Current Pharmaceutical Design, 2007, 13, 829-843
1381-6128/07 $50.00+.00 © 2007 B4entham Science Publishers Ltd.
Technological Options for the Production of Health-Promoting Proteins
and Peptides Derived from Milk and Colostrum
H. Korhonen* and A. Pihlanto
MTT Agrifood Research Finland, Biotechnology and Food Research, FIN-31600 Jokioinen, Finland
Abstract: Milk proteins are known to exert a wide range of nutritional, functional and biological activities. Apart from
being a balanced source of valuable amino acids, milk proteins contribute to the consistency and sensory properties of
various dairy products. Furthermore, many milk proteins possess specific biological properties which make them potential
ingredients of health-promoting foods. These properties are attributed to both native protein molecules and to physiologi-
cally active peptides encrypted in the protein molecules. Considerable progress has been made over the last twenty years
in technologies aimed at separation, fractionation and isolation in a purified form of many interesting proteins occurring in
bovine colostrum and milk. Industrial-scale methods have been developed for native whey proteins such as immunoglobu-
lins, lactoferrin, lactoperoxidase, ?-lactalbumin and ?-lactoglobulin. Their large-scale manufacture and commercial ex-
ploitation is still limited although validated research data about their physiological health benefits is rapidly accumulating.
Promising product concepts and novel fields of use have emerged recently, and some of these molecules have already
found commercial applications. The same applies to bioactive peptides derived from different milk proteins. Active pep-
tides can be liberated during gastrointestinal digestion or milk fermentation with proteolytic enzymes. Such peptides may
exert a number of physiological effects in vivo on the gastrointestinal, cardiovascular, endocrine, immune, nervous and
other body systems. However, at present the industrial-scale production of such peptides is limited by a lack of suitable
technologies. On the other hand, a number of bioactive peptides have been identified in fermented dairy products, and
there are already a few commercial dairy products enriched with blood pressure-reducing milk protein peptides. There is a
need to develop methods to optimise the activity of bioactive peptides in food systems and to enable their optimum utilisa-
tion in the body. This review highlights existing modern technologies applicable for the isolation of bioactive native pro-
teins and peptides derived from bovine colostrum, milk and cheese whey, and discusses aspects of their current and poten-
tial applications for human nutrition and promotion of human health.
Key Words: Milk, colostrum, bioactive proteins, bioactive peptides, novel technologies, functional ingredients, health promo-
ognised. In many countries dairy products contribute signifi-
cantly to daily protein intake. Also, the multiple functional
properties of the major milk proteins are now well character-
ised . Today, milk protein concentrates - particularly whey
protein concentrates (WPC) and whey protein isolates (WPI)
– and the enzymatic hydrolysates of milk proteins are manu-
factured industrially and have found extensive use in a wide
range of foodstuffs, such as infant formulas, dietary supple-
ments, and clinical and sports diet formulations [2,3].
Over the past two decades, increasing scientific and in-
dustrial interest has been focused on the biological properties
of milk proteins. It has been demonstrated that intact protein
molecules of both major milk protein groups, caseins and
whey proteins, exert distinct physiological functions in vivo,
as reviewed recently in many articles [4-13]. Many of the
bioactive whey proteins, notably immunoglobulins, lactofer-
rin and growth factors, occur in colostrum in much greater
concentrations than in milk, thus reflecting their importance
The high nutritional value of milk proteins is widely rec-
*Address correspondence to this author at the MTT Agrifood Research
Finland, Biotechnology and Food Research, FIN-31600 Jokioinen, Finland;
Tel: + 358 3 4188 3271; Fax: + 358 3 4188 3244;
to the health of the new-born calf [14,15]. Table 1 lists the
major proteins found in bovine colostrum and milk and pro-
vides information about their concentration, molecular
weight, and suggested biological functions. Furthermore,
research carried out over the past decade reveals that milk
proteins possess additional physiological functions due to the
numerous bioactive peptides that are encrypted within intact
proteins. A great number of bioactive peptides have been
identified, exhibiting various activities including antioxida-
tive, anti-hypertensive, antimicrobial, immunomodulatory,
opioid or mineral-carrying activity [16-22]. Increasing
knowledge of the biological properties of individual milk-
derived proteins and their fractions has prompted a need to
develop technologies for obtaining these components in a
purified or enriched form to enable their use as ingredients in
various functional foods . This article reviews the cur-
rently available and emerging technological options for the
isolation and enrichment of bioactive milk proteins and their
fractions, and features their potential fields of application for
the promotion of human health.
FRACTIONATION OF CASEINS
which casein constitutes about 80% and whey proteins 20%.
Bovine casein is further divided into ?-, ?- and ?-casein .
Normal bovine milk contains about 3.5% of protein, of
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830 Current Pharmaceutical Design, 2007, Vol. 13, No. 8 Korhonen and Pihlanto
The current technologies employed in the large-scale frac-
tionation of whole casein from milk are based either on
acidic coagulation at an isoelectric point of pH 4.6 or on en-
zymatic hydrolysis of caseins with rennet. The chemical
composition and functional properties of these casein prepa-
rations differ from each other . Whole casein is consid-
ered to have nutritional functions primarily because it is a
carrier of calcium and phosphate ions and also an ample
source of amino acids. Moreover, individual casein fractions
have proven biologically active and a good source of differ-
ent bioactive peptides that can be released, e.g., during gas-
trointestinal digestion of caseinates . Whole casein and
casein fractions have also been shown to have immuno-
modulatory activities, e.g., by regulating the proliferation of
lymphocytes and the production of different antibodies and
cytokines [24-26]. Various chromatographic and membrane-
separation techniques have been developed for fractionation
of ?-casein in view of its potential use, e.g., in infant formu-
lae. These methods exploit the fact that the ?-casein fraction
exists in solution as monomers at low temperatures, e.g., at
+4ºC. Under such conditions ?-casein remains partly soluble,
while ?s1-, ?s2- and para-?-caseins coagulate, and ultrafiltra-
tion (UF) or microfiltration (MF) can be used to separate the
soluble ?-casein from the aggregated other casein fractions
in milk or sodium caseinate . In another method 
which was successfully scaled up, the ?s1-, ?s2- and para-?-
caseins were removed from renneted Ca-caseinate by cen-
trifugation and the precipitated ?-casein by heating the su-
pernatant to 30ºC. Industrial manufacture of casein fractions
for dietary purposes has not progressed to any significant
extent so far.
