Review
Recent advances on the role of process variables affecting gelatin yield and characteristics with special reference to enzymatic extraction: A review
Graphical abstract
Introduction
Gelatin is a natural macromolecule material extracted from skins, bones and connective tissue of animals (Karim & Bhat, 2009). It is a high molecular weight biopolymer obtained by partial hydrolysis of collagen (Mohtar, Perera, & Quek, 2010).It exhibits different functional properties, such as water binding capacity, film-forming properties, foaming and emulsifying abilities, making it a versatile ingredient in food, pharmaceutical, photography and cosmetic industries (Gimenez et al., 2005a, Gómez-Guillén et al., 2011). Table 1 shows the uses of gelatin in different food categories with their recommended level and bloom strength.
Gelatin is obtained by controlled hydrolysis of collagen from various sources (hides, skins, ossein from bones, or sinews). Collagen is a structural protein having three α-chains intertwined and the stability to the triple helix structure is provided by inter-chain hydrogen bonding (Johnston-Banks, 1990, Te Nijenhuis, 1997). There are also very short terminal regions, called telopeptides, which do not form triple helical structures, where intermolecular attachment is mainly brought about by lysine and hydroxy-lysine crosslinks (Orgel, Irving, Miller, & Wess, 2006). There are different types of collagen. Type I collagen, found primarily in connective tissue such as skin, bone, and tendons (Schrieber & Gareis, 2007), is unique in forming a right-handed triple super helical structure consisting of three α (similar size) left-handed helical polypeptide chains. The collagen chain is also characterized by a Gly-X–Y repeating motif with the X and Y positions being mostly occupied by Pro and Hyp, respectively (Gelse, Poschl, & Aigner, 2003). The Type II, Type III and other types of gelatin are found in cartilage tissue, very young skin and organs, respectively.
During the gelatin manufacturing process, collagen is denatured and loses its native structure. Its helixes are partially reformed and are different from that of collagen. It forms a gel by trapping water in the mesh of chains. The structure of gelatin changes during gelation. The chains have different space arrangements and different interactions depending on the state of the gel (Guo, Colby, Lusignan, & Whitesides, 2003). These two characteristics are determined by the gelatin concentration, temperature and the energy required for the formation of the secondary structure. Two α-chains or one α-chain, which creates a loop, can form one double strand structure. Similarly, three different α-chains, or two α-chains one of which forms a loop, or only one α-chain with two loops can form a triple strand structure (Guo et al., 2003). The structure is determined by the molecular weight distribution of gelatin chains. α-chains present in the collagen have molecular weight of around 110 000 g/mole. In gelatin, these α-chains form β and γ chains have molecular weights of 200 000 g/mole and 300 000 g/mole, respectively (Guo et al., 2003). These β and γ chains are formed by linking the α-chains together by covalent bonds which are different from the double or triple strand structures forming helixes stabilized by weak bonds (Guo et al., 2003). β-chain is a double strand structure formed when two α-chains covalently link together while γ-chain is a triple strand structure, stabilized by covalent bonds, formed when three α-chains form a triple helix (Diaz et al., 2011, Taheri et al., 2009, Gómez-Guillén et al., 2011).
The gelatin manufacturing process consists of three major steps: i) removal of non-collagenous material from the collagenous material ii) controlled hydrolysis of collagen to gelatin and iii) the recovery and drying of the final product (Fig.1). The typical process of gelatin production can be classified into two methods: the acid process and the liming process. The acid process uses acid solution to hydrolyze collagen and the obtained product is called type A gelatin. Pigskins are the typical raw materials for this method and the processing duration is around 10–45 h. On the contrary, alkalis used to hydrolyze protein in the liming process yielding type B gelatin. Raw materials are pretreated with either acid or alkaline to allow the swelling of collagen in order to increase the efficiency of gelatin extraction during thermal hydrolysis. Typical raw materials in the liming process are bones and hides that usually take around 30–100 days (Damrongsakkul, Ratanathammapan, Komolpis, & Tanthapanichakoon, 2008). Apart from low gelatin yield and quality, the aforementioned gelatin production processes have several disadvantages, especially, the large amount of wastes generated and the limitation of raw materials. The isoelectric point of Type A gelatin is between 6.5 and 9 and that of Type B gelatin is between 4.8 and 5.2 (Gómez-Guillén et al., 2011). A helix-to-coil deformation of the triple helices of the collagen molecule is brought to produce loosely coiled gelatin polypeptide chains during thermal hydrolysis extraction step (Djabourov, Lechaire, & Gaill, 1993). In the industry, the thermal extraction step is usually carried out in several steps with gradually increasing temperatures starting from 50 to 60 °C at about 5–10 °C temperature increment, until boiling temperatures (Hinterwaldner, 1977).
