The document discusses bioactive peptides derived from milk proteins, their properties, significance, and applications across various physiological functions, including antihypertensive, antioxidative, antithrombotic, and immunomodulatory effects. It highlights the mechanisms through which these peptides are released and their implications for health, as well as the impact of different factors on the bitterness of cheese associated with peptide breakdown. The presence of growth factors in milk and their role in cell growth and differentiation is also outlined, noting differences between human and bovine milk.

1. MILK PROTEIN DERIVED BIOACTIVE PEPTITEDS -THEIR
PROPERTIES; SIGNIFICANCE AND APPLICATION;
BITTER PEPTIDES IN CHEESE;
GROWTH FACTORS IN MILK
KARTHIKA R
PHD DAIRY CHEMISTRY

2. MILK PROTEIN DERIVED BIOACTIVE PEPTITEDS
Bioactive peptides are present in an inactive state in their native protein molecules and are liberated by
(1) Gut microbial enzymes
(2) gastrointestinal enzymes
(3) milk-derived proteases
(4) microorganisms secreting enzymes (used as starter cultures)
which are then added during the processing of milk
(Park & Nam, 2015).

3. Bioactivity of milk components have been categorized as four major areas:
(1) gastrointestinal development, activity, and function;
(2) immunological development and function;
(3) infant development; and
(4) microbial activity, including antibiotic and probiotic action
Gobbetti et al., 2007

4. Park, 2009b

5.  BPs have been defined as specific protein fragments that have a positive influence
on physiological and metabolic functions or condition of the body.
 Most of the bioactivities of milk proteins are being absent or incomplete in the
original native state.
 BPs are inactive within the sequence of the parent protein, and they can be released in
three ways:
(i) through hydrolysis by digestive enzymes,
(ii) through hydrolysis of proteins by proteolytic microorganisms, and
(iii) through the action of proteolytic enzymes derived from plants

7. Antihypertensive
peptides
The angiotensin is a peptide hormone that causes vasoconstriction and an increase in
blood pressure.
It is part of the renin-angitensin system, which regulates blood pressure.
The ACE causes elevation of blood pressure by converting angiotensin-I to the
angiotensin-II.
The ACE-inhibitors derived from milk proteins are attributed to different fragments of
casein, named casokinins, or whey proteins, named lactokinins.

8. Milk protein-derived antihypertensive peptides which act as
angiotensin I-converting enzyme inhibitors (CEI)

9. Antioxidative peptides
 Peptides derived from αs-casein have free radical-scavenging activity and inhibit
enzymatic and nonenzymatic lipid peroxidation.
 Proteolytic enzymes can release antioxidative peptides from caseins, soybean and
gelatine by enzymatic hydrolysis.
 Low temperature-processed whey protein contained high levels of specific dipeptides
(glutamylcysteine).These dipeptides can promote the synthesis of glutathione, which is
an important antioxidant for cellular protection and repair processes.
 Antioxidant activities of hydrolysates were evaluated using DPPH radical scavenging
activity.

10. Antithrombotic
peptides
 These peptides reduce or inhibit the formation of blood clots.
 Caseinomacropeptide (CMP) is a peptide split from k-casein when the milk
protein is coagulated by rennin enzyme.
 This CMP has functions of inhibiting the aggregation of blood platelets and
binding of the human fibrinogen.
 There are two reported antithrombotic peptides, derived from human and bovine
k-caseinoglycopeptides, which were identified in the plasma of 5-d-old newborns
after breast-feeding and feeding cow milk based formula.

11. Hypocholesterolemic
peptides
A novel peptide (Ile-Ile-Ala-Glu-Lys) from tryptichydrolysate of β-lactoglobulin
showed hypocholesterolemic effect.
Antiappetizing peptides
These peptides have functions of suppressing the appetite, whereby they prevent gaining weight and
obesity. Total whey protein in the diet has been associated with a lowering of LDL cholesterol, and also
related to the increased release of an appetite-suppressing hormone, cholecystokinin
Immunomodulatory
peptides
Immunomodulatory peptides generated from milk include αs1-CN f194-199 (αs1-
immunocasokinin) and β-CN f193-202, f63-68, f191-193 (immunopeptides), which are
synthesized by hydrolysis with pepsin-chymosin.

