIDS
MFGM
CASEINS
MILK
WHEY
LACTOSE
CALCIUM
NUTRITION
PHOSPHORUS
DEVELOPMENT PATH
FOR A
VALUABLE
DAIRY PROTEIN
INGREDIENT
ALPHA-LACTALBUMIN
Juan M. Gonzalez, PhD, MBA

2. Milk is a complete nutritional food designed
by nature to support the growth of infant
mammals.
Milk is synthesized and secreted by
mammary glands upon hormonal
stimulation (Prolactin).
Ref:
• Nutrient Data Lab. USDA
NUTRIENT % (w/w)
Water 88.3
Protein 3.2
Total lipid (fat) 3.3
Ash 0.7
Carbohydrate, by
difference (Lactose)
4.5
Water
Carbohydrates
Protein
Total lipid (fat)
Ash
milk: composition

3. A model of premium quality food protein:
• All essential amino acids are present
in appreciable amounts.
CASEINS constitute about 80% of total
bovine milk proteins
WHEY PROTEINS constitute about 20%
of total bovine milk proteins.
Milk protein is product of:
• Synthesis in the mammary gland (Caseins)
• Filtration from the blood (serum albumin,
immunoglobulins)
CASEINS: The portion of the milk proteins
that coagulate into a curd after adding
rennet or after acidifying milk to pH 4.6.
WHEY PROTEINS: The portion of the milk
proteins that remain in solution in the liquid
portion.
Three major type of caseins that self
assemble into porous spheres known as
micelles
Casein
micelles
a s1
a s2
b k
a-lactalbumin
b-lactoglobulin
serum albumin
immunoglobulin
Whey
proteins
milk proteins

4. Ref:
• Nutritional Changes in Milk Comp. Virginia Tech Cooperative Ext. 1998.
• Science of Providing Milk to Man. Campbell & Marshall, 1975
• Kunz C, Lönnerdal B. Re-evaluation of the whey protein/casein ratio of human milk. Acta Paediatr 1992;81:107–12
Infant mammal growth rate and milk composition
HUMAN COW DOG
WEIGHT x2, days 180 47 8
PROTEIN , % 1.6 3.3 7.1
FAT , % 3.7 4.0 8.3
LACTOSE , % 7.0 5.0 3.7
milk proteins
HOLSTEIN JERSEY
PROTEIN, % 3.1 3.7
FAT , % 3.7 5.1
LACTOSE , % 4.9 5.0
OTHERS , % 0.7 0.8
Milk composition changes within species
Ratio of milk proteins changes with lactation period.

5. Ref: Kunz C, Lönnerdal B. Re-evaluation of the whey protein/casein ratio of human milk. Acta Paediatr 1992;81:107–12.
milk proteins
casein
a-lac
other
proteins
b-lacto
globulin
Human Milk Bovine Milk
LF
casein
a-lactalbumin
Lactoferrin (LF)
other
proteins
a- lactalbumin
molecular weight, kDa 14
isolectric point 4.2- 4.5
disulfide bonds 4 (no SH)
presence in (% total prot):
human milk 28
bovine milk < 4

6. milk ingredients
MILKcheese cream
skim
milk
filtration
milk fat
acid
precipitation
acid whey
sweet whey
deproteinized
whey *
whey prot
conc, WPC &
buttermilk
butter serum
MFGM
whole milk
powder
skim milk
powder
oligosaccharides
galactooligosaccharide GOS
Lactulose
lactitol
tagatose
filtration
whey protein isolate, WPI
glycomacropeptide, GMP
protein
hydrolyzates
enzymes
milk fat
fractions
caseinates
milk protein
isolate
MPI
lactose
crystals
crystallization
filtration, resins
filtration
whey protein
concentrates, WPC
deproteinized
whey
filtration,
resins
chemical /
enzymatic
modification
centrifugationenzyme churn
dry
... same &... same *
Ref: Milk protein ingredients for infant and clinical nutritional products: Challenges and opportunities. Gonzalez, J.M.. In: High
Quality Dairy Ingredients Spray drying, Functionality and Application. 2014. South Dakota State University - NIZO Symposium.
dehydrate
fractionation

7. Physical-
chemical
Nutritional
Law -
Economics
Enabler
INDUSTRY
DRIVERS
Sensory Health
 Food Ingredient development could be traced across inter-related drivers
 These drivers are a conjunction of Societal – as its regulations and market
forces - and Science & Technology advances
 Ingredient functionality is identified in the physical-chemical, sensorial,
nutritional, and health impact drivers
drivers for food ingredient
development and functionality
Ref: The role of Food Science in Ingredient Functionality and Food Systems. JM Gonzalez. In: Consumer
Behavior and Food Science Innovations for Optimal Nutrition. 2014. Sackler Int Nutr Sci. NY Acad Sci.

