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SCIENTIFIC BULLETIN OF THE TECHNICAL UNIVERSITY OF LODZ
No. 1058 Food Chemistry and Biotechnology, Vol. 73 2009
KATARZYNA DYBKA
PIOTR WALCZAK
Institute of Fermentation Technology and Microbiology
Technical University of Lodz
COLLAGEN HYDROLYSATES AS A NEW
DIET SUPPLEMENT
Review: Professor Marianna Turkiewicz, Ph.D., D.Sc
Paper reviews literature data connected with properties of
collagen hydrolysates applied as diet supplements. Biological and
health promoting activity of collagen derived peptides has been well
documented in many studies, especially for the therapeutical
treatment of bones and joints diseases as well as for the
improvement of skin, hair and nails conditon. High tolerance of
patients for long-term ingested collagen hydrolysates make them
attractive for use as health promoting diet supplement.
1. Introduction
Modern lifestyle characterized with permanent lack of time results in
consumption of a highly processed food which does not have any beneficial
effect on our health. Inbalanced and incomplete diets can be a reason of many
diet depended diseases. If we care about healthy lifestyle, we have to include
nutrient-rich food to our normal diet. Diet supplements are such kind of health
beneficial substances which contain concentrated source of nutrients or other
components causing positive physiological effects. Diet supplements are
produced in the form of powder, capsules, powder in sachets, liquid in bottles
with droppers or in other forms suitable for proper dosage. It is well recognized
that diet supplements are not medicines and their use is not regulated by the
pharmaceutical law [25].
K. Dybka, P. Walczak 84
2. Collagens characteristic
Collagen proteins are the most abundant in the human and animal body.
They are the major proteins of connective tissue, skin, tendons, cartilage,
ligaments, cornea, teeth, nails and hair [8]. Proteins of collagen family represent
a group of varied extracellular matrix molecules linked by the occurrence of the
collagen triple-helical domain as a common structural element [5]. In vertebrates
organisms, at least 27 types of collagen with 42 distinct polypeptide chains has
been reported [19]. According to similarieties in their structure and
supramolecular organization, they are classified into fibril-forming, fibril-
associated collagens with interruptions in triple helix (FACITs), network-forming
collagens, anchoring fibrils or transmembrane collagens [22]. The different
collagen types are characterized by considerable complexity and diversity in their
structure, their splice variants, the presence of additional, non-helical domains
and their function. All members of the collagen family have one characteristic
feature – a right-handed triple helix composed of -chains (Fig. 1). Triple-helix
can be formed by three identical chains (homotrimers) as in types II, III, VII,
VIII, X, XIII, XV, XVII, XXIII, XXV collagen and by two or three different
chains (heterotrimers) as in types I, IV, V, VI, IX, and XI collagen [19, 22]. Each
of the three collagen -chains coils into a left-handed helix which assemble to
rope-like figure bordered by the C- and N-propeptides [8].
Fig. 1. Molecular structure of fibrillar collagens
Collagens consist of a high amount of glycine (about 33% amino acid
residues), proline (12-14%), 4-hydroksyproline (<14%) and 4-hydroksylysine
(1.5%) [17]. Tryptophan and cysteine were not noticed [21]. Collagens are
known to share a repeating pattern Gly-X-Y in which the X and Y positions are
frequently occupied by proline (Pro) and 4-hydroksyproline (Hyp) residues
Collagen hydrolysates as a new diet supplement 85
[2, 7, 17]. Research have reported that the content of Hyp plays an especially
important role in stabilizing the triple-helical conformation in collagen and in
peptides with collagen-like domains [2, 4]. Hydroksyproline residues stabilize
triple helical conformation by sharing direct hydrogen bonds[2]. The most
common motif in fibril-forming collagens is repeating sequence Gly-X-Y
resulting in triple helical domains of 300 nm in length which corresponds to
about 1000 amino acids [8]. The three residues in the repeating triplet occupy
distinct positions within the supercoiled helix. The central possition of Gly
residues makes this residue not suitable for interacting with other residues.
Interactions caused by proximity between neighboring chain are related with less
solvent accessibility of Y position residues. In contrast to Gly and Y positions in
triplet motif, the greatest exposure for interactions, shows the X position [5].
Bella et al. [2] suggested that the water molecules aggregate as a shell to the
carbonyl and hydroxyprolyl groups resulting diverse conformation with a
specific motifs of water bridges bonding oxygen atoms within a single chain or
between different chains of triple helix.
