P3. Milk proteins Flashcards
7 common sources of proteins?
- milk
- meat
- eggs
- fish
- cereals
- soybean
- pulses (pea, lentil, chickpea)
each protein system is _________ and its properties also depend on what? (2)
- unique
- processes used to extract or isolate it
fluid milk contains __% solids, of which ___% is protein
12% solids
- 3.5% proteins
milk proteins have high _________ value (what does it mean?), surpassed only by ____ _________ and are a good source of ________ (an _________ aa)
- nutritive value (good aa balance)
- by egg albumin
- lysine (essential aa)
2 groups of milk proteins?
- casein
- whey proteins
_________ is a general term for milk proteins and have an isoelectric point at pH _____ –> isolated by adjusting pH of milk to _____ using _______
- proteins left in solution are termed ______ proteins
- casein
- 4.5
- 4.5 using acid
- whey
% of total milk protein:
- caseins
- whey proteins
- casein: 83%
- whey proteins: 17%
what are the main types of casein in order of highest to lowest percentage of caseins?
- a(s1) = 44%
- b = 25%
- k (kappa) = 14%
- a(s2) = 11%
- y (gamma) = 5%
main types of “whey proteins” in order of highest to lowest percentage?
- beta-lactoglobulin = 58%
- a - lactalbumin = 13%
- immunoglobulin = 12%
- minor proteins = 12%
- serum albumin
which whey protein are 7% of population allergic to?
beta-lactoglobulin
in 1877, O Hammarsten distriguished (how many) proteins in milk: name them
- 3!
- casein + lactalbumin +lactoglobulin
O Hammarsten outlined the following procedure for the isolation of casein: (2 steps ish)
- skim milk is diluted, then acidified with acetic acid
- casein flocculates, while whey proteins stay in solution
specific property of casein: it is __________ in ______ _______ media
insoluble in weakly acidic media
casein was subsequently established to be a ___________ with _______ being attached to the aa ________
- phosphoserine
- phosphate being attached to serine
in 1939, casein was shown by _________ to consist of 3 fractions: ?
- electrophoresis
- alpha, beta and kappa
how are different types of casein differentiated? (3 ish)
- isoelectric point
- molecular weight (different aa sequence)
- phosphorus content
North American cows have which variants?
A and B (vs C and D are more Australian)
y-casein are the products of proteolysis of __________
beta-casein
in milk, most of casein proteins are present in _________ particles called ________ __________
- colloidal particles
- casein micelles
casein micelles are responsible for the ________ ______________ of milk, an optical effect due to ______ ___________
- opaque whiteness of milk
- due to light scattering
micelles are very complex structures: ____________ assemblies made up of the different _______ _________
- supramolecular assemblies
- different casein fractions
casein micelles __________ if Ca2+ is dialyzed from solution –> will reform if Ca2+ is ??
- dissociate
- if Ca2+ is added back
what is a submicelle? vs a micelle?
- submicelle: 1 b + 1 a(s1) + 1 a(s2) + 1 k –> 10 nm
- micelles = lots of submicelles together –> 20-300 nm
alpha (s1) - casein:
- how many aa?
- which aa distributed all along peptide chain –> hindering formation of ?
- contain ___ phosphoserine residues
- how many phosphoserine residues localized btw positions ____ and _____ –> this portion also contains 12 ? = very _________
- aa 100-199 are _______ = responsible for ?
- 186-199 aa
- proline –> hinder formation of ordered structures
- 8 phosphoserine residues
- 7 localized between 43 and 80 –> also contains 12 carboxyl groups (glutamic and aspartic residues) = extremely polar
- apolar = responsible for strong self-association tendencies of a(s1)-casein/hydrophobic interactions (despite repulsive forces btw phosphate groups and lack of cysteine residues)
where can Ca2+ aggregate in a(s1) casein?
in the polar portion of the peptide chain (btw 43-80)
in the presence of Ca2+ ions at levels found in milk, a(s1) casein forms ?
insoluble Ca-salt
a(s2) casein
- how many aa?
- ordered or not?
- ___ phosphoserine and ___ cysteine residues
- precipitates with Ca2+ more/less easily than a(s1) casein
- 207
- more ordered structure than a(s1), more alpha helices
- 11 phosphoserine + 2 cysteine
- more easily!
b-casein:
- most ___________ casein
- has (2) thus resembling _____-like molecules
- ___ phosphoserine residues are localized btw positions __ and ____ of the peptide chain
- precipitates in presence of Ca2+ are what levels?
