EXTRACTION OF GELATIN FROM FISH SKINS BY HIGH PRESSURE TREATMENT

Transcription

1 EXTRACTION OF GELATIN FROM FISH SKINS BY HIGH PRESSURE TREATMENT M.C. Gómez-Guillén*, B. Giménez and P. Montero Instituto del Frío (CSIC). Ciudad Universitaria. 0 Madrid, Spain. * Corresponding author. Tel: ; fax: address: cgomez@if.csic.es Abstract High pressure, at 0 and 00 MPa, for or 0 min, was applied at either of two stages: during pre-treatment in acid at ºC to facilitate destabilization of acid labile crosslinks, or during extraction in water at ºC to accelerate collagen hydrolysis. The resulting gelatins were evaluated in terms of yield of extraction, molecular weight distribution by SDS-PAGE, and viscoelastic properties of gelatin newly dissolved and after overnight cold maturation. Pressure level and time of treatment induced noticeable changes in molecular weight distribution and consequently affected viscoelastic properties. The use of high pressure to extract gelatin from fish skins is a useful alternative to the conventional procedure. Its utility lies basically in that the longest phase of the treatment can be drastically shortened, thus making it possible to produce a gelatin of high gelling quality in only a few minutes. Keywords: fish gelatin, extraction, high pressure, viscoelastic properties

2 Introduction Collagen, from which gelatin is derived, is the major structural protein found in skin and bones of animals. Collagen molecules, composed of three -chains intertwined in the so-called collagen triple helix, adopt a D structure which is an ideal geometry for interchain hydrogen bonding (Johnston-Banks, 10; te Nijenhuis, 1). In fact, the hydroxyproline content, through its OH group, has been reported to be largely responsible for the triple helix stabilisation (Burjandze, 1; Ledward, 1). There are also very short terminal regions, called telopeptides, which do not form triple helical structure, in which intra- and intermolecular covalent crosslinks, mainly involving lysine and hydroxylysine residues, are located. Because of the acid lability of cross-linking in fish skin collagen, mild acid treatment is normally enough to produce adequate swelling and cleavage of noncovalent intra- and inter-molecular bonds (Stainsby, 1; Montero, Borderías, Turnay & Lizarbe, 10; Norland, 10). Subsequent thermal treatment above 0ºC cleaves hydrogen and a number of covalent bonds; this destabilises the triple helix by means of a helix-to-coil transition, leading to conversion into soluble gelatin (Djabourov, Lechaire & Gaill, 1). High molecular weight polymers may arise in the resulting gelatin through the possible persistence of cross-links depending on the nature and degree of solubilisation. High pressure above 10 MPa has been shown to induce protein denaturation by disturbing the balance of the non-covalent interactions that stabilise the native conformation of many proteins (Masson, 1). However, collagen has been reported to be scarcely affected by high pressure, given that hydrogen bonds are chiefly pressure-insensitive (Gekko & Koga, 1; Heremans, 1). At relatively low pressure they could appear to be even somewhat strengthened, due to an associated small reduction in volume (Cheftel & Culioli, 1). No significant effects by high pressure treatment around MPa were found on intramuscular collagen solubility from beef (Suzuki, Watanabe, Ikeuchi, Saito &

3 Takahasi, 1) or pacific blue whiting (Fernández-Martín, Pérez-Mateos & Montero, 1). However, changes in bovine collagen solubility have been shown to varied with applied pressure; 00 MPa increased solubility, whereas 00 MPa reduced it (Kwiatkowska, Jankowska & Korzeniowski, 001). Regarding high pressure utilisation in gelatin, there are a few works focused on aspects related to induced gelation (Walkenstrom & Hermansson, 1; Montero, Fernández-Díaz & Gómez-Guillén, 00). However, no published articles are available in relation to gelatin extraction assisted by high pressure. Gómez-Guillén and Montero (001) reported a procedure for extracting gelatin with high gelling capacity from fish skins; the procedure was essentially based on a mild acid pre-treatment for collagen swelling followed by extraction in water at moderate temperature (ºC). The entire process takes around hours. The point of applying high pressure in the fish gelatin extraction process is mainly to cut down the treatment times and/or to improve the quality of the gelatin produced. In conventional gelatin manufacturing procedure, high pressure can be applied at two stages: either during pre-treatment in acid at ºC to facilitate destabilization of acid labile crosslinks, or during extraction in water at ºC to accelerate collagen hydrolysis. The aim of this work was to analyse the yield, molecular weight distribution and viscoelastic properties of gelatin extracted from fish skins using high pressure (0 or 00 MPa) either during mild acid pre-treatment at ºC or during extraction in water at ºC. Material and methods Gelatin extraction Skins from Dover sole (Solea vulgaris) were collected and stored frozen at 0ºC until use. Cleaning of thawed fish skins and gelatin extraction procedure was