3. Biosynthesis and degradation of collagen
Permanent collagens exchange processes in our body takes place during the
whole human life. Old fibrils are replace by new one all the time. When we are
young, collagen production and degradation are in dynamic balance, but during
maturation of tissues, degradation is being more intenssive. UV radiation,
smoking cigarettes, stress and unhealthy diet lead to the degradation of natural
collagen structure and to earlier senility.
3.1. Biosynthesis of collagens
The biosynthesis of fibril-forming collagen is a multisteps and complicated
process which takes place in intracellular and extracellular spaces.
It begins with transcription of the genes and ends with assemble of a triple helix
collagen fibrils into fibers with their final distinctive functions in tissues (Fig. 2).
Cell type, growth factors and cytokines are considered as particularly agents in
the system of transcriptional regulation during collagen biosynthesis. It is well
konown that the major group of collagen genes assemble into a complex of 3 to
117 exons and introns, characterised with more than 50 exons encoding the
mRNAs of fibrillar collagens. It was reported that other mRNA species could be
found. They are related with mulitple initiation sites of transcription or
alternative splicing of exons. The process of mRNA translation into synthesized
polypeptide chains (preprocollagen) takes part in membrane-bound ribosomes.
K. Dybka, P. Walczak 86
Fig. 2. The main steps in biosynthesis of fibril-forming collagens
In the endoplasmic reticulum preprocollagen is involved in several
posttranslational modifications. Three vitamin C-depended enzymes, prolyl
3-hydroxylase, prolyl 4-hydroxylase and lysyl hydroxylase catalyze hydroxylation
Collagen hydrolysates as a new diet supplement 87
of proline and lysine residues. Presence of 4-hydroxyproline is critical for
hydrogen bonding within molecule [8, 19]. Hydroksylysine residues are recognized
as bonding agents within fibril chains. The 3-hydroxyproline function has not been
reported, yet. Other action is glycosylation of some of hydroksylysine residues and
asparagine residues in C and/or N propeptides. After the association of C
propeptides and formation of disulfide bonds, three chains form molecule
called procollagen, this precursor of collagen is secreted and released into the
extracellular space in transport vesicles of Golgi apparatus.
Then procollagen trimers are processed in different ways which depend on
the collagen type. The C-propeptides and N-propeptides are removed by specific
metalloproteases. Following the procollegen modifications, the tropocollagen
fibrils are assembled. It was found that some of fibril-forming collagens (e.g. I,
II, III, V, XI) associate spontaneously into fibrillar structures during in vitro test.
It has been compared to the crystalization process. Several models described self-
assembly structure encoded in collagens and formation mechanism of periodic
fibrils. Fully formed fibers are stabilized by hydrophobic and electrostatic
interactions between collagen monomers and covalent cross-links joining
differently orientated fibrils in tissues [8, 28].
3.2. Degradation of collagen
Collagen is a very stable protein in normal healthy conditions. Collagen
degradation may proceed in different ways, but generally it is belived that there
are two possibilities − intracellular and extracellular. The main cause of intercellular
degradation process are proteolytic enzymes, particularlly cathepsins. Cathepsins
are various proteolytic enzymes found in animal tissue that catalyze the
hydrolysis of proteins into polypeptides in acid environment. In the extracellular
way there are several stages including depolymerisation which effects with
deterioration of molecular structures; activity of collagen-specific enzymes −
tissue collagenases; heat-disintegration at body temperature of products of
collagenases degradation, which lose triple helices structure and become
available for non specific proteinases. Collagenases can be synthesized by many
cells of human body (e.g. fibroblasts, neutrophils, and tumor cells [28].
4. Collagen hydrolysates production
The main source of collagen peptides are bovine hide, bone, pigskin or
fishbones and fish skin. Marine sources are an alternative to bovine or porcine
and they are not associated with the prions related to risk of Bovine Spongiform
Encefalopathy (BSE) [12]. Collagen hydrolysates are manufactured in controlled
hydrolysis process to obtain soluble peptides. The raw material is washed,
K. Dybka, P. Walczak 88
homogenized and demineralized with diluted mineral acid or alkaline. The raw
material is extracted in several stages with warm water. Further enzymatic
degradation of gelatin results in a final product which is collagen hydrolysate
[18, 24, 26]. Clemente [6] has presented enzymatic hydrolysis as the most
appropriate method for preparation of tailor-made peptides. Collagen
hydrolysates vary from each other with respect of peptides molecular weight,
mostly their molecular weight range from 2 to 6 kDa [18, 26]. Its molecular
weight is less than the average molecular weight of peptones. After purification,
the product is concentrated and dried. The most common post-dried procedures
are related to the control of molecular size and the elimination or reduction of
bitterness in the resulting hydrolysates. The most efficient procedure to remove
residual high-molecular weight peptides and proteins or to reduce the antigen
content of hypoallergenic formulas, is ultrafiltration [6].
