Gelation of surimi pastes treated by high isostatic pressure

R. Hayashi and C. Balny (Editors), High Pressure Bioscience and Biotechnology 9 1996 Elsevier Science B.V. All rights reserved.

357

Gelation ofsurimi pastes treated by high isostatic pressure T.C. Lanier Food Science Department, North Carolina State University Raleigh NC, USA 27695-7624

Abstract

Surimi, a myofibrillar concentrate of fish muscle, gels at 25~ (for Alaska pollock) due to crosslinking of proteins by endogenous transglutaminase, at 90~ by heat-induced protein denaturation, and at pressures near 300 Mpa due to pressure-induced denaturation. The latter two treatments doubtless induce extensive gel network formation via formation of intermolecular hydrophobic associations, and heat is known to induce disulfide bonding. We have shown that pressure-induced gels evidence disulfide bonding as well. Endogenous transglutaminase evidently survives the pressure treatment, and subsequent setting at 25~ with or without subsequent cooking at 90~ results in very strong gels as compared to those prepared without prior pressurization. I. INTRODUCTION Fish muscle proteins are typically heat-gelled in the manufacture of crab analog products. High temperatures (40-60~ depending on the protein source) are normally required to denature fish myosin, leading to intermolecular covalent and non-covalent interactions including disulfide bond formation and hydrophobic group interactions that result in gelation (1). Proteins are destabilized and may also be induced to gel at low temperature by pressure treatment ranging from 100-1000 MPa (2). Gels formed by pressure treatment generally possess increased glossiness and deformability, as well as a more natural flavor (3) as compared to heat-induced gels. Fish protein gels can be formed with pressure at ambient or lower temperatures at pressures ranging from 200 to 500 MPa (4). Heremans and Heremans (5) proposed that the process of pressure-induced protein denaturation could be thought of as a cascade effect. Initially, hydrophobic interactions which stabilize the native structure of the protein are disrupted under the influence of pressure. This causes an opening of the protein structure, allowing hydrophobic groups to be exposed to the aqueous environment. Changes result from pressure-induced destabilization of the protein and produce further volume decreasesdue to electrostriction around charged groups, water structuring around exposed apolar groups, and solvation of

358 polar groups through hydrogen bonding. It was considered that disulfide bond formation might occur at this time. Upon release of pressure, the hydrophobie groups interact once again to minimize exposure to the aqueous environment. Along with other protein-protein interactions such as possibly disulfide bonds formed under pressure and hydrogen bonds that form upon release of pressure, the intermoleeular hydrophobie interactions result in the formation of a gel structure when protein concentration is sufficient. Even at atmospheric pressure muscle pastes from most species of fish can gel at low temperatures (0-40~ but the time required for gelation is greater than by heat or pressure (6). Endogenous transglutaminase is thought to induce this low temperature gelation (termed "setting"), which also imparts added strength to the gel upon subsequent cooking at higher temperatures. Transglutaminase forms intermolecular covalent e-(T-glutamyl) lysine bonds between myosin heavy chains (MHC), the polymerization of which can be measured by SDS-PAGE (7). The endogenous transglutaminase requires Ca 2§ to be active, and thus can be inhibited with EDTA (8). Since pressure causes denaturation and gelation of proteins at low temperatures, it may also affect the activity of transglutaminase (9). 2. RECENT EXPERIMENTS We subjected pastes of Alaska pollock (Theragra chalcogramma) surimi (refined myofibrillar protein containing 4*/, sucrose, 4*/. sorbitol and 0.3% sodium tripolyphosphate), adjusted to 78% moisture content and 2% NaCI, to cooking (90~ for 30 min.), setting (25~ for 2 hr), pressure treatment (300 MPa isostatic pressure for 30 min at 5~ or a combination of these treatments, setting or cooking always being carried out at atmospheric (10) (Fig. 1). Stress (strength) and strain(deformability) of gels were determined at the tensile failure point, measured at 25~ It is apparent that the various methods of processing the paste into a gel had dramatic effects on tensile strength, but little effect on tensile deformability. The setting treatment strengthened the gel, particularly when preceded by a pressure treatment. This enhancement of the setting effect by a prior pressure treatment raises the question of whether the endogenous transglutaminase presumed to be responsible for gdation during setting is also active during the pressure treatment, and survives pressure-induced denaturation. Since the enzyme is known to be calcium activated, we can inhibit its action by addition of EDTA (Fig. 2, setting only treatment) (10). EDTA addition had no effect on the pressure-only treatment; thus it is logical to conclude that transglutaminase is not involved in pressure-induced gelation. The dramatic effect of EDTA on gel stress in the pressure+setting treatments indicates that the transglutaminasr survives a pressure treatment of this magnitude, despite assertions to the contrary by Shoji et al.. (11). The data of Fig. 1 and 2 were corroborated by SDS-polyacrylamide gel dectrophoresis of these gels, which indicated polymerization of myosin heavy chain as a result of inclusion of setting as part of the treatment. The test used was more qualitative than quantitative with

359 respect to polymerization; however, loss of myosin heavy chain monomer was approximately the same for setting with or without a prior pressure treatment. (10). Figure l. Effect of gelling treatment on tensile stress and strain at failure of surimi gels. Treatments: P = 300Mpa isostatic pressure, 30 min, 5~ S = 25~ 2 hr, atmospheric pressure; C = 90~ 30 min, atmospheric pressure.

1000

A

(U n

750

(/) (/) UJ hI'U)

5OO

1.5

Z

"--I

~r~

1

250 0 . 5 84

C

S

S/C

P

P/C

P/S

P/S/C

O-

i

i

I

C

S

S/C

P

PIe

PIS

PIS/C

Figure 2. Effect of EDTA and gelling treatment on tensile stress and strain at failure of surimi gels. Treatments are same as in Fig. 1.