FRACTIONATION OF WHEY PROTEINS
proteins which differ from each other in their chemical struc-
ture, functional properties and biological functions. These
characteristics have been used to separate individual pro-
teins, but the purity and biological activity of such fractions
have proven critical factors. Membrane-separation processes,
such as ultrafiltration (UF), reverse osmosis (RO) and diafil-
tration (DF), are now industrially applied in the manufacture
of ordinary whey powder and whey protein concentrates
(WPCs) with a protein content of 30-80%. Gel filtration and
ion-exchange chromatography techniques can be employed
in the manufacture of whey protein isolates (WPI) with a
protein content of 90-95% [29-32]. The introduction of nan-
ofiltration (NF) allows a selective separation of salts and
ions from whey, and has made it possible to manufacture
industrially demineralised and fractionated whey protein
ingredients. The chemical composition and functionality of
whey protein preparations are largely affected by the method
used in the process. The individual whey proteins differ
widely in their functional properties, e.g. acid stability, adhe-
sion, binding properties, emulsification, film formation,
foaming, gelation, organoleptic properties, solubility, viscos-
ity and water holding capacity . Also, the biological
properties of whey protein preparations are affected but are
difficult to standardise due to the complex nature of the bio-
activities exerted by different proteins . Therefore, there
is growing interest to develop specific techniques for the
isolation of pure whey protein components.
The whey fraction of milk contains a great variety of
Table 1. Concentration, Molecular Weight and Potential Biological Functions of Major Proteins of Bovine Colostrum and Milk
Concentration (g/L) Molecular Weight
Colostrum Milk Daltons
(?s1, ?s2, ? and ?)
26 28 14,000-22,000 Ion carrier (Ca, PO4, Fe, Zn, Cu), precursor for bioactive peptides, immunomodulator
Retinol carrier, potential antioxidant, precursor for bioactive peptides, binds fatty acids
Lactose synthesis in mammary gland, Ca carrier, immunomodulator, precursor for
bioactive peptides, anticarcinogenic
Specific immune protection (antibodies and complement system), potential precursor
for bioactive peptides
Antimicrobial, antithrombotic, bifidogenic, gastric regulator
Antimicrobial, antioxidative, anticarcinogenic, anti-inflammatory, iron transport, cell
growth regulation, precursor for bioactive peptides, immunomodulator
Antimicrobial, synergistic effect with immunoglobulins and lactoferrin
Antimicrobial, synergistic effect with immunoglobulins and lactoferrin
Serum albumin 1.3
Precursor for bioactive peptides
Potential mineral carrier
N.A. = not announced
References used in compilation of this table: [5, 11, 14, 23, 70]
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Technological Options for the Production of Health-Promoting Proteins Current Pharmaceutical Design, 2007, Vol. 13, No. 8 831
FRACTIONATION OF ?-LACTALBUMIN AND ? ?-
plied on a pilot or large scale to isolate enriched ?-lacta-
lbumin (?-la) and ?-lactoglobulin (?-lg) fractions from either
milk or whey, as reviewed by Mulvihill and Ennis . Tech-
niques used for this purpose include anion-exchange chro-
matography [35,36], high-performance liquid chromatogra-
phy [37,38], fast-protein liquid chromatography, isoelectric
focusing , electrochromatographic , membrane-
separation  and ultra-high pressure  techniques. Pres-
sure treatment in the last-mentioned novel method offers an
opportunity to denature ?-lg selectively without severe de-
naturation of ?-la because it is more pressure-resistant than
the previous protein. Denatured ?-lg can be precipitated by
acidification and separated by centrifugation or membrane
filtration. This method seems to be more suitable for skim
milk than WPI, because skim milk contains calcium which
may have a protective effect by stabilising the ?-la molecule.
More research is needed to establish the technological feasi-
bility and cost-efficiency of this method.
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Many methods have been developed and are being ap-
of ?-la and ?-lg is a combination of the selective denatura-
tion of proteins by heat treatment and subsequent separation
by membrane filtration [43-45]. In the case of milk, casein
micelle fractions will be firstly removed either by adjusting
the pH to 4.6 and centrifugation, or by the use of MF with an
average pore-size distribution of 0.1-0.2 μm. The next step
consists of concentrating the casein-free milk serum by UF
with a molecular weight cut-off of 10-25 kDa. The obtained
milk serum or whey fraction will then be subjected to
isoelectric precipitation at pH 4.2 and at 55-65ºC, a treatment
which precipitates ?-la while ?-lg remains soluble. The pre-
cipitate can further be separated from the soluble fraction by
membrane separation or centrifugation, concentrated and
The most frequently used technique for the fractionation
by this process ranges between 50-80% and 60-99%, respec-
tively . Another novel method developed for the separa-
tion of the above major whey proteins is based on the enzy-
matic hydrolysis of whey proteins by pepsin and concentra-
tion of ?-lg, which is resistant to pepsin, by UF [46-48].
Other new techniques developed recently are based on radial
flow chromatography columns, i.e., the Sepralac TM-process
 and ion-charged chromatographic membranes and beads
in columns .
The purity of the ?-la and ?-lg preparations manufactured
many industrial processes have been developed for the en-
richment and purification of ?-la . Although purification
methods have worked on a laboratory scale, scaling up to
pilot and industrial scale has proven challenging due to the
low purity of the preparations. Gezan-Gizou et al.  de-
veloped a modified heat precipitation method in which solu-
ble ?-lg was separated from precipitated ?-la using MF. The
precipitated ?-la was washed, solubilised, concentrated and
finally purified by UF. The purity of the resulting ?-la and ?-
lg fractions was in the range of 52-83 and 85-94%, respec-
tively. The recovery rates of the fractions were, however,
poor (6 and 51 %, respectively).
In view of potential dietary and pharmaceutical uses,
low purity of the ?-la and ?-lg fractions obtained from sweet
whey by selective precipitation methods is due to the high
concentration of caseinomacropeptide (CMP) in this source
material. CMP is a peptide sequence (f 106-169) cleaved
from ?-casein by rennet and remains in the whey after co-
agulation of casein. The high resistance of CMP to heat
treatment and proteolysis complicates its fractionation from
sweet cheese whey by selective denaturation or enzymatic
pretreatment. In studies by Tolkach and Kulozik  the
treatment of sweet whey and whey protein concentrates by
the transglutaminase enzyme followed by MF was found to
be a useful tool for separating native whey proteins from
CMP. This may allow the production of CMP-free whey
protein fractions with a high degree of purity. Another recent
technological innovation for the separation of milk proteins
is the application of MF using a uniform transmembrane
pressure (UTP) in combination with an UF-based DF process
. This technology seems to offer a new way to obtain
highly enriched, almost pure native milk protein fractions
with decreased formation of a deposited layer on the mem-
brane surfaces. Deposit formation frequently compromises
the efficiency of separation in membrane technology.
Tolkach and Kulozik  suggest that the reason for the
dairy protein ingredients with retention of bioactivity is a
continuous chromatographic separation technique (CSEP)
based on adsorption chromatography. This simulated mov-
ing-bed system was successfully adapted recently by an Aus-
tralian dairy ingredient manufacturer for the production of
ingredients enriched in lactoferrin (LF), glycomacropeptide
(GMP) and ?-lg. The technology has been found to provide
several advantages over conventional chromatography, in-
cluding efficiency, productivity and flexibility . Moreo-
ver, a novel separation technology called expanded-bed ad-
sorption (EBA) chromatography was introduced some years
ago for the high-purity separation of different unclarified
fluids such as milk and cheese whey . Unlike traditional
separation technology which employs packed beds of ad-
sorbent, EBA adsorbents are expanded by the upward flow
of the liquid. EBA performs a selective adsorption of bio-
molecules to porous beads based on a selective ligand chem-
istry. Subsequently, unwanted purities can be removed by
washing. During elution the adsorbed proteins are released
again by a change of buffer conditions, typically a change of
pH or ionic strength. The eluate contains the product in a
purified and concentrated form. Biomolecules of interest can
be separated in a serial set-up, each step capturing a specific
molecule. According to the supplier, LF and immunoglobu-
lins (Igs) can be separated stepwise from whey and the run
through can be further treated to produce WPI.