Several analysis may be done for the quality control of these products: the
osmolarity, analysis of the hydrolysis degree, the molecular weight distribution,
the total nitrogen, amino acid composition and the presence of toxic compounds
(e.g. biogenic amines or pathogens). Protein hydrolysate qualitative analysis use
different techniques based on spectrophotometric, chromatographic and
electrophoretic methods (UV-spectrophotometry, HPLC, SDS-PAGE) [23].
5. Properties and applications of collagen hydrolysates
Gelatin and collagen hydrolysates have been reported to have beneficial
biological functions. Hydrolyzed gelatin products have been designated as
generally recognized as safe (GRAS) food products or food additives by the
Food and Drug Administration (FDA) [1, 18]. Despite the fact that collagen
hydrolysate has been generally regarded as having a low biological value,
because it does not contain all of the essential amino acids, its a reputable
nutritional component often used to supplement other proteins because of its
superb digestibility and high consumer tolerance [26].
5.1. Beneficial role of collagen hydrolysate in health
According to the opinion of many researchers, beneficial effects of oral
administration of collagen hydrolysates results of crossing the intestinal barrier,
by a dietary bioactive peptides, which reach the blood circulation and become
available for metabolic processes [26]. Collagen hydrolysates are used in medical
applications, such as high-energy supplements, geriatric products and enteric,
therapeutic or weight-control diets. Applification of protein hydrolysates in
treatment of patients with specific disorders of digestion, absorption and amino
acid metabolism. Tests also included clinical cure of patients with malnutrition
Collagen hydrolysates as a new diet supplement 89
attached with trauma, burns, cancer and hepatic encephalopaties [6]. Collagen
hydrolysates are good source of amino acids for people suffering from anorexia,
anaemia and for vegetarians (because of absence of meat in their diet). Diet
supplements conataining collagen hydrolysates are considered as improvement
agents in tendon or joint regeneration in physically active athlets with activity-
related joint pain [18, 26].
Orally consumed collagen hydrolysate has been shown to be absorbed
intestinally and to accumulate in cartilage. Speciffically, collagen hydrolysate
ingestion stimulates a significant increase in the synthesis of extracellular matrix
macromolecules by chondrocytes [3]. According to medical data clinical
investigations suggest that ingestion of collagen hydrolysates reduces pain in
patients suffering from osteoarthritis and osteoporosis. It is considered that about
15% of world population suffer from joint pain-related diseases. In Poland an
increasing problem become joint-related diseases connectet with other high risk
disorders agents which are abundant. Increasing risks agents are senility (over 50%
of elderly people suffer from rheumatism), gender (a high amount of patients are
women, particularlly after menopause), body weight (huge body weight is
a reason of joint overload and results in joint pain), constantly excessive sport
activity, joint injury (e.g. dislocations), metabolic diseases (e.g. diabetes) [24].
Collagen hydrolysates are involved in cartilage matrix synthesis [26]. Over
almost two decades scientists have studied a relationships between therapeutic
trials in joint diseases and collagen, gelatin or collagen hydrolysates. In numerous
studies researchers accepted dose of 10 g of collagen hydrolysates daily as a safe
and well tolerated by patients. Additionaly clinical tests have proved that this
level of daily ingested proteins can reduce the pain in comparison with placebo
group patients [18].
Several scientific reports have presented good bioavailability of hydrolyzed
collagen, after oral administration by animals and human beings. Oesser et al. [20]
discovered that about 95% of orally applied collagen hydrolysate was absorbed
within the first 12 h. Wu et al. [26] described the high safety of eating collagen
hydrolysates in an animal model (1.66 g/kg of body weight per day). Studies
related with preparations consisting gelatin derivated peptides showed good
tolerance and little side effects including a sensation of unpleasant taste, a feeling
of heaviness in the stomach, and a bloated feeling or pyrosis after oral
administration [20].
According to opinion of Zague [26] some studies described chemotactic
activity of short peptides (Pro-Hyp and Pro-Hyp-Gly) to human fibroblast,
peripheral blood neutrophils and monocytes in the cell culture. Collagen-
degradation peptides might attract these cells and result in repair of damaged
tissue. It is believed that collagen hydrolysates can not be absorbed from skin and
the basis of the skin effectiveness of collagen hydrolysate depends on a gradual
improvement of water absorption to skin as a result of possitive effect of the oral
K. Dybka, P. Walczak 90
administration of supplement. A beneficial effects has been also observed for
skin-related organs and for hair and nail quality.
