Interaction of thaumatin with carrageenans. I. Effects of pH, temperature and competing cations

Food Hydrocolloids VolA no.2 pp.105-l19, 1990

Interaction of thaumatin with carrageenans. I. Effects of pH, temperature and competing cations Shiro Ohashi, Fumiko Ura, Takashi Ochi, Hiroki Iida and Shigeo Ukai 1 SAN-EI Chemical Industries, Ltd, 1-11, Sanwa-cho l-chome, Toyonaka, Osaka 561, Japan and 1 Gifu Pharmaceutical University, 5-6-1, Mitahora higashi, Gifu-shi, Gifu502, Japan Abstract. The effect of temperature and pH on the interaction of thaumatin with carrageenans were examined by turbidity (absorbance) measurements. The maximum ratio of thaumatin reacting with carrageenans correlated with pH, increasing particularly at pH ;;. 5. The sweetness intensity of thaumatin-carrageenan complexes was markedly reduced at pH 3-4, and aduced to a lesser extent at pH 25. There was also a temperature effect, with a change in absorbance at 60°Cgreatly different from that at 20°e. When Na", K+ and Ca2+ were added to screen the negative charge of the carrageenans, there was no interaction with thaumatin. Since turbidity disappeared when these cations were added to suspensions of the thaumatin-x-carrageenan complex, it was assumed that thaumatin interacts with carrageenan via electrostatic bonding.

Introduction

The sweetener, thaumatin, obtained from the seed coat of Thaumatococcus daniellii Benth (1,2), is a basic protein with an isoelectric point at pH 11.5-12.5. It consists of thaumatin I and II (3) and trace amounts of thaumatins a, band c (generically called thaumatin 0) (4). Thaumatin I is composed of a polypeptide chain consisting of 207 amino acid residues and has a mol. wt of 22 209 daltons. It has eight intramolecular disulfide bonds (5,6), and a stable tertiary structure (7). Thaumatin II also consists of 207 amino acid residues and has a mol. wt of 22 293 (Figure 1). Thaumatin I and II have the same amino acid sequence, except that five amino acid residues of thaumatin I (Asn46, Ser63, Lys67, Arg76 and Asn113) are replaced with Lys, Arg, Arg, GIn and Asp, respectively (8,9). The sweetness intensities of both proteins are reported to be comparable (9). Thaumatin is 105 times sweeter than sucrose on a molar basis (3); its sweetness intensity factor, corresponding to a sucrose concentration of 8%, is 2500-3000 (5). Since thaumatin has a flavor-enhancing effect when used at a concentration below the sweetness threshold, it is used as a flavor enhancer with other sweetening agents in various foods, feeds and drugs (9-13). Carrageenans are sulfated polysaccharides extracted from red algae (Rhodophyceae) of the Gigartinaceae and Solieraceae families. The A-, K- and Lcarrageenans are widely used as food additives. The idealized carrageenan structures are shown in Figure 2. A-Carrageenan consists of repeating disaccharide composed of a-(1---,)4)linked D-galactose-2,6-disulfate residues and 13-(1---,)3)-linked D-galactose-2sulfate residues. The sulfate residues at the C-2 and C-6 positions are oriented inside the molecular structure. At room temperature, A-carrageenan molecules

© Oxford University Press

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exist as random coils; they do not gel but have a thickening effect. K- and Lcarrageenans consist largely of a repeating disaccharide composed of a-(1~4)­ linked 3,6-anhydro~D-galactose residues (the C-2 position is substituted by S04 residues in c-carrageenan) and 13-(1~3)-linked D-galactose-4-sulfate residues. The sulfate residues on the C-2 and C-4 positions are oriented outside the molecular structure. At room temperature, both carrageenans form a double helix structure and gel (14-17). All carrageenans interact with almost all proteins directly via an electrostatic bond at pH below the isoelectric point (14). Interaction of gelatin with carrageenans depends on the net charge ratio of gelatin to carrageenan, the pH of the system, and the weight ratio of carrageenan to gelatin (18). Carrageenans interact with 13-lactoglobulin at pH 4 or below (19). Thaumatin has been reported to lose its sweetness in the presence of an excessive amount of xanthan, pectin, carboxymethyl cellulose, carrageenan, guar, locust bean gum or alginates (13). Since there have been no detailed studies concerning interaction between thaumatin and acidic polysaccharides, we examined the interaction of thaumatin 106

Interaction of thaumatin with carrageenans. I.