Another novel technology for the isolation of functional
Isolation of Lactoferrin and Lactoperoxidase
neutral pH, while the other whey proteins are negatively
charged. Many methods exploit this property to separate
these proteins from the remaining whey proteins. Again, LF
and LP can be separated from each other by chroma-
toghaphic methods, as reviewed by Mulvihill and Ennis .
Hereunder, a few methods are referred to as examples for
isolation of LF and LP.
LF and lactoperoxidase (LP) are positively charged at
832 Current Pharmaceutical Design, 2007, Vol. 13, No. 8 Korhonen and Pihlanto
milk and many other body secretions . LF is known to
exert many biological activities and is considered to play an
important role in the body’s innate defence system against
microbial infections and degenerative processes induced,
e.g., by free oxygen radicals . Hydrolysis of LF by pep-
sin produces antimicrobial peptides, called lactoferricins
which display a wider range of antimicrobial activity than
the native LF . For these reasons, LF has attracted com-
mercial interest, and a few products (infant formula, yoghurt)
containing added LF have already been launched on the
market in Japan. Also, LF is produced industrially by a num-
ber of companies worldwide and it is expected that its use as
an ingredient in functional foods and pharmaceutical prepa-
rations will increase drastically in the near future. The total
annual production of LF is now estimated at more than 70
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LF is an iron-binding glycoprotein found in colostrum,
for the isolation and purification of LF from colostral or
cheese whey based on different gel filtration and chroma-
tographic methods [59-61]. Patented large-scale production
techniques based on cation-exchange resins followed by gel
filtration or UF have been developed to isolate both LF and
LP from cheese whey [62,63]. Also, a microporous mem-
brane containing immobilised sulphonic acid has been ap-
plied to fractionate LF and LP from cheese whey . The
average recovery rates were 73% for LP and 50% for LF.
Recently, Zhang et al.  developed a simple rapid method
for the purification of LF from bovine colostrum based on
high-performance liquid chromatography (HPLC) with an
SP-Sepharose cation exchanger. It was concluded that the
method could be employed to purify LF from bovine colos-
trum on a large scale. Other methods developed for the isola-
tion of LF, in particular, include monoclonal antibody im-
munoaffinity, copper ions immobilised on Sepharose 6B
(metal chelate interaction chromatography) and affinity
chromatography using immobilised ferritin, heparin, DNA or
textile dyes .
Since the 1970s, several methods have been introduced
applications, the production of human LF in transgenic cows
and plants has also been studied lately. Van Berkel et al. 
reported that transgenic cows harbouring the genomic hLF
gene under the regulatory control of the bovine ?s1-casein
promoter produced rhLF at 0.4-3.0 g/ l of milk. rhLF and
bLF in transgenic cow’s milk could be separated by Mono S
chromatography. Although rhLF and native hLF underwent
differential N-linked glycosylation, they were equally effec-
tive in in vivo infection models. Bethell and Huang 
found that the amount of rhLF expressed in rice reached 25%
of total soluble protein and 0.5% of grain weight. Apart from
the rhLF produced in rice with plant-pattern glycosylation,
the physicochemical and biochemical properties of rhLF and
native hLF were similar.
In view of potential pharmaceutical and clinical nutrition
in the colostrum, milk and many other secretions of humans
and animals. LP is known to catalyse an antimicrobial sys-
tem consisting of the thiocyanate anion (SCN) and hydrogen
peroxide. This system is considered to have many biological
defence functions in the body and provide a natural method
for preserving raw bovine milk [11,68]. LP concentration is
The enzyme LP is a glycoprotein which occurs naturally
relatively low in colostrum, but it represents the most abun-
dant enzyme in milk. As mentioned above, LP can be recov-
ered in substantial quantities from whey using cation-
exchange chromatography, which allows its separation from
LF in appropriate elution conditions .
ISOLATION OF IMMUNOGLOBULINS
antibodies, are present in colostrum and milk of all lactating
species. Five major classes of Igs have been characterised in
mammals: IgG, IgM, IgA, IgD and IgE. The basic chemical
structure of all Igs is similar but their biological functions
differ, although in principle they all contribute to the major
defence mechanism against foreign materials recognised by
the body’s immune system . Igs account for up to 70-
80% of the total protein content in colostrum, whereas in
milk they account for only 1-2% of total protein. IgG1 is the
predominant Ig class in bovine lacteal secretions as com-
pared to IgA in human milk .
Immunoglobulins, which carry the biological function of
preparations for promoting the health of farm animals and
humans is an interesting research subject which has been
studied for decades. One of the challenges in this task is the
enrichment or isolation of active Igs from colostrum due to
its complex composition. Also, Igs are relatively heat-
sensitive and will not survive heat sterilisation processes. A
number of patented methods have been reported for pilot or
industrial-scale separation of Igs from colostral or cheese
whey (for a review see ). Separation technologies such
as membrane filtration and chromatography have been em-
ployed alone or in combination. With these methods, the
recovery rate of Igs has varied from 40 to 70% of the level
present in the starting material . Specific chromato-
graphic techniques such as immobilised metal chelate chro-
matography, immunoaffinity chromatography and cation-
exchange chromatography have been applied to improve the
yield and purity of immunoglobulin preparations further [73-
75]. More recently, MF (0.1 μm porosity) of transgenic goat
milk whey was demonstrated to increase Ig yield to over
95% . MF combined with UF of bovine, equine and ca-
prine colostrum has led to IgG/total solids purity of more
than 90% . Using a 0.1 μm membrane, cheese whey and
the concept of selective membrane separation through pH
manipulation, Mehra and Kelly  produced an Ig-rich
preparation with a protein composition similar to that pro-
duced from colostral whey . When EBA was applied to
isolate Igs from whey, Ig purity of between 50 and 70%
could be achieved . Korhonen et al.  used various
MF methods, such as UF, MF and reverse osmosis, and a
cation-exchange resin as a molecular sieve to concentrate Igs
from colostral whey. The Ig level of the final freeze-dried
concentrates varied from 45 to 75%. Additionally, colostral
Ig supplements have been developed to protect farm animals
against diarrhea, and colostrum-based products are nowadays
marketed for human use as dietary supplements . It is
also possible to raise the concentration of specific antibodies
against pathogenic microorganisms in colostrum and milk by
immunising cows with a vaccine made from these microbes
or their antigens . In clinical trials such immune milk
preparations have proven effective against many human mi-
crobial diseases caused, e.g., by Escherichia coli, Coryne-
The use of bovine colostrum as a source of antibody
Technological Options for the Production of Health-Promoting Proteins Current Pharmaceutical Design, 2007, Vol. 13, No. 8 833
bacterium, Streptococcus mutans, Helicobacter pylori, Clos-
tridium difficile and rotavirus (for reviews see [82-85]). Al-
though the potential of such immune milk products has been
recognised and demonstrated in many clinical studies, there
still are very few commercial products on the global market.