8. PROTEIN INGREDIENT
DEVELOPMENT &
FUNCTIONALITY
SENSORY
PROPERTIES
• Flavor
• Texture
• Aroma
• Color
NUTRITIONAL
PROPERTIES
• Digestibility
• Compositional
Profile
• Absorption
Mechanism
HEALTH
• Obesity
• CV disease
• Immunological
• Allergy
• Life-stage
LAW /ECONOMICS
• Market: Need-Want,
Price, Supply-Demand
• Regulation: Law, Safety
• Sustainability: energy,
environment, pollution
PHYSICAL-CHEMICAL
PROPERTIES
• Structure
• Surface
• Hydration
• Reactivity
ENABLER
• Processes- make ingredient
• Products - ingredient make
• Attributes – define
ingredient/ product
(Convenience, Portable,
Ready-to-eat, Shelf-life, Life
Style)
drivers for food ingredient development and functionality
Ref: The role of Food Science in Ingredient Functionality and Food Systems. JM Gonzalez. In: Consumer
Behavior and Food Science Innovations for Optimal Nutrition. 2014. Sackler Int Nutr Sci. NY Acad Sci.

9. drivers for protein ingredient development and functionality
PHYSICAL-CHEMICAL
PROPERTIES
• Structure
• Surface
• Hydration
• Reactivity
PHYSICAL-CHEMICAL property of proteins is the result of the interaction
between intrinsic and extrinsic factors:
• Intrinsic - pertain to the protein itself:
Structure, Size, Shape, Charge, Hydrophobicity, Flexibility
• Extrinsic - pertain to the environment where the protein interacts:
pH, Temperature, Pressure, Ionic Strength, Components (Water, Fat,
Salts), Energy used in the system.
Hydration - relate to how a
water-bound protein unit
interacts with itself and other
components present in the
bulk.
Example of those:
Cohesion
Elasticity
Water binding
Thermal denaturation
Structural - relate to how the
protein molecule is folded,
cross-linked, linked to other
subunits, or to other non-
protein molecules.
Example of those:
Viscosity
Gelation
Film formation
Antimicrobial
Metal chelation
Surface - relate to how the bulk
solvent – water – interacts at
the interface with the protein
molecule.
Example of those:
Solubility
Wettability
Dispersibility
Emulsification
Foaming
Fat and Flavor binding
Functional properties may be:

10. PHYSICO-CHEMICAL
PROPERTIES
• Structure
• Surface
• Hydration
• Reactivity
Ref: Denaturation and aggregation of three a-Lactalbumin preparations at neutral pH. McGuffey, M.K., Epting, K.L., Kelly,
R.M., Foegeding, E.A. J. Agric. Food Chem. 2005, 53, 3182-3190
• Heating (95C) pure a-lactalbumin produced distinct monomers and dimer
aggregates at pH 7.0
• a-lac monomers are formed via intra-molecular disulfide interchange, while
dimers are formed via inter-molecular disulfide linkages.
• Hydrophobic interactions may be responsible for these interactions.
• Presence of other whey protein impurities requires ionic screening (via
higher phosphate or calcium presence) to produce similar aggregates.
• a-lac aggregates dissociated with cooling below 95 °C.
U H U H U H
Denaturation and aggregation of a-lactalbumin at neutral pH
drivers for protein ingredient development and functionality

11. PHYSICAL-CHEMICAL
PROPERTIES
• Structure
• Surface
• Hydration
• Reactivity
a-Lactalbumin structure:
• a large helical domain connected by a loop to a small beta-sheet domain.
• four disulfide bridges and one hydrophobic pocket.
• a calcium ion is bound to a high affinity binding site in the loop connecting
two domains
• Oleic and palmitic acids bind to the a-lac-apo but not to the a-lac-holo form.
Apo a-lac has one binding site for vitamin D3
The binding is mostly hydrophobic, with one hydrogen bond.
The binding of vitamin D3 :
• induces a-lac conformational changes (random coil increase).
• Increases a-lac surface hydrophobicity.
• Decreases a-lac heat stability.
• Induces formation of a-lac aggregates (125 nm) that
encapsulate the vitamin D3
• a-lac complexes (aggregates) are stable in presence of high
vitamin D3 concentration.
Ref: Alpha-lactalbumin: A new carrier for vitamin D3 food enrichment.
Delavari, B., Saboury, A.A., Atri, M.S., Ghasemi, A., Bigdeli, B., Khammari, A., Maghami,
P., Moosavi-Movahedi, A., Haertle, T., Goliaei, B. Food Hydrocolloids 45 (2015) 124-131
Alpha-lactalbumin: A new carrier for vitamin D3 food enrichment
drivers for protein ingredient development and functionality

