Biotechnology & Biotechnological Equipment

ISSN: 1310-2818 (Print) 1314-3530 (Online) Journal homepage: http://www.tandfonline.com/loi/tbeq20

Antibacterial Peptides from Goat and Sheep Milk

Proteins

J. Atanasova & I. Ivanova

To cite this article: J. Atanasova & I. Ivanova (2010) Antibacterial Peptides from Goat and Sheep
Milk Proteins, Biotechnology & Biotechnological Equipment, 24:2, 1799-1803, DOI: 10.2478/
V10133-010-0049-8

To link to this article: http://dx.doi.org/10.2478/V10133-010-0049-8

2010 Taylor and Francis Group, LLC

Published online: 15 Apr 2014.

Submit your article to this journal

Article views: 1145

View related articles

Citing articles: 23 View citing articles

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=tbeq20

Download by: [150.165.227.40] Date: 31 July 2017, At: 08:56

 Review DOI: 10.2478/v10133-010-0049-8 FB

ANTIBACTERIAL PEPTIDES FROM GOAT AND SHEEP MILK PROTEINS

J. Atanasova1, I. Ivanova2
1
LB Bulgaricum PLC., R&D Center, Sofia, Bulgaria
2
Sofia University, Biological Faculty, Department of General and Applied Microbiology, Sofia, Bulgaria
Correspondence to: Jivka Atanasova
E-mail: atanasova.j@lbbulgaricum.bg