12. Opioid peptides
 An opioid is any chemical such as morphine that resembles opiates in its pharmacological
effects.
 Opioid peptides are opioid receptor ligands with agonistic or antagonistic activities.
 The αs1-casein-exorphin (αs1-CN f90-96), β-casomorphins-7 and -5 (β-CN f60-66 and f60-64,
respectively), and lactorphins (α-lactalbumin f50-53 and β-lactoglobulin f102-105) act as
opioid agonists.
 whereas casoxins (i.e., k-CN f35-42, f58-61, and f25-34) act as opioid antagonists.
 The hydrolysis of Lactobacillus fermented UHT milk by the pepsin/trypsin has shown to
release several opioid peptides from αs1- and β-CN, and α-lactalbumin.

13. Antimicrobial peptides
Among antimicrobial peptides, the lactoferricins are studied the most,
which are derived from bovine and human lactoferrin.
Lactoferricins have been shown to have antimicrobial activity against
various Gram-positive and -negative bacteria, yeasts and filamentous
fungi.
Lactoferricin is an amphipathic, cationic peptide with anti-microbial
and anti-cancer properties.
Lactoferricin can be generated by the pepsin-mediated digestion of
lactoferrin.

14. Lactenin may have been the first antibacterial factor found in milk, which
has been released from rennet hydrolysis of milk.
Casecidins are a group of basic, glycosylated and high molecular weight (about
5 kDa) polypeptides, which possess bactericidal properties against lactobacilli
and also against various pathogenic bacteria such as Staphylococcus aureus.
Isracidin is another antibacterial peptide derived from αs1-CN, which is
hydrolyzed with chymosin.

15. peptides
 Mineral-binding phosphopeptides or caseinophosphopeptides (CCPs) have the
function of carriers for different minerals by forming soluble organophosphate
salts, especially Ca++ ion.
 About 1 mol of CPP can bind 40 mol of Ca2+
.
 The αs1-, αs2- and β-CN of cow milk contain phosphorylated regions which can be
released by digestive enzymes.
 Specific CPPs can form soluble organophosphate salt and increase Ca absorption
by limiting Ca precipitation in the ileum.
 The negatively charged side chains, particularly the phosphate groups, of these
amino acids of CPPs become the specific binding sites for minerals.
 Chemical phosphorylation of αs1- and β-CN increased the binding capacity and
the stability of these proteins in the presence of Ca2+

18. Schanbacher et al. (1998), Meisel (1998), and Clare and Swaisgood (2000), Park (2009b)

21. MARCONE et al., 2017.

22. APPLICATION:

24. GROWTH FACTORS IN MILK
Many growth factors in milk have been identified, such as
1. Epidermal growth factors (EGF),
2. Colony - stimulating factors
3. Insulin-like growth factor (IGF),
4. Transforming like growth factors (TGF),
5. Mammary derived growth factors (MDGF),
6. Platelet-derived growth factors (PDGF),
7. basic fibroblast growth factors (bFGF)
8. Lactulose from lactose,
9. Nucleotides,
10. Somatotropin

25. The term 'growth factor' is applied to a group of potent hormone-like polypeptides
which play a critical role in the regulation and differentiation of a variety of cells
acting through cell membrane receptors.
Quantitatively, the relative concentrations of growth factors in milk are
IGF-I > TGF-β2 > EGF ≈ IGF-II > bFGF.
The concentrations of growth factors are highest in colostrum and gradually
decrease through lactation, except for BTC (betacellulin).

