functional & bioactive collagen and gelatin form alternative sources 2011 rew.pdf

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Review

Functional and bioactive properties of collagen and gelatin from alternativesources: A review

M.C. Gómez-Guillén*, B. Giménez, M.E. López-Caballero, M.P. MonteroInstituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN, CSIC), C/José Antonio Novais 10, 28040 Madrid, Spain

a r t i c l e i n f o

Article history:

Received 30 November 2010Accepted 8 February 2011

Keywords:

CollagenGelatinBioactive peptidesFishPoultryFunctional properties

a b s t r a c t

The rising interest in the valorisation of industrial by-products is one of the main reasons why exploringdifferent species and optimizing the extracting conditions of collagen and gelatin has attracted theattention of researchers in the last decade. The most abundant sources of gelatin are pig skin, bovine hideand, pork and cattle bones, however, the industrial use of collagen or gelatin obtained from non-mammalian species is growing in importance. The classical food, photographic, cosmetic and pharma-ceutical application of gelatin is based mainly on its gel-forming properties. Recently,and especially in thefood industry, an increasing number of new applications have been found for gelatin in products suchas emulsiers, foaming agents, colloid stabilizers, biodegradable lm-forming materials and micro-encapsulating agents, in linewith thegrowing trend to replacesyntheticagents with more natural ones. Inthelast decade,a large numberof studieshave dealt with theenzymatic hydrolysis ofcollagen orgelatin forthe production of bioactive peptides. Besides exploring diverse types of bioactivities, of an antimicrobial,antioxidant or antihypertensivenature, studieshavealso focusedon theeffectof oral intakein both animaland human models, revealing the excellent absorption and metabolism of Hyp-containing peptides. Thepresent work is a compilation of recent information on collagen andgelatin extraction from newsources,aswellas new processingconditionsand potential novel or improved applications,manyof which arelargelybased on induced cross-linking, blending with other biopolymers or enzymatic hydrolysis.

2011 Elsevier Ltd. All rights reserved.

1. Introduction

Gelatin is a soluble protein compound obtained by partialhydrolysis of collagen, the main brous protein constituent inbones, cartilages and skins; therefore, the source, age of the animal,and type of collagen, are all intrinsic factors inuencing the prop-erties of the gelatins ( Johnston-Banks, 1990). Although to date, upto 27 different types of collagen have been identied, type Icollagen is the most widely occurring collagen in connective tissue.Interstitial collagen molecules are composed of three a-chains

intertwined in the so-called collagen triple-helix. This particularstructure, mainly stabilized by intra- and inter-chain hydrogenbonding, is the product of an almost continuous repeating of theGly-X-Y- sequence, where X is mostly proline and Y is mostlyhydroxiproline (Asghar & Henrickson, 1982). Only the very shortN- and C-terminal regions, called telopeptides (15e26 amino acidresidues), do not form triple helical structures as they are largelymade up of lysine and hydroxylysine (Hyl) residues, as well as their

aldehyde derivatives, in both intra- and inter-molecular covalentcross-links (Bateman, Lamandé, & Ramshaw, 1996). Four to eightcollagen molecules in cross-section are stabilized and reinforced bycovalent bonds to constitute the basic unit of collagen brils. Thus,the typical strong, rigid nature of skins, tendons and bones is due tothe basic structure formed by many of these cross-linked collagenbrils.

Insoluble native collagen must be pre-treated before it can beconverted into a form suitable for extraction, which is normallydone by heating in water at temperatures higher than 45 C. A

chemical pre-treatment will break non-covalent bonds so as todisorganize the protein structure, thus producing adequateswelling and collagen solubilization (Stainsby, 1987). Subsequentheat treatment cleaves the hydrogen and covalent bonds todestabilize the triple-helix, resulting in helix-to-coil transition andconversion into soluble gelatin (Djabourov, Lechaire, & Gaill, 1993;Gómez-Guillén, Turnay et al., 2002). The degree of collagenconversion into gelatin is related to the severity of both the pre-treatment and the warm-water extraction process, as a function of pH, temperature, and extraction time ( Johnston-Banks, 1990). Twotypes of gelatin are obtainable, depending on the pre-treatmentprocedure and are known commercially as type-A gelatin

* Corresponding author. Tel.: þ34 31 5492300; fax: þ34 91 5493627.E-mail address: [email protected] (M.C. Gómez-Guillén).

Contents lists available at ScienceDirect

Food Hydrocolloids

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . co m / l o c a t e / f o o d h y d

0268-005X/$ e see front matter 2011 Elsevier Ltd. All rights reserved.

doi:10.1016/j.foodhyd.2011.02.007

Food Hydrocolloids 25 (2011) 1813e1827

mailto:[email protected]://www.sciencedirect.com/science/journal/0268005Xhttp://www.elsevier.com/locate/foodhydhttp://dx.doi.org/10.1016/j.foodhyd.2011.02.007http://dx.doi.org/10.1016/j.foodhyd.2011.02.007http://dx.doi.org/10.1016/j.foodhyd.2011.02.007http://dx.doi.org/10.1016/j.foodhyd.2011.02.007http://dx.doi.org/10.1016/j.foodhyd.2011.02.007http://dx.doi.org/10.1016/j.foodhyd.2011.02.007http://www.elsevier.com/locate/foodhydhttp://www.sciencedirect.com/science/journal/0268005Xmailto:[email protected]


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(isoelectric point at pHw 8e9) and type-B gelatin (isoelectric pointat pH w 4e5) obtained under acid and alkaline pre-treatmentconditions respectively. Industrial applications call for one or theother gelatin type, depending on the degree of collagen cross-linking in the raw material. Because of the acid lability of cross-linking in immature collagens, such as in sh skins, reasonably mildacid treatment is enough to effect collagen solubilisation (Montero,Borderías, Turnay, & Leyzarbe, 1990; Norland, 1990).

Gelatin quality for a particular application depends largely on itsrheological properties (Stainsby, 1987). Apart from basic physico-chemical properties, such as composition parameters, solubility,transparency, colour, odour and taste, the main attributes that bestdene the overall commercial quality of gelatin are gel strength andthermal stability (gelling and melting temperatures). For stan-dardizing purposes, measurement of gel strength is determinedusing the so-called Bloom test, which consists of performing a well-dened protocol at a given gelatin concentration (6.67%), temper-ature (10 C) and maturation time (17 h), thus allowing gel strengthto be expressed in the normalized “bloom value” (Wainewright,1977). Both, gel strength and thermostability, are largely depen-dent on the molecular properties of gelatin, especially with respectto two main factors: (i) the amino acid composition, which is

species-specic, and (ii) the molecular weight distribution, whichresults mainly from processing conditions (Gómez-Guillén, Turnayet al., 2002). The proline and hydroxyproline content is particularlyimportant for the gelling effect. Triple helical structure stability inrenatured gelatins has been reported to be proportional to the totalcontent in pyrrolidine imino acids, given that it is the Pro þ Hyprich zones of the molecules that are most likely tobe involved in theformation of nucleation zones (Ledward,1986). However, althoughPro is important, Hyp is believed to play a singular role in thestabilization of the triple-stranded collagen helix due to itshydrogen-bonding ability through its eOH group (Burjandze,1979;Ledward, 1986). Moreover, it has also been observed that thetotal Gly-Pro-Hyp sequence content is one of the main factorsaffecting collagen thermostability (Burjandze, 2000). With regard

to molecular weight distribution, gelatin consists of a mixture of polypeptides frequently presenting a band pattern distributiontypical of type I collagen, with a characteristic a1/a2 chain ratio of around 2, the presence of b- and g-components (covalently linkeda-chain dimers and trimers, respectively), together with highermolecular weight forms as well as low-molecular weight proteindegradation fragments (Stainsby, 1987). A strong decrease in theprevalence of b- and g-components and the near-disappearanceof higher molecular aggregates, with an increased presence of degradation fragments, are normally the result of the application of more intense extracting conditions (pH, temperature, time), whichis normal industrial practice for yield improvement ( Johnston-Banks, 1990).

Physical properties of gelatin inuence its quality and potential

application, since they are related to gelatin structure (Yang &Wang, 2009). Structural properties of gelatin have been studiedby different methodologies such as Size Exclusion Chromatog-raphy-Multi Angle Laser Light Scattering (SEC-MALLS) (Haug,Draget, & Smidsrød, 2004), viscoelasticity ( Jamilah & Harvinder,2002), confocal uorescence microscopy (Nordmark & Ziegler,2000), scanning and transmission electron microscopy (SEM andTEM) (Djabourov, Bonnet et al., 1993; Saxena, Sachin, Bohidar, &Verma, 2005), circular dichroism (Gómez-Guillén, Turnay et al.,2002), electrophoretic analysis (Zhou, Mulvaney & Regenestein,2006), dielectric analysis (Lefebvre et al., 2006), differential scan-ning calorimetry (Badii & Howell, 2006), Fourier transform infraredspectroscopy (FTIR) (Muyonga, Cole, & Duodu, 2004) and FT-Ramanspectroscopy (Badii & Howell, 2006). Recently, nanotechnology has

received much attention in studying structure at nanoscale level in

food science, including gelatins. In this area, atomic force micros-copy has been successfully applied to the study of gelatin structureobtained from mammalian sources (Benmouna & Johannsmann,2004; Saxena et al., 2005), and more recently from sh skins(Wang, Yang & Regenstein, 2008; Yang & Wang, 2009; Yang, Wang,Regenstein, & Rouse, 2007; Yang, Wang, Zhou & Regenstein, 2008).