ABSTRACT
Milk is an excellent source of well balanced nutrients and also exhibits a range of biological activities that influence digestion,
metabolic responses to absorbed nutrients, growth and development of specific organs, and resistance to disease. Goat milk is as
close to perfect food as possible in nature.
Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions or conditions and
may ultimately influence health. It can be generated during milk fermentation by the proteolytic activity of starter cultures. The
beneficial health effects may be classified as antimicrobial, antioxidative, antithrombotic, antihypertensive and immunomodulatory.
Sheep and goat milk proteins are also important sources of bioactive ACE inhibitory peptides and antihypertensive peptides.
They can provide a non- immune disease defence and control of microbial infections. The activity of these biofunctional peptides
is based on their inherent amino acid composition and sequence. The size of active sequences may vary from two to twenty amino
acid residues. The total antibacterial effect in milk is greater than the sum of the individual contributions of immunoglobulin
and nonimmunoglobulin defence proteins such as LF, LP, lysozyme, and other peptides. A variety of naturally formed bioactive
peptides have been found in fermented dairy products, such as yoghurt, sour milk and cheese. BP have the potential to be used in
the formulation of health-enhancing nutraceuticals, and as potent drugs with well defined pharmacological effects.
Biotechnol. & Biotechnol. Eq. 2010, 24(2), 1799-1803 and k- CN. The main forms of caprine and ovine CMP are the
Keywords: milk proteins, bioactive peptides, antibacterial soluble C-terminal derivatives from the action of chimosin on
peptides, goat milk proteins k-casein during the milk clotting process of cheesemaking.
These main forms are identified and determined to be a good
Abbreviations used: lactoperoxidase: LP; lactoferin: LF;
source of antithrombotic peptides.
Bioactive peptides: BP; angiotensin I converting enzyme:
Whey protein is composed of - lactoglobulin, -
ACE; calcium-binding phosphopeptides: CBPs; lactoferricin:
lactalbumin, immunoglobulins, glycomacropeptides, bovine
LFcin; minimal inhibitory concentrations: MICs; caseins: CN;
serum albumin and minor proteins such as LP, lysozyme and
caseino-macropeptides: CMP; calmodulin: calcium binding
LF.
protein; Non-protein nitrogen: NPN;
Sheep and goat milk proteins are also important sources
Goat milk as close to perfect food as possible in of bioactive ACE inhibitory and antihypertensive peptides.
They can provide a non-immune disease defence and control
nature of microbial infections. Important minor milk proteins include
Milk is an excellent source of well balanced nutrients and immunoglobulins, lactoferrin, transferring, ferritin, proteose
also exhibits a range of biological activities that influence peptone, calmodulin (calcium binding protein), prolactin, and
digestion, metabolic responses to absorbed nutrients, growth folate-binding protein. NPN contents of goat and human milks
and development of specific organs, and resistance to disease. are higher than in cow milk. Taurine in goat and sheep milk
Goat milk is as close to perfect food as possible in nature. Its derived from sulphur-containing amino acids has important
chemical structure is amazingly similar to mothers milk. It is metabolic functions as does carnitine, which is a valuable
a complete protein that contains all the essential amino acids nutrient for the human neonate. Mineral and vitamin contents
without the heavy fat content and catarrh (mucus) producing of goat and sheep milk are mostly higher than these in cow
materials of cow milk. milk (29).
Source of bioactive peptides
Milk protein is comprised of about 80% caseins and 20% Production of bioactive peptides
whey proteins. These biological activities are mainly due to the BP have been defined as specific protein fragments that have
peptides and proteins in milk. However, some of the biological a positive impact on body functions or conditions and may
activity of milk protein components is latent, and is released ultimately influence health. (12). BP are encrypted in milk
only upon proteolytic action. proteins and are only released by enzymatic hydrolysis in
Caseins are divided into - , - and k- caseins (13). Principal vivo during gastrointestinal digestion, food processing or by
CN in goat, sheep and cow milk are s1- CN, s2- CN, - CN microbial enzymes in fermented products. BP are formed from
Biotechnol. & Biotechnol. Eq. 24/2010/2 1799
the precursor inactive protein (18). Due to their physiological
and physico-chemical versatility, milk peptides are regarded as
highly prominent components for health promoting foods or
pharmaceutical applications. At present significant research is
undertaken on the health effects of BP. A variety of naturally
formed bioactive peptides have been found in fermented dairy
products, such as yoghurt, sour milk and cheese.
Physiological effects of bioactive peptides
Effects of these peptides on the cardiovascular, nervous and
immune systems are described, altogether with bioactive
peptides that possess antimicrobial activity, effects on the
nutritional status and dental health and other functional
roles. The beneficial health effects may be classified as
antimicrobial, antioxidative, antithrombotic, antihypertensive
and immunomodulatory (27, 34).
Major antimicrobial proteins in milk
The antimicrobial properties of milk have been widely
acknowledged for many years. The antimicrobial activity of
milk is mainly attributed to immunoglobulins, and to non-
immune proteins, such as lactoferrin, lactoperoxidase and
lysozyme. In some species minor proteins, including a folate
binding protein, are also regarded.
Lysozyme is a relatively small basic protein and is classified
as a 1,4-- N-acetylmuramidase (6).
The function of Lf in vivo is not well understood.
However, two main roles for Lf in vivo have been postulated:
its antibacterial effect and role in the iron metabolism. The
antibacterial effect of Lf in vitro is well established. The
antibacterial mechanism has been assigned to the ability of
capacity to chelate iron and thereby depriving bacteria of the
iron that is essential for their growth. This is supported by
the fact that Lf saturated with iron does not demonstrate an
antibacterial effect (3).
LP is basic glycoprotein that has a molecular weight of 78