12. ENABLER relates to:
• A technology – that allows ingredients to be developed
• An ingredient – that allows products to be developed
Enabling technologies allow for the development of enabling ingredients or
products with needed or wanted attributes.
Seek for Enabling Innovations !!!!
Technologies:
• Membrane Filtration
• Chromatography
• Extrusion
• Encapsulation
• Hydrolysis
Ingredients:
• Protein fractions
 WPC
 WPI
• Isolated proteins:
 a-lactalbumin
 Lactoferrin
• Texturized proteins
• Encapsulated oils,
probiotics
• Bioactive peptides
Products:
• Infant formula
• Sport beverages
• Geriatric foods
• Premature foods.
• Medical foods
• Snacks
• Beverages
ENABLER
• Processes- make ingredient
• Products - ingredient make
• Attributes – define
ingredient/ product
(Convenience, Portable,
Ready-to-eat, Shelf-life, Life
Style)
drivers for protein ingredient development and functionality

13. Ref: Evaluation of commercially available, wide-pore ultrafiltration membranes for production of α-lactalbumin–enriched whey
protein concentrate. Marella, C., Muthukumarappan, K., Metzger, L.E., J. Dairy Sci. 94(2011):1165-1175
Evaluation of ultrafiltration membranes for production of
α-lactalbumin–enriched whey protein concentrate
drivers for protein ingredient development and functionality
Step 1: PVDF (0.2 mm), pH 6.3,
TMP 76 kPa.
Step 2: PVDF50. pH 6.3, TMP
207 kPa.
Step 3: PES 5 kDa membrane.
PVDF: polyvinylidene
fluoride membrane
PES: polyethersulfone
membrane
TMP: trans-membrane
pressure
Process produces a WPC80 enriched
a-lactalbumin with:
• 63% purity
• 1.4 ratio a-lactalbumin/b-
lactoglobulin
• 21% yield a-lactalbumin

14. intact
selective
hydrolysis
partial
hydrolysis
 Selective hydrolysis increases a-lactalbumin content in model infant formula
 Increases ratio a-lactalbumin/b-lactoglobulin
 The increase in a-lactalbumin:
• Reduced the viscosity of wet-mixes
• Created stable emulsions
• Reduced fouling during processing
prior
after
heating & homogenization
Ref: Physical stability of infant milk formula made with selectively hydrolysed whey proteins.
Murphy, E.G., Roos, Y.H., Hogan, S.A., Maher,P.G., Flynn, C.G., Fenelon, M.A. Intl Dairy J. 40(2015):39-46
drivers for protein ingredient development and functionality
Physical stability of infant milk formula made with selectively
hydrolysed whey proteins

15. drivers for protein ingredient development and functionality

16. SENSORY
PROPERTIES
• Flavor
• Texture
• Aroma
• Color
SENSORY PROPERTIES define the sensory experience of the
consumer and influence the interaction with the product.
It relates to:
• Liking
• Repeat purchase
• Convenience
Sensory properties may become enablers as they may:
• Satisfy consumer expectations
• Create new experiences
• Supports life-style
• Supports compliance
• Supports quality of life
drivers for protein ingredient development and functionality

17. NUTRITIONAL
PROPERTIES
• Digestibility
• Compositional
Profile
• Absorption
Mechanism
drivers for protein ingredient development and functionality
NUTRITIONAL properties of proteins relate to the digestibility and
the absorption mechanisms that allow the protein to be utilized
by the body for growth, maintenance or medical purposes.
Nutritional properties also relate to intrinsic and extrinsic factors:
• Intrinsic – relate to the protein itself, such as:
• amino acid composition (protein quality)
• Amino acid sequence (peptides)
• Conformation (domains)
• Extrinsic – relate to the external environment where the protein
interacts:
 Processing
• Heat treatment (denaturation)
• Hydrolysis (digestibility, bioactivity)
 Interaction with other food components:
• Complexation
• Glycosylation
 Hydration (enzyme accessibility)

18. Study effect of whey with a-lactalbumin and lactoferrin on:
- Weight - Body composition
- Food intake - Energy expenditure
- Glucose tolerance - Meal-induced hormone response
a-lactalbumin and Lactoferrin seem to be more
beneficial than just whey itself in improving :
- energy balance
- glucose tolerance
At 65 days:
a-Lactalbumin is more effective than whey in:
• increasing energy expenditure
Lactoferrin is more effective than whey in:
• inducing hypophagia
• promoting fat loss
Ref: Lactoferrin and lactalbumin are more effective than whey protein in improving energy balance and glucose tolerance in diet-
induced obese rats. Zapata, R., Pezeshki, A., Singh, A., Chou, M., Chelikani, P. ADSA 2015. #784. Protein & Enzyme Symp
Diet-induced obese rats:
• 65 day study.
• Feed - isocaloric and
isonitrogenous
 30% calories - protein.
 40% calories – fat.
Diets:
• Whey
• a-lactalbumin
• Lactoferrin
NUTRITIONAL
PROPERTIES
• Digestibility
• Compositional
Profile
• Absorption
Mechanism
drivers for protein ingredient development and functionality
Lactoferrin and α-lactalbumin for improving energy balance
and glucose tolerance