The most abundant sources of gelatin are pig skin (46%), bovinehide (29.4%) and pork and cattle bones (23.1%). Fish gelatinaccounted for less than 1.5% of total gelatin production in 2007, butthis percentage was double that of the market data for 2002,indicating that gelatin production from alternative non-mamma-lian species had grown in importance (Gómez-Guillén, Pérez-Mateos et al., 2009). Apart from the well-known socio-culturaland sanitary aspects, the rising interest in putting by-products fromthe sh industry to good use is one of the reasons why exploringdifferent species and optimizing the extraction of sh gelatin hasattracted the attention of researchers in the last decade (Gómez-Guillén, Turnay et al., 2002; Karim & Bhat, 2009). The main draw-back of sh gelatins is that gels based on them tend to be less stableand have worse rheological properties than gelatins from landmammals, and this may limit their eld of application. Generallyspeaking, this is true in the case of cold-water sh species, such as

cod, salmon, Alaska pollack, etc. Nevertheless, relatively recentstudies havepointed out that tropical and sub-tropical warm-watersh species (tilapia, Nile perch, catsh) might have similar rheo-logical properties and thermostability to that of mammal gelatins,depending on the species, type of raw material and processingconditions (Gilsenan & Ross-Murphy, 2000; Gómez-Guillén, Pérez-Mateoset al., 2009; Jamilah & Harvinder, 2002; Karim & Bhat, 2009;Muyonga et al., 2004; Rawdkuen, Sai-Ut, & Benjakul, 2010).

Scientic literature about different alternative sources and newfunctionalities of collagen and gelatin has experienced a boom inthe last 10e15 years, in part due to the growing interest in theeconomical valorisation of industrial by-products (from the meatand sh industry), the environmental friendly management of industrial wastes, and the search for innovative processing condi-

tions as well as potential novel applications. Many of theseimproved functional properties are largely based on chemical- andenzymatic-induced cross-linking, as well as blending with otherbiopolymers.

The classical food, photographic, cosmetic and pharmaceuticalapplication of gelatin is based mainly on its gel-forming andviscoelastic properties. Recently, and especially in the foodindustry, an increasing number of new applications have beenfound for gelatin in products such as emulsiers, foaming agents,colloid stabilizers, ning agents, biodegradable packaging materialsand micro-encapsulating agents, in line with the growing trend toreplace synthetic agents with more natural ones. Moreover, inmany cases, these studies are dedicated to using collagens andgelatins from alternative sources to land-based animals.

On the other hand, enzyme-hydrolysed collagen plays anincreasingly important role in various products and applications. Itsdifferent properties and functionalities benet the end consumernow in ways which were not present ten years ago. Over the pastdecade, a large number of studies have investigated enzymatichydrolysis of collagen or gelatin for the production of bioactivepeptides. Besides exploring diverse types of bioactivity, studiesfocused on the effect of oral intake in both animal and humanmodels have revealed the excellent absorption and metabolism of Hyp-containing peptides.

2. Alternative sources of collagen and gelatin

The most common raw materials for collagen and gelatin

extraction are skins or hides, bones, tendons and cartilages. Pig skin

M.C. Gómez-Guillén et al. / Food Hydrocolloids 25 (2011) 1813e1827 1814


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was therst raw material used for the manufacture of gelatin in the1930s and continues to be the most important material for large-scale industrial production. Raw materials from sh and poultryhave received considerable attention in recent years, but their stilllimited production makes them less competitive in price thanmammalian gelatins. As far as sh gelatin is concerned, the hugenumber of species having very different intrinsic characteristics,has aroused the interest of the scientic community in optimisingthe extracting conditions as well as characterising the yields, andphysico-chemical and functional properties of the resulting gela-tins, obtained mainly from skin and bone residues. Examples of extracting procedures and gelling properties of gelatins from theskins of different typical cold-water species, such as cod, Atlanticsalmon, haddock, Alaska pollack or hake; tropical or sub-tropicalspecies, such as black or red tilapia, Nile perch, channel catsh,yellown tuna, sin croaker, shortn scad, skate or grass carp; atspecies,such as megrim, Doversole; as well as cephalopods,such asgiant squid have been reviewed by Gómez-Guillén, Pérez-Mateoset al. (2009) and Karim and Bhat (2009). Although strict compari-sons are dif cult since methodologies may differ considerably fromone work to another, cold-water sh gelatins are characterised asgenerally having low gelling and melting temperatures (w4e12 C

and 90%, witha gelatin yield of 22% and gel strength of 152 g, values considerablyhigher than those obtained with 0.20 mol/L HCl or 1.2 g/L citricacid.

Pre-cooked n, an important waste product from canned tunaprocessing, has been proposed as a promising source for highperformance gelatin extraction, though with a low yield (w2%)(Aewsiri, Benjakul, Visessanguan, & Tanaka, 2008). Tuna n pre-sented an ash content as high as 40%, consequently an exhaustivedemineralisation step was needed. Moreover, during the steamheating pre-cooking treatment, collagen might have undergonesome denaturation, in detriment to the yield and the properties of the extracted gelatin, which exhibited inferior gelling, emulsifying,foaming and lm-forming properties to those of commercial pigskin gelatin.

The solid waste from surimi processing, which may range from50 to 70% of the original raw material (Morrissey, Lin, & Ismond,2005), could also be the initial material for obtaining gelatin or

collagen from under-utilized sh resources. More specically,rener discharge from the Pacic whiting surimi process, repre-senting around 4e8% of whole sh, consisted of muscle (95%), skin(2.1%), bone (2.9%) and trace amounts of scale fragments (Kim &Park, 2005). Crude collagen from rener discharge, extracted inthe form of acid-soluble collagen or recovered in a simpler way aspartially puried collagen, was found to present higher functionalproperties (emulsifying activity, cooking stability, water and oilabsorption capacity) compared with the other by-products (skinsand frames) generated in the same manufacturing process (Kim &Park, 2004; 2005). In a later study, the acid-soluble collagen fromAlaska pollack surimi rener discharge, having a thermal dena-turation temperature slightly higher than that for Alaska pollackskin, was also proposed as a potentially functional food ingredient

(Park, Lee, Kang, Park, & Kim, 2007).The offal from further processing of semi-processed sh prod-

ucts, such as skins from salted and marinated herring or cold-smoked salmon, has been studied as a source of gelatin(Ko1odziejska, Skierka, Sadowska, Ko1odziejski, & Niecikowska,2008). Smoking did not signicantly alter collagen susceptibilityto thermal denaturation, as the resulting gelatin was considerablyless degraded, and with higher gel strength than gelatins from theskins of marinated and salted herrings.

Farm-raised alligator bones ( Alligator mississippiensis), whichrepresent a highly signicant part of the wastage from alligatormeat processing industries in the southern states of the USA, Chinaand Thailand, were also investigated in an effort to nd additionalnon-land-based sources of highly thermostable collagen (Wood

et al., 2008). Most of the collagen from alligator bone was identi-ed as type I collagen, and the imino acid contents of the acid- andpepsin-solubilized collagen were 194 and 196 per 1000 residues,respectively, values which were very close to sub-tropical shspecies (black drum and sheepshead) and slightly lower than thatfor calf skin.

Giant red sea cucumber (Parastichopus californicus) has beenconsidered as a potential source of collagen for nutraceutical andpharmaceutical applications (Liu, Oliveira, & Su, 2010). Type Icollagen in the skin and connective tissue of giant red sea cucum-bers was isolated by pepsin digestion procedures with yields of 20.8% from skin and 24.3% from connective tissue on a dry weightbasis. The lower gel-forming ability of collagen from giant red seacucumber in comparison to that from calf skin was attributed to the

low content of imino acid residues in both skin and connective



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tissue (153 and 142 residues/1000 total, respectively), which wereeven lower than in walleye pollock or cod.

Poultry by-products are also increasingly processed intocollagen-based high-value products. Pepsin or ethylene diaminewas used for extracting type I and type III collagens from chickenskins, with a yield (collagen content in the solid phase) of 38.9% forpepsin extraction and 25.1% for extraction with ethylene diamine(Cliche, Amiot, Avezard & Gariépy, 2003). A pepsin-aided processwas proposed to extract type II collagen from chicken sternalcartilage, which is another important by-product of the poultryprocessing industry, in order to eliminate the presence of immu-nogenic response-inducing telopeptides, while affecting onlyminimally collagen functional properties (Cao & Xu, 2008).

