Development of New, Low Calorie Sweetener: New Aspartame

Chapter 30

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 13, 2016 | http://pubs.acs.org Publication Date: March 4, 2008 | doi: 10.1021/bk-2008-0979.ch030

Development of New, Low Calorie Sweetener: New Aspartame Derivative 1

1

2

YusukeAmino ,KenichMori ,YasuyukiTomiyama , HiroyukiSakata ,andTakeshiFujieda 2

1

1

2

AminoScience Laboratories and Food Products Development Center, Ajinomoto Company, Inc., 1-1, Suzuki-Cho, Kawasaki-Ku, Kawasaki 210-8681, Japan

Since its launch in the market over 20 years ago, the amino acid based sweetener Aspartame has substantially contributed to the improved taste quality of uncountable number of reduced calorie food and beverage products around the globe. During this period, value added functionalities of Aspartame was developed for a wide spectrum of food products. In parallel, the Ajinomoto Company has explored a great number of molecules to develop a novel sweetener. B y using advanced technology, in conformational analysis, molecular design, and receptor model building, Ajinomoto has developed the next generation sweetener. In this paper, we will describe Ajinomoto's research and development work on the new sweetener (Laboratory code name: ANS9801).

© 2008 American Chemical Society

In Sweetness and Sweeteners; Weerasinghe, Deepthi K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

463

464


Introduction A brief history of research on sweet molecules, especially aspartyl-based molecules can be summarized as follows. In the later 1960s, after Aspartame was accidentally discovered (1), almost at the same time, the Shallenerger and Aeree's " A H - B " model of the chemical basis of sweetness was proposed. In the 1970s through the 1980s, other new peptide sweeteners such as Alitame were discovered by trial and error (2). In the later 1970s, Dr. Ariyoshi proposed a novel idea o f structural modeling of Aspartame derivatives as sweeteners (3). Since then, some mode of interaction between sweet peptides and sweet taste receptor has been hypothesized. In the 1990s, computational chemistry was applied to the analysis of sweet molecules and construction of sweet molecular models. In this century, the sweet taste receptor was identified by several groups. Our research on sweeteners started in the late 1960s together with the development of Aspartame as a sweetener. Through these experiences, we have continued to find and develop new sweet molecules, such as aspartyl-based sweeteners, applying advanced theory and technique.

Discovery Strategy The following strategy was utilized to explore new aspartyl-based sweet molecules. • Select lead compounds • Conduct lead optimization and S A R study • Apply computer aided molecular modeling • Synthesize compounds and screen potential new sweeteners

Lead compounds Aspartame and compounds discovered by The Coca-Cola Company (4) and Dr. Nofre (5) were chosen as lead compounds for this study (Figure 1).

Lead optimization and S A R study Lead optimization and structure-activity relationships studies were carried out based on the sweet taste recognition models of Dr. Ariyoshi or Dr. Goodman


465 (6, 7), (Figure 2 and 3). Computer aided molecular modeling was also applied to synthesize sweet compounds (8).


H

2

N ^ C O N H ^ C 0

2

C H

3

Aspartame

H 0

2

C

Hfi^

y

*

The Coca-Cola Company (1994)

2

Hfi£"° \

Dr. Nofre (1994)

Figure 1. Lead compounds.

A: sweet

B: not sweet

R >Ri 2

B: not sweet L>M>S

Figure 2. Ariyoshi modelfor aspartyl dipeptide sweeteners.

Applying the Ariyoshi model, we could predict whether the aspartyl dipeptide derivative is sweet or not sweet (Figure 2). Namely, in the Fisher projection formulae " A " and " B " , the sweet dipeptide ester group is uniformly of " A " type while the inactive analogues are of the " B " type molecular topography.



466 Goodman model was proposed based on the conformational analysis of sweet-tasting, tasteless and bitter-tasting aspartyl-based peptides and peptidemimetics. The taste recognition model shown in Figure 3 describes the relationship between topochemical array and taste in aspartyl-based ligands. The zwitterionic glucophore (termed A H / B ) of the Shallenberger-Kier model is oriented on the +y axis, and the hydrophobic group plays a decisive role in determining the taste class (I-VI Class). In the taste recognition model o f Figure 3, of the two conformers that contribute to the sweet taste of the aspartyl-based ligands, the "L-shaped" hydrophobic glucophore occupies the +x axis region of space (Class I) and the extended glucophore lies along the - y axis (Class VI).

+y

Figure 3. Goodman model. Class-I : an L-shaped structure with the AH- and B-containing zwitterionic ring of the N-terminus forming the stem of the L in they axis, and the hydrophobic X group projecting along the base of the L in the +x axis. Sweet.Classes I,VI; Bitter.Class V; Tasteless: Classes II, III, IV; D zone: key to the enhancement ofsweet potency. (Reproduced with permission from reference 14. Copyright 2006 Wiley.)