=1] ~"

9

~t

NOEDTA

S/C

i

I

No EDTA

I II .'~t ~

L"~ I:~I,.IEDTA

S

I

~

P

P/S

P/S/C

9

S

S/C

P

P/S

PIS/C

360 The activity of the endogenous transglutaminase is dependent upon denaturation of the substrate myosin to expose available binding (7). Thus it is reasonable to believe that the greater strength of gels prepared by the pressure+setting+cook treatment as compared to those prepared by setting + cooking (Fig. 1) might be attributed to a more available substrate, which facilitates more active crosslinking of myosin, in the former treatment. However, we did not quantitate numbers of e-(g-glutamyl) lysine bonds directly to verify this hypothesis, and it should be noted that we have previously seen instances where the rate of bond formation, rather than total bonds formed, seemed to correlate better with ultimate gel strength (12). Of course the pressure treatment, having induced gelation of the fish proteins, is certainly introducing intermolecular bondings, most likely hydrophobic and possibly even covalent/disulfide (5). Solubility of the gels for which fracture data are presented in Fig. 1 were determined in the manner of Buttkus (13) in 2% SDS-SM urea-20 mM Tris-HCl (pH 8.0), with or without added 2% 13-mercaptoethanol (13-ME) (Table 1). These data indicate Table 1: Solubility results (% solubility) on samples prepared +/- EDTA (raw= starting paste, P=300 MPa/30 min, C=90~ min, S=25~ min w/o EDTA Gel Type

w/EDTA

wlo 13-Me

w/ ~Me

w/o w/ 13-Me 13-Me

Raw

90.9

100

90.9

100

P

42

100

41.6

100

P/C

38.8

100

35.7

100

C

86.3

100

88.5

100

P/S

17.7

93.6

39

100

S

70.8

93.2

89.6

100

P/SIC

15

93.2

36.1

100

SIC

63.7

93.2

85.3

100

that the treatments decreased solubility in SDS-urea in the absence of ~3-ME, but this loss was restored upon inclusion of 13-ME in the dissolving medium for all treatments except those which included a setting step. The remaining insoluble fraction in gels subjected to setting can be attributed to formation of non-disulfide of e-(T-glutamyl) lysine bonds by transglutaminase. Note that no such remaining insolubility occurred in samples subjected to

361 setting which contained added EDTA. Comparison of solubility data for samples prepared +/- EDTA also indicates that loss of solubility in gels subjected to only a setting treatment can largely be attributed to the action of transglutaminase, not disulfide bonding.

The remarkable loss of solubility in SDS-urea for pressure-treated gels indicates that substantial disulfide bonding likely occurred, in excess of that which resulted from cooking at 90~ Note that EDTA addition had little or no effect on solubility of these gels. Berg et al. (14) had noted a dramatic increase in the numbers of readily reacting SH-groups of myosin at pressures of 300 Mpa. 3. C O N C L U S I O N S

These experiments thus indicated that gelation of surimi pastes by high pressure does likely involve formation of intermolecular disulfide bonds, but doubtless is also greatly stabilized by intermolecular hydrophobic bondings facilitated by weakening of intramolecular hydrophobic associations under pressure. Transglutaminase-mediated covalent crosslinking proceeds unabated after a 300 Mpa/30 min treatment when warmed to 25~ and the effects of this subsequent setting treatment on gel strength are quite synergistic with the prior pressure treatment. Conformational changes in the proteins must occur that, after pressure release, increase the effectiveness of transglutaminase to catalyze covalent crosslinking of MHC. This opens the possibility of enhancing the gelling effects of added microbial transglutaminase in protein foods by a prior pressure treatment, a hypothesis we are presently investigating. 4. REFERENCES

1 2 3 4 5 6 7 8 9 10 ll

H. Lee and T.C. Lanier, J. Muscle Foods, 6 (1995) 125. T. Ohshima, H. Ushio and C. Koizumi, Trends in Food Sci. and Technol., 4 (1993) 370. M. Okamoto, Y. Kawamura and R. Hayashi, Agric. Biol. Chem., 54 (1990) 183. T. Shoji, H. Saeki, A. Wakameda, M. Nakamura and M. Nonaka, Nippon Suisan Gakkaishi, 56 (1990) 2069. L. Heremans and K. Heremans, Biochim. Biophys. Acta, 999 (1989) 192. G. Kamath, T.C. Lanier, E. Foegeding and D.D. Hamann, J. Food Biochem., 16 (1992) 151. D. Joseph, T.C. Lanier and D.D. Hamann, J. Food Sci., 59 (1994) 1019. Y. Kumazawa, T. Numazawa, M. Motoki and M. Takamura, Abstr., Annual Meeting, Institute of Food Technologists, Chicago, IL. (1993). P. Low and G. Somero, Comp. Biochem. Physiol., 52B (1975) 67. M.G. Gilleland and T.C. Lanier, J. Food Sci. (1996), in press. T. Shoji, H. Saeki, A. Wakameda and M. Nonaka, Nippon Suisan Gakkaishi, 60 (1994) 101.

362 12 13 14 15

H.G. Lee, T C. Lanier, D.D. Hamann and J.A. Knopp, J. Food Sci. (1996) in press. H. Buttkus, Can. J. Biochem., 49 (1971) 97. Y.N. Berg, N.A. Lebedeva, E.A. Markina and I.I. Ivanov, Biokhimiya, 30 (1965) 277. P.M. Nielsen, Food Biotechnol. 9 (1995) 119.