3. Methods for species differentiation

Frequently, it may be necessaryto differentiate between gelatinsfrom different sources not only for safety and religious reasons, butbecause it has been reported that most gelatin-allergic patientsdevelop allergic reactions to bovine and porcine gelatin, but do notreact to sh gelatin (Sakaguchi et al., 2000). Despite labellingprecautions, bovine and porcine gelatins pose a high degree of risk

for sensitized patients because they are often present in commer-cial foods and food ingredients, due to cross-contamination duringprocessing. Some studies have reported on differentiation betweenbovine and porcine gelatins based on amino acid analysis usingprincipal component analysis (Nemati, Oveisi, Abdollahi, &Sabzevari, 2004) or the pH drop method after calcium phosphateprecipitation (Hidaka & Liu, 2002). Nevertheless, more accuratemethodologies have nowbeen developedto trace the species originof gelatin contained in commercial food products.

The close similarity between collagen amino acid sequencesfrom different species makes their immunochemical differentiationdif cult when using polyclonal antibodies against the wholemolecule. Venien and Levieux (2005) reported on the successfulproduction of bovine specic antibodies by immunization of rabbits

with synthetic peptides mimicking a short putative species-specicsequence of the bovine alpha 1(I) chain. Using these antibodies, anindirect ELISA was developed to allow a quick and easy differenti-ation between bovine and porcine gelatins and for the sensitivequantitation of their mixtures. Similarly, two sandwich ELISAmethods, based on antibodies from rabbits and goats immunizedwith bovine gelatin were found to be highly specic for bovine andporcine gelatin, but had little reactivity with sh gelatin (Doi,Watanabe, Shibata, & Tanabe, 2009).

Zhang et al. (2009) proposed a new method for species differ-entiation using high performance liquid chromatography/tandemmass spectrometry (HPLC-MS/MS), based on detection and iden-tication of specic marker peptides in tryptic-digested bovine andporcine gelatins, and found proline hydroxylation to be a key factor

affecting peptide identication.Recently, a method based on the application of Fourier trans-form infrared spectroscopy (FTIR) in combination with attenuatedtotal reectance (ATR), principal component analysis and discrim-inant analysis, has been developed to provide simple, rapid quali-tative differentiation of bovine and porcine gelatins (Hashim et al.,2010). The IR-regions found to give information about the origin of the gelatin ranged from 3290 to 3208 cm1 and from 1660 to1200 cm1, and were related to NeH bond deformation.

4. Functional properties of collagen and gelatin

Besides their basic hydration properties, such as swelling andsolubility, the most important properties of collagen and gelatin

can be divided into two groups: i) properties associated with their

gelling behaviour, i.e. gel formation, texturizing, thickening andwater binding capacity, and ii) properties related to their surfacebehaviour, which include emulsion and foam formation and sta-bilisation, adhesion and cohesion, protective colloid function, andlm-forming capacity (Schrieber & Gareis, 2007). Sikorski, Scott,and Buisson (1984) reviewed the possibilities of sh collagen asa functional material, and Montero and Borderías (1991) andMontero et al. (1990) examined the effects of concentration, pH andionic strength on optimization of its functional properties.

4.1. Gelling and water binding properties

Gel formation, viscosity and texture are closely related proper-ties determined mainly by the structure, molecular size andtemperature of the system. Although collagen and gelatin aredifferent forms of the same macromolecule, gelatin and collagengels are often confused by non-specialists. The gelatin gelationmechanism as well as the formed gel network structure differsconsiderably from that of collagen, as described by Djabourov,Bonnet et al. (1993). During collagen gelation the process of collagen molecule aggregation and bril formation takes place,induced by changes in ionic strength, pH and temperature. During

the collagen gelation process, there is a lag phase where theprimary aggregates (dimers and trimers of collagen molecules) arenucleated. Then, microbrillar aggregation starts with the lateralaggregation of sub-units until equilibrium is reached. The self-assembly process of type I collagen from vertebrates occurs whenthe gelation temperature is raised from 20 to 28 C. In contrast, thebasic mechanism of gelatin is related to the reverse coil-to-helixtransition triggered by cooling solutions below 30 C, during whichthe helices that are created are similar to the collagen triple-helix,but in this case, no equilibrium is reached. The gelation process forboth collagen and gelatin is thermo-reversible, but in oppositedirections: collagen gels melt by lowering the temperature, whilegelatin gels melt by raising the temperature.

The most widespread single use of gelatin in food products is in

water gel desserts, due to its unique melt-in-the-mouth property.Certain other hydrocolloids also have thermo-reversible charac-teristics, but they generally melt at higher temperatures. Gelatindesserts made from various gelatins may provide variety in textureand gel melting behaviour, offering new product developmentopportunities. By increasing gelatin concentrations or by usinggelatin mixtures (of cold and warm-watersh), desserts made fromsh skin gelatin were found to be more similar to desserts madefrom high bloom pork skin gelatin. Furthermore, the lower meltingtemperature in gel desserts made from sh gelatins may accelerateavour release (Zhou & Regenstein, 2007).

New textures and appearance, offering ample versatility inproduct development, can be provided by introducing a gas phaseinto gelatin-gel-based products, such as fruit jellies or marshmal-

lows. Moreover, the dispersed air makes portions less calorie-dense, by reducing the energy consumed per unit volume. Theincorporation of gas (air, nitrogen or helium) in the form of bubbleshas been found to weaken the structure of gelatin gels, resulting inunique mechanical properties and an opaque white appearance(Zúñiga & Aguilera, 2009). Among the three gases tested, air-lledgels presented the lowest densities, the largest bubble diametersand the weakest structures in comparison with the other gasiedgels.

Gelatin or collagen chains in solution may be covalently cross-linked to form matrices capable of swelling in the presence of aqueous solutions, forming what are commonly known as gelatinhydrogels. Hydrogels, which are characterized by their hydrophi-licity and insolubility in water, have the ability to swell to an

equilibrium volume while preserving their shape. The chemical



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cross-linkers used may be either relatively small bifunctionalmolecules or polyfunctional macromolecules like, for instance,glutaraldehyde (Deiber, Ottone, Piaggio, & Peirotti, 2009). Atpresent, the use of natural polyampholytic hydrogels in applica-tions such as gelatin-casings, with the ability to absorb largequantities of water, are becoming more important in the eld of medicine, pharmacy, agriculture and biodegradable food pack-aging. Gelatin is particularly attractive for forming hydrogel pack-aging because it is relatively inexpensive and biodegradable, and itsstructure facilitates multiple combinations of molecular interac-tions. The properties of gelatinepectin hydrogels have been shownto depend strongly on the pH in the reaction mixture and on thecharge balance (determined by the gelatinepectin ratio), whichwill inuence the degree of electrostatic associations and ionicinteractions in the gelling system (Farris, Schaich, Liu, Piergiovanni,& Yam, 2009). These authors proposed the development of inte-grated hydrogel lms from gelatin and pectin, based on theformation of permanent polyion-complex hydrogels composed of a primary entangled network of chemically-crosslinked gelatinchains that connect and encase physical hydrogel regions in whichpectin is linked to gelatin by ionic interactions.

The super-swelling properties of gelatin hybrid hydrogels

produced by mixing with synthetic polymers have been exhaus-tively revised, especially with reference to their swelling capacity,degradation rate and controlled release of drugs (Zohuriaan-Mehr,Pourjavadi, Salimi, & Kurdtabar, 2009). In this work, a number of organic (PEG-dialdehyde, acrylamines, EDTAD, poly(acrylic acid))and inorganic (kaolin, silica gel) compounds have been shown toproduce modications in the gelatin backbone, thus affecting thestrength, solubility, surface hydrophilicity and morphology of thecomposite hydrogels. The suitability of highly hydrolysed collagen(w2000e20000 Da) to synthesize superabsorbent hybrid hydro-gels was also revised.

A possible means of manipulating the characteristics of a low-gelling gelatin to achieve greater similitude with the properties of mammalian gelatins is to induce enzymatic cross-linking through

the action of an enzyme called transglutaminase (TGase), usually of microbial origin. The enzyme acts by catalysing an acyl transferreaction between the g-carboxamide group of glutamine residuesand the 3-amine group of lysine residues of peptide chains (Folk,1983). Extensive covalent cross-linking during the cooling of setgelation may cause an almost complete loss of thermo-reversibilityin the resultant gelatin gel, depending on whether covalent cross-linking occurs predominantly before or after formation of thehydrogen-bonded triple-helix junction zones (Babin & Dickinson,2001). The thermo-reversibility of the gelatin gel may be modu-lated depending on enzyme concentration, incubation time and thedegree of enzyme heat-inactivation. Increasing concentrations of TGase were reported to raise the melting temperature, and toincrease the elasticity and cohesiveness of megrim skin gelatin gels,

but due to excessively rapid gel network formation there wasa lowering of gel strength and hardness (Gómez-Guillén, Sarabia,Solas, & Montero, 2001). For gelatin extracted from Baltic codskins, the addition of TGase has proven to be effective for producingstable gels at room temperature, as well as after heating in boilingwater for a period inferior to 30 min (Ko1odziejska, Kaczorowski,Piotrowska, & Sadowska, 2004).