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with carrageenans in order to expand the application range of thaumatin and to improve its utilization . Materials and methods

Thaumatin and A-carrageenan

Thaumatin was a product of Tate & Lyle, Ltd and A-carrageenan was a pure material supplied by FMC Co. Purification of K- and v-carrageenan

K-Carrageenan (FMC Co. , Gelcarin HMR) and c-carrageenan (FMC Co., Gelcarin DG) were respectively dissolved in distilled water at a concentration of stirring at 80°C. After cooling , the carrageenans were dialyzed through a membrane (Viskase Sales Co .) for ;;.48 h, and precipitated by adding 95% alcohol. The precipitate was separated and dried. Determination of sulfate content in A-,

K-

and v-carrageenans

After addition of 2 ml of 35% HCI and 100 ml of deionized water to 2 g of purified carrageenan, the mixture was heated with refluxing for 1 h. Following addition of 20 ml of 30% H2 0 2 , the mixture was refluxed for a further 3 h. The mixture was filtered and 20 ml of 2 N BaCI2 added to the filtrate. The 107

S.Ohashi et al.

precipitate was separated by filtration, and ignited at 600°C in an electric furnace. The remaining ash was weighed and the sulfate content was calculated from the weight of BaS04 generated. Results are given in Table I. Preparation of carrageenan soLution

Carrageenan, purified as described above, was added to distilled water, heated to 80°C with vigorous stirring and maintained at that temperature for 10 min. The solution was then allowed to stand until it had cooled to room temperature. After pH adjustment by addition of 0.01 N NaOH and/or HCI, solutions of the required concentrations were prepared. Table II shows additional amounts of 0.01 N HCI and/or 0.01 N NaOH required to adjust pH; these did not influence later experiments since small amounts were used. Preparation of thaumatin soLution

Thaumatin was gently dissolved in distilled water, care being taken to avoid bubble formation. After pH adjustment using NaOH and/or HCI, solutions with the required concentrations were prepared. Measurement of turbidity (absorbance)

A carrageenan solution of 0.002% (w/v) was prepared. Concentrations of thaumatin solutions were set so that the ratios of thaumatin to carrageenan were 1:1,2:1, ... ,50:1 (g/g). Samples (20 ml) of carrageenan solution were shaken with 1 ml of thaumatin solution at each reaction temperature (20°C, 40°C and 60°C) in incubators for 5 min. The mixture was allowed to stand until cooled to room temperature, and its absorbance at 550 nm was measured to determine the turbidity. Absorbance was measured on a Shimadzu UV-260 UV visible recording spectrophotometer with a quartz cell. Table I. Sulfate contents of carrageenans

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Interaction of thaumatin with carrageenans. I.

Electrophoresis

Aliquots (2 ,...1) of 1 mg/ml of carrageenan solution and 4 ,...1 of 2 mg/ml of thaumatin solution were applied to a cellulose acetate membrane and electrophoresis was conducted at 200 V for 15 min. Buffers used were 0.05 M citrate buffer for pH 3 and 5,0.05 M Tris buffer for pH 7 and 0.05 M glycine-NaOH buffer for pH 9. Carrageenan was stained with 0.25 gil of methylene blue solution, and thaumatin with 0.20 gil of amide black lOB solution. The membrane was washed with distilled water to remove excess dye. Viscosity measurement

Viscosities of 10 gil of A-carrageenan solution,S gil of c-carrageenan solution and 10 gil of x-carrageenan solution (pH adjusted using HC1) were measured at 70°C, 60°C, 50°C, 40°C, 30°C and 20°C with a viscometer Type B from Tokyokeiki-Siezosho Ltd. Reduction of thaumatin's sweetness intensity