Antihypertensive and antioxidative activities of bioactive peptides isolated
from collagen hydrolysates have been discovered [26]. Collagen and gelatin
digests contain angiotensin-converting enzyme (ACE) inhibitory peptides. ACE
play an important role in blood pressure regulation and inhibition of this enzyme
can cause an antihypertensive effect [14, 15, 16]. Protein supplements (e.g.
collagen hydrolysates) may be useful to enhance nitrogen retention [10].
5.2. Industrial application of collagen hydrolysates
Gelatin and hydrolyzed collagen are utilized in food industry in confectionery
(to improve texture, chewiness and foam stabilization), dairy (as stabilisation
and texturization agents), bakery (to provide stabilization, emulsification and
gelling), low-fat spreads (to provide fat reduction, creaminess and mouthfeel), in
meat-processing (to provide water-binding e.g. in reconstituted hams), in wine
and fruit juices production (fining agent) [1, 12, 13, 27]. Collagen hydrolysates,
like all protein hydrolysates show technological advantages such as good
solubility, heat stability and relatively high resistance to precipitation by many
agents, such as metal ions or pH [6]. Protein hydrolysates have an excellent
solubility at high degree of hydrolysis, which is a substantially useful
characteristic for many food applications and influences other functional features
such as emulsifying and foaming properties [9, 15].
Collagen hydrolysate has a high water-binding capacity and can be used as
an essential product low-calorie carbohydrates or low fat food production.
In the pharmaceutical industry gelatin and collagen hydrolysates are used to
manufacture capsules, implants and intravenous infusions [11, 12].
6. Conclusions
Collagens are the most abundant group of organic macro-molecules in
human and animal body. Because of their tensile strength, they perform
numerous important structural functions within the body, especially in connective
tissues. Collagen proteins are essential in connective tissues of such organs as
heart, intestines, lungs or parenchymal organs like liver and kidneys; as protein
matrix of the skeleton and its related structures (e.g. bones, teeth, tendons,
cartilage and ligaments); in fibrous matrix of skin and blood vessels [6, 7, 18, 26]. Its
excellent properties are result of their amino acid composition and molecular
structure. Collagens are also involved in the management of cellular mediators.
Collagen protein (in the form of collagen hydrolysate) has been shown to
improve skin hydration, reduce wrinkles and decrease pain and functionality
Collagen hydrolysates as a new diet supplement 91
disorders in joint diseases. In addition, collagen hydrolysate seems to be a relatively
inexpensive and widespread available protein source.
Collagen hydrolysate and gelatin can be used in food, cosmetics or
pharmaceutical industry as a natural additive revealing an antioxidant properties
with competitive foaming and emulsifying functionalities [9,15]. Finally, such
properties like excellent biodegradability, low immunogenicity and the
possibilities for large-scale production make them interesting compounds for
a wideespread industrial use in food industry, cosmetics industry or medicine.
7. References
[1] Baziwane D., He Q.: Gelatin: the paramount food additive. Food Rev. Int. 19,
423-435, (2003).
[2] Bella J., Brodsky B., Berman H.M.: Hydration structure of a collagen peptide.
Structure. 9, 893-906, (1995).
[3] Bello A.E., Oesser S.: Collagen hydrolysate for the treatment of osteoarthritis and other
joint disorders: a review of the literature. Cur. Med. Res. Opin. 22(11), 2221-2232, (2006).
[4] Bornstein P.: Covalent cross-links in collagen: a personal account of their
discovery. Matrix Biol. 22, 385-391, (2003).
[5] Brodsky B., Ramshaw J.A.M.: The collagen triple-helix structure. Matrix Biol.
15, 545-554, (1997) .
[6] Clemente A.: Enzymatic protein hydrolysates in human nutrition. Trends Food
Sci.Techn. 11, 254-262, (2000).
[7] Dioguardi F.S.: Nutrition and skin. Collagen integrity: a dominant role for amino
acids. Clin. Dermatol. 26, 636-640, (2008).
[8] Gelse K., Pöschl E., Aigner T.: Collagens-structure, function, and biosynthesis.
Adv. Drug Deliv. Rev. 55, 1531-1546, (2003).
[9] Giménez B., Alemán A., Montero P., Gómez-Guillén M.C.: Antioxidant and
functional propertioes of gelatin hydrolysates obtained from skin of sole and squid.