Different polysaccharides, such as k-carrageenan and/or gellan(Haug et al., 2004; Pranoto, Lee, & Park, 2007), or hydrox-ypropylmethylcellulose (Chen, Lin, & Kang, 2009) have also beenused to increase sh gelatin gel strength, setting time and ther-mostability in order to enhance their cold and thermal gelationproperties, without the loss of their thermo-reversible character.Similarly, gelatin or hydrolyzed collagen materials could be hard-

ened by reaction with chemically modi

ed polysaccharides, such as

dialdehyde starch (Langmaier, Mokrejs, Kolomaznik, & Mladek,2008) or dextran dialdehydes (Schacht et al., 1993).

The high swelling and water binding capacity of solubilizedcollagen and gelatin makes them suitable materials for reducingdrip loss and impairing juiciness in frozen sh or meat productswhen thawed or cooked, and where denatured protein has suffereda partial loss of its water-holding capacity. Borderías, Martí, andMontero (1994) used collagenous material from freeze-driedplaice skin to improve the water-holding ability and the sensoryproperties of cod mince during frozen storage. The improvementsin sensory characteristics were due not only to the prevention of drip loss, but also because upon heating the sh mince portion, thecollagenous material gelatinized, giving it a much more desirabletexture than portions without this material.

The application of sh gelatinas an additive in surimi processingto improve water retention in heat-set gels has also been examined.Alaska pollack surimi gels containing 7.5e15 g/kg of sh gelatinshowed improved expressible moisture; however, when 15 g/kgwere added it produced a disruptive effect which proveddetrimental to the mechanical properties of the gel, more so whenusing surimi of maximum quality (Hernández-Briones, Velázquez,Vázquez, & Ramírez, 2009). The gel-forming capability of

threadn bream (Nemipterus japonicus) mince was substantiallyincreased by adding gelatin (0.5%) from the skin of bigeye snapper(Binsi et al., 2009). In contrast, gelatin additions of 5% and 10%reduced the elastic modulus (G0) values considerably, possibly dueto water molecule entrapment by high gelatin concentrationsmaking them unavailable for protein gelation. Accordingly, gelatinwas considered to be an inactive binder with little or no interactionbetween ller particles and gel matrix. However, when examiningthe gelling prole of chicken myosin and pork skin gelatin mixturesupon heating from 25 to 80 C, Yang, Zhou, Xu and Wang (2007)found that G 0 values were signicantly higher than those of puremyosin, which meant that a possible myosinegelatin interaction,likely of an electrostatic nature, had led to higher gel elasticity,suggesting the usefulness of this gelatin application in restructured

(chicken) meat products. In a much more simplied application,washed and defatted poultry skin was included in the formulationof bologna (an emulsied meat product) (Bonifer, Froning,Mandigo, Cuppett, & Meagher, 1996). The authors found that theincorporation of skin (10%) signicantly improved sensory prop-erties (texture, avour and appearance) without affecting nega-tively the emulsion stability or textural parameters in the nalproduct.

4.2. Surface properties

Collagen and gelatin surface properties are based on the pres-ence of charged groups in the protein side chains, and on certainparts of the collagen sequence containing either hydrophilic or

hydrophobic amino acids. Both hydrophobic and hydrophilic partstend to migrate towards surfaces, hence reducing the surfacetension of aqueous systems and forming the required identicallycharged lm around the components of the dispersed phase, whichcan be additionally strengthened by gel formation (Schrieber &Gareis, 2007).

Type A gelatins, with a relatively high isoelectric point (pI 7.0),are suitable for creating oil-in-water emulsions with a positivecharge over a wider range of pH values than is possible withconventional protein emulsiers, such as soy, casein or wheyproteins (Dickinson & Lopez, 2001). Early studies by Kim, Jeon, Lee,and Lee (1996) reported that cod bone gelatin had emulsifyingproperties similar to those of a commercial emulsier, such asTween-80. As is often the case with gelling properties, the emulsion

capacity of gelatin from

sh species is frequently lower than that



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from mammalians. Apart from the distribution of charge, animportant criterion in selecting a suitable gelatin type is gel rm-ness, because, at the same temperature and concentration, thehigher the gel rmness is, the rmer the gel-like protective sheathis around the oil droplets (Schrieber & Gareis, 2007). For example,the emulsion activity index of tuna n gelatin was lower than forpig skin gelatin at the same protein concentration (Aewsiri et al.,2008). With both types of gelatin, the emulsion capacityincreased with increasing protein concentration from 2 to 5%, withhigh protein concentrations facilitating more protein adsorption atthe interface. However, oil-in-water emulsions could be preparedusing a relatively low concentration of gelatin (0.05%), extractedfrom the skin of bigeye snapper sh (Binsi et al., 2009). Theaforementioned authors attributed the higher value of emulsioncapacity recorded by increasing gelatin concentrations (0.1 and0.2%) to a higher degree of polypeptide unfolding during theshearing involved in the emulsifying process.

Besides protein concentration, the molecular weight could bea key factor inuencing the ability of gelatin to form and stabilizeoil-in-water emulsions. In this respect, low-molecular weight shgelatin (w55 kDa) emulsion contained more large droplets andexhibited more oil destabilization than high molecular weight sh

gelatin (w120 kDa) (Surh, Decker, & McClements, 2006). Theseauthors also observed the presence of a small population of largedroplets in the emulsions after homogenization, which wasattributed to the relatively low surface activity of sh gelatincompared with globular proteins such as b-lactoglobulin.

Being an insoluble protein, collagen may be presumed to havelittle signicance as an emulsier. However, as early as 1973,Satterlee, Zachariah, and Levin (1973) pointed out that, whenadequately processed, the use of collagen afforded advantages withrespect to milk proteins. Forthisreason, isolated or partially puriedcollagen needs to be in a soluble form (acid-soluble or pepsin-soluble collagen) in order to display its surface active properties,whichmaydifferconsiderablyfromonerawmaterialtoanother.Theemulsifying capacity of acid-soluble collagen from hake skin was

found tobe higher than that for trout skin, in both cases being lowerthan in the corresponding muscle connective tissues (Montero &Borderías, 1991). In a later study, the acid-soluble collagen fromPacic whiting surimi renerdischarge was found to present higheremulsifying activity than both acid-soluble collagen from skin andthe commercial emulsier, Tween-80 (Kim & Park, 2004).

Gelatin and soluble collagen exhibit suitable foaming properties,even without gelling, because they are able to reduce the surfacetension at the liquid/air interface by increasing the viscosity of theaqueous phase (Schrieber & Gareis, 2007). Foaming propertiesdepend to a great extent on the characteristics of the raw material.For adsorption at the airewater interface, molecules should containhydrophobic regions which become more exposed upon proteinunfolding, thus facilitating foam formation and stabilisation

(Townsend & Nakai, 1983). In this respect, the foam capacity of gelatin from farmed giant catsh was found to be higher than thatfrom calf skin, the difference possibly being due to the highercontent of hydrophobic amino acid residues in the former. More-over, the almost 4 times greater viscosity of the farmed giant catshskin gelatin was also one of the main reasons for its better foamstability ( Jongjareonrak et al., 2010).

4.3. Film-forming properties

Gelatin has been extensively studied on account of its lm-forming ability and its usefulness as an outer lm to protect foodfrom drying and exposure to light and oxygen (Arvanitoyannis,2002). Furthermore, there is an important trend in favour of the

use of biodegradable

lms made from edible biopolymers from

renewable sources to combat the environmental impact of plasticwaste (Tharanathan, 2003). The highly hygroscopic nature of gelatin is its main drawback when considering the use of gelatinlms as protective barriers, because they tend to swell or dissolvewhen in contact with the surface of foodstuffs with high moisturecontent. Consequently, the current trend in designing gelatin-basedbiodegradable materials for food packaging or biomedical appli-cations is focused on developing lms with improved mechanicaland water resistance properties, by combining gelatin withbiopolymers with different characteristics, such as lipids (Bertan,Tanada-Palmu, Siani, & Grosso, 2005; Limpisophon, Tanaka, &Osako, 2010; Pérez-Mateos, Montero, & Gómez-Guillén, 2009),soy protein isolates (Cao, Fu, & He, 2007; Denavi et al., 2009),polysaccharides as gellan (Lee, Shim, & Lee, 2004), konjac gluco-mannan (Li, Kennedy, Jiang, & Xie, 2006), chitosan (Arvanitoyannis,Nakayama, & Aiba, 1998) pectins (Farris et al., 2009; Liu, Liu,Fishman, & Hicks, 2007), new hydrophobic or hydrophilic plasti-cizers (Andreuccetti, Carvalho, & Grosso, 2009; Cao, Yang, & Fu,2009), synthetic polymers such as poly(vinyl) alcohol (Carvalhoet al., 2009) or polyethylene (Haroun, Beherei, & Abd El-Ghaffar,2010a), as well as cross-linking agents, such as glutaraldehyde(Bigi, Cojazzi, Panzavolta, Rubini, & Roveri, 2001), TGase or 1-ethyl-

3-(3-dimethylamino-propyl) carbodiimide (EDC) (Ko1odziejska &Piotrowska, 2007; Yi, Kim, Bae, Whiteside, & Park, 2006).