Samples of 0.02% (w/v) thaumatin solution were adjusted to pH 3, 4, 5, 6 and 7 using NaOH and/or HCl. A carrageenan solution was prepared at a concentration which give the maximum reaction ratio of thaumatin to carrageenans (Table III), and adjusted to pH 3, 4, 5, 6 and 7 with NaOH and/or HCl. A 200 ml sample of carrageenan solution at each pH and 200 ml of thaumatin solution at each pH were mixed and shaken in incubators at 20,40 and 60°Cfor 5 min. The mixture was permitted to stand until cooled to room temperature, and subjected to a sensory test. For the sensory test, 0.01% (w/v), 0.0095, 0.009, 0.0085, ... ,0.001,0.0005 and 0% thaumatin solutions were prepared and used as sweetness intensity standard solutions after pH adjustment to pH 3,4,5,6 and 7 by NaOH and/or HCl. Sweetness intensity of the 0.01% (w/v) solution was assessed at 100%. The remaining sweetness intensity was determined by comparing sweetness between reaction mixtures at each pH and standard solutions for each pH. The sweetness intensity reduction rate was calculated by subtracting this remaining rate of sweetness intensity from 100%. Effect of metal ions

Carrageenan was dissolved in distilled water at 80°C by stirring for 10 min and permitted to stand until cooled to room temperature. After NaCl, KCl or CaCh had been added to the solution, the pH was adjusted using NaOH and/or HC1, and the carrageenan concentration was adjusted to 0.002% (w/v). Thaumatin solutions were prepared at concentrations which gave the maximum reaction ratio with carrageenan, and 1 ml of these solutions were mixed with 20 ml of carrageenan solution at 40°C in an incubator for 5 min; turbidity of the reaction mixture was measured. To test the solubility of the gelatin-like white turbidity obtained, pH-adjusted 109

S.Ohashi et al,

0.002% (w/v) carrageenan and thaumatin solutions (20 ml of x-carrageenan solution and 1 ml of thaumatin solution) were mixed at 40°C in an incubator for 5 min. After NaC!, KCI and CaCl z had been added, the reaction mixture was incubated for a further 5 min. Absorbance was measured by using a Shimadzu UV-260 UV visible recording spectrophotometer with a quartz cell. Results and discussion

Effects of pH and temperature on interaction Figure 3 shows the effects of pH and reaction temperature on the interaction of thaumatin with x.-, K- and c-carrageenans. 1.6

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110

Interaction of thaumatin with carrageenans, I.

At all temperatures tested, the reaction ratio of thaumatin to 'JI.-, K- or Lcarrageenan (hereinafter abbreviated as Th/Cg ratio) increased with increasing pH, and the reaction mixture turbidity increased in parallel. At a Th/Cg ratio of 5-10, the turbidity became maximum at pH 3-4; at a Th/Cg ratio of 15-30, the turbidity became maximum at pH 5-7. Figure 4 summarizes the effect of pH on electrophoretic mobilities of carrageenans and thaumatin. The mobility of thaumatin was highest at pH 3, and became low and almost constant at pH ;;04. The electrophoretic mobilities of all carrageenans were greatest at pH 9, and decreased with decreasing pH. The pK of carrageenans is known to increase with elevations of pH (20). These results indicate that the cationic charge of thaumatin decreases with increasing pH, while the anionic charge of carrageenans increases. Therefore, the ThlCg ratio also increases with increasing pH. At each pH level, the interaction of thaumatin and carrageenans may become maximum and turbidity also peak when the net charge ratio of thaumatin to carrageenans is 1:1. (em)