Structure-Activity Relationships We have found hundreds of high-potency Aspartame derivatives from the structure-activity relationships study (9-18). Conformational analysis of aspartyl dipeptide amide (the compound of the Coca-cola company and its analogue), N alkylated aspartame (the compound of Dr. Nofre and its analogue) and N alkylated aspartyl dipeptide amide was carried out and the detailed descriptions given in the published papers (11, 12, 13). When examining the preferred conformations in solution as determined by N M R and molecular modeling, these



467 compounds can adopt "L-shaped" conformation. In addition, it was found that a hydrophobic substituent of N-terminal residue which is positioned above the base of the " L " in the +x, +y quadrant of space might be importent for enhancement of sweet potency. The Tinti-Nofre model for sweet taste ligands assigns special regions to a number of pharmacophoric groups considered to be essential for sweet taste (15). When the Tinti-Nofre model is compared with the Goodman model, the G domain of this model can be viewed as equivalent to the X domain in the Goodman model since it accommodates the hydrophobic group. The D zone of the Tinti-Nofre model remains unexplored in term of molecular arrangement. We assumed that the hydrophobic substituent (the second hydrophobic binding domain) above the base of the " L " in the Goodman model oriented to the D zone of the Tinti-Nofre model. As a result, we sought to design and synthesize sweet ligands to probe D zone (the second hydrophobic substituent ) and to determine its role in sweet taste. A n example of S A R study on the N-substituted aspartyl-based sweet molecules is shown in Figure 4. By introducing an "arylpropyl" substitution on the N-atom of aspartic acid moiety of Aspartame, the sweetening potency of Aspartame was increased. Moreover, comparison of the structure of hydrophobic substitution and sweetening potency of each compounds clearly showed that die replacement of aromatic hydrogen atoms at 3,4-positions by hydroxy 1, alkoxy or alkyl group and substitution of hydrogen atom(s) at 3-position of propyl group by alkyl group(s) can lead to dramatically high sweetness potencies. Among them, the sweetest compound has the sweetening potency 50,000 times more than that of sucrose. The extraordinary potency of the N-arylalkylated compounds can be explained by the effect of a second hydrophobic binding domain in addition to interactions arising from the "L-shaped" structure of the original ligand Aspartame. The "arylpropyl" substitution of this series of compounds is arrayed above the basfe of " L " (D zone) in the +x, -fy plane and might be responsible for the increased sweetness potency of the original molecule (Figure 3). We believe that the "arylpropyl" substitution play a fundamental role in enhancing the sweet potency because of introduction of orientational constrains on the whole molecule by hydrophobic (aromatic)-hydrophobic (aromatic) interactions (7, 14).

Screening for Development At the first screening, about 10 compounds were chosen from hundreds of compounds based on ; • • •

sweet potency taste availability of synthesis



468

Figure 4. The SAR study on the hydrophobic binding domain of aspartyl-based sweet molecules.

At the secondary screening, the candidate was decided based on following viewpoints; • • • •

sweet potency detail of taste profile physico-chemical property feasibility of industrial production

•

estimation of metabolic dynamics in human body by in vitro assay

the

New Sweetener Through these processes, a novel non-nutritive sweetener, N-[N-[3-(3hydroxy-4-methoxyphenyl)propyl-a-aspartyl]-L-Phenylalanine 1-methyl ester, monohydrate (hereafter referred to as ANS9801) was discovered (Figure 5). ANS9801 has a structural similarity to natural sweeteners. The functional group 3-hydroxy-4-methoxyphenyl exists as part o f a component of Phyllodulcin, a Japanese traditional sweetener obtained from Saxifragaceae amateur's leaf and Neohesperidin dihydrochalcone, a precursor obtained from citrus fruits (Figure 6).



469

Lab. Code name: ANS9801 Chemical names ( I U P A C ) : N-[yV-[3-(3-hydroxy-4-methoxyphenyl) propyl] -α-aspartyljL-phenylalanine 1-methyl ester C A S No.: 714229-20-6 C24H30N2O7.H2O

Molecular formula:

Relative molecular mass: 476.52 Structural formula:

H0

*

Figure 5. Characteristics

Ammm

c

ofANS980I.

Aqpamne

Figure 6. Structural formulas ofÂNS9801 and other sweeteners


470

Synthesis


ANS9801 is synthesized from Aspartame and (3-hydroxy-4-methoxyphenyl)propylaldehyde in a one step process by reductive N-alkylation, which is carried out by treating A P M and the aldehyde with hydrogen in the presence of a platinum (Pt/C) in a methanolic solution. The important intermediate, (3hydroxy-4-methoxyphenyl)propylaldehyde, is derived from vanillin via four steps (Figure 7).

reductive N-alkylation

ANS9801 Figure 7. Preparation

ofANS9801.

Properties ANS9801 is an odorless white crystalline compound and obtained as a hydrate (empirical formula C24H oN 07-H 0; formula weight 476.52). The melting point of the ANS9801 hydrate is 101.5°C. The advanced analysis results to set the specification for Good Manufacturing Practice are summarized in Table