The effect on lm properties of gelatin attributes, especiallyfrom sh origin, was reviewed recently by Gómez-Guillén, Pérez-Mateos et al. (2009). The molecular weight distribution andamino acid composition, which are the main factors inuencing thephysical and structural properties of gelatin, are also believed toplay a key role in the mechanical and barrier properties of theresulting lms. Weaker and more deformable lms are normallyobtained when low-molecular weight fragments predominate ina given gelatin preparation, which may be caused by protein heatdegradation during the water extraction step (Muyonga, Cole, &Duodu, 2004a) or during the evaporation step (Carvalho et al.,2008). Furthermore, lms made using a lower-molecular weight

gelatin were found to be more plasticized, as a result of the higherplasticizer:biopolymer molar ratio (Thomazine, Carvalho, & Sobral,2005). When gelatins come from different species, attention mustalso be paid to the amino acid composition, especially that of themost characteristic amino acids Gly, Pro and Hyp. A recent studycompared lms made from tuna-skin gelatin and bovine hidegelatin, and found that the tuna gelatin lms, having a loweramount of Pro þ Hyp residues (185 vs 210), presented breakingdeformation values approximately 10 times higher than those forbovine-hide gelatin lms (Gómez-Estaca, Montero, Fernández-Martín, & Gómez-Guillén, 2009). The pyrrolidine rings of theimino acids may impose conformational constraints, impartinga certain degree of molecular rigidity that can affect lm deform-ability. Films made from warm-water sh species, such as Nile

perch or channel catsh, have exhibited mechanical and watervapour barrier properties comparable to those of lms made frommammalian gelatin (Muyonga et al., 2004b; Zhang, Wang, Herring,& Oh, 2007). Avena-Bustillos et al. (2006) observed that the watervapour permeability (WVP) of cold-water sh-gelatin lms wassignicantly lower than that of lms made from warm-water shgelatin or mammalian gelatin, and based their explanation forthese differences on the higher amounts of hydrophobic aminoacids and lower levels of hydroxiproline in cold-water sh gelatins.A higher hydrophobic amino acid content in blue shark gelatin hasalso been put forward as the main reason for the lower WVP in theresultinglms, as compared to otherlms fromsh and land-basedanimal gelatins (Limpisophon, Tanaka, Weng, Abe, & Osako, 2009).

Gelatin has been reported to be one of therst materials used as

a carrier of bioactive components (Gennadios, McHugh, Weller, &



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Krochta, 1994). Enriching gelatin lms with natural antioxidantsand/or antimicrobial substances will extend the functional prop-erties of these biodegradable lms and provide an active packagingbiomaterial. Because of “clean labelling” concerns, there is growinginterest in using plant extracts as natural sources of polyphenoliccompounds in the formulation of gelatin lms. Aqueous extractsfrom leaves of murta (Ugni molinae) (Gómez-Guillén, Ihl, Bifani,Silva, & Montero, 2007), oregano (Origanum vulgare) and rose-mary (Rosmarinus of cinalis) (Gómez-Estaca, Montero, Fernández-Martín, Alemán, & Gómez-Guillén, 2009; Gómez-Estaca, Bravo,Gómez-Guillén, Alemán, & Montero, 2009), as well as a water/alcohol extract from borage (Borago of cinalis) seeds (Gómez-Estaca, Giménez, Montero, & Gómez-Guillén, 2009) have beenused to add antioxidant capacity to sh gelatin lms. In thesestudies, a slight decrease in either breaking force or breakingdeformation was observed depending on the added extract,differences being attributed to the qualitative and quantitativecomposition of the plant extracts used, which might have inu-enced the degree of proteinepolyphenol interactions (Gómez-Guillén, Pérez-Mateos et al., 2009). Furthermore, proteineproteininteraction capacity specic to the type of gelatin, could also bea determinant factor inuencing the polyphenol contribution to

protein matrix reinforcement or disruption. Thus, Gómez-Estaca,Montero, Fernández-Martín, Alemán et al. (2009) found thatbovine-hide gelatin lm properties were much less affected by theinclusion of oregano and rosemary aqueous extracts than by tuna-skin gelatin lms, in which proteinepolyphenol interactions weremore evident, causing a slight decrease in deformability andincreased water solubility.

Based on their capacity to induce non-disulde covalent inter-actions with gelatin polypeptide chains, oxidized phenols fromoxygenated seaweed extracts have been proposed as natural non-toxic cross-linking agents for improving the elongation of shgelatin lms(Rattaya, Benjakul, & Prodpran, 2009). In line with this,grapefruit seed and green tea extracts were found to increase thetensile strength and decrease WVP when included in blend lms

made of Gelidium corneum, red seaweed rich in agarose, and gelatin(Hong, Lim, & Song, 2009). Another polyphenolic extract from

Acacia nilotica bark, rich in catechin and gallic acid derivatives, hasbeen reported to induce hydrogen bonding and hydrophobicinteractions, the two major forces involved in the stabilization of cross-linked gelatin-poly(acrylamide)co-acrylic acid compositelm (Haroun & El Toumy, 2010).The A. nilotica bark extract stabi-lized the gelatin molecules in the blended networks and promoteda reduction in both swelling and degradation of the polymericbiocomposite lms.

4.4. Microencapsulation

Gelatin coacervates complexed with anionic polymers in theform of microcapsules are of special interest as they can entrapfunctional components in a carrier and provide protection againstoxidation or degradation during storage. Moreover, encapsulationcan be used to control the release of functional components fromthe food product or the bioactive packaging when ingested in thebody. Coacervates are formed when a mixed dilute solution of gelatin and an anionic polyelectrolyte are brought to a pH at whichthe polyelectrolytes have opposite net charges. Under theseconditions the solution separates into a highly concentrated coac-ervate phase in the form of microdroplets and a dilute bulk phase.The liquid coacervate envelopes the liquid drops or solid particlesthat are present in the solution, encapsulating them on a micro-scale; hence the term “microencapsulation” (Schrieber & Gareis,

2007).

The use of complex systems and natural cross-linkers has alsobeen reported as a means of reinforcing gelatin microcapsules. Inthis respect, plant-derived polyphenols and avonoids (containedin instant coffee and grape juice) have been shown to react withgelatinepectin-based microparticular coacervates under oxidizingconditions to form covalent cross-links. The resulting microparti-cles present a structure with greater mechanical strength andthermal stability, and less capacity to expand and absorb water,which could nd a practical application as a reduced calorie fatreplacer, avour binder or texturizer (Strauss & Gibson, 2004).A complex coacervation microcapsule system has been used toencapsulate baking avour oil in a complex of gelatin-gum Arabicsystem to improve the appeal of frozen baked foods upon heatingbycontrolling the rate of oil release (Yeo, Bellas, Firestone, Langer, &Kohane, 2005).

Lycopene extract from tomato pulp waste, which is highlysusceptible to oxidation and isomerization reactions, could havea possible application in the manufacture of functional foodformulations as it has been microencapsulated, producing anemulsion system with porcine skin type-A gelatin and poly(g-glu-tamic acid) as carriers (Chiu et al., 2007). The release of lycopenefrom the microcapsules occurred rapidly at pH 5.5 and 7.0, while no

lycopene was released at pH 2.0 and 3.5, which means that theunstable constituents can remain intact in the stomach and then bereleased into the intestine over a range of physiological pH values.

Incorporation of probiotic bacteria in functional food productshas become an increasingly popular trend because of their ability toexert benecial effects on intestinal microora (Gobbetti, Di Cagno,& de Angelis, 2010). Microcapsules based on gelatin, using extru-sion and spray-drying technology, have also been developed toimprove the survival of lactic acid bacteria (Li, Chen, Cha, Park, &Liu, 2009; Weissbrodt & Kunz, 2007) and bidobacteria (Lian,Hsiao, & Chou, 2002) against harmful conditions during process-ing as well as against the aggressive conditions of the stomach.