4 carried out as previously described (Gómez-Guillén & Montero, 000). Briefly, the method consist in a mild acid (0 mm acetic acid) swelling step for h and subsequent overnight (1-1h) gelatin extraction in distilled water at moderate temperature (ºC). Extracted gelatin was air-dried until moisture was less than 1%. This procedure was considered as conventional treatment and the resulting gelatin was called control gelatin, in order to compare with the gelatins obtained by means of high pressure. For the pressure-assisted extractions, fish skins were frozen, thawed and cleaned as mentioned above. Pretreatment in 0 mm acetic acid at ºC was performed during min at 0 MPa, followed by extraction in water at ºC during 1-1ºC at atmospheric pressure (S 0 ). Samples extracted in water at ºC by high pressure were all preceded of a pre-treatment (at atmospheric pressure) in 0 mm acetic acid at ºC during hours. In substitution of a conventional overnight extraction during 1-1 h, high pressure was applied at 0 MPa for min (E 0 - ) or 0 min (E 0-0 ), and at 00 MPa for min (E 00 - ) or by two pulses of min each (E 00 -x ). Electrophoretic analysis (SDS-PAGE) Dry gelatin was dissolved in distilled water at 0ºC and then -fold-concentrated loading buffer containing mercaptoethanol was added until reach a final concentration of mg/ml of gelatine, as described previously by Gómez Guillén et al. (00). Protein samples were heat-denatured min at 0ºC and analysed by PAGE-SDS according to Laemmli (10) using % stacking gels and % resolving gels in a Mini Protean II unit (Bio Rad Laboratories, Hercules, CA) at ma/gel. The loading volume was 1 µl in all lines. Protein bands were stained with Coomassie brilliant Blue R0. Type I and type III collagens from fetal calf skin (Sigma Aldrich) were used as markers of -chain, - and -component mobilities. Viscoelastic properties Dynamic studies were performed on a Bohlin CRS- controlled stress rheometer rotary viscometer (Bohlin Instrumments Ltd., Gloucestershire, U.K.) using a cone-

5 plate geometry (cone angle º, gap=0.1mm), according to Gómez-Guillén, Turnay, Fernández-Díaz, Olmo, Lizarbe & Montero (00). Solutions were prepared by mechanical stirring for 1 0 min of pre-weighed (.% w/v) dry gelatin in water at 0ºC. Also the samples were cooled in a refrigerator at ºC (maturation temperature) for 1 1 h overnight to analyse the effect of maturation time on gelatin. Temperature ramps were performed at a scan rate of 1ºC/min, frequency 1Hz, and oscillating applied stress of.0 Pa. The phase angle (º) was represented as a function of temperature. Values of elastic modulus (G ) and viscosity modulus (G ) were taken at ºC. The ramps were implemented from 0ºC to ºC and back to 0ºC for newly dissolved gelatin, to study gelatin gelation and subsequent melting. Gelatin matured overnight (1 1 h) at -ºC was also subjected to temperature ramps from ºC to 0 and then from 0 to ºC, to evaluate the effects of maturation time on the gelatin gel. Measurements were done at least in triplicate and results were expressed as mean values and standard deviation. Results and discussion The utility of the high pressure treatment was tested at two stages: during acid swelling to facilitate the conversion of the insoluble collagen into a form suitable for subsequent extraction; and during the actual gelatin extraction step in water at ºC. As a control for comparison of the gelatins from different extraction procedures, gelatin was extracted conventionally following the procedure described by Gómez-Guillén and Montero (000), with a mild acid pre-treatment followed by overnight extraction at ºC. Yield of extraction Pressure-assisted acid swelling was performed at 0 MPa/ minutes at ºC (S 0 ), followed by an overnight extraction in water. These conditions were assayed with 0 mm acetic acid as in the control treatment, and also with mm acetic