19. drivers for protein ingredient development and functionality
HEALTH properties of proteins relate to the benefits that the organism
derives from consuming the protein food.
Protein ingredients and foods may be developed to manage particular
life stages, life styles, or diseases:
• Muscle building (sports nutrition)
• Muscle maintenance and recovery (elderly or convalescence)
• Allergy management (protein allergy)
• Digestion deficiencies (PKU)
• Weight control
Peptides have been shown effective:
 ANTI-OXIDANTS
 ANTI-HYPERTENSIVE
 ANTI-MICROBIAL
 IMMUNO-MODULATORY
 FAST DIGESTION RATE
 ANTI-TUMOR
 HYPOCHOLESTEROLEMIC
 ANTI-THROMBOTIC
 ENHANCES MUSCLE RECOVERY
 ENHANCE MINERAL ABSORPTION
HEALTH
• Obesity
• CV disease
• Immunological
• Allergy
• Life-stage

20. Ref: Evening intake of -lactalbumin increases plasma tryptophan availability and improves morning alertness and brain measures of
attention. Markus, R., Jonkman, L.M., Lammers, J., Deutz, N., Messer, M., Rigtering, N.; Am J Clin Nutr 2005; 81:1026 –33
HEALTH
• Obesity
• CV disease
• Immunological
• Allergy
• Life-stage
• Brain serotonin function is thought to promote sleep regulation and cognitive processes
• Brain uptake of tryptophan, a serotonin precursor, is dependent on nutrients that influence
the availability of tryptophan in blood plasma.
Evening intake of a-lactalbumin increases plasmaTrp
availability, improves alertness and brain measures of attention
drivers for protein ingredient development and functionality
 Study tested evening consumption of a-lactalbumin to increase plasma Trp level, to
improve alertness and performance on the morning after on patients with sleep
complaints.
 Evening a-lactalbumin intake caused a 130% increase in plasma Trp levels before
bedtime; the morning after it reduced sleepiness, improved brain-sustained attention
processes and also improved behavioral performance.

21. Ref: α-Lactalbumin in the regulation of appetite and food intake. Nieuwenhuizen, A.G., Hochstenbach-Waelen, A., Westerterp-
Plantenga, M. Chp. 7. In: “Weight Control and Slimming Ingredients in Food Technology” Susan S. Cho. 2010 Blackwell Pub
HEALTH
• Obesity
• CV disease
• Immunological
• Allergy
• Life-stage
α-Lactalbumin in the regulation of appetite and food intake
drivers for protein ingredient development and functionality
Trp
• Serotonin plays a role in the regulation of human appetite and food intake
• Serotonin is synthesized from its precursor, the amino acid L-tryptophan
• Studies have shown that supplementation with L-tryptophan reduces food intake.
 Patients consuming a breakfast with either α-lactalbumin or casein as the sole
protein source, voluntary energy intake of a subsequent lunch was lower after
the α-lactalbumin-containing breakfast.
 Trp-rich α-lactalbumin may be useful in controlling appetite or food intake;
however, its mechanism of action remains to be elucidated.

22. Regulatory Aspects:
• Demonstrate safety and efficacy
• Health claims: Generic vs. Innovative
• Scientific validation – animal and human intervention studies
Quality:
• Strict manufacturing standards
• Strict ingredient specifications
• Strict control microbial and chemical contaminants
• Consistent and validated nutrient levels
Economics:
• Supply and demand balance
• Specialty ingredients: Capital investment for commercial isolation
• Specialty premium ingredients demand premium prices
• Possible low recovery yields – unfavorable pricing proposition
• Sourcing high quality dairy feed
• Investments to meet high quality standards
• Investments to support health claim
drivers for protein ingredient development and functionality
LAW /ECONOMICS
• Market: Need-Want,
Price, Supply-Demand
• Regulation: Law, Safety
• Sustainability: energy,
environment, pollution

23. drivers for protein ingredient development and functionality
LAW /ECONOMICS
• Market: Need-Want,
Price, Supply-Demand
• Regulation: Law, Safety
• Sustainability: energy,
environment, pollution

24. at the
ALPHA SUMMIT
August 4-5, 2015
Jerome, Idaho
Organized by
Davisco®
Agropur
Presented by
Juan M Gonzalez, PhD, MBA
a-lactalbumin:
Development path for a valuable dairy protein ingredient