The treatment of gelatin raw materials with acid or alkali brings about thermal solubilization of collagen molecules due to the cleavage of a number of intra- and intermolecular covalent cross-linked bonds in collagen. Hence, the gelatin obtained has molecular weights lower than the native collagen and constitutes a mixture of fragments with molecular weights in the range of 16–150 kDa (Sai-Ut, Jongjareonark, & Rawdkuen, 2012). A sufficient number of the covalent and non-covalent crosslinks in collagen must be broken in order to enable the release of free α-chains and oligomers (Johnston-Banks, 1990). Oligomers composed of three α-chains may remain as intact triple helices, but a significant percentage of extended polymers α -chains bonded randomly by end-to-end or side-to-side-bonds also exit. Apart from covalent links, there may be additional interactions of hydrophilic and hydrophobic nature between the gelatin chains.
The properties of gelatin are greatly influenced by the species or tissue from which it is extracted, and by the extraction process, which may depend on pH, temperature, and time during both pre-treatment and extraction process (Gómez-Guillén & Montero, 2001). These extraction variables also influence the length of the polypeptide chains formed and thus, the functional properties of gelatin (Kołodziejska, Skierka, Sadowska, Kołodziejski, & Niecikowska, 2008). The effects of different process variables on the gelatin yield, gel strength, viscosity, protein pattern and emulsion activity index are summarized in Table 2. Apart from these extraction variables, protease inhibitors are known to affect the properties of the gelatin from the skin of some fish species (Kaewruang et al., 2013, Nagarajan et al., 2012). The pretreatment process required for gelatin manufacture is dependent on the degree of collagen cross-linking present in it (Gimenez, Turnay, Lizarbe, Montero, & Gomez-Guillen, 2005b). The chemical pretreatment cleaves non-covalent bonds of the protein structure and thus effecting swelling and collagen solubilization (Stainsby, 1987). Subsequent heat treatment destabilizes the triple-helix, resulting in helix-to-coil transition and conversion into soluble gelatin by breaking the hydrogen and covalent bonds (Djabourov et al., 1993;; Gómez-Guillén et al., 2002). Swelling properties and subsequent solubilization of collagen is greatly influenced by the type and concentration of acid used depending on the persistence of some of the cross-links between collagen chains. This leads to variations in molecular weight distribution in the resultant gelatins. According to Asghar and Henrickson (1982), the lyotropic effect of non-ionized carboxylic acids (swelling agent) on collagen seems to dominate the swelling activity. This activity is achieved by competing with the peptide group through acid hydrogen bond. During acid pretreatment, the acid destabilizes the triple helical structure of collagen by disrupting acid labile cross-links at the telopeptide region and amide bonds of the triple helix as well as non-covalent intra and inter-molecular bonds (Benjakul, Oungbho, Visessanguan, Thiansilakul, & Roytrakul, 2009). Hence, the type of acid, acid concentration and pretreatment time influenced the physicochemical properties of the gelatin obtained (; Zhou and Regenstein, 2005, Boran and Regenstein, 2009) which led to variations in the distribution of molecular weight of the resultant gelatins.