ISOLATION OF GROWTH FACTORS
number of growth factors, hormones and cytokines. The
concentration of these compounds is highest in colostrum
and decreases gradually during lactation.They are all in-
volved in cell proliferation and differentiation but exert also
specific physiological functions . Among growth factors,
insulin-like growth factors IGF-I and IGF-II, platelet-derived
growth factor PDGF, fibroblast growth factor FGF, trans-
forming growth factor TGF-?, epidermal growth factor EGF
and betacellulin have been identified and characterised .
These factors have been shown to be potent growth stimu-
lants and mediators for a range of mammalian cells, both in
vitro and in vivo . In recent years many methods have
been developed for the extraction of growth factors from
bovine colostral and cheese whey. Cation-exchange chroma-
tography has been mostly used because of the basic nature of
growth factor molecules [87,88]. Also, MF has been applied
for the concentration of some growth factors from colostrum
[72,77] and UF has been employed for separation of IGF-I
from IGF-II in whey . Many of these methods have been
patented, for example Maubois et al.  described a com-
bination of acidification and heat treatment to precipitate a
TGF- ?2-rich fraction from native whey. The precipitate was
further concentrated using 0.1 μm MF membrane to yield a
product characterised by a predominant content of ?-la and
70 % of the initial TGF-?2. Smithers  described a pat-
ented process to isolate an extract of growth factors from
Cheddar cheese whey. The process involves MF for pre-
treatment of whey, cation-exchange chromatography, con-
centration and activation using UF and DF, and sterile filtra-
tion and packaging. The product has demonstrated efficacy
in a number of in vitro and in vivo models of the tissue-repair
process and is being evaluated in the treatment of oral mu-
cositis and chronic ulcers. These trials have demonstrated the
safety of the extract when taken orally or when administered
topically . It has been proposed that dairy-derived
growth factors have potential applications in a number of
lucrative biotechnological, biomedical and functional food
areas, for example topical treatment of chronic wounds and
gastrointestinal diseases, e.g. Crohn’s disease [23,92] and
prevention of side effects of non-steroid anti-inflammatory
Bovine colostrum and milk (and cheese whey) contain a
PRODUCTION OF BIOACTIVE PEPTIDES
from precursor milk proteins in the following ways: (a) en-
zymatic hydrolysis by digestive enzymes (b) fermentation of
milk with proteolytic starter cultures (c) proteolysis by en-
zymes derived from microorganisms or plants and (d) prote-
olytic action by indigenous proteases present in milk. In
many studies, a combination of a) and b) or a) and c), respec-
tively, has proven effective in generating short functional
peptides . Databases on the identified bioactive peptides
are available . Quantitative structure activity relationship
Basically, biologically active peptides can be produced
(QSAR) modelling provides a methodology for finding
mathematical expressions for relationships that may be use-
ful in estimating the activities of any related compound and
in predicting structures of high activity. Predicting the bioac-
tivity of peptides can help to identify food proteins that con-
tain encrypted peptides with potential for functional foods
and specific enzymes to liberate those peptides. However,
the bioactivity of the predicted peptides should be measured
to validate the findings . The main technological chal-
lenges lie, firstly, in the manufacture of fermented dairy
products with a high concentration of particular bioactive
peptides or their precursors which, upon digestion in the gas-
trointestinal tract, would give rise to bioactive peptides, and
secondly, on the production of milk protein hydrolysates
with a high concentration of peptides having a specific bio-
activity and functionality that makes them suitable as ingre-
dients in other foods, including dairy products.
FERMENTATION OF MILK TO PRODUCE BIOAC-
highly proteolytic. The production of bioactive peptides by
making use of microbial sources, starter and non-starter bac-
teria, will aid in the development of new fermented dairy
products. The proteolytic system of lactic acid bacteria
(LAB), such as Lactococcus lactis, Lactobacillus helveticus
and Lactobacillus delbrueckii ssp. bulgaricus, is already well
characterised. This system consists of a cell wall -bound pro-
teinase and a number of distinct intracellular peptidases, in-
cluding endopeptidases, aminopeptidases, tripeptidases and
dipeptidases . Lb. helveticus is known to possess high
proteolytic activities , causing the release of oligopep-
tides from the digestion of milk proteins . These oli-
gopeptides can be a direct source of bioactive peptides fol-
lowing hydrolysis by gastrointestinal enzymes. Rapid pro-
gress has been made in recent years to elucidate the bio-
chemical and genetic characterisation of these enzymes. The
fact that peptidase activities are affected by growth condi-
tions makes it possible to manipulate the formation of pep-
tides to a certain extent . Table 2 gives a list of experi-
mental studies on the release of bioactive peptides upon fer-
mentation of milk using different live proteolytic microor-
ganisms or proteolytic enzymes derived from such microor-
Many of the industrially utilised dairy starter cultures are
have reviewed the release of different bioactive peptides
from milk proteins through microbial proteolysis [20, 101-
104]. Most of these studies report the production of angio-
tensin I-converting enzyme (ACE)-inhibitory or antihyper-
tensive peptides, but immunomodulatory, antioxidative and
antimicrobial peptides have also been identified. Lb. helveti-
cus is widely used as a dairy starter in the manufacture of
traditional fermented milk products like Emmental cheese.
Milks fermented with Lb. helveticus have been shown to
possess antihypertensive peptides [105-107], antimutagenic
properties , an immunomodulating effect on lympho-
cyte proliferation in vitro  and the ability to stimulate
the phagocytic activity of pulmonary macrophages .
Several studies have demonstrated that Lb. helveticus strains
are capable of releasing ACE-inhibitory peptides, in particu-
lar, the best known of which are the ACE-inhibitory tripep-
Over the last few years, many articles and book chapters
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834 Current Pharmaceutical Design, 2007, Vol. 13, No. 8 Korhonen and Pihlanto
tides Val-Pro-Pro and Ile-Pro-Pro [111,112]. Pihlanto-
Leppälä et al.  studied the potential formation of ACE-
inhibitory peptides from cheese whey and caseins during
fermentation with various commercial dairy starters used in
the manufacture of yoghurt, ropy milk and sour milk. No
ACE-inhibitory activity was observed in these hydrolysates.
However, further digestion of the above samples with pepsin
and trypsin resulted in the release of several strong ACE-
inhibitory peptides derived primarily from ?s1-casein and ?-
casein. Gobbetti et al.  demonstrated the formation of
ACE-inhibitory peptides with two dairy strains, Lb. del-
brueckii ssp. bulgaricus and Lc. lactis ssp. cremoris, after
fermentation of milk separately with each strain for 72 hours.