- hydrophobic
- polar head + apolar tail –> soap-like molecule
- 5 phosphoserine btw 1 and 40
- at levels found in milk
y-casein:
- _________ products of __-casein formed by milk _______
- obtained by cleavage of residues __-___
- if you have y-casein = not good ?
- degradation products of b-casein
- residues 1-28
- pasteurization: proteases not deactivated enough
kappa-casein:
- how many aa residues
- ____ prolines + ___ cysteines + ___ phosphoserine
- normally occurs as a ______ or higher _________
- self-association involves formation of ______ ______ btw the _______ residues
- what molecules (3 examples) are bound to peptide chain through ________ residues (at position _____, _____ and ____ in variant __)
- 169 aa
- 20 prolintes, 2 cysteine, 1 phosphoserine
- trimer or higher oligomer
- disulfite bonds btw cysteine residues
- carbohydrates (galactose, galactosamine, N-acetylneuramic acid) –> through threonine residues (131, 133, 135 of variant A)
numb of carb molecules bound to k-casein peptide chain is ________
- ie for N-acetylneuramic acid, galactose, galactosamine
- variable:
- N-acetylneuramic acid: 0-3 moles
- galactose: 0-4 moles
- galactosamine: 0-3 moles
kappa-casein is the only constituent of casein that remains ______ in the presence of ___ ions at concentrations found in milk
soluble
- Ca2+ ions
3D model for k-casein –> described as ? model (3 parts)
- this model suggests that kappa-casein contains approx __% a-helix and __% b-structure
- horse and rider
- N-terminal = horse
- C-terminal = rider
- 2 legs = beta sheets
- 16% a-helix + 27% b-structure
which sections of k-casein are very hydrophobic? –> role?
- leg sections
- postulated to be area of interaction with other caseins
only up to __% of the total casein present in milk is in the form of _________, usually designated as _______ caseins
- 10%
- monomers
- serum caseins
the majority of casein proteins __________ to form casein complexes and micelles. the extent of __________ is dependant on ____ concentration and ___
- aggregate
- aggregation
- Ca2+ concentration and on pH
which constituent of casein remains soluble in present of Ca2+ ions at concentrations found in milk?
k-casein
aggregation of (2 types of casein) with __-casein prevents/accelerates their coagulation in present of Ca2+ ions
- a(s1) and b-caseins with k-casein
- prevents (the formation of really really big micelles that could hurt the cow when milking)
what is the utmost important property for formation of stable casein complexes and casein micelles as occurs in milk?
the property of k-casein to prevent coagulation in presence of calcium ions
3 forms of casein structure (NOT a, b, k and y)
________ –> ________ –> ________
5 properties that will increase formation on 1 side vs the other
- monomers (soluble caseins) –> casein complex –> micella
- to form casein complex and micella:
1. lower pH (increase H+)
2. increase Ca2+
3. remove citrate
4. remove phosphate
5. increase temperature
(inverse of these 5 steps to dissociate casein complex)
dialysis of casein complexes against a _______ agent (ex?) would shift equilibrium towards __________ vs against a high Ca2+ concentration, shift would be to _______ ________
- chelating agent (EDTA)
- monomers
- high ca2+ concentration –> large micelles
diameter of micelles ranges from __ to ____ nm, with a mean of _____, corresponding to volume of ____ x 10^__ nm^3
- 10-300 nm
- 150 nm
- 1.6 x 10^6 nm^3
average of how many monomers per micelle?
400 - 40 000 monomers per micelle
micelles are highly _______ and hence are _____
- highly solvated
- are porous
casein micelles are kept together/casein stability:
(3 interactions)
- hydrophobic interactions: minimal at temperature below 5°C
- electrostatic interactions, mostly as calcium and calcium bridges between phosphoserine and glutamic acid residues
- hydrogen bond
what disrupts the electrostatic interactions between calcium & PO4 and calcium & COO-? how?
additions of acetic acid! (decrease in pH)
- neutralizes negative charges –> adding acid will protonate the side chains
what disrupts the electrostatic interactions between calcium & PO4 and calcium & COO-? how?
additions of acetic acid! (decrease in pH)
- neutralizes negative charges –> adding acid will protonate the side chains
what are the 2 types of submicelles that exist?
- one contains k-casein
- the other does not
characteristics of submicelles that contains k-casein (2)
- aggregation of submicelles proceeds until what?
