多糖酸水解对分子量的影响

ELSEVlER PII: SOl44-8617(96)00054-9 Curhoh,dm Polwm 31 (1996) 83-92 Copyright 0 1996 Elsevw Science Ltd Printed in Great Britain. All rights reserved 0144-8617/96/$15.00 + .OO Effect of acid hydrolysis on the molecular weight of kappa carrageenan by GPC-IS* David E. Myslabodski”, Dimitri Stancioffh & Ruth Anna Heckertta “FMC Cor f., Chemical Research and Development Center, P.0. Box 8, Princeton, NJ 08543, USA FMC Corp., Food Ingredients Division, Box 308, Rockland, ME 04841, USA (Received 3 July 1995; revised version received 1 April 1996; accepted 8 April 1996) Kappa carrageenan was subjected to acid-catalyzed hydrolysis under closely controlled pH and temperature conditions. The effect of acid hydrolysis was followed by measuring the change in molecular weight by gel permeation chroma- tography (GPC) coupled to a triple detector system consisting of a single (90”) angle laser light scattering (RALLS) detector, a viscometer and a refractometer. The changes in the weight average (M,+) molecular weight of kappa carrageenan can be described by a first-order random hydrolysis process involving selective attack at carrageenan glycosidic linkages. Kinetic parameters are also calculated and a general mathematical model is presented relating hydrolysis rate to pH and temperature. The model was used to compare carrageenan molecular weight change to published data on gel strength variation. Copyright 0 1996 Elsevier Science Ltd INTRODUCTION Carrageenans are water soluble polysaccharides extrac- ted from red seaweeds. These biopolymers consist of alternating 4-linked a-D-galactosyl- and 3-linked B-D- galactosyl residues. The commercial types include furcellaran, kappa, iota, and lambda carrageenans. Structurally, they differ from one another by the amount and location of ester sulfate, presence of pyru- vate, or formation of a 3,6 anhydro derivative in the 4- linked sugar. which occurs either naturally or is formed chemically by alkali treatment of the native poly- saccharide. These structural differences, along with variances in molecular weight, account for the gelling and other rheological properties that make carragee- nans useful in food and other applications. The functionality of carrageenans has traditionally been evaluated by Brookfield viscometers and by various gel testers (Marine Colloids, Stevens, etc.). These tests, although very useful, give only an indirect estimation of carrageenan molecular weight. A direct measurement of molecular weight will improve our understanding of the functionality of carrageenans. This will enable better process control in manufacturing and more effective product development. *Presented in part by DEM at the Fall Meeting of the Amer- ican Chemical Society, Washington DC, August, 1994 and at The International Food Hydrocolloid Corzference, Columbus, OH, September, 1994. ‘To whom correspo ndence should be addressed. 83 In this study, gelling carrageenans, kappa and iota. were subjected to acid hydrolysis under various pH and temperature conditions. The effect of acid hydrolysis of carrageenans was followed by measuring the change in molecular weight and intrinsic viscosity by gel permea- tion chromatography (GPC). The GPC unit was coupled to a triple detector system consisting of a single (90”) angle laser light scattering (RALLS) detector, a viscometer and a refractometer. In the present commu- nication, we report the changes in the weight average (M,) molecular weight of kappa carrageenan under closely controlled conditions of time. temperature and pH. A future report will deal with the results obtained with iota carrageenan. MATERIALS AND METHODS Chemicals A sample of commercial kappa carrageenan (Gelcarin GP 9 1 I, Lot 86 1304) extracted from Eucheuma cottonit’ was obtained from FMC Corp. (Food Ingredients Division, Rockland, ME). Tri-potassium citrate mono- hydrate, citric acid monohydrate, potassium chloride, lithium nitrate, sodium azide and imidazole (all Purum grade) were obtained from Fluka Chemical Corp. Nitric acid (Reagent Grade) was obtained from VWR Scien- tific. All reagents were prepared in distilled (Barnstead) and polished water (Milli-Q System, Millipore Corp.) 84 D. E. Myslabodski et al. and filtered through a 0.2pm (Gelman VacuCap) bottle-top vacuum filter. Hydrolysis reactor Three (1 I) jacketed glass reactors were connected in series and temperature was regulated by a constant temperature circulator. The reactors were provided with high torque (Caframo) mechanical stirrers and conden- sers to prevent evaporation. Initial solution volume was 600ml. At each sampling interval, 100 ml of solution were withdrawn. pH was measured either on-line or at the time of sampling. In the first case, a DPAS (Ingold) electrode was mounted inside the reactor. In the latter case, a Ross Sure-Flow (Orion) electrode was used. In