The most inhibitory fractions of the fermented milk mainly
contained ?-casein-derived peptides with inhibitory concen-
tration (IC50) values ranging from 8.0 to 11.2 μg/ml. Yama-
moto et al.  identified an ACE-inhibitory dipeptide
(Tyr-Pro) from a yoghurt-like product fermented with Lb.
helveticus CPN4 strain. The concentration of this peptide
sequence, which is present in all major casein fractions, was
found to increase during fermentation, reaching a maximum
concentration of 8.1 μg/ml in the product. Fuglsang et al.
 tested a total of 26 strains of wild-type LAB, mainly
belonging to Lc. lactis and Lb. helveticus, for their ability to
produce a milk fermentate with ACE-inhibitory activity. All
of the test strains produced ACE-inhibitory substances in
varying amounts, and two of them exhibited high ACE inhi-
bition and a high OPA index, which correlates well with
peptide formation. Studies with normotensive rats demon-
strated that Lb. helveticus produces substances which in vivo
can give rise to ACE inhibition. More recently, Ashar and
Chand  identified an ACE-inhibitory peptide from milk
fermented with Lb. delbrueckii ssp. bulgaricus. The peptide
showed the sequence Ser-Lys-Val-Tyr-Pro-Phe-Pro-Gly-
Pro-Ile from ?-casein with an IC50 value of 1.7 mg/ml. In
combination with Str. salivarius ssp. thermophilus and Lc.
lactis biovar. diacetylactis, a peptide structure with a se-
quence of Ser-Lys-Val-Tyr-Pro was obtained from ?-casein
with an IC50 value of 1.4 mg/ml. Both peptides were mark-
edly stable to digestive enzymes and to acidic and alkaline
pH, and also during storage at 5 and 10ºC for four days.
Robert et al.  identified several known antihypertensive
peptides from sodium caseinate hydrolysates produced by
Lb. helveticus NCC 2765.
Table 2. Release of Bioactive Peptides from Food Proteins by Various Micro-Organisms and Microbial Enzymes
Active Sequence Fermented by/ Microbial Enzymes Bioactivity Ref.
Lactobacillus helveticus LBK16H
Attenuates the development
of hypertension in SHR
enzymes + pepsin &
Whey proteins Tyr-Pro
Lactobacillus helveticus CPN 4 ACE inhibitory 
Lactobacillus delbrueckii subsp. bulgaricus SS1
Lactococcus lactis subsp. cremoris FT4
ACE inhibitory 
Lactobacillus delbrueckii subsp.
Lactobacillus rhamnosus +digestion with p
epsin and Corolase PP
Lactobacillus delbrueckii ssp. bulgaricus
ACE inhibitory 
Str. salivarius ssp. thermophilus +
Lactococcus lactis biovar. diacetylactis
ACE inhibitory 
Lb. helveticus R938
Kluyveromyces marxianus var. marxianus ACE inhibitory 
Tritirachium album derived proteinase K Antihypertensive 
Lactobacillus helveticus CP90 proteinase ACE inhibitory 
?s-cn, ?-cn Several active peptides
Lb. helveticus NCC2765
Not For Distribution
Technological Options for the Production of Health-Promoting Proteins Current Pharmaceutical Design, 2007, Vol. 13, No. 8 835
peptides Val-Pro-Pro and Ile-Pro-Pro in Lb. helveticus CM
was elucidated in a study by Ueno et al. . An endopep-
tidase which seems to process the carboxyl terminal of the
peptides has a sequence homology in the amino terminal
sequence to a previously reported pepO gene product. Kilpi
et al.  found higher ACE inhibition in milk fermenta-
tion using peptidase-deletion mutants compared to a wild-
type Lb. helveticus strain. Unlike with the wild-type strain,
ACE inhibition remained constant during the course of fer-
mentation with the proline-specific peptidase mutant. The
mutant strains also had different peptide profiles than the
Matar et al.  fed milk fermented with a Lb. helveti-
cus strain to mice for three days and detected significantly
higher numbers of IgA secreting cells in their intestinal mu-
cosa, compared with control mice fed with similar milk in-
cubated with a non-proteolytic variant of the same strain.
The immunostimulatory effect of fermented milk was attrib-
uted to peptides released from the casein fraction. Leblanc et
al.  investigated the effect of peptides released during
milk fermentation with Lb. helveticus R938 on the system-
atic immune system and on the growth of fibrosarcomas. The
fermented milk was fractionated by size-exclusion HPLC
and the fractions containing different molecular weight com-
pounds were tested in vivo. The results suggested that LAB
may modulate the immunological properties of milk proteins
prior to or after oral ingestion of the product. Such modula-
tion may be beneficial, e.g., in down-regulating hypersensi-
tivity reactions to ingested proteins in patients with food
An antioxidative peptide derived from ?-casein was de-
tected in milk after fermentation with L. delbrueckii subs.
bulgaricus . The 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical-scavenging activity of the peptide was lower than
that of butylhydroxyltoluene (BHT), but when the beta-
carotene decolorisation system was used the peptide showed
about five times stronger antioxidative activity as compared
In addition to live microorganisms, proteolytic enzymes
isolated from LAB have been successfully used for the pro-
duction of bioactive peptides from milk proteins. Yamamoto
et al.  reported that casein hydrolysed with a cell wall -
associated proteinase from Lb. helveticus CP790 showed
antihypertensive activity in spontaneously hypersensitive
rats (SHR). Several ACE-inhibitory peptides and one anti-
hypertensive peptide were isolated from the hydrolysate.
Utilising the same proteinase, Maeno et al.  identified a
?-casein-derived antihypertensive peptide from the casein
hydrolysate. The antihypertensive effect of this peptide (Lys-
Val-Leu-Pro-Val-Pro-Gln) was dose-dependent in SHR at a
dosage level from 0.2 to 2 mg of peptide per kg body weight.
The enzymatic process generating the antihypertensive
LYSATES WITH A HIGH CONCENTRATION OF
OF MILK PROTEIN HYDRO-
a number of bioactive peptides that have been characterised.
Until now, enzymatic hydrolysis of whole protein molecules
The enzymatic cleavage of milk protein molecules yields
has been the most common way to produce bioactive pep-
tides. Pancreatic enzymes, preferably trypsin, have been used
to liberate many known bioactive peptides from milk pro-
teins. For example, ACE-inhibitory peptides and calcium-
binding phosphopeptides (CPPs) are most commonly pro-
duced by trypsin [104, 123-126] Moreover, ACE-inhibitory
peptides have recently been identified in tryptic hydrolysates
of bovine ?s2-casein  and in bovine, ovine and caprine
?-casein macropeptides . Other enzymes and different
enzyme combinations of proteinases including alcalase,
chymotrypsin, pancreatin and pepsin, as well as enzymes
from bacterial and fungal sources have been applied to gen-
erate bioactive peptides from various protein sources [20,
129]. McDonagh and FitzGerald  clearly demonstrated
that a range of proteinase activities from plant, animal and
bacterial sources could be used to generate CPPs from so-
dium caseinate. Antimicrobial peptides can be obtained by
chymosin-mediated digestion of casein at neutral pH 
or by pepsin digestion of ?s2-casein and LF . Hydroly-
sis by pepsin alone or followed by trypsin hydrolysis is
needed to produce opioid-like peptides from caseins and
whey proteins . Sequential digestion brought about by
pepsin and trypsin increases the overall degree of proteoly-
sis, which generates hydrolysates with stronger antimicrobial
or ACE-inhibitory activity [134, 135]. In a recent study, Mi-
zuno et al.  measured the ACE-inhibitory activity of
casein hydrolysates upon treatment with nine different com-
mercially available proteolytic enzymes. Among these en-
zymes, a protease isolated from Aspergillus oryzae showed
highest ACE-inhibitory activity in vitro per peptide. When
Bitri et al.  incubated whole casein with pepsin at low
pH, the release of bioactive material occurred over time and
correlated with acidity rather than with enzymatic activity.