Furthermore, encapsulation technology could be used for thedevelopment of active packaging materials based on the incorpo-

ration of bioactive compounds, for example, plant polyphenolicextracts in gelatin lms. Apart from protecting the active potentialof the natural extract and modulating its release into the coveredfood product, encapsulation may be useful for taste and odourmasking purposes, since vegetal extracts often have very strongavours. Bao, Xu, and Wang (2009) incorporated chitosan nano-particles containing tea polyphenol in sh skin gelatin lms toprovide them with antioxidant capacity, and found sh oil oxida-tion to be effectively retarded when packed with this compositematerial. The nanoparticles greatly reduced the tensile strengthand oxygen permeability in the resulting lms but increasedits water vapour permeability. A gelatin/poly(L -lactide) (PLLA)composite has been suggested for producing homogeneous andwell-shaped nanobres by electrospinning, using gelatin extracted

from channel catshskins, asa rst step in the development of newmaterial with applications in the biomedical eld (An, Liu, Guo,Kumar, & Wang, 2010). The addition of PLLA signicantlyincreased the diameters of the e-spun gelatin bres, as well as thestrength and elastic behaviour of the composite brous material.

5. Bioactive properties of hydrolysates

Dietary proteins are a source of biologically active peptides,which are inactive in the parent protein sequence but can beliberated during gastrointestinal digestion, food processing orfermentation. Once they are released, bioactive peptides can affectnumerous physiological functions of the organism. Collagen andgelatin have been focused on as a source of biologically active

peptides with promising health bene

ts for nutritional or



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pharmaceutical applications. Usually, collagen and gelatin hydro-lysates and peptides have been produced from pig skin or bovinehide ( Jia et al., 2009). However, outbreaks of mad cow disease andthe banning of collagen from pig skin and bone in some regions forreligious reasons have made it necessary to nd new marine orpoultry sources, that are safer and healthier for consumers. Nor-mally discarded collagenous materials from the poultry and shprocessing industries, have been found to be valuable sources of hydrolysates and peptides with bioactive properties (Cheng, Liu,Wan, Lin, & Sakata, 2008; Cheng et al., 2009; Nam, You, & Kim,2008; Saiga et al., 2008). These collagenous materials mayinclude skins, tunics, bones, ns and scales. Furthermore, theisolation of peptides with important biological activities has beenreported for collagen and gelatin obtained from other sources like

jellysh, bullfrog or sea cucumber (Qian, Jung, & Kim, 2008; Wanget al., 2010; Zhuang, Sun, Zhao, Hou, & Li, 2010; Zeng et al., 2007;Zhao et al., 2009; Zhuang et al., 2009; Zhuang, Zhao & Li, 2009).

Gelatin and collagen-derived hydrolysates and peptides aregenerally obtained by enzymatic proteolysis. A number of commercial proteases have been used for the production of thesehydrolysates and peptides, including trypsin, chymotrypsin, pepsin,alcalase, properase E, pronase, collagenase, bromelain and papain

(Kim, Kim, Byun, Nam, Joo & Shahidi, 2001; Lin & Li, 2006; Mendis,Rajapakse, Byun, & Kim, 2005; Yang, Ho, Chu, & Chow, 2008).Besides commercial proteases, enzymatic extracts from sh viscerahave been used to obtain bioactive hydrolysates from skin andbones of different sh species ( Jung, Park, Byun, Moon, & Kim,2005; Phanturat, Benjakul, Visessanguan, & Roytrakul, 2010).Protease specicity affects size, amount, free amino acid composi-tion and, peptides and their amino acid sequences, which in turninuences the biological activity of the hydrolysates (Chen,Muramoto, & Yamauchi, 1995; Jeon, Byun, & Kim, 1999; Wu,Chen, & Shiau, 2003). Alcalase, which is a commercial proteasefrom a microbial source, has been used in numerous studies dealingwith gelatin/collagen hydrolysis because of its broad specicity aswell as the high degree of hydrolysis that can be achieved in

a relatively short time under moderate conditions (Benkajul &Morrissey, 1997; Diniz & Martin, 1996). This enzyme manifestedextensive proteolytic activity during the hydrolysis of skin gelatinfrom Alaska pollack, squid Todarodes paci cus and giant squid,producing hydrolysates with low average molecular weight whichexhibited high antioxidant activity and ACE inhibitory capacity(Giménez, Alemán, Montero, & Gómez-Guillén, 2009; Kim, Kim,Byun, Nam et al., 2001; Nam et al., 2008).

The average molecular weight of protein hydrolysates is one of the most important factors which determines their biologicalproperties ( Jeon et al., 1999; Park, Jung, Nam, Shahidi, & Kim, 2001).Peptide fractions from protein hydrolysates may vary in theireffectiveness for a given biological activity. An ultraltrationmembrane system could be a useful and industrially advantageous

method for obtaining peptide fractions with a desired molecularsize and a higher bioactivity, depending on the composition of thestarting hydrolysate and the activity being studied (Cinq-Mars & Li-Chan, 2007; Jeon et al., 1999; Korhonen & Pihlanto, 2003; Picotet al., 2010). This system has been successfully applied in thefractionation and functional characterization of gelatin hydroly-sates from squid or cobia skins (Lin & Li, 2006; Yang, Ho, et al.,2008;Yang, Wang, et al., 2008);and alsoas a rst step in the isolation andfurther purication of bioactive peptides from both non-collage-nous and collagenous sources (Alemán, Giménez, Pérez-Santín,Gómez-Guillén, & Montero, 2011; Hai-Lun, Xiu-Lan, Cai-Yun, Yu-Zhong, & Bai-Cheng, 2006; Hernández-Ledesma, Amigo, Ramos, &Recio, 2004; Hernández-Ledesma, Dávalos, Bartolomé, & Amigo,2005; Kim, Kim, Byun, Nam et al., 2001; Mendis, Rajapakse, Byun

et al., 2005; Saiga et al., 2008; Zhao et al., 2007).

Most of the studies about collagen and gelatin-derived peptidesin the area of food science and technology have dealt with theirantioxidant and antihypertensive/ACE inhibitory activity. Thesepeptides have repeated unique Gly-Pro-Hyp sequences in theirstructure, and the observed antioxidative and antihypertensiveproperties have presumably been associated with this uniqueamino acid composition (Kim & Mendis, 2006). Moreover, collagenand gelatin-derived peptides have exhibited numerous otherbioactivities, namely: antimicrobial activity, mineral bindingcapacity, the lipid-lowering effect, immunomodulatory activity andbenecial effects on skin, bone or joint health (Gómez-Guillén et al.,2010; Hou et al., 2009; Jung et al., 2005; Jung et al., 2006;Moskowitz, 2000; Zhang, Kouguchi, Shimizu, Sato, Takahata &Morimatus, 2010).

Some studies have been performed to conrm the in vivo bio-logical activity of collagen and gelatin peptides, and someconvincing data have been obtained for animal models. Moreover,human studies have also been carried out with positive results,especially those related to the capacity shown by collagen andgelatin hydrolysates to improve joint conditions. Because of thisability, collagen and gelatin hydrolysates, mainly obtained frommammalian sources, have long been used in pharmaceutical and

dietary supplements (Adam, 1991; Benito-Ruiz et al., 2009; Beuker& Rosenfeld, 1996; Krug, 1979; Moskowitz, 2000; Zuckley et al.,2004). Fish skin collagen hydrolysates have been reported toaffect lipid absorption and metabolism in rats (Saito, Kiyose,Higuchi, Uchida, & Suzuki, 2009). Besides the lipid-loweringeffect, chicken bone collagen hydrolysates have been shown toreduce proinammatory cytokine production in mice (Zhang et al.,2010). Bone mineral density in osteoporotic rats and joint disease indogs were improved by ingesting chicken and porcine gelatin andcollagen hydrolysates (Beynen, van Geene, Grim, Jacobs, & van derVlerk, 2010; Han et al., 2009; Watanabe-Kamiyama et al., 2010). Incontrast, bovine collagen hydrolysate consumption did not produceany effects on bone metabolism as measured by biochemicalindices of bone remodelling in postmenopausal women (Cúneo,

Costa-Paiva, Pinto-Neto, Morais, & Amaya-Farfan, 2010). Recently,some peptides from marine sources were reported to act protec-tively against ultraviolet radiation-induced damage on mice skin(Hou et al., 2009; Zhuang, Hou, Zhao, Zhang, & Li, 2009).

5.1. Antimicrobial properties

Published information on the antimicrobial properties of hydrolysates or peptides from collagen or gelatin is very scarce.Gómez-Guillén et al., 2010 reported antimicrobial activity inpeptide fractions from tuna and squid skin gelatins within a rangeof 1e10 kDa and


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such as amino acid composition, sequence, molecular weight andtype of bacteria need to be taken into account (Di Bernardiniet al., 2011). The hydrophobic character of amino acids wouldlet peptides enter the bacterial membrane, as the positive chargewould initiate the peptide interaction with the negatively chargedbacteria surface (Wieprecht et al., 1997). On the other hand, thedifferences existing in membrane composition have implicationsin the mode of action and the specicity of the antibacterialcompounds (Floris, Recio, Berkhout, & Visser, 2003). Patrzykatand Douglas (2005) reported that the degree of lipopolysaccha-ride (LPS) binding is neither directly nor inversely proportional topeptide activity because, following the rupture of the outermembrane, peptide activity would depend on its ability tointeract with the bacterial cytoplasmic membranes. Thus, boththe sequence and concentration of the peptide and the compo-sition of the bacterial membranes would inuence the mode of interaction.