- k-casein coverage is around __ nm thick = provide _________
1) k- casein are arranges on surface of submicelles
2) hydrophilic c-terminal domains protrude like hairs from surface, preventing further aggregation
- until entire surface of forming micelle is covered with k-casein
- 5nm –> provide stability
are k-caseins also found inside the micelle?
yes
cleavage of k-casein causes casein proteins to _______ = ______ formation
- this is the underlying principle of use of ________ in cheese making
coagulate
- curd formation
- rennet
rennet is a complex of _______ produced in _____ stomach of cow that contains what?
- enzymes
- fourth stomach
- contains precursor of enzyme chymosin (rennin)
what is chymosin?
chymosin = acid protease having specificity for k-casein
(cleaves k-casein from micelle and casein complex)
chymosin specifically recognizes sequence in k-casein chain from _____-___ to ___-_____ and selectively cleaves the peptide bond between _____-____ and ____-____
- His-98 to Lys-111
- cleaves Phe-105 and Met-106
k-casein cleaved into which 2 fragments?
- para-k-casein
- glycomacropeptide
2 fragments of k-casein: which is soluble and which one precipitates?
- glycomacropeptide is soluble
- para-k-casein precipitates in presence of Ca2+ ions
2 ways to precipitate casein:
1. how? produces ________ = first step in ?
2. how? produces __________ –> for use as ?
- chymosin reaction –> produces paracasein –> first step in cheese making
- addition of acetic acid. produces acid casein or isoelectric casein
- for use as functional ingredient
one similar characteristics for acid/isoelectric casein and paracasein?
- similar physically
- not temp sensitive
- can boil them bc very little 2° structure
which types of whey proteins are mainly used in food science? (2) what are the other 3 types?
- b-lactoglobulin
- a-lactalbumin
NOT used: - bovine serum albumin
- immunoglobulin
- minor proteins
percentage of total milk protein, isoelectric point and molecular weight of b-lactoglobulin and a-lactalbumin
b-lactoglobulin:
- 10% of total milk protein
- 5.34 isoelectric point
- 18.3 kDa
a-lactalbumin:
- 2% of total milk protein
- 4.8 isoelectric point
- 14.1 kDa
which milk protein is also sold as a nutraceutical/medical claims?
alpha-lactalbumin
beta-lactoglobulin:
- ____% of whey protein fraction
- how many aa? molecular weight?
- what differs between genetic variant A and B?
- ___ disulfite bond + ___ free sulfhydryl group + ____ phosphorus
- 58%
- 162 aa –> 18.3 kDa
- glycine in variant B replaced by aspartic acid in variant A
- 2 disulfite bond + 1 free sulfhydryl group + 0 phosphorus
describe the 2 disulfite bonds of b-lactoglobulin
one is fixed between Cys-66 and Cys 160
- the second one is dynamic: either Cys-106 with Cys-119 (+ free sufhydryl Cys 121) OR Cys-106 with Cys 121 (+ free sufhydryl Cys 119)
the 2° structure of beta-lactoglobulin is homologous to that of __________-binding proteins
retinol
beta-lactoglobulin:
- ___ strands of beta-structure –> ___ of them arranged to form a ________ and a single ________ that resembles a _____ on the ___________
- the center of the _________ is hydrophobic/hydrophilic and can be involved in binding of what?
9 strands of beta structure
- 8 of them form a beta-barrel
- single alpha helix resembles a lid on the beta-barrel
- center of the barrel is hydrophobic + involved in binding of hydrophobic molecules (pharma tried to hide drugs in the barrel)
although b-lactoglobulin is manufactures specifically in _________ ______ for inclusion in _______, its biological role is ___________
- mammary gland for inclusion in milk
- unknown
b-lactoglobulin’s __________ pocket is quite effective in binding __________ and it has been speculated that the binding of ________ may have a regulatory role in __________ gland
- hydrophobic
- retinol
- binding of vit. A
- mammary
because of its prevalence in _______ milk, to a large extent, the properties of whey protein ________ and whey protein ________ employed in the food industry are in effect, the properties of ____________
- bovine
- concentrates
- isolates
- beta-lactoglobulin
alpha-lactalbumin:
- ___% of total milk protein vs ____% of total whey protein
- ____ aa + MW of ?
- ___ disulfite linkages and _____ phosphate groups
- 2% of total milk VS 13% of total whey protein
- 123 aa + 14.16 kDa
- 4 disulfite links and no phosphate groups
alpha-lactalbumin variant A vs variant B?
variant A: glutamine (Gln)
variant B: arginine (Arg)
disulfide bonds of alpha-lactalbumin are between which cys residues?