The hydrolysate also showed an opioid-like effect.
A number of studies have demonstrated that hydrolysis
of milk proteins by digestive and/or microbial enzymes can
produce peptides with immunomodulatory activities .
Sütas et al. [138, 139] found that digestion of casein frac-
tions with both pepsin and trypsin produced peptides that
provoked immunomodulatory effects on human blood lym-
phocytes in vitro. When the caseins were hydrolysed by en-
zymes isolated from a probiotic strain of Lactobacillus GG
var. casei prior to pepsin-trypsin treatment, all hydrolysate
fractions were immunosuppressive and the highest activity
was again found in ?s1-casein. The same hydrolysates also
down-regulated in vitro the generation of interleukin-4 by
lymphocytes. In a recent study Lorenzen and Meisel 
demonstrated that trypsin treatment of yoghurt milk prior to
fermentation with yoghurt cultures resulted in a release of
phosphopeptide-rich fractions. In particular, the release of
the CPP sequences ?-CN(1-25)-4P and ?s1 –CN(43-79)-7P
during trypsin treatment was pronounced whereas the prote-
olysis caused by peptidases of the yoghurt cultures was not
Fractionation and Enrichment of Hydrolysates Contain-
ing Bioactive Peptides
often intermediates and are isolated from very complex pep-
tidic hydrolysates in which their concentrations are often
low. The preparation of such peptide or peptide-containing
The bioactive peptides derived from food proteins are
Not For Distribution
836 Current Pharmaceutical Design, 2007, Vol. 13, No. 8 Korhonen and Pihlanto
fractions generally requires time-consuming chromatogra-
phic steps. Accordingly, the commercial production of bio-
active peptides from milk proteins has been limited by a lack
of suitable large-scale technologies. Until now, membrane-
separation techniques have provided the best technology
available for the enrichment of peptides with a specific mo-
lecular weight range . UF is routinely employed to en-
rich bioactive peptides from protein hydrolysates. Other re-
searchers have developed biphasic extraction systems with
organic solvents to isolate bioactive peptides from hydro-
lysates (Table 3) .
Hydrolysis can be performed by conventional batch hy-
drolysis or by continuous hydrolysis using UF membranes.
The traditional batch method has several disadvantages, such
as the relatively high cost of the enzymes and their ineffi-
ciency compared with a continuous process. The enzymatic
membrane reactor, which integrates enzymatic hydrolysis,
product separation and catalyst recovery into a single opera-
tion, is an attractive approach for this purpose. The use of
Not For Distribution
enzymatic membrane reactors for continuous production of
specific peptide sequences was introduced during the 1990s.
It has already been widely applied for total conversion of
food proteins of various origins to hydrolysates with im-
proved nutritional and/or functional properties [142, 143].
UF membrane reactors have been shown to improve the effi-
ciency of enzyme-catalysed bioconversion and to increase
product yields, and they can be easily scaled up. Further-
more, UF membrane reactors yield a consistently uniform
product with desired molecular mass characteristics. Con-
tinuous extraction of bioactive peptides in membrane reac-
tors has been mainly applied to milk proteins. Bouhallab and
Touzè  employed this technique for the recovery of
antithrombotic peptides derived from hydrolysed CMP.
Gauthier and Pouliot  combined enzymatic hydrolysis
and UF to produce emulsifying peptides from ?-lg. On the
other hand, they noted severe fouling problems with peptide-
membrane interactions, especially in UF of casein hydro-
lysates. Bordenave et al.  demonstrated that ?-lacto-
rphin could be successfully generated by continuous hy-
Table 3. Technological Means to Hydrolyse Proteins and Fractionate as well as Enrich Peptide Fractions to Enhance Bioactivity
Bioactivity Active Form Released by Fractionation/Enrichment Method Ref.
Strong ion exchange chromatography, continuous
membrane reactor with nanofiltration, large scale
?s2-cn f(183-207) Pepsin Batch-wise electro-membrane filtration 
Lactoferrin Antibacterial Lf f(17-41/42) Pepsin
Ion-exchange chromatography In situ enzymatic
hydrolysis on an ion-exchange membrane
?-cn f(193-209) Chymosin
Continuous membrane reactor with
cellulosic type membrane
Goat whey Opioid
?-la f(50-53) Pepsin Continuous ultrafiltration reactor 
Opioid b-hemoglobin f(31-40) Pepsin
Continuous enzymatic membrane reactor,
Phosphorylated ?s1, ?s2
and ?-cn peptides
Acid precipitation, diafiltration and anion ex-
Ca-binding Phosphorylated peptides
Hydrolysis using fluidized bed bioreactor
containing immobilized enzyme, chromatography
?s1-cn f(1-12) Trypsin Ultrafiltration and ionic separation 
ACE-inhibitory Not identified Trypsin
Batch hydrolysis, ultrafiltration 30 kDa and
1 kDa molecular mass cut-off membranes
?-la, ?-lg ACE-inhibitory
?-la f(104-108), f(99-108); ?-
lg f(22-25), f(32-40), f(81-83)
Batch hydrolysis, 2 step ultrafiltration
30 and 1 kDa cut-off membranes
Corn zein ACE-inhibitory Not identified Thermolysin
Aqueous two-phase system, with
dextran rich bottom phase
tory, mineral binding
Several peptides Trypsin
Continuous multicompartment enzyme
reactor operating under electric field
Continuous stirred tank reactor
using immobilized enzyme
Technological Options for the Production of Health-Promoting Proteins Current Pharmaceutical Design, 2007, Vol. 13, No. 8 837
drolysis of goat whey in UF without a previous purification
step of ?-lactalbumin (?-la). Righetti et al.  proposed a
multicompartment enzyme reactor operating under an elec-
tric field for the continuous hydrolysis of milk proteins. This
technique allowed for the continuous harvesting of some
biologically active peptides, such as phosphopeptides and
precursors of casomorphins from the tryptic digest of ?-
mass have been found useful in separating out small peptides
from high molecular mass residues and remaining enzymes.
Turgeon and Gauthier  used a two-step UF process and
were able to produce a mixture of polypeptides and a frac-
tion rich in small peptides with a molecular mass below 2000
Da. Pihlanto-Leppälä et al.  applied selective UF mem-
branes (30 kDa and 1 kDa) to enrich opioid peptides (?-
lactorphin and ?-lactorphin) from pepsin-hydrolysed ?-la
and from pepsin- and trypsin-hydrolysed ?-lg, respectively.