An alternative approach to acid treatment is the use of proteolytic enzymes. Compared with the liming and acid processes, the enzymatic process has several advantages such as short processing time and much less waste generation (Damrongsakkul et al., 2008;; Pitpreecha & Damrongsakkul, 2006). Up to 80%–90% (depending on the source) of native skin collagens can be extracted by limited pepsin digestion using a defined enzyme to substrate ratio and low temperature (Kern, Menasehe, & Robert, 1991). Norziahn, Kee, and Norita (2014) used bromelain to extract gelatin from surimi processing waste with encouraging results. The extraction of gelatin from rawhide with papain enzyme enhanced the yield of gelatin (Damrongsakkul et al., 2008). Other studies have utilized pepsin and/or protease inhibitor to generate gelatin from bigeye snapper skin (Ahmad, Benjakul, Ovissipour, & Prodpran, 2011; Nalinanon, Benjakul, Visessanguan, & Kishimura, 2008 and; Intarasirisawat et al., 2007). Nalinanon et al. (2008) demonstrated that enzymatic extraction using bigeye snapper pepsin (BSP) enhanced the gelatin extraction efficiency but the resulting gelatin showed complete degradation of β, α 1 and α 2-chains. Pepsin has been reported to cleave peptides in the telopeptide region of native collagen, which contains the intra and inter molecular covalent crosslinks (Chomarat, Robert, Seris, & Kern, 1994). Therefore, pepsin can solubilize the collagen in the skin matrix during the acid-swelling process, by cleaving some peptide bonds, resulting in a higher efficacy in gelatin extraction. There was an improvement in the yield of gelatin extracted from fish using crude proteases extracted from the viscera of different fish (Balti et al., 2011, Bougatef et al., 2012; and; Ktari et al., 2014). However, the procedure caused the degradation of α and β chains (dimer of α chain) of the gelatin thereby decreasing the gelatin properties (Balti et al., 2011, Bougatef et al., 2012; and; Ktari et al., 2014). Pretreatment with pepsin led to higher gelatin yield but reduced gel strength (Lassoued et al., 2014). The presence of endogenous peptidases in the skin of different fish has been reported (Intarasirisawat et al., 2007; Nalinanon et al., 2008, Ahmad et al., 2011). Addition of endogenous protease inhibitor prevented the degradation of the resulting gelatin chains by inhibiting the endogenous proteases found in the different fish skin but it caused reduced gelatin yield (Intarasirisawat et al., 2007; Nalinanon et al., 2008 and; Ahmad et al., 2011). In view of the above facts, this review tries to cover the role of these processing variables on the gelatin extraction and its characteristics.
Section snippets
Fish skin
Gelatin extracted from splendid squid (Loligo formosana) skin at 50, 60, 70 and 80 °C, had a yield (dry weight basis) of 8.8%, 21.8%, 28.2%, and 45.3% respectively (Nagarajan et al., 2012). A relatively higher free amino group content was obtained for gelatin extracted at higher temperature (80 °C) than gelatin extracted at lower temperatures (50, 60 and 70 °C). High proline and hydroxyproline contents were obtained for the gelatin extracted at 50 and 60 °C. Significant loss of molecular order
The future research: the way forward
Acid or alkali is used to treat the gelatin raw materials resulting in the partial cleavage of the crosslinks resulting in breaking of the structure to the extent that “warm-water-soluble collagen”, i.e. gelatin, is formed (Schrieber & Gareis, 2007). Since collagen cross-links are stable to thermal and acid treatment (Galea et al., 2000), a low yield of the resulting gelatin is generally obtained. However, as an alternative to chemical pretreatments, which often take longer time and pollute the
Conclusions
Pretreatment processes using enzymes can produce a higher gelatin yield. Papain, neutrase, bromelain, pepsin, proctase and crude proteases extracted from the viscera of different fish used as pretreatment agents during gelatin extraction increased the gelatin yield but degradation the α and β chains of the resulting gelatin thereby lowering the gelatin quality particularly the gel strength. The degradation was caused by the continued action of added protease enzymes. This scenario justifies the
Acknowledgement
The first author acknowledges the Indian Council of Agricultural Research, New Delhi, India for providing ICAR-International Fellowship vide letter no F. No. 29-1/2009-EQR/Edn (pt.III) dated October 28, 2014 and Department of Agricultural Research & Education, Ministry of Agriculture, Government of India for granting study leave to him.
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