- 6 and 120
- 28 and 111
- 61 and 77
- 73 and 91
(not important though)
role of alpha-lactalbumin?
- what happens if absence of a-lactalbumin?
modify activity of enzyme galactosyl transferase (a-lactalbumin is a cofactor ish)
- galactosyl transferase adds UDP-galactose to N-acetylglucosamine groups that are attached to proteins
enzyme galactosyl transferase can transfer _______ to _______, but the Km for glucose is _____ mM and thus the reaction proceeds slowly/fast
- alpha-lactalbumin serves to increase/lower the Km for _________ to __ mM and the enzyme complex will not add ________ to ________ to produce ________ and UDP
- UDP-galactose to glucose
- 1400 mM
- slowly, if at all
- lower the Km for glucose to 5 mM
- add UDP-galactose to glucose to produce lactose and UDP
milk of all mammals that contains lactose also contains ___________
alpha-lactalbumin
40% of residues btw a-lactalbumin and _________ are the same, including all _______ residues
- another ___% have similar ___________
- lysozyme
- cysteine
- 20% have similar structures
similar structure btw a-lactalbumin and lysozyme coupled with the fact that a-lactalbumin helps what? suggests that the molecules are closely related
that a-lactalbumin helps to synthesize same linkage that lysozyme cleaves
- glycosyl linkage
do a-lactalbumin and lysozyme work on same substrates and are related antigenetically?
no and no
a-lactalbumin is unusual in that the molecule is more/less stable to heat in presence of ________ whereas proteins show increased/decreased heat sensitivity in the presence of _________
- more stable in presence of calcium
vs
increased heat sensitivity in presence of calcium (ie b-lactoglobulin will precipitate with calcium and heat)
increased sensitivity to heat of most proteins is probably due to ability of calcium to promote what? with most proteins. these __________ hold the molecules in proximity and increase/decrease the likelihood of aggregation upon heating
VS a-lactalbumin uses calcium to form what?
- promote formation of ionic intermolecular cross links with most proteins
- these crosslinks
- increase
- to form intramolecular ionic bonds that tend to make molecule resistant to thermal unfolding
under favorable conditions of ______ and ____, a-lactalbumin can remain soluble after exposure to temperatures up to ______°C
- calcium and pH
- 100°C
a-lactalbumin has a lot of _________ for 2° structure
helical structures
bovine serum albumin (BSA) isolated from milk is identical to BSA isolated from _______ _______
blood serum
how does BSA enter the milk?
- BSA not synthesized in mammary gland but rather enters milk through bloodstream (diffusion)
BSA:
- MW?
- ___ phosphorus + ___ disulfides + __ free sylhydryl group
- 69 kDA
- no phosphorus + 17 disulfides + 1 free sylhydryl group
function of albumin in blood plasma?
- molecule has specific binding sites for ______ molecules and may bin them in _____ as well
- carrier of free fatty acids
- hydrophobic
- milk
milk contains a great number of ________. the extent of heat activation/inactivation of enzymes indicates type and extent of heating to which milk has been subjected
- ex.:
- enzymes
- inactivation
- lipase
casein and caseinates are widely used in food processing for (2)
- protein enrichment and/or
- achieve stabilization of certain physical properties of processed meats, baked products, candies, cereal products, ice creams, whipping creams and coffee whiteners (and cheese)
what are the non-food uses of casein and caseinates? (4)
- manufacture of paper as sizing/coating to improve adhesion of printer’s ink to surface of paper
- glue manufacturing as a type of waterproof glue
- textile industry to make fabrics water-repellent
- production of certain plastics
for the recovery of casein from milk, coagulation is achieved by _________ or __________ __________
- acidification
- enzymatic treatment
how is acid coagulation achieved (to recover casein from milk)?
- results in what type of casein?
- achieved by lactic acid fermentation
- OR by direct addition of acids such as HCl, H2SO4, lactic acid or H3PO4 to milk at 35-50°C to attain pH of 4.2-4.6, at which casein precipitates out as coarse grains
- acid casein
what is the process of enzymatic treatment of milk? (2 steps)
- results in what type of casein?
- addition of protease enzymes such as chymosin and pepsin to the milk at 45°C
- in cheese production, the enzyme causes coagulation of casein
- to terminate the process, milk is heated to 65°C to inactivate enzyme (pepsin)
- rennet casein
what happens after coagulation of casein?