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111

S.Ohashi et al,

The turbidity of the mixture of thaumatin and carrageenans at 60°C was different from that at 20 and 40°C. This is explained by the fact that all A-, K- and L-carrageenans have random coil-like structures at 60°C (14,15,21,22). On the other hand, at ~55°C, the stable tertiary structure of thaumatin (7) changes reversibly; this heat-denatured structure may interact with random coil-like carrageenans. At 40°C, the turbidities of mixtures of thaumatin with A- and L-carrageenans were markedly different from those at 60°C. The turbidity of the mixture with Kcarrageenan at 40°C was similar to that at 60°C. This difference may be attributed to the difference in the structure of L- and K-carrageenans at 40°C; Lcarrageenan forms a double helix at 40°C, observed as optical rotation is increased, while x-carrageenan is a random coil at this temperature (23). This idea agrees with the results of our experiment on the viscosity change of the carrageenan solutions with changes in temperature. As shown in Figure 5, at pH 3-7, the viscosity of i-carrageenan solution steeply increased at
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112

Interaction of thaumatin with carrageenans, I.

The difference in turbidity between the two carrageenans is, therefore, attributed to the fact that c-carrageenan is a double helix and x-carrageenan is a random coil when they react with thaumatin. A-Carrageenan does not form a double helix; that is, its structure does not change with temperature (23); A-carrageenan is believed to have a random coil structure when it interacts with thaumatin. At 20°C, turbidities of mixtures for A-, K- and t-carrageenans were similar. It is notable that the turbidity of the reaction mixture with c-carrageenan at 20°C was comparable with that for A-carrageenan at 40°C, and that the turbidity of the reaction mixture with K-carrageenan at 20°C was comparable with that for c-carrageenan at 40°C. This finding suggests that sulfate residue sites of carrageenan, which interact with thaumatin, may become similar among carrageenans because of structure changes at various temperatures. Figure 6 shows the change in reaction mixture turbidity with changes in Th/Cg ratio at various pH values. At reaction temperatures of 20,40 and 60°C, various amounts of thaumatin were added to carrageenan solution at pH 3-7 to determine the Th/Cg ratio at which the turbidity became maximum. When turbidity peaked and then obviously decreased with increasing Th/Cg ratio, the ratio at which turbidity peaked was regarded as the maximum. When turbidity increased to a certain limit, and thereafter was largely unchanged by further addition of thaumatin, the ratio to which turbidity elevated with the increase of thaumatin was regarded as the maximum reaction ratio. At 60°C, all A-, K- and c-carrageenans showed similar charges in turbidity, suggesting that thaumatin and carrageenans interacted via structures reversibly changed by heat. At 20 and 40°C, however, turbidities for the three carrageenans varied between pH 3-4 and pH 5-7. This variation may be attributed to differences in ionic charge densities of thaumatin and carrageenans, and structural changes due to pH change. Table III shows the maximum reaction ratios of thaumatin to carrageenans at which turbidity, as determined by measuring the absorbance at 550 nm, became maximum, at various pH values and reaction temperatures. The data were examined for correlation by regression analysis. At reaction temperatures of 20, 40 and 60°C, the amount of thaumatin reacting with carrageenan almost correlated with pH in the range 3-7. The correlation was particularly good at 60°C. The reaction ratio of thaumatin to carrageenans increased with the elevation of pH, particularly at pH ;?;5. The maximum reaction ratio was not necessarily related with the sulfate content (see Table I) of carrageenans (A, Kand L). This finding may be explained by the configuration of sulfate residues in carrageenan molecules as well as changes in conformation of carrageenans and thaumatin dependent on temperature and pH. At 20°C, for example, K- and i-carrageenans showed comparable maximum reaction ratios although sulfate contents of the two carrageenans differed. This finding can be explained as follows: at this reaction temperature, both carrageenans have double helix structure. Sulfate residues at the C-4 113

S.Ohashi et al. 1.8

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A-Carrageenan 20°C 40°C ThICg ThlCg

60°C ThICg

K-Carrageenan 20°C 40°C ThICg ThICg

60°C Th/Cg

c-Carragee nan 40°C 20°C ThICg ThlCg

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3.951 7.108 17.971 22.244 21.299