000 and contains one heme group. Buffalos milk, ewes milk
and goats milk generally also contain high levels of LP (4).
Goat milk can have significant levels of folate binding
protein and it may therefore be advisable to supplement infant
formulas with folic acid in addition to the normal humanizing
compositional adjustments when caprine milk is used in
human neonate. One of the most potent antimicrobial peptides
described so far corresponds to a fragment of the whey protein
lactoferrin, named lactoferricin (LFcin). More recently, other
whey proteins such as -lactalbumin and -lactoglobulin have
also been considered as potential precursors of bactericidal
fragments. Similarly, antibacterial fragments have also been
derived from s1-, s2- and k- casein.
Occurrence of bioactive peptides in dairy
products
It is now well documented that bioactive peptides can
be generated during milk fermentation by the proteolytic
1800
activity of starter cultures. As a result, peptides with various
bioactivities can be found in the end- products, such as various
cheeses and fermented milks. These traditional dairy products
may under certain conditions have specific health effects when
ingested as part of the daily diet.
The activity of these biofunctional peptides is based on
their inherent amino acid composition and sequence. The size
of active sequences may vary from two to twenty amino acid
residues, and many peptides are known to have multifunctional
properties (27) e.g., peptides from the sequence 60-70 of
-casein show immunostimulatory, opioid and ACE-inhibitory
activities. This sequence has been defined as a strategic zone
(26, 28). The sequence is protected from proteolysis because
of its high hydrophobicity and the presence of proline residues.
Other examples of the multifunctionality of milk-derived
peptides include the s1-casein fraction 194-199 showing
immunomodulatory and ACE-inhibitory activity, the opioid
peptides - and - lactorphin also exhibiting ACE inhibitory
activity and the CPPs, which possess immunomodulatory
properties (34).
Antimicrobial peptides from goat and sheep
milk proteins
Bioactive proteins and peptides derived from milk have been
reported to provide a non-immune disease defence and control
of microbial infections. It is generally accepted, that the total
antibacterial effect in milk is greater than the sum of the individual
contributions of immunoglobulin and nonimmunoglobulin
defence proteins such as LF, LP, lysozyme, and peptides. This
may be due to the synergistic activity of naturally occurring
proteins and peptides in addition to peptides generated from
inactive protein precursors (9). It has been proved, that milk
proteins can also act as antimicrobial peptide precursors, and
in this way might enhance the organisms natural defences
against invading pathogens. Consequently food proteins can
be considered as components of nutritional immunity (30).
Antimicrobial peptides derived from whey
proteins
Peptides derived from LF are antibacterial peptides from
proteins. Tomita et al. first reported the enzymatic release
of antibacterial peptides with more potent activity than the
precursor LF in 1991 (40). Shortly afterwards the antibacterial
domains of bovine LF f(17-41) and human LF f(1-47), that are
called bovine and human LFcin, were purified and identified
(2). These peptides showed a potent antimicrobial activity
against a wide range of Gram-positive and Gram-negative
bacteria (44).
Concerning goat and sheep species, the chemical synthesis
of fragment f(17-41) of caprine LF, consisted the first
investigation, in which this fragment displayed antibacterial
activity, although to a lesser extent than the bovine counterpart
(42). Hydrolysis of caprine and ovine LF by pepsin resulted
in antibacterial hydrolysates, and a homologous peptide to
Biotechnol. & Biotechnol. Eq. 24/2010/2
LFcin, corresponding to fragment f(14-42) was identified
in the caprine LF hydrolysate. Caprine LFcin showed lower
antibacterial activity than bovine LFcin against Escherichia
coli but comparable activity against Micrococcus flavus
(Table 1).
Antibacterial peptides have been found to be active against
a broad range of pathogenic organisms e.g. Escherichia,
Helicobacter, Listeria, Salmonella and Staphylococcus, yeasts
and filamentous fungi. Depending on the target microorganism,
inhibitory concentrations of peptides vary, e.g.: antimicrobial
peptides s2-CN f183-207 and f164-179 exhibited inhibition
against Gram-positive and -negative bacteria with MICs
ranging from 8 to 95 mol/l.
Antimicrobial peptides derived from caseins
In the same way as whey proteins, caseins are also a source of
antimicrobial peptides (21, 22). In a preliminary study, an ovine
-casein hydrolysate with pepsine, trypsin and chymotrypsin
has showed inhibition of bioluminescent production by
Escherichia coli JM103, but the peptides responsible for this
activity have not been identified (10, 11).
Four antibacterial peptides were identified from a pepsin
hydrolysate of ovine s2-casein (21). The peptides corresponded
to s2-casein fragments f(165-170), f(165-181), f(184-208)
and f(203-208), being the fragments f(165-181) and f(184-
208) homologous to those previously identified in the bovine
protein (36, 37) (Table 1).
These peptides showed a strong activity against Gram-
negative bacteria. Of them, the fragment f(165-181) was
the most active against all tested bacteria. The peptide
corresponding to ovine s2-casein f(203-208) is a good
example for a multifunctional peptide, because it exhibited not
only antimicrobial activity, but also potent antihypertensive
and antioxidant activity (35). These results can be extended
to caprine proteins, because the amino acid sequence of these
peptides is the same (Table 1).
Future perspectives for bioactive peptides
Milk provides the newborn (neonate) with nutrients and an
array of antimicrobial factors. These are believed to help
protect the neonates from infection until their own immune
system has developed.
Developments in nano-encapsulation (39) and nano-
emulsion technology, as well as research into delivery systems,
may ultimately lead to the generation of specific formulations
containing bioactive peptides with anti-bacterial activity.
Antibacterial peptides from food protein deserve attention
due to their mechanism of activity, which makes microbial
resistance improbable (8), and for increasing the functional
values of foods.