both cases, pH readings were corrected by an automatic temperature compensator probe attached to the pH meter (Orion 720A). Hydrolysis procedure The solution was formulated so it would be possible to study carrageenan hydrolysis without interference from carrageenan interaction with other components normally found in food systems (i.e. proteins). It was also required to quantitatively recover the carrageenans for further GPC analysis. These conditions restricted the formulation to a simple system, which consisted of 10 mM potassium citrate-citric acid buffer. Potassium ion concentration was maintained at a constant 30mM by the addition of the necessary amount of potassium chloride. The potassium citrate level was that typically found in food dessert gels. Solutions were prepared at the required pH levels (room temp.) of 3, 4, 5, 6 and 7. To minimize hydrolysis during sample dissolution (see Singh & Jacobsson, 1994) 0.3g carrageenan was initially dispersed in 500 ml of potassium citrate solution and left to hydrate for I5 min. It was then rapidly dissolved by heating in a microwave oven with occa- sional stirring, and then added at temperature to the reactor containing the citric acid and potassium chloride solution. The contents were stirred for 1 min to stabilize temperature and the first aliquot was then retrieved. Subsequent samples were retrieved at the required times (see Table 1). Time, temperature and pH were recorded. The aliquot was split into two 50ml sub samples (one retained, one processed) and quenched at -86°C (dry ice in acetone). All samples were stored at -20°C. GPC-triple detector system The GPC system consisted of the following compo- nents: A 201 glass reservoir containing the eluent, a four-channel in-line degasser (Hewlet Packard 1050 Series), an HPLC quaternary pump (Dionex Model 4500) a high-pressure pulse dampener (Dionex part Table 1. Sampling schedule (the times under 200 min include the lag period between sampling and final temperature quench- ing to stop the hydrolysis reaction) Target temp. (“C) 55 75 95 Sample time TO Tl T2 T3 (min) (min) (min) (min) 5 300 600 1200 5 64 94 184 5 9 14 24 No. 43945) a column oven (Perkin-Elmer Model LClOO), a six-port sample injector (Valco Model C6W) with a 100~1 sample loop (Valco), and a Viscotek triple detector system. The injector was triggered by a micro- electronic valve actuator (Valco). The columns’ bank consisted of a TSK PWXL column guard and a set of TSK-Gel Columns (300 x 7.8 mm connected in sequence): G6000 + G4000 PWxL (TosoHaas). The triple detector system consisted of a right angle laser light scattering (Viscotek RALLS Model 600) unit, a viscometer (Viscotek Model H502B) and a differential refractometer (Knauer Model 298). All components were maintained at 60°C. Data were acquired with an A/D Data Manager (Viscotek Model 4000). Raw signals were processed with TriSEC GPC Software (Viscotek, Version 2.25). Samples were dialyzed to minimize ionic aggregation prior to chromatographic analysis. Polarimctry controls were examined to deter- mine the onset of thermal aggregation. In order to determine sample loss during a chromatography run, eluent fractions were collected and analyzed for total anionic hydrocolloid content following an established procedure (Soedjak, 1994). Typical recoveries of about 90% were found. The development of the sample preparation protocol and the operational conditions during GPC will be published elsewhere. Dialysis and chromatography eluent Buffered 0.2~ LiN03 dialysis solution and chromato- graphy eluent were prepared by dissolving 275.8 g of LiNOs, 13.62 g of imidazole, and 5.Og of NaNs (as preservative) in 2 1 of water and diluting to 20 1. pH was adjusted to 7.5 with nitric acid. Eluent was stored in containers having a 0.2g venting filter (PolyVent, Whatman) to prevent particulate contamination. Dialysis procedure In preparation for dialysis, samples were thawed, the gel was broken and a 5 ml sample was diluted with 5 ml of GPC eluent and transferred into a dialysis bag (Spectra/ Por ” 3,500 MWCO, Spectrum). The bag was then placed in a capped bottle containing 500ml of eluent and equilibrated under constant stirring for a total of 4 days, with daily solution change. After dialysis, samples Effect of acid hydrolysis on the molecular weight of kappa carrageenan by GPC-LS 85 were removed from the dialysis bag and stored in small glass vials at 4°C. Samples were dialyzed to convert carrageenan to the lithium form. This eliminates gelling ions that would interfere in the determination of molecular weight. The use of the appropriate membrane resulted in a fast ion exchange without sample loss (Knutsen et al., 1993). Potassium and magnesium concentrations (data not shown) were reduced to background level after a single exchange. Due to its high affinity with carrageenan, calcium