5.689 12.035 16.687 19.770 23.711

5.840 9.602 17.379 16.911 17.878

4.544 7.239 16.973 21.622 18.277

5.981 5.897 16.789 20.801 20.814

5.093 9.507 18.171 18.708 16.533

4.336 8.345 14.676 16.832 17.545

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114

Interaction of thaumatin with carrageenans, I.

positrons of f3-(1~3)-D-galactose-4-su1fate residues of K- and c-carrageenans mainly may combine with basic residues of thaumatin (tertiary structure), while sulfate residues at the C-2 positions of a-(1~4)-D-galactose-2-sulfate residues of i-carrageenans rarely may combine with thaumatin. At 60°C and pH 7, the maximum reaction ratios of carrageenans and their sulfate contents almost correlated. This result is presumed that the three carrageenans and thaumatin are in random coil-like forms at this temperature and this pH value , and thaumatin is evenly linked with all sulfate residues of carrageenan molecules.

Reduction rate of thaumatin 's sweetness intensity

A fixed concentration of thaumatin solution was treated at pH 3, 4, 5, 6 and 7 with carrageenan solutions of concentrations at which the maximum reaction ratio (ThlCg ratio), as determined by turbidity (absorbance) measurement, was obtained at pH 3, 4, 5, 6 and 7. After incubation at 20,40 or 60°C, the mixtures were subjected to a sensory test to determine the reduction rate of thaumatin's sweetness intensity. Results obtained are given in Figure 7. Whilst the sweetness intensity of thaumatin markedly reduced at pH 3-4, the reduction was small at pH ~5 ; the effect of temperature on the reduction rate of sweetness intensity was also small. Among the three carrageenans, the reduction rate was greatest in A-, followed by L- and K- in this order. The bonding of thaumatin to the sweetness receptor of the taste bud involves lysine residues of the protein; the tertiary structure and ionic charge density of thaumatin are, therefore, important for sweet taste. Sweetness is said to be reduced by cleavage of disulfide bonds (3,24), and by decreasing ionic charge density of lysine residues (24-26) . Bonding of Domains II and III of thaumatin to the membrane bond sweetness receptor (27) is also reported to be essential for sweet taste (28). The marked reduction of sweetness intensity of thaumatin at pH 3 and 4 may be attributable to the decrease in the ionic density of the protein caused by binding of its lysine residues (in Domains II and III , in particular) to sulfate residues of carrageenans. The small reduction in sweetness intensity at pH ~5 is thought to arise from the fact that, at this pH, sulfate residues of carrageenan do not combine with many lysine residues, but instead mainly combine with arginine residues of thaumatin. This speculation is supported by the report that the sweetness intensity of thaumatin was not lost even if the charge density was reduced by blocking six of 11 arginine residues with 1,2~cyclo'hexane diane (29). Cys121 and Cys126 of thaumatin make up most of the hydrophobic portion of the protein molecule (7). At pH 5-7, sulfate residues of carrageenans may combine with arginine residues linking around Cys121 and Cys126 via peptide bonds, enhancing the hydrophobic tendency of thaumatin . In a separate experiment (unpublished), we examined the reduction of thaumatin's sweetness by reacting thaumatin and A-, K- and c-carrageenans at 40°C at pH 3, 4, 5, 6 and 7. For A-, K- and i-carrageenans, the concentrations were fixed at levels at which the reaction ratio of thaumatin (the fixed 115

S.Ohashi et al.

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10O-L-=~L.J=:;PL.J"""'F'--__;P'--__;-_:::d

pH

Fig. 7. Reduction rate of sweetness intensity of thaumatin reacted with carrageenans at the maximum reaction ratio determined in Table III. ~: 20°C; ~: 40°C; Ill: 60°C.

concentration) to carrageenan became maximum at pH 3 and 4, respectively. This experiment, varying only pH, indicated that sweetness of the thaumatincarrageenan complex markedly reduced at pH 3 and 4, and that reduction of sweetness intensity was small at pH ~5. These results suggest that amino acid residues of thaumatin, with which sulfate residues of carrageenan combine, varied with the change in pH value.