TABLE 1
Some examples of bioactive peptides from goat and sheep milk with antibacterial activity

Protein Peptide fragment Biopeptide Reference

-Lactalbumin -La 1-5
/17-31/S-S/109-114/ - lactorfin Pellegrini A. et al. (1999) (32)
/61-68/S-S/75-80/ - lactorfin Pellegrini A. (2003) (30)
Complex -La and Lz Clare D.A. and Swaisgood H.E. (2000) (5)
Partially unfolded protein complexed Meisel H. (1998) (26)
with oleic acid
Lactoferrin Ovine and caprine LF f/17-41/ lactofericin Pihlanto-Leppala A. (2001) (33)
Ovine and caprine LF f/17-42/ lactofericin Vorland L.H. et.al. (1998) (42)
Recio I. and Visser S. (2000) (38)
s1-casein f/1-23/ isracidin Hill R.D. et al. (1974) (15)
f/99-109/ McCann K.B. et al (2006) (25)
s2-casein ovine s2-CNf/165-170/ multifunctional Lopez-Exposito I. et al. (2006) (21)
ovine s2-CNf/165-181/ peptides Lopez-Exposito I. et al. (2006) (22)
ovine s2-CNf/165-170/
ovine and caprine s2-CNf/203-208/
k-casein f/106-169/ Liepke C. Et al. (2001) (20)
f/18-24/ Malkoski M. et al. ( 2001) (23)
f/30-32/ McCann K.B. et al (2006) (25)
and other peptides Lopez-Exposito I. et al. (2006) (22)
-Lactoglobulin -Lgf/15-20/ Pellegrini et al. (2001) (31)
-Lg f/25-40/
f/78-83/
f/92-100/