Collagen per se has also been described as a biomaterial withbactericidal and fungicidal properties when applied as a coating towool-based materials ( Jus, Kokol, & Guebitz, 2009). However, ascollagen takes part of the extracellular matrix it may be involved inbinding some bacteria to the host tissues, for instance, Pseudo-monas aeruginosa, which has been shown to readily adhere toa matrix rich in type I collagen (Plotkowski et al., 1991).

5.2. Antioxidant activity

Since the antioxidant effect of peptides was rst reported(Marcuse, 1960), numerous studies have investigated hydrolysateand peptide antioxidant properties from both plant and animalsources such as rice bran (Revilla et al., 2009), sunower protein(Megías et al., 2008), alfalfa leaf protein (Xie, Huang, Xu, & Jin,2008), casein (Suetsuna, Ukeda, & Ochi, 2000), egg-yolk protein(Sakanaka & Tachibana, 2006), mackerel muscle protein (Wu et al.,2003), squid skin gelatin(Mendis, Rajapakse, Byun et al., 2005), shskin gelatin (Mendis, Rajapakse & Kim, 2005), bovine skin gelatin

(Kim, Kim, Byun, Park & Ito, 2001), tuna backbone ( Je, Qian, Byun, &Kim, 2007), tuna cooking juice (Hsu,Lu, & Jao, 2009),etc. Due totheenormous volume of sh processing waste generated annually,a great deal of attention has been paid to the production of

antioxidant hydrolysates and peptides from skin gelatin of differentsh species, such as Alaska pollack (Kim, Kim, Byun, Nam et al.,2001), hoki ( Johnius belengerri) (Mendis, Rajapakse & Kim, 2005),cobia (Rachycentron canadum) (Yang, Ho, et al., 2008; Yang, Wang,et al., 2008) and sole (Giménez et al., 2009), as well as fromseveral squid species, such as giant squid (Dosidicus gigas)(Giménez et al., 2009; Mendis, Rajapakse, Byun et al., 2005), Jumboying squid (Dosidicus eschrichitii Streenstrup) (Lin & Li, 2006) orsquid (Todarodes paci cus) (Nam et al., 2008). Some of the antiox-idant peptide sequences isolated from sh skin gelatin and othercollagenous sources are shown in Table 1.

The exact mechanism underlying the antioxidant activity of peptides is not fully understood, but various studies have shownthat they are lipid peroxidation inhibitors, free radical scavengersand transition metal ion chelators. According to some studies,gelatin peptides could inhibit lipid peroxidation more ef cientlythan antioxidative peptides derived from many other proteinsources (Kim, Kim, Byun, Park,& Ito, 2001). In addition, it has beenreported that collagen and gelatin antioxidative peptides mayprotect living cells against free radical mediated oxidative damage.Therefore, scavenging of free radical species is an importantmechanism by which antioxidant peptides enhance cell viability

against oxidation-induced cell death. Kim, Kim, Byun, Nam et al.(2001) reported that a peptide isolated from Alaska pollack skingelatin was able to protectrat liver cells from oxidant injury inducedby organic hydroperoxide t-BHP. In the same manner, two puriedpeptides from squid skin gelatin exhibited a dose-dependent cellviability enhancement effect when their ability to overcome t-BHP-induced cytotoxicity was tested in human lung broblasts (Mendis,Rajapakse, Byun et al., 2005). Moreover, results from one studyrevealed that peptides isolated from hoki skin gelatin were capableof enhancing the expression of antioxidative enzymes such asglutathione peroxidase, catalase and superoxide dismutase inhuman hepatoma cells (Mendis, Rajapakse & Kim, 2005).

Peptide antioxidative properties are related to their amino acidcomposition, structure and hydrophobicity. The amino acid

composition of collagen and gelatin hydrolysates is very similar tothat of the parent proteins, being rich in residues of Gly, Ala, Pro,Hyp, Glx and Asx, but poor in Met, Cys, His and Tyr ( Alemán,Giménez, Pérez-Santín et al., 2011; Gómez-Guillén et al., 2010;

Table 1

Antioxidant peptides derived from collagenous sources.

Source Characteristic Preparation Activity Reference

Fish skin gelatin(Alaska Pollack)

Gly-Glu-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly

Serial digestion (Alcalase,Pronase E, collagenase)

Inhibition of lipid peroxidation Kim, Kim, Byun,Park et al. (2001)

Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly

Increase of cell viability exposedto t -BHP

Squid skin gelatin(Dosidicus gigas)

Phe-Asp-Ser-Gly-Pro-Ala-Gly-Val-Leu Trypsin Radical scavenging Mendis, Rajapakse,Byun et al. (2005)Asn-Gly-Pro-Leu-Gln-Ala-Gly-Gln-Pro-

Gly-Glu-ArgIncrease of cell viability exposedto t -BHP

Squid tunic gelatin(Dosidicus gigas)

Gly-Pro-Leu-Gly-Leu-Leu-Gly-Phe-Leu-Gly-Pro-Leu-Gly-Leu-Ser

Alcalase Radical scavenging Alemán, Giménez,Pérez-Santín et al. (2011)Ferric reducing power

Fish skin gelatin( Jonius belengerii)

His-Gly-Pro-Leu-Gly-Pro-Leu Trypsin Radical scavenging Mendis, Rajapakse, andKim (2005)Inhibition of lipid peroxidation

Increase of antioxidativeenzyme levelsin hepatoma cells

Porcine skin collagen Gln-Gly-Ala-Arg Mixture of proteases(bovine pancreas, Streptomycesand Bacillus spp)

Radical scavenging Li et al. (2007)

Bovine skin gelatin Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly

Pronase E Inhibition of lipid peroxidation Kim, Byun,Park et al. (2001)Increase of cell viability exposed

to t -BHPTuna backbone Val-Lys-Ala-Gly-Phe-Ala-Trp-Thr-

Ala-Asn-Gln-Gln-Leu-SerPepsin Radical scavenging Je et al. (2007)

Inhibition of lipid peroxidation



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Kim, Kim, Byun, Nam et al., 2001; Mendis, Rajapakse, Byun et al.,2005). Dávalos, Miguel, Bartolomé, and López-Fandiño (2004)studying individual amino acid activity reported that Trp, Tyr andMet showed the highest antioxidant activity, followed by Cys, Hisand Phe. The rest of the amino acids did not show any antioxidantactivity. However, many peptides have been described as havingantioxidant capacity without containing anyof the above mentionedproton-donating amino acid residues in their sequences. Thus, Kim,Kim, Byun, Nam et al. (2001) isolated two peptides composed of 13and 16 amino acid residues, respectively from Alaska pollack skin,both of which contained a Gly residue at the C-terminus and therepeating motif Gly-Pro-Hyp. Li, Chen, Wang, Ji, and Wu (2007)identied the peptide which exhibited the highest antioxidantactivity from porcine skin collagen hydrolysates as Gln-Gly-Ala-Arg. The peptide Asn-Gly-Pro-Leu-Gln-Ala-Gly-Gln-Pro-Gly-Glu-Arg was puried from squid skin gelatin with a valuable free radicalquenching capacity (Mendis, Rajapakse, Byun et al., 2005).Furthermore, the antioxidant activity of collagen and gelatinpeptides has been linked to the high content of hydrophobic aminoacids, which could increase their solubility in lipids and thereforeenhance their antioxidative activity (Kim, Kim, Byun, Nam et al.,2001). Rajapakse, Mendis, Byun, and Kim (2005) found that sh

skin gelatin peptides showed higher antioxidant activity thanpeptides from meat protein, probably because of the higherpercentage of Gly and Pro.

Not only is the presence of proper amino acids essential, buttheir correct positioning in the peptide sequence plays an impor-tant role too in antioxidant activity. Peptide conformation has alsobeen claimed to inuence antioxidant capacity, showing bothsynergistic and antagonistic effects, as far as the antioxidantactivity of free amino acids is concerned (Hernández-Ledesmaet al., 2005). As mentioned above, protease specicity used forhydrolysis may determine the size and the sequence of thepeptides, and as a result their antioxidant activity. In differentstudies, it has been found that alcalase gelatin-derived hydrolysateantioxidant activity was higher than that of other enzyme hydro-

lysates such as those obtained by collagenase, pepsin, trypsin,chymotrypsin, papain or neutrase (Alemán et al., 2011; Qian et al.,2008). Moreover, antioxidant activity is strongly related topeptide molecular weight as demonstrated by Gómez-Guillén et al.(2010) who found antioxidant activity in all the peptide fractionsfrom squid skin hydrolysate, but that it was higher in the fractionswith lower-molecular weight. Similarly, the peptide fraction fromcobia skin hydrolysate, with most of the molecular mass valuesbelow 700Da showed the highest radical scavenging activity, beingapproximately 20% higher than that of the non-fractionatedhydrolysate (Yang, Ho, et al., 2008; Yang, Wang, et al., 2008 ).