- casein is separated form milk in a sedimentation centrifuge, washed and dried (whirlwind drier)
to produce a casein product that is soluble or readily dispersible: casein is dispersed in _____ and treated with ______ (5 ex.) at ___-____°C and pH ___-___.
- the solubilized product (2) is then ________.
- water (20-25% w/w)
- treated with alkali (NaOH, Ca(OH)2, alkali or alkaline-earth carbonates or citrates
- at 89-90°C
- pH 6.2-6.7
- sodium caseinate or calcium caseinate
- spray dried
why accumulates in considerable amounts in production of ________. until recently, whey was considered a __________ by-product of __________ production, in part bc whey is too high in ______ to be consumed directly in large amounts and was dumped as _____________
- cheese
- valueless by product of cheese
- too high in salt
- wastewater
whey byproducts of cheese-making:
although _______ methods were available, costs were too high/low in relation to value of product. however, in recent years tighter __________ regulations have required expensive waste treatment in most jurisdictions
- desalting
- too high
- environmental
what allowed recovering proteins from whey?
- take advantage of (2) of whey protein
- what are spray-dried whey and whey produces used in? (7)
- improved technological processes
- high nutritional quality + excellent functional properties of whey protein
- infant formula, baked goods, confectionery, candies, beverages, animal feed and pet food
whey’s solids composition is especially high in ________ –> making them super _______
- what has higher protein and lower lactose?
- what is mostly protein and no lactose?
- lactose –> super cheap
- whey protein concentrates (1-2x ultrafiltration fo lower lactose)
- whey protein isolates
3 steps for drying whey
- processing costs may be reduced by preliminary concentration of why to __-___% dry matter by what?
- concentration of whey to 50-55% dry matter in falling-film evaporator (thermal or mechanical vapour compression)
- spray drying (1 or 2 step)
- subsequent drying on a fluidized bed drier
- 21-25% dry matter by reverse osmosis using a perm-selective membrane that holds back the protein, lactose and salts
what is reverse osmosis? (other word for it?)
- goal?
- water is forced through membrane by applying pressure exceeding osmotic pressure (which would normally water back across the membrane)
- hyperfiltration
- to force lactose out! but keep protein
how are whey protein concentrates produced?
2 methods
- downfall of the 2nd method?
more porous membrane employed to hold back proteins but allows lactose and salts to pass through with water
VS
less gentle method: heating whey (95°C for 3-4min) by direct steam injection followed by precipitation of denatured proteins at pH 4.5 + separation in a sedimentation centrifuge and drying
BUT highly detrimental to functional properties of whey proteins
how is lactose recovered from whey protein concentrates?
- filtrate obtained in ultrafiltration step can be concentrated by reverse osmosis using a membrane that selectively holds back lactose while allowing salts/smaller molecules to pass through
once whey protein concentrates and lactose are isolated, what is done to them?
- whey protein concentrates: seeded and cooled slowly to induce sugar crystallization
- lactose may be recrystallized to yield a raffinade (pharmaceutical-grade lactose), which is used as a filler in tablets
Lactose (isolated from whey protein) is used in manufacturing of (5)
- dietary food products
- baked products dehydrated food
- cocoa products
- beverages
- ice creams
whey protein isolates require extensive processing (3) to obtain highly purified protein
- ultrafiltration
- microdialysis
- and/or ion exchange
whey protein isolates are relatively __________ to produce + are used primarily in high value-added production of _____ ________ ___________
- expansive
- whey protein hydrolysates
how are whey protein hydrolysates produces?
- enzymatic treatment of whey protein isolates to break proteins down into smaller peptides that are more easily digested while nutritional quality of protein remains very high
- cut down b-lactoglobulin into smaller fragments
why are whey protein hydrolysates often used as protein source in infant formulas?
bc believed to be less likely to trigger allergic reactions than non-hydrolyzed whey protein
- bc beta lactoglobulin is cut down
do dairy proteins have a lot of properties as ingredients in food processing?
yes!
emulsification, stabilization, aeration, film formation, opacity, water/fat binding, texturization, thickening, heat stability, gelation, acid solubility, flavor development, browning
typical formulation of imitation cheese? (6 ingredients)
- water (51.1%)
- Ca/Na caseinate (26%)
- vegetable oil (18%) to give texture
- glucono-delta-lactone (emulsifier and flavor) (2.8%)
- salt (2%)
- color and aroma substances