required three exchanges for reduction to background levels. Polarimetry Carrageenan conformational changes as a function of temperature were measured by following changes in optical rotation of the polysaccharide (Perkin-Elmer Polarimeter Model 241 MC). Hot solution was trans- ferred into the polarimetry cell (100 mm path length) and left to equilibrate for 15 min between measure- ments. The sample was prepared following the same procedure as for the GPC samples. It was determined (data not shown) that the onset of thermal aggregation of kappa carrageenan in the GPC eluent occurs near 30°C. The same type of experiments were also carried out with iota carrageenan, which indicated that the GPC experiments had to be carried out at 60°C. To standardize the experiments, all GPC runs were conducted at 60°C. GPC sample preparation Dialyzed samples were heated to 80°C over a 10 min period under constant stirring and then successively filtered through two syringe filters, 1 pm (glass fiber, Puradisc 25GD, Whatman) and 0.45 pm (polysulfone, Puradisc 25 AS, Whatman); filtered solutions were stored in glass vials held at 65” C until chromato- graphed. Determination of molecular weight The determination of molecular weight by light scatter- ing requires an accurate determination of the refractive index of the solvent. It also requires the measurement of the specific refractive index increment (dn/dc) of the solvent-polymer combination. The refractive index value for the present solvent was found to be 1.334. The dn/dc value was obtained by reference to the Pullulan value of O.l47ml/g (Viscotek Corp., per. comm.). The average value obtained for kappa and iota carrageenan was 0.110 ml/g. In the calculation of molecular weight, the contribution of the second virial coefficient was neglected. In addition, the axial dispersion correction is included as an integral component in calculations with the Viscotek TriSEC software. The procedure for using viscometric measurements to correct the scattering data to zero angle with the TriSEC software relies on an iterative process of calcu- lation. An initial estimate of molecular weight, Mest, is first obtained from the Rayleigh equation, Me,, = R(d)IKc (1) where R(8) is the light scattering constant for the right angle light scattering detector, c is the injected concen- tration of carrageenan, and K = 2n2R1212/A4N~, (dn/dc)’ (2) where A is wavelength, RI is refractive index of the LiN03 solvent, NA is Avogadros Number, (dn/dc) is the differential refractive index increment for carrageenan in solution. This estimate of molecular weight is inser- ted, along with the measured intrinsic viscosity, v, into the Flory-Fox equation to obtain an estimate of the radius of gyration, Rgest, Rg est = [1/(6)“‘](7jM est /f)“j (3) where f is the Flory Constant equal to 2.86 x 1023. The estimated radius of gyration is inserted into the Debye equation to estimate the angular scattering probability function P(O), P(B) = (2/x2)(e-” +x - 1) (4) where x is (8/3) {(nRl)/n)Rgestsin(Q/2)}2 and 0 is 90”. The estimated P(90) is used in equation (5) below to calculate an improved estimate of molecular weight M: A4 = MJP(90) (5) The molecular weight calculation is iterated, beginning with equation (3), until the values of M, Rg, and P(90) converge. RESULTS Proper determination of the molecular weight of carrageenans requires conditions that avoid aggrega- tion. Aggregation will result in artificially high mole- cular weight values. Aggregation is caused by inter- molecular interactions that accompany conformational changes in carrageenans. These changes depend on the total ion content and on the temperature of the system. The choice of hydrolysis buffer therefore cannot be a fortuitous one made at random (see Perrin et al., 1974; Gueffroy, 1975). The buffer should be fully characterized regarding its pK values, buffer capacity, dilution effects, temperature dependence and complexing association constants with different coun- terions. The buffer should be an inert component in the system and must not interact with the reactants. It has to have enough buffering capacity to hold pH constant during long periods of hydrolysis. When 86 D. E. Myslabodski et al. working with systems at varied temperatures, the change in buffer pH as a function of the temperature (ApH/“C) must be known. Some buffers are known to undergo large shifts (Gueffroy, 1975) due to changes in individual ion dissociation constants. The addition of the sample (carrageenan is a polyelectrolyte) and its accompanying salts will also cause shifts in the pH of the system. All of these facts underscore the necessity of fully characterizing the buffer to be used in hydro- lysis studies. When operating at multiple temperatures and with viscous samples, careful techniques must be used during the determination of solution