6 acid. Preliminary results showed that no significant amount of gelatin could be extracted at the low acetic acid concentration, even with 0 min pressurising. All the preliminary collagen swelling (pre-treatment) assays in the extracting step were therefore performed with 0 mm acetic acid. As Table 1 shows, the yield (expressed as grams of dry gelatin per 0 g of clean skin) was slightly higher when pressurisation was used for collagen presolubilization (S 0 ), than in conventional gelatin extraction (control). To the contrary, the yield was considerably lower in all cases when pressurisation was applied in the extraction step. The general impression was that increasing the pressure or prolonging pressurisation tended to reduce the yield. As regards the pressure level, more gelatin was extracted with 0 MPa than with 00 MPa. Prolonging of treatment with 0 MPa from min to 0 min also caused a reduction in the extraction yield. In the case of pressurising at 00 MPa, when samples were pressurised in two consecutive -min cycles, the amount of gelatin extracted was only % greater than when samples were pressurised continuously for min in the same conditions. Molecular weight distribution The electrophoretic (SDS-PAGE) profiles of the various gelatin preparations are shown in Fig. 1. Samples extracted with high pressure at 00 MPa (E 00 - and E 00 -x ) were characterised by a typical gelatin/collagen type I profile, with a notable amount of -chains ( 1 / ratio around ), a considerable presence of - components ( -chain dimers), and particularly of higher molecular weight polymers including -components ( -chain trimers) (Gómez-Guillén et al., 00). There were noticeably more of these high molecular weight peptides than with the conventional extraction procedure; the reason may be either that high pressure favours more polymer extraction, or else that there is some aggregation during hydrolysis. Both in the swelling step (S 0 ) and in the water extraction step (E 0 - ), pressurisation at 0 MPa for min led to gelatin preparations in which peptides of MW 0 kda predominated and dimers and trimers were practically absent. The

7 electrophoretic profiles and the higher yield of these pressure-assisted gelatin preparations indicate a higher degree of collagen solubilization or hydrolysis. When pressurisation at 0 MPa was prolonged to 0 min (E 0-0 ) during extraction in water, the profile contained appreciably more high molecular weight polymers, which suggests a degree of pressure-induced aggregation. Viscoelastic properties of newly dissolved gelatins Dry gelatins were dissolved at 0ºC. Figure shows changes in the phase angle ( ) of the newly dissolved gelatin preparations (concentration.% w/v) upon cooling to ºC and subsequent heating. As the cooling ramp shows, the transition curve from solution to gel was rapid and similar in all samples, the only variation being in the gelling temperatures. All samples presented low phase angles at ºC ( < º), indicating good gelling gelatins. The same was true during subsequent heating; the studied gelatins differed in melting temperature but all presented a sharp melting transition. The onset of gelling and melting occurred at slightly higher temperatures than in the control in gelatins extracted under 00 MPa pressure, but at considerably lower temperatures than the control in gelatins extracted under 0 MPa pressure for min (S 0 and E 0 ). However, transition temperatures were higher when the pressurisation time was prolonged (E 0-0). As reported by Johnston-Banks (10), the increase in the gelling/melting temperatures is directly related to the increase in the average molecular weight of the studied gelatins. Table shows gelling and melting temperatures and elastic modulus (G ) and viscous modulus (G ) values at ºC and 0ºC. The samples pressurised at 0 MPa for min, both during swelling (S 0 ) and during extraction in water (E 0 - ), presented significantly lower G and G values as measured at ºC than the other gelatins. In samples pressurised at 00 MPa in two -min cycles, viscoelastic properties were again lower than with continuous pressurisation for min. On the other hand, values of G in 0ºC samples were low upon subsequent heating, indicating that the gels were all perfectly thermo-reversible and melted completely. 1

8 Viscoelastic properties of overnight cold matured gels The transition curves of the matured gelatin gels upon melting and subsequent cold renaturation (Fig. ) again revealed differences among the samples, predominantly in the thermal transition temperatures rather than in the slope. Both melting and gelling temperatures of S 0 and E 0 - gels were again notably lower than in the others. Also, phase angle values at ºC were higher in these samples, indicating poorer overall gel forming capacity as a result of cold maturation. In sample pressurised at 00 MPa for min (E 00 - ), the cooling phase angle transition indicated the onset of collagen renaturation at around 0ºC, which was considerably higher than with the conventional treatment. As shown in Table, values of G measured after overnight maturation at refrigeration temperature (around ºC) in E 0-0 and E 00 - gelatin gels were significantly higher than in the control gelatin. Again, all gels were completely melted at 0ºC. Upon subsequent cold renaturation, the E 00 - sample attained the highest values of G and re-gelling temperature, which was in fact higher than in the corresponding newly dissolved gelatin. There is clearly a relationship between the effect of pressure and the molecular weight of gelatin fractions. According to Stainsby (1), the greater presence of -chain dimers and trimers in these gelatins should make them more susceptible to renaturation to the full native collagen form. In addition, the presence of a significant amount of higher molecular weight components acts also in favour of their slightly higher melting/gelling temperatures (Johnston-Banks, 10). On the other hand, Sims et al. (1) indicated that the generation of short triple helices by many interactions of free chains, rather than rapid completion of a longer triple helix, will form a more stable gel. In this sense, the way of triple helix formation upon cooling in E 00 - and E 00 -x samples having similar molecular weight profile, may explain the difference in their renaturation ability. High pressure-assisted extraction at 00 MPa for min produced considerably less amount of gelatin than conventional extraction over 1-1h. However, the