The same technique has been successfully used to enrich
ACE-inhibitory peptides from purified ?-la and ?-lg .
Not For Distribution
Stepwise UF using cut-off membranes of low molecular
small charged biomolecules, like peptides, has been the de-
velopment of the NF membrane, because of its high selectiv-
ity in the separation of components in the molecular mass
range of 300-1000 Da. Inorganic NF membranes, in particu-
lar, possess substantially high separative properties due to
their amphoteric behaviour as a function of pH. It has been
shown that transmission of selected peptides through NF
membranes results from a combination of size and charge
effects and is strongly affected by physico-chemical condi-
tions (pH and ionic strength) [150-152]. Lapointe et al. 
identified two bioactive peptides (?-lg f(102-105); f(142-
148) that might interact in the polarised layer during the fil-
tration process, as their transmission decreased with time
under specific conditions. These peptides are thought to in-
teract with the high molecular weight aggregates that accu-
mulate in the polarized layer and ultimately impair transmis-
sion. Using a more selective membrane for basic peptides of
MW <1000 g mol-1, the peptide ?-lg f(142-148) was selec-
tively transmitted . Electro-membrane filtration (EMF)
seems to be a promising alternative technology to produce
charged peptides. EMF combines conventional membrane
filtration (MF, UF or NF) with electrophoresis. Compared
with pressure-driven membrane filtration, an increase in se-
lectivity for the isolation of charged components can there-
fore be anticipated. Bargeman et al.  used bath-wise
EMF to isolate positively charged peptides with antimicro-
bial activity from ?s2-casein hydrolysate. Antimicrobial pep-
tides were enriched from 7.5% of total protein in the feed to
25% in the permeate product. Isolation of this and other
charged bioactive peptides could not be achieved by conven-
tional membrane DF using the same membrane.
One of the most important advances in the separation of
phase system to produce ACE-inhibitory peptides from
thermolysin-catalysed hydrolysis of a corn protein (zein).
They found that the mixture of peptides which was selec-
tively recovered from the dextran-rich bottom phase had
higher ACE-inhibitory activity than native zein. Thus, this
method is useful to obtain a mixture of biologically active
peptides for use in foodstuffs.
Murakami and Hirata  employed an aqueous two-
in a continuous process would be protein hydrolysis in a het-
erogeneous system in which the protease is immobilised on a
support. So far, only a few scaling up processes are known to
transform food proteins with immobilised enzymes in the
context of bioactive peptide production. Enzymes are better
stabilised in an immobilised form than in membrane-ultra-
filtration reactors in which the protease is free in solution.
Coletti-Previero et al.  developed a simple method for
pepsin immobilisation on a mineral support, acidic alumin-
ium oxide, through the natural phosphoserine residue of this
enzyme which forms an alumina phosphate complex. Ticu et
al.  hydrolysed haemoglobulin with immobilised pepsin
in a continuous stirred tank reactor and were able to produce
an enriched hydrolysate with three intermediate bioactive
peptides at a stationary concentration. Calcium-binding phos-
phopeptides have been prepared using immobilised trypsin
or glutamic acid-specific endopeptidase. The peptides were
prepared using a fluidised-bed bioreactor and separated by
anion-exchange chromatography [159, 160].
Another approach to the preparation of bioactive peptides
late bioactive peptides, such as ?-casein f(114-169), from
tryptic casein hydrolysate . Membrane-assisted solvent
extraction appears to be an efficient means of extracting con-
tinuously specific intermediate opioid peptides from bovine
Precipitation by adjustment of pH may be useful to iso-
been developed for the enrichment of CPPs from casein hy-
drolysates, but the production costs of this technique are
prohibitive for large-scale operation. Ellegård et al. 
developed a process-scale method for the isolation of high-
purity CPPs using acid precipitation, DF and anion-exchange
chromatography. Ion-exchange membrane chromatography
has emerged as a promising technique for enriching peptide
fractions from protein hydrolysates. Recio and Visser 
described a method where the protein of interest was concen-
trated in a chromatographic medium and hydrolysed in situ
by an appropriate enzyme. The resulting active peptides were
retained on the ion exchanger, while the other peptides were
washed out. Finally, the fraction containing the active pep-
tides was eluted from the chromatographic medium. With
this method it was possible to isolate and enrich cationic
antibacterial peptides from LF and ?s2-casein, as well as
negatively charged phosphopeptides from ?-casein. The ad-
vantages of the process are that isolation of the precursor
protein is unnecessary and the enzyme used in the process
can be recovered. This technique offers new possibilities for
enriching peptides with a low molecular mass, and it can be
easily up-scaled to yield gram or even kilogram quantities
Several ion-exchange chromatographic methods have
Isolation of Glycomacropeptide
terminal glycopeptide released from ?-casein by the action
of chymosin at 105Phe-106Met during cheese making. Because
of its unique carbohydrate composition and biological activi-
ties, GMP is thought to be a potential ingredient for func-
tional foods and nutraceuticals . Much attention has,
therefore, been given to the development of techniques to
isolate and purify GMP from cheese whey or rennet casein
The GMP present in cheese whey is a 64 residue C-
838 Current Pharmaceutical Design, 2007, Vol. 13, No. 8 Korhonen and Pihlanto
for commercial purposes. GMP has been isolated and puri-
fied from cheese whey or rennet whey by using various
techniques including UF, gel chromatography, hydrophobic
interaction chromatography, and ion-exchange chromatogra-
phy . Saito et al.  purified GMP using a combined
method of heat treatment, ethanol precipitation and anion-
exchange chromatography. Anion-exchange chromatography
is one of the most practical methods applicable to large-scale
production of GMP. Nakano and Ozimek  found that
with this technique it was possible to separate GMP, which
has a lower isoelectric point than do the major whey pro-
teins, with a relatively high yield and purity. When chitosan
resins were used as anion exchangers, it was observed that
resins with primary amine had a high binding capacity of
GMP. This technique might be useful for industrial-scale
production of GMP due to the low cost of resin . Li and
Mine  compared the chromatographic profiles of GMP
isolated by three methods (trichloracetic acid fractionation
(TCA), ethanol precipitation and UF). The TCA pretreat-
ment recovered only sialo-GMP (glycosylated) and elimi-
nated all contaminating proteins; however, the recovery rate
was the lowest (6.7 % of the initial WPI). Ethanol precipita-
tion recovered 20.4 % of GMP from WPI and 75.7 % was
glycosylated. UF was found to be the most effective in re-
covering GMP, the recovery rate being 33.9 % with 81.6 %
of sialo-GMP. It was proposed that the combination of UF
with anionic chromatography might be a suitable and practi-
cal approach for an industrial-scale production of GMP, be-
cause this process retains the carbohydrate structures which
are claimed to exert many of the bioactivities attributed to
GMP. Lieske et al.  employed a two-stage UF process
for isolation of caseinomacropeptide (CMP), the non-
glycosylated form of GMP. The molecular weight cut-off
limits of the UF membranes used were 30 kDa and 50 kDa,
respectively. The results showed that in an aqueous solution
CMP existed as dimer and/or trimer with the molecular
weight between 20.3 and 17.5 kDa depending on the pH of
solution (pH 3.0 and 7.0, respectively). It is, therefore, likely
that CMP occurs in whey as a polymer structure and is frac-
tionated with ?-lg at pH 3.5 and with ?-la at pH 7.0 when a
single UF process in used. A further purification of CMP can
be obtained with the second UF stage. In another recently
developed method  the whey proteins were firstly re-
moved from skim milk by MF-diafiltration and the resulting
casein concentrate was treated with chymosin to obtain a
CMP enriched whey. Coagulated casein was separated by
MF and the whey was concentrated by UF to yield a CMP-
rich preparation. The purity of the CMP product was not
given but it was reported to possess good foaming and emul-
OTHER TECHNOLOGICAL OPTIONS
nant DNA techniques have also been employed to produce
specific peptides or their precursors in microorganisms. For
example, the sequence encoding the antihypertensive peptide
Arg-Pro-Leu-Lys-Pro-Trp has been introduced into the gene
for soybean ?-conglycin ?’subunit. This subunit was ex-
pressed in E. coli, recovered from the soluble fraction and
purified by chromatography. The Arg-Pro-Leu-Lys-Pro-Trp
peptide was released from the recombinant containing the
In addition to the techniques described above, recombi-
subunit after digestion by trypsin and chymotrypsin .
