alpha lactalbumin - development path for a valuable dairy protein ingredient

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

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

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

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.

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-l

ac

oth

er

pro

tein

s

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

milk ingredients

MILK cheese 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

centrifugation enzyme 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

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.

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.

drivers for protein ingredient development and functionality



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:

PHYSICO-CHEMICAL PROPERTIES


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




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


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)


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


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

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


Physical stability of infant milk formula made with selectively

hydrolysed whey proteins

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





Mechanism


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)

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




Mechanism


Lactoferrin and α-lactalbumin for improving energy balance

and glucose tolerance


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


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


• 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


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.

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


α-Lactalbumin in the regulation of appetite and food intake


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.

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


LAW /ECONOMICS





LAW /ECONOMICS




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