26.  EGF and BTC stimulate proliferation of epidermal, epithelial and embryonic cells, inhibit the secretion of
gastric acid and promote wound healing and bone reabsorption while TGF-βs are important in
embryogenesis, tissue repair and control of immunity.
 IGF-I and II are important for cell proliferation ( reproduction of new parts or cells).
 The role of EGF, IGFs-I and II, insulin and TGF-β in stimulating GI tissue growth and repair in the
suckling neonate.
 PDGF growth factors are involved in embryonic development and proliferation of many cell types.
 FGF is important for the proliferation and differentiation of epithelial, endothelial and fibroblast cell,
promote collagen synthesis and are involved in angiogenesis and wound healing.

27. Bovine mammary secretions contain many compounds that stimulate the growth of cells in cultures :
1. IGFs, part of the insulin family which includes insulin, IGF-I, IGF-II and relaxin. IGFs act as
mediators of growth, development and differentiation. They are heat and acid-stable and hence have
potential bioactivity in the GIT of consumers.
2. Insulin occurs in bovine milk and colostrum at much higher concentrations than in blood and
higher in turn in pre partum secretions than in those post partum.
3. Transforming growth factors (TGF α and β), are present in bovine milk. TGF-β is important for
cell proliferation and differentiation.

28.  Human milk contains physiologically active levels of growth factor, whereas bovine milk
has much lower levels of growth factor activity.
 Colostrum of most mammals usually contains high concentrations of growth factors and
others bioactive compounds, while the high levels of these growth factor compounds drop
rapidly during the first 3 d postpartum.
 Goat milk is shown to be a great source of physiologically active growth factors.
 Caprine milk has yet to be studied in this premise.

29. BITTER PEPTIDES IN CHEESE;
 Bitterness is a common flavor attribute of aged cheese associated with the peptide
fraction, but excessive levels are a defect leading to consumer rejection.
 Known bitter peptides in cheese are derived from the breakdown of αs1-, αs2-, β- and
κ-casein due to enzymatic hydrolysis from rennet.
 Some studies have indicated that αs1-casein is the primary source of bitter peptides
in cheese, whereas others have indicated that β-casein is the primary source of bitter
peptides
 Compared with αs1-casein and β-casein, αs2-casein, κ-casein and whey proteins are
rarely referenced in the literature as sources of bitter peptides
 The cause of a peptide’s bitterness has been linked to physical properties, including
hydrophobicity, molecular weight, peptide length and amino acid (AA)
composition.

30. Factors that affect bitterness in
cheese
Bitterness in cheese has been linked to many factors in the cheese making process,
• Initial milk quality,
• pH,
• Fat,
• Salt (NaCl) and
• Cheese mineral content (MgCl2 and CaCl2);
• Quantity and type of bacterial cultures and rennet applied,
• The degree of sanitation used in processing and packaging conditions

31.  Reduced pH in milk is often linked to poor milk quality and has been associated with
the formation of bitter flavor in cheese.
 Cheeses with lower fat have a higher tendency of being bitter.
 Low salt concentrations in cheese are associated with bitterness.
 A more recent analysis indicated that the presence of MgCl2 and CaCl2 are critical for
the perception of bitterness for peptides and amino acids in aged gouda cheese

32. 1. Excessive action of rennet or rennet substitutes (either via excess addition to milk or
prolonged exposure to rennet) can lead to overproduction of bitter peptides.
2. Hydrolysis of casein by rennet releases large peptides that may not initially be bitter but
can become precursors to bitter peptides through additional proteolytic cleavage.
3. Proteolytic starter bacteria, such as Lactococcus lactis subsp. lactis and Lactococcus
lactis subsp. cremoris, have high proteolytic activity - can cause the formation of bitter
peptides.
4. Nonstarter bacteria can also produce proteases that can hydrolyze milk proteins and
produce bitter peptides.
5. Endogenous milk proteinases, such as plasmin, can act on proteins during cheese making
and contribute to bitterness. Plasmin is resistant to high temperatures and survives
pasteurization, which enables it to remain active during cheese making.

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