Feeney et al.  reported that the construction of glutenin
genes and their expression in E. coli is a viable method for
producing peptides. In addition, Kim et al.  succeeded
in expressing recombinant human ?s1-casein in E. coli and
purifying it. The trypsin digest of this protein contained sev-
eral ACE-inhibitory peptides. Rao et al.  demonstrated
the release of a bioactive peptide (H-Arg-Tyr-Leu-Pro-Thr-
OH) in a transgenic tobacco plant, expressing a specifically
designed precursor gene. Antimicrobial peptides derived
from shrimp have been successfully expressed in the yeast
Sacchramoyces cerevisiae, but the expression levels were
relatively low . The methylotrophic yeast Pichia pas-
toris has emerged as a powerful and inexpensive expression
system for the production of high levels of functionally ac-
tive recombinant proteins and several antimicrobial peptides
. Despite significant advances, synthesis of short se-
quences by means of genetic engineering methods often re-
mains impractical due to the low expression efficiencies ob-
tained and the difficulties encountered in product extraction
and recovery. On the other hand, genetic engineering can be
used to produce enzymes with specific activity to release the
desired peptides from the precursor proteins.
tives have attracted great interest in recent years due to their
potential applications in functional foods and nutraceuticals.
Industrial or semi-industrial scale processing techniques are
available for fractionation and isolation of major proteins
from colostrum and milk and a few whey derived native pro-
teins, e.g. ?-la, Igs, LF and GMP are manufactured commer-
cially . On the other hand, there is a growing need to
develop large-scale separation technologies for other bioac-
tive compounds which occur in colostrum or milk in rela-
tively minor quantities but entail emerging potential as func-
tional ingredients. Such compounds include e.g. growth fac-
tors, hormones, cytokines, proteose-peptone fraction, milk
basic protein and milk fat globule membrane proteins [11,
Furthermore, colostrum and milk have in recent years
proven as an interesting model to demonstrate the nutritional
needs of the neonate for optimal growth and development
. Screening of lacteal secretions for the diversity of
different bioactive molecules has emerged as a new approach
to understand the role of nutrition. The advancement of this
concept may in the future lead to a new type of biomining
and bioguided processing industries exploiting bovine colos-
trum and milk as a major raw material [181,182]. In this con-
text, future research is expected to also reveal how such
biomolecules affect the expression of genes. It is envisaged
that in the future colostrum and milk derived biomolecules
will be utilised to develop functional dairy foods that are
specifically designed to improve genome health maintenance
in humans . For this purpose, new technologies are ob-
viously needed, particularly for non-thermal processing of
biomolecules. Such emerging technologies include e.g. high
hydrostatic pressure processing and pulsed electric field
technology offering a potential alternative to existing heat-
preservation processes without inactivating the biological
Biologically functional dairy proteins and their deriva-
Not For Distribution
Technological Options for the Production of Health-Promoting Proteins Current Pharmaceutical Design, 2007, Vol. 13, No. 8 839
crypted in milk and other dietary proteins is well docu-
mented. Their optimal exploitation for human nutrition and
health poses a great scientific and technological challenge,
but at the same time it offers ample potential for commer-
cially successful applications. The industrial-scale produc-
tion of bioactive peptides is, however, currently limited by a
lack of suitable technologies. For this purpose, specific
chromatographic and membrane-separation techniques have
to be developed and up-scaled using the standards employed
by pharmaceutical industries. Also, in view of demonstrating
the clinical efficacy of bioactive peptides it is important to
study the technological properties and bioavailability of ac-
tive peptide fractions using criteria applied for pharmaceuti-
cal compounds. These studies must include the safety as-
pects, in particular allergenic potential which is often associ-
ated with protein based materials.
Microbial fermentation is one option applicable for the
large-scale production of bioactive peptides either from ani-
mal or plant proteins. This potential is already well demon-
strated by the presence of bioactive peptides in fermented
milks and different varieties of cheese . Controlled
fermentation of protein-rich raw materials with known LAB
strains may be developed on a commercial scale utilising
continuously operating fermentors or bioreactors. Commer-
cial production of specific peptide sequences could alterna-
tively be enabled through recombined enzyme technology
utilising certain production strains, or through the use of pu-
rified proteolytic enzymes isolated from suitable microor-
ganisms . In recent years sophisticated metabolic engi-
neering strategies have been developed for many LAB which
allow the generation of genetically engineered strains that
can over-express different nutrients . In the future, it is
likely that these food grade bacteria will be employed as cell
factories to produce a variety of biogenic compounds, in-
cluding specific bioactive peptides, which can positively
benefit human health.
The occurrence of many biologically active peptides en-
?-la = ?-lactalbumin
?-lg = ?-lactoglobulin
= Angiotensin I-converting enzyme
CMP = Caseinomacropeptide
DPPH = 1,1-diphenyl-2-picrylhydrazyl
= Expanded-bed adsorption
= Epidermal growth factor
EMF = Electro-membrane filtration
= Fibroblast growth factor
HDL = High-density lipoprotein
HPLC = High-performance liquid chromatography
Ig = Immunoglobulin
IGF = Insulin growth factor
= Lactic acid bacteria
LF = Lactoferrin
NF = Nanofiltration
PDGF = Platelet-derived growth factor
QSAR = Quantitative structure activity relationship
RO = Reverse osmosis
= Spontaneously hypertensive rat
= Transforming growth factor
UF = Ultrafiltration
= Uniform transmembrane pressure
= Whey protein concentrate
WPI = Whey protein isolate
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