Effect of metal ions As shown in Figure 8, the amounts of metal ions required to make the turbidity (absorbance at 550 nm) comparable to that of pure thaumatin solution (absorbance = 0) varied with pH in the order pH 3 > 4 > 5 = 6 = 7 except for Na" and K+ with A-carrageenan, which were in the order pH 3 > 4 > 7 > 5 = 6. This result indicates that thaumatin and carrageenans bond more firmly as the reaction solution becomes acidic, and the bond becomes weak as the solution 116

Interaction of thaumatin with carrageenans, I. 1.4£

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Fig. 8. Absorbance change by the interaction of thaumatin with carrageenan solution containing metal ions. The reaction ratio of thaumatin to carrageenans used was the maximum reaction ratio determined in Table III. The amount of added metal ion (N) is plotted on the abscissa. 0: pH 3; !'c.: pH 4; 0: pH 5; .: pH 6; A: pH 7.

becomes neutral. At pH 5-7, the binding of metal ions to carrageenans, blocking sulfate residues, was stronger in the order Ca 2+ > K+ > Na+. At pH 3 and 4, the order was K+ > Ca 2+ > Na+ with the exception of i-carrageenan which was Ca 2+ > K+ > Na+. The amounts of metal ions required to release thaumatin from the thaumatin-X-carrageenan complex were also determined (Figure 9). The required amounts of Ca2+ , K+ and Na+ were almost equal to those necessary for inhibiting formation of the complex. Turbidity tended to increase with the increase in added metal ions at pH 3 (Figures 8 and 9). This turbidity change is observed when metal ions are added to thaumatin solution. 117

S.Ohashi et al,

1.6

f

La m b d a (Nil)

I.'

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L

O.

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c

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.

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I,

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.

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~

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Fig. 9. Absorbance change by the addition of metal ions to the product of inte raction of thauma tin with A-carrageenan. The reaction ratio of thaumatin to A-carr ageenan used was the maximum reaction ratio determined in Ta ble III . The amount of added metal ion (N) is plolted o n the abscissa. 0 : pH 3; 6. : pH 4; 0 : pH 5; . : pH 6; . : pH 7.

It is assumed from these findings that thaumatin interacts with carrageenans via electrostatic bonding. Conclusion In the interaction of thaumatin with carrageenans, the reaction ratio of thaumatin to carrageenans proportionally increased with increa sing pH in the range pH 3-7. At pH 3-4, the mixture of thaumatin and carrageenans at the maximum reaction ratio almost completely lost its sweetness. At pH ;;':5, reduction of sweetness of the mixture was small ; this may be explained by the difference in amino acid residues of thaumatin reacting with carrageenans. It is thought that carrageenans bond primarily with lysine residues , the functional group for sweet taste, at pH 3-4, while they mainly react with arginine residues at pH ;;':5. The turbidity change of the mixture of thaumatin and carr ageenans at 60°C was different from tho se at 20 and 40°C. It is believed that structures of thaumatin and carrageenans are reversibly changed by heat at 60°C, and that heat-changed molecules interact at this temperature. When sulfate residues of carrageenans were blocked by Na+, K+ and Ca 2 +, carrageen ans did not interact with thaumatin . The blocking potency was greater in the order Ca 2 + > K+ > Na+ except in cases where ;..- and K-carrageenans were at pH 3 and 4. Since the addition of Na +, K+ and Ca2+ to the thaumatin

118

Interaction of thaumatin with carrageenans. I.

and X-carrageenan complex caused release of thaumatin from the complex, it can be assumed that interaction between thaumatin and carrageenans is attributed to electrostatic bonding. Acknowledgements