Food Chem. 114, 976-983, (2009).
[10] Hays N.P., Kim H., Wells A.M., Kajkenova O., Evans W.J.: Effects of whey and
fortified collagen hydrolysate protein supplements on nitrogen balance and body
composition in older women. J. Amer. Diet. Assoc. 109, 1082-1087, (2009).
[11] Karim A.A., Bhat R.: Gelatin alternatives for the food industry: recent developments,
challenges and prospects. Trends Food Sci. Techn. 19, 644-656, (2008).
[12] Karim A.A., Bhat R.: Fish gelatin: properties, challenges, and prospects as an
alternative to mammalian gelatins. Food Hydroc. 23, 563-576, (2009).
[13] Kim S.K., Kim Y.T., Byun H.G.: Purification and characterization of
antioxidative peptides from bovine skin. J. Bioch. Mol. Biol. 34, 219-224, (2001).
[14] Korhonen H., Pihlanto-Leppälä A., Rantamäki P., Tupasela T.: Impact of
processing on bioactive proteins and peptides. Trends Food Sci. Techn. 9, 307-319, (1998).
[15] Li B., Chen F., Wu Y., Wang X., Ji B.: Isolation and identification of
antioxidative peptides from porcine collagen hydrolysat by conescutive
xhromatography and electrospray ionization-mass spectrometry. Food Chem. 102,
1135-1143, (2007).
K. Dybka, P. Walczak 92
[16] Mendis N. Rajapakse N., Kim S.K.: Antioxidant properties of a radical-
scavening peptide purified from enzymatically prepared fish skin gelatin
hydrolysate. J. Agric. Food Chem. 53, 581-587, (2005).
[17] Minakowski W., Weidner S.: Biochemia krgowców. PWN. Warszawa 2005.
[18] Moskowitz R.W.: Role of collagen hydrolysate in bone and joint disease. Semin.
Arthritis Rheum. 30, 87-99, (2000).
[19] Myllyharju J., Kivirikko K.I.: Collagens, modifying enzymes and their
mutations in humans, flies and worms. Trends Genet. 20, 33-43, (2004).
[20] Oesser S., Seifert J., Adam M., Babel W.: Oral administration of
14
C labeled
collagen hydrolysate leads to an accumulation of a radioactivity in cartilage of
mice (C57/BL). J. Nutr. 129, 1891-1895, (1999).
[21] Pytrus-Sdłak B.: Kosmetyka ozdobna i pielgnacja twarzy. Medpharm Polska.
Wrocław 2007.
[22] Ricard-Blum S., Ruggiero F.: The collagen superfamily: from the extracellular
matrix to the cell membrane. Path. Biol. 53, 430-442, (2005).
[23] Silvestre M.P.C.: Review of methods for the analysis of protein hydrolysates.
Food Chem. 60, 263-271, (1997).
[24] widerski F., Czerwonka M., Waszkiewicz-Robak B.: Hydrolizat kolagenu
nowoczesny suplement diety. Przem. Spo. 4, 42-44, (2009).
[25] Wrzeniewska-Wal J.: Suplementy diety w nowej ustawie o bezpieczestwie
ywnoci. Przem. Spo. 6, 49-51, (2007).
[26] Zague V.: A new view concerning the effects of collagen hydrolysate intake on
skin properties. Arch. Derm. Res. 300, 479-483, (2008).
[27] Zhang Z., Li G., Shi B.: Physicochemical properties of collagen, gelatin and
collagen hydrolysate derived from bovine limed split wastes. J. Soc. Leath. Techn.
Chem. 90, 23-28, (2005).
[28] http://www.osteoforum.org.pl/kolagen.html; 4.08.2009
HYDROLIZAT KOLAGENU JAKO NOWY
SUPLEMENT DIETY
Streszczenie
W artykule dokonano przegldu literatury dotyczcej właciwoci hydrolizatów
kolagenu stosowanych jako suplementy diety. Aktywno biologiczna i oddziaływanie
prozdrowotne hydrolizatów białek kolagenowych zostały udowodnione naukowo,
zwłaszcza w leczeniu chorób zwyrodnieniowych koci i stawów oraz poprawie
kondycji skóry, włosów i paznokci. Wysoka tolerancja pacjentów na spoywane
hydrolizaty kolagenu w długim czasie powoduje, i s one atrakcyjnym, prozdro-
wotnym suplementem diety.
Instytut Technologii Fermentacji i Mikrobiologii
Politechnika Łódzka