9 quality of the gelatin extracted in that short time was high, comparable to that of conventionally- extracted gelatin as reported by Montero and Gómez-Guillén (000). The molecular weight distribution of the gelatin extracted by high pressure was characterised by a high presence of -chain, together with β- and γ- components (dimers and trimers of -chain, respectively), as well as a certain amount of greater polymers. All of them are key factors for high viscoelastic properties, gel strength and melting/gelling temperatures. After maturing overnight at -ºC, the modulus of elasticity (G ) of the gel from pressure-processed gelatin was greater than that of a conventional gelatin, and the melting temperature was slightly higher. This gelatin also presented greater renaturation ability upon subsequent cooling, but only with continuous extraction (E 00 - ); when extraction was carried out at 00 MPa in two -min cycles (E 00 -x ) there was no improvement in renaturation ability even although the molecular weight profile was similar to that of gelatin E 00 -, from which we deduce that it is not only the amount but also the configuration of these fragments that matters. Pressurising at 0 MPa for 0 minutes produced a gelatin with less amount of dimers and trimers than with 00 MPa, however, viscoelastic properties were similar, and the value of G after overnight maturation was even higher. With pressurising at 0 MPa, time was a key factor in the quality of the extracted gelatin: with min pressurisation in these conditions, the peptides extracted were largely of relatively low molecular weight and there were insufficient -chains for proper annealing of collagen molecules upon cooling. Although the yield was higher, this gelatin exhibited considerably poorer gel-forming ability than any of the others, including the control. In light of these results, we conclude that the use of high pressure to extract gelatin from fish skins is a useful alternative to the conventional procedure, essentially in that it drastically reduces the duration of the longest part of the process, so that a high-quality gelatin can be produced in a matter of minutes. Successive extractions at atmospheric pressure and increasing temperatures would improve the yield from

10 original raw material. Further studies would be needed to optimise pressure/time/temperature settings so as to increase the yield in the first extraction and in successive extractions from the same original material. Acknowledgements This research was financed by the Comunidad de Madrid under project 0G/ 00/00, and by the Spanish Comisión Interministerial de Ciencia y Tecnología under project AGL000-1.

11 References Burjandze, T.V. (1). Hydroxy-proline content and location in relation to collagen thermal stability. Biopolymers, 1, 1-. Cheftel, J.C. & Culioli, J. (1). Effects of high pressure on meat: a review. Meat Science, (), -. Djabourov, M., Lechaire, J. & Gaill, F. (1) Structure and rheology of gelatin and collagen gels. Biorheology 0, -0. Férnandez-Martín, F., Pérez-Mateos, M. & Montero, P. (1). Pressure/heat combinations on blue whiting (Micromesitius poutassou) thermal and mechanical behaviour. Journal of Agriculture and Food Chemistry, (), -. Gekko, K. & Koga, S. (1). The effect of pressure on thermal stability and in vitro fibril formation collagen. Agriculture and Biological Chemistry,, -. Gómez-Guillén, M.C. & Montero, P. (001). Method for the production of gelatin of marine origin and product thus obtained. International Patent PCT/ S01/00. Gómez-Guillén, M.C., Turnay, J., Fernández-Díaz, M.D., Olmo, N., Lizarbe, M.A. & Montero, P. (00). Structural and physical properties of gelatin extracted from different marine especies: a comparative study. Food Hydrocolloids, 1,. Heremans, K. (1). High pressure effects on biomolecules. In D.A. Ledward, D.E. Johnston, R.G. Earnshaw & A.P.M. Hasting, High pressure processing of food (pp 1-). Nottingham University Press,Notthingham, U.K. Kwiatkowska, A., Jankowska, b. & Korzeniowski, W. (001). Changes in solubility of the bovine semitendinosus muscle collagen under the influence of high pressure. Polish Journal of Food and Nutrition Sciences, /1(), -.