Biotechnol. & Biotechnol. Eq. 24/2010/2 1801

 BP have the potencial to be used in the formulation of
health-enhancing nutraceuticals, and as potent drugs with well
defined pharmacological effects (12).
REFERENCES
1. Bargeman G., Dohmen-Speelmans M., Recio I., Timmer
M., van der Horst C. (2000) Lait, 80, 175-185.
2. Bellamy W., Takase M., Wakabayashi H., Kawase K.,
Tomita M. (1992) Journal of Applied Bacteriology, 73,
472-479.
3. Bullen J.J. and Armstrong J.A. (1979) Immunology, 36,
781-791.
4. Carlstrom A. (1969) Acta Chem. Scand., 23, 171-184.
5. Clare D.A. and Swaisgood H.E. (2000) Journal of Dairy
Science, 83, 1187-1195.
6. Dubois M.-A., Zoll A., Bouillant M.-L., Chopin J. (1982)
Phytochemistry, 21, 1141-1143.
7. Fiat AM, Migliore-Samour D, Jolles P, Drouet L, Bal
dit Sollier C, Caen J. (1993) Journal of Dairy Science, 76,
301-310.
8. Floris R., Rcio I., Berkhout B., Visser S. (2003) Curr.
Pharm. Des., 9, 1257-1273.
9. Gobbetti M., Minervini F., Rizzello C.G. (2004)
International Journal of Dairy Technology, 57, 172-188.
10. Gomez-Ruiz J.A., Recio I., Pihlanto A. (2005)
Milchwissenschaft, 60, 41-44.
11. Gomez-Ruiz J.A., Taborda G., Amigo L., Recio I.,
Ramos M. (2006) Eur. Food Res. Technol. 223, 561-595.
12. Haque E. and Chand R. (2001) home page address: http://
www.dairyscience.info/bio-peptides.htm
13. Haque E. and Chand R. (2006) home page address: http://
www.dairyscience.info/bio-peptides.htm
14. Hakansson A., Svensson M., Mossberg A.K., Sabharwal
H., Linse S., Lazou I., Lonnerdal B., Svanborg C. (2000)
Molecular Microbiology, 35, 589-600.
15. Hill R.D., Lahov E., Givol D. (1974) Journal of Dairy
Research, 41, 147-153.
16. Hoek K., Milne J.M., Grieve P.A., Dionysius D.A.,
Smith R. (1997) Antimicrobial Agents and Chemotherapy,
41, 54-59.
17. Ibrahim H., Taniyama N., Aoki T. (2004) Letters in Drug
Design & Discovery, 1, 101-109.
18. Korhonen H. and Pihlanto A (2003) Australian Journal of
Dairy Technology, 58, 129-134.
19. Lahov E. and Regelson W. (1996) Federal Chemistry
Toxicology, 34, 131-145.
1802
20. Liepke C., Zucht H.D., Forssman W.G., Standker L.
(2001) Chromatography B, 752, 369-377.
21. Lopez-Exposito I., Gomez-Ruiz J.A., Amigo L., Recio I.
(2006) International Dairy Journal, 16, 1072-1080.
22. Lopez-Exposito, I., Minervini, F., Amigo, L & Recio, I.
(2006) Journal of Food Protection ( in press)
23. Malkoski M., Dashper S.G., O Brien-Simpson N.M.,
Talbo G.H., Macris M., Cross K.J., Reynolds E.C.
(2001) Antimicrobial Agents and Chemotherapy, 45, 2309-
2315.
24. McCann K.B., Shiell B.J., Michalski W.P., Lee A., Wan
J., Roginski H., Coventry M.J. (2005) International Dairy
Journal, 15, 133-143.
25. McCann K.B., Shiell B.J., Michalski W.P., Lee A., Wan
J., Roginski H., Coventry M.J. (2006) International Dairy
Journal, 16, 316-323.
26. Meisel H. (1998) International Dairy Journal, 8, 363-373.
27. Meisel H. and FitzGerald R.J. (2003) Current
Pharmaceutical Design, 9, 1289-1295.
28. Miqliore-Samour D., Floch F., Jolles P. (1989) Journal of
Dairy Research, 56, 357-362.
29. Park Y.W., Juarez M., Ramos M., Haenlein G.F.W.
(2006) available online at www.sciencedirect.com
30. Pellegrini A. (2003) Current Pharmaceutical Design, 9,
1225-1238.
31. Pellegrini A., Dettling C., Thomas U., Hunziker P.
(2001) Biochimica et Biophysica Acta, 1526, 131-140.
32. Pellegrini A., Thomas U., Bramaz N., Hunziker P., von
Fellenberg R. (1999) Biochimica et Biophysica Acta,
1426, 439-448.
33. Pihlanto-Leppala A. (2001) Trends in Food Science &
Technology, 11, 347-356.
34. Philanto A. and Korhonen H. (2003) In: Advances in
Food and Nutrition Research (S.L. Taylori ed.) Elsevier
Inc., San Diego, CA, 175-276.
35. Recio I., Quiros A., Hernandes-Ledesma B., Gomez-Ruiz
J.A., Miguel M., Amigo L., Lopez-Exposito I., Ramos
M., Alexandre A. (2005) Bioactive peptides identified in
enzyme hydrolysates from milk caseins and procedure for
their obtention, European Patent 2005011373.
36. Recio I. and Visser S. (1999a) Journal of Chromatography
A, 831, 191-201.
37. Recio I. and Visser S. (1999b) Biochimica et Biophysica
acta, 1428, 314-326.
38. Recio I. and Visser S. (2000) International Dairy Journal,
10, 597-605.
39. Tadros T., Izquierdo P., Esquena J., Solans C. (2004)
Adv. Colloid Interface Sci., 108-109, 303-318
Biotechnol. & Biotechnol. Eq. 24/2010/2
40. Tomita M., Bellamy W., Takase M., Yamanchi K.,
Wakabayashi H., Kawase K. (1991) Journal of Dairy
Science, 74(12), 4137-4144.
41. Tomita M., Shimamura S., Kawase K., Fukuwatari Y.,
Takase M., Bellamy W.R., Hagiwara T., Matukuma H.
(1992) A process for large-scale production of antimicrobial
peptide in high purity, European patent 92305622.0.
42. Vorland L.H., Ulvatne H., Andersen J., Haukland
H., Rekdal O., Svendsen J.S., Gutteberg T.J. (1998)
Scandinavian Journal of Infectious Diseases, 30, 513-517.
Biotechnol. & Biotechnol. Eq. 24/2010/2
43. Visser S. and Recio I. (1998) Process for producing
peptides from biological fluids and peptides obtainable by
said process, European patent 98203107.2.
44. Wakabayashi H., Takase M., Tomita M. (2003) Current
Pharmaceutical Design, 9, 1277-1287.
45. Zucht H.D., Raida M., Adermann K., Magert H.J.,
Forssmann W.G. (1995) FEBS Letters, 372, 185-188.
1803