pH (see Bates, 1964: Galster, 1991). Temperature compensated pH values obtained under operational conditions are critical if these values are to be used in the determination of acid hydrolysis reaction constants. The citrate buffer employed in the present study (Table 2a) had minimal pH shifts due to changes in temperature and the addition of carrageenan. The largest shifts observed were between -0.22 and +0.27pH units (Table 2b). Moreover, after a rapid initial stabilization period, pH values remained constant for all runs, even after 20 h. The weight average molecular weight (M,) (see Tanford, 1961, for a discussion of the different mole- cular weight averages and distributions; Schroder et al., 1989 for the various experimental methods) of the different sample aliquots was obtained by GPC-light scattering (LS). An independent determination was conducted to obtain M, for carrageenan prior to hydrolysis. The value found was 523 000 Daltons (Da) (M,), which compares favorably with previously reported values determined by GPC-LS (340-575, 353 and 690 kDa) for commercial kappa carrageenans (Lecacheux et al., 1985; Slootmaekers et al., 1991; Rochas et al., 1989, respectively). A typical elugram is shown in Fig. 1. The elugram shows the reduction in the molecular weight of the sample due to acid hydrolysis as seen by a larger retention volume as the hydrolysis proceeds. The M, values for the different runs are summarized in Table 3a. It is noteworthy to observe the very small loss of molecular weight at pH 6. The changes at pH 7 were even smaller. Therefore, values obtained at pH 6 and 7 were not used in the subsequent data analysis. Statistical and kinetic analyses were performed with the JMP@ statistical software package from the SAS Institute. The triple detector system used in this study also gives an inde- pendent measure of intrinsic viscosity, n, used to calculate Rgest, as previously discussed. Values of n corresponding to each determined value of M, are listed in Table 3b. A linear correlation was obtained between the reci- procal of the molecular weight and hydrolysis time (l/ M, vs time). A typical result is presented in Fig. 2. This represents a first-order random hydrolysis process (Mark & Tobolsky, 1950). For such a reaction, the kinetics of hydrolysis can be described by equation (6) (Masson, 1955). 1 /M, - 1 /MO = kt/m (6) where M, and M, (kDa) are the molecular weights at time t and time zero, respectively, k (min-‘) is the first-order rate constant, t (min) is the reaction time, and m (kDa) is the molecular weight of the repeating Table 2a. Buffer composition PH 0.1 M Citric acid (ml) 2.0 M KC1 (ml) 0.1 M Tripotassium citrate (ml) Water (ml) 3 52.3 7.85 7.7 531.3 4 37.4 5.62 22.6 534.3 5 23.5 3.53 36.5 536.5 6 10.6 I .58 49.4 538.5 I 1.8 0.27 58.2 539.7 Table 2b. pH values for the 10m~ potassium citrate-citric acid buffer (constant potassium content). Buffer values recorded without carrageenan. System values recorded in the presence of carrageenan at operating hydrolysis temperature Target PH Buffer Buffer System pH at (temp. “C) System pH at (temp. “C) System pH at (temp. “C) 3.0 2.95 2.98 3.00 at 59 3.10 at 78 3.11 at 84 4.0 3.98 4.02 3.89 at 55 4.03 at 75 3.98 at 85 5.0 4.96 5.07 4.66 at 54 4.14 at 75 4.78 at 85 6.0 5.97 6.18 5.81 at 78 5.87 at 85 7.0 6.96 7.15 7.23 at 74 7.22 at 85 Effect of acid hydrolysis on the molecular weight ofkappa carrageenan by CPC-12 87 90 80 70 60 SO 40 30 20 10 0 -10 c I ‘ 1 8 10 13 16 19 22 Elution volume (mL) 5 Fig. 1. GPC-RALLS &gram for the hydrolysis of kappa carrageenan at pH 4, 75°C. Sampling intervals at 5, 64, 94 and 184 min. unit for kappa carrageenan (0.392 kDa). This equation holds very precisely for all but the very lowest degrees of polymerization (Mark & Tobolsky, 1950). The use of the dimer as the weight of the ‘monomer’ in the kinetic calculation is justified based on the very different response towards acid attack of the two bridging bonds in kappa carrageenan (see below). Statistical data analysis indicated that pH and temperature were key independent variables affecting hydrolysis rate. The pH and temperature dependence of the first-order reaction rate constant can be expressed by equation (7): LogkjpH, T) = LogA’ - BpH - C/T (7) which at constant pH can be reduced to equation (8): Logk(T) = Log A - E/2.303RT (8) which is the well-known Arrhenius equation, account- ing for the effect of temperature on reaction rates. Figure 3 shows the good fit of the values from the present study to the Arrhenius relation. The lines having the same slope indicate that the activation energy, E, (30 kcal) is the same for the hydrolysis reaction at different pH values. The expected increase in hydrolysis rate with increase in temperature is dearly seen. The different intercepts on the Y axis correspond to the different values