5.3. Antihypertensive/ACE inhibitory activity

Antihypertensive peptides are peptide molecules which maylower blood pressure when ingested through inhibition of vaso-active enzymes such as the angiotensin converting enzyme (ACE).ACE plays an important role in the regulation of blood pressure bymeans of the rennin-angiotensin system, and inhibition of thisenzyme is considered to be a useful therapeutic approach in thetreatment of hypertension (Ondetti, Rubin, & Cushman, 1977; Chenet al., 2007).

Since the discovery of ACE inhibitory peptides in snake venom,many studies have tried to synthesize ACE inhibitors, such ascaptopril, enalapril, alacepril and lisinopril, which are used exten-sively in the treatment of hypertension and heart failure in humans(Ondetti, 1977; Patchett et al., 1980). However, synthetic ACEinhibitors are believed to produce certain side effects such ascoughing, taste disturbances, skin rashes or angioneurotic edema(Atkinson & Robertson, 1979). Therefore, in the last 10 years manyresearchers worldwide have concentrated on nding naturalsources of ACE inhibitors such as food proteins, which though lesspotent that the synthetic ones are without known side effects.Although the major natural source of ACE inhibitory peptides

identied to date is milk, these peptides have been isolated frommany other animal and plant protein sources, like blood proteins(Mito et al., 1996), ovalbumin (Miguel, Recio, Gómez-Ruiz, Ramos, &López-Fandiño, 2004), maize (Miyoshi et al., 1991), chickpea (Yustet al., 2003), soy (Wu & Ding, 2001) and muscle proteins frompig, cattle, sh and chicken (Ahhmed & Muguruma, 2010; Fujita &Yoshikawa, 1999; Fujita, Yokoyama, & Yoshikawa, 2000; Jang &Lee, 2005; Katayama et al., 2003). Collagen and gelatin have alsobeen shown to be good sources of antihypertensive peptides byenzymatic digestion, despite not having been so extensivelystudied as other sources. Potent ACE inhibitory hydrolysates andpeptides have been obtained from collagenous materials, not onlyfrom land-based sources such as porcine skin collagen (Anzai et al.,1997; Ichimura, Yamanaka, Otsuka, Yamashita, & Maruyama, 2009),

bovine skin gelatin (Kim, Byun, Park, & Shahidi, 2001), chicken legsand leg bone (Cheng et al., 2009; Saiga et al., 2008), but also frommarine sources such as sh skins (Byun & Kim, 2001; Nagai,Nagashima, Abe, & Suzuki, 2006; Park, Kim, Kang, Park, & Kim,2009), sh cartilage (Nagai et al., 2006), scales (Fahmi et al.,2004), squid tunics (Alemán, Giménez, Pérez-Santín, Gómez-Guillén & Montero, 2011) and sea cucumbers (Zhao et al., 2007).The peptide sequences identied from collagenous materials areshown in Table 2.

Although the relationship between the structure and theactivity of ACE inhibitory peptides has not yet been established,

Table 2

Antihypertensive/ACE inhibitory peptides derived from collagenous sources.

Source Characteristic Preparation Activity in vitro/in vivo Reference

Fish skin gelatin(Alaska Pollack)

Gly-Pro-Leu Serial digestion (Alcalase,Pronase E, collagenase)

IC50 2.6 mM Byun and Kim (2001)Gly-Pro-Met IC50 17.13 mM

Bovine skin gelatin Gly-Pro-Val Serial digestion (Alcalase,Pronase E, collagenase)

IC50 4.67 mM Kim, Byun, Park et al. (2001)Gly-Pro-Leu IC50 2.55 mM

Chicken leg collagen Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro

Protease FP IC50 29 mM Saiga et al. (2008)Decrease blood pressure in SHR Shimizu et al. (2010)Stimulation of nitric oxide synthesis

Chicken bone Tyr-Tyr-Arg-Ala Pepsin IC50 33.9 mm/ml Nakade et al. (2008)Porcine skin gelat in Gly-Phe-Hyp-Gly-Pro Protease ( Aspergillus oryzae) IC50 91 mM Decrease blood pressure

in SHR (10e30 mg/ml)Ichimura et al. (2009)

Gly-Pro IC50 360 mM Decrease blood pressurein SHR (500 mg/ml)

Squid tunic gelatin(Dosidicus gigas)

Gly-Pro-Leu-Gly-Leu-Leu-Gly-Phe-Leu-Gly-Pro-Leu-Gly-Leu-Ser

Alcalase IC50 90.03 mM Alemán, Giménez,Pérez-Santín et al. (2011)



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these peptides have certain common features. Most of them arerelatively short sequences with low-molecular mass, as the activesite of ACE cannot accommodate large peptide molecules. BindingtoACE is strongly inuenced by the C-terminal tripeptide sequence,which may interact with subsites at the active site of the enzyme(Ondetti & Cushman, 1982). ACE prefers substrates or inhibitorsthat contain hydrophobic amino acid residues (aromatic orbranched side chains) at each of the three C-terminal positions(Cheung, Wang, Ondetti, Sabo, & Cushman, 1980; Murray &FitzGerald, 2007). The presence of Arg or Lys on the C-terminalposition has also been reported to contribute substantially to theinhibitory activity (Ariyoshi, 1993; Cheung et al., 1980; Meisel,2003). The ACE inhibitory activity described for collagen andgelatin hydrolysates and peptides may be related to the highconcentration of hydrophobic amino acids, as well as to high Prolevels. This amino acid seems to be one of the most effective forincreasing ACE inhibitory activity and has been identied in manyof the naturally occurring ACE peptide inhibitors (Contreras,Carrón, Montero, Ramos, & Recio, 2009; Gómez-Ruiz et al., 2004a,2004b; Pihlanto, Akkanen, & Korhonen, 2008; Quirós et al., 2007),especially in those derived from collagenous sources (Table 2)(Alemán, Giménez, Pérez-Santín, Gómez-Guillén & Montero, 2011;

Byun & Kim, 2001; Ichimura et al., 2009; Kim, Byun et al., 2001;Saiga et al., 2008; Shimizu et al., 2010).

The in vivo effects of antihypertensive peptides are usuallytested in spontaneously hypertensive rats (SHR), which constitutean accepted model for human essential hypertension (FitzGerald,Murray, & Walsh, 2004). Many of the ACE inhibitory peptides iso-lated from collagenous sources have already been tested in vivo,and an antihypertensive effect has been reported. SHR experienceda signicant decrease in blood pressure following oral adminis-tration of both a chicken leg collagen hydrolysate and the isolatedoctapeptide Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro (Cheng et al., 2009;Iwai et al., 2008; Saiga et al., 2008). Furthermore, Faria, da Costa,Gontijo, and Netto (2008) found that bovine and porcine collagenhydrolysates produced a considerable reduction in blood pressure

in SHR after oral administration. In another study, the blood pres-sure of renal hypertensive rats was signicantly reduced byadministering the lowest fraction of sea cucumber gelatin hydro-lysate (Zhao et al., 2007). ACE inhibitory peptides Gly-Pro and Gly-Phe-Hyp-Gly-Pro, isolated from porcine skin collagen hydrolysatealso had an antihypertensive effect on SHR (Ichimura et al., 2009).

6. Conclusions

Gelatin production from alternative non-mammalian specieshad grown in importance, largely as a way to valorise by-productsfrom sh and poultry industrial processes. Regarding sh origin,tropical and sub-tropical warm-water species might have similarrheological properties and thermostability to that of mammal

gelatins; therefore, they could be used for similar applications.Recently, an increasing number of new applications have beenfound for gelatin or collagen in products such as emulsiers,foaming agents, colloid stabilizers, hydrogels, ning agents,biodegradable packaging materials, micro-encapsulating agents, aswell as bioactive peptides, in line with the growing trend to replacesynthetic agents with more natural ones. The main source forcollagen or gelatin extraction from sh are skins and bones,however more recently they have been also extracted from scalesand ns, processed sh offal, as well as from other aquatic organ-isms such as red sea cucumber or alligators. The huge number of available species had made necessary to adapt the extractionprocedures in order to optimize the properties of the resultingmaterial (in the form of collagen or gelatin). In the last decade,

further improvement of the characteristics of low-gelling gelatins

from alternative sources has been achieved by using complexsystems and natural cross-linkers. Additionally, the functionalproperties of collagen or gelatin hydrolysates have been focused onthe production of bioactive peptides with a number of biologicalactivities which have been previously described in other proteinsources. On the other hand, the great development of modernanalytical methods has allowed a deeper characterization of gelatinand collagen properties from all these new sources and of specialinterest could be their application for species identication.

Acknowledgements

This work was supported by the Spanish Ministry of Science andInnovation under projects AGL2008-00231/ALI and AGL2008-02135/ALI.

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