12 Johnston-Banks, F.A. (10). Gelatin. In P. Harris, Food Gels. (pp -) London: Elsevier Applied Science Publ. Ledward, D.A. (1). Gelation of gelatin. In J.R. Mitchell, & D.A. Ledward, Functional Properties of Food Macromolecules (pp -01). London: Elsevier Applied Science Publishers. Masson, P. (1). Pressure denaturation of proteins. In C. Balny, R. Hayashi, K. Heremans & P. Masson, High Pressure and Biotechnology (Vol. ), (pp -). Colloque INSERM/John Libbery Eurotext Ltd. Montero P. & Gómez-Guillén M.C. (000). Extracting conditions for megrim (Lepidorhombus boscii) skin collagen affect functional properties of the resultant gelatin. Journal of Food Science,, -. Montero, P., Borderías, J., Turnay, J., & Lizarbe, M.A. (10). Characterization of hake (Merluccius merluccius L.) and trout (Salmo irideus Gibb.) collagen. Journal of Agricultural and Food Chemistry,, 0-0. Montero, P., Fernánez-Díaz, M.D. & Gómez-Guillén, M.C. (00). Characterization of gelatin gels induced by high pressure. Food Hydrocolloids, 1, 1-0. Norland, R.E. (10). Fish gelatin. In M.N. Voight, & J.K. Botta, Advances in Fisheries Technology and Biotechnology for Increased Profitability (pp -). Lancaster: Technomic Publishing Co. Sims, T.J., Bailey, A.J & Field, D.S. (1). The Chemical Basis of Molecular Weight Differences in Gelatins. The Imaging Science Journal,,

13 1 1 Stainsby, G. (1). Gelatin gels. In A.M. Pearson, T.R. Dutson, & A.J. Bailey, Advances in Meat Research (Vol. ), Collagen as a Food (pp. 0-). New York: Van Nostrand Reinhold Company Inc. Suzuki, A., Watanabe, M., Ikeuchi, Y., Saito, M. & Takahashi, K. (1). Effects of high-pressure treatment on the ultrastructure and thermal behaviour of beef intramuscular collagen. Meat Science,, 1-. Te Nijenhuis, K. (1). Thermoreversible networks: Viscoelastic properties and structure of gels. Advances in Polymer Science, 10, 1. Walkenstrom, P. & Hermansson, A.M. (1). High-pressure treated mixed gels of gelatin and whey proteins. Food Hydrocolloids (),

14 LEGEND TO FIGURES Figure 1.- Electrophoretic analysis of gelatin preparations (SDS-PAGE) in the presence of - -mercaptoethanol. (a) control, (b) S 0, (c) E 0 -, (d) E 00 -, (e) E 00 -x and (f) E 0-0. Figure.- Changes in phase angle, monitored upon cooling from 0ºC to ºC and subsequent heating from ºC to 0ºC, of newly dissolved gelatins at.% (w/v). after overnight maturation at ºC, phase angle was monitored from ºC to 0ºC and subsequent renatured from 0ºC to ºC (ii) Figure.- Changes in phase angle, monitored upon heating from ºC to 0ºC and subsequent cooling from 0ºC to ºC, of gelatin gels (.% w/v, 1- hours maturation at -ºC)

15 Table 1.- Yield of extraction (expressed as g dry gelatin per 0 g of cleaned skins SAMPLES YIELD OF EXTRACTION (%) Control 1. S 0. E 0. E 0 0. E 00. E 00 X. 1

16 Table.- Viscoelastic properties of newly dissolved gelatins Cooling ramp Heating ramp SAMPLES G ºC G ºC g.t G 0ºC G 0ºC m.t (ºC) Control S E E E E 00 X g.t.: gelation temperature m.t: melting temperature 1

17 Table.- Viscoelastic properties of overnight matured gelatins Heating ramp Cooling ramp SAMPLES G ºC G ºC G 0ºC G 0ºC m.t G ºC G ºC g.t (ºC) Control S E E E E 00 X g.t.: gelation temperature m.t: melting temperature 1

18 a b c d e f Agregados FIG. 1 1

19 0 0 Phase angle (º) Phase angle (º) Control S0 E0- E0-0 E00- E00-* Temperature (ºC) Temperature (ºC) FIG. 1

20 Phase angle (º) Control S0 E0- E0-0 E00- E00-* Temperature (ºC) Temperature (ºC) FIG. 0