Grateful acknowledgement is made to the late Mr Arthur Moirano and Dr Norman Stanley of the Marine Colloids Division of FMC Co. for helpful advice. References Inglett,G.E. and May,J.F. (1968) Econ. Bot., 22, 326-331. van der Wel,H. (1972) Olfaction and Taste, 4, 226. van der Wel,H. and Loeve,K. (1972) Eur. J. Biochem., 31, 221-225. Higginbotham,J.D. (1979) In Hough,C.A.M., Parker,K.J. and Vlitos,A.J. (eds), Developments in Sweeteners 1. Applied Science, London, pp. 87-123. 5. Iyenger,R.B., Smits,P., van der Wel,H., van der Ouderaa,F.J.G., van Broucwershaven,J., Ravestein,P., Richters,G. and van Wassenaar,P.D. (1979) Eur. J. Biochem., 96,193-204. 6. van der Wel,H., Iyengar,R.B., van Brouwershaven,J., van Wassenaar,B.D., Bel,W.J. and van der Ouderaa,F.J.G. (1984) Eur. J. Biochern., 144,41-45. 7. De Vos,A.M., Hatada,M., van der Wel,H., Karbbendam,H., Peerdemann,A.F. and Kim,S.H. (1985) Proc. Natl. A cad. Sci. USA, 82, 1406-1409. 8. Edens,L., Heslinga,L., Klok,R., Ledeboer,A.M., Matt,J, ,Toonen,M.Y., Visser,C. and Verrips,C.T. (1982) Gene, 18, 1-12. 9. Higginbotham,J.D. (1986) In Nabors,L.O. and Gelardi,R.C. (eds), Alternative Sweeteners. Marcel Dekker, Inc., New York, pp. 103-134. 10. Higginbotham,J.D., Lindley,M. and Stephens,P. (1981) In Inglett,G.E. and Charalambous,G. (eds), The Quality of Food and Beverages. Academic Press, New York, Vol. 1, pp. 91-111. 11. Ochi,T. (1980) New Food Inc., 22,1. 12. Ohashi,S. (1981) Food Sanitat. Res., 31, 9-26. 13. Higginbotham,J.D. (1983) In Grenby,T.J., Parker,K.J. and Lindley,M.G. (eds), Developments in Sweeteners 2. Applied Science, London, pp. 119-155. 14. Moirano,A.L. (1977) In Graham,H.D. (ed.), Food Colloids. AVI Publishing Co., Westport, CT, pp. 347-381. 15. McKinon,A.A., Rees,D.A. and Williamson,F.B. (1969) Chern. Commun., 701-702. 16. Rees,D.A., Steele,I.W. and Williamson,F.B. (1969) J. Polyrn. Sci., 28, 261-276. 17. Morris,V.J. (1982) Int. J. Macromol., 4, 436. 18. McMullan,E.A. and Eirich,F.R. (1963) J. Colloid Sci., 18, 526-537. 19. Hidolgo,J. and Hansen,P.M.T. (1969) Agric. Food Chem., 17, 1089-1092. 20. Sarkar,R. (1974) Master Thesis, Salford. 21. Rinaudo,M. and Rochas,C. (1986) Polyrn. Prep., 27, 246. 22. Morris,E.R., Rees,D.A. and Robinson,G. (1980) J. Mol. Bio!., 138, 349-362. 23. Snoeren,T.H.M. and Payens,T.A.J. (1976) Biochem. Biophys. Acta, 437, 264-272. 24. van der Wel,H. and Bel,W.J. (1980) Eur. J. Biochem., 104, 413. 25. van der Wel,H. and Bel,W.J. (1976) Chern. Senses Flavor, 2, 211. 26. Morris,R., Cagan,R., Martens,R. and Deiber,G. (1978) Proc. Soc. Exp. Bio!. Med., 157, 194. 27. Edens,L. and van der WeI,H. (1985) Trends Viatechnol., 3, 61. 28. De Vos,A. and Kim,S.H. (1987) NATO ASI Ser., Ser. A, 126,395-402. 29. van der Wel,H., Wiersama,A. and Brouwer,J.N. (1978) J. Labelled Compo Radeopharm., 14, 735. 1. 2. 3. 4.

Received on January 3, 1990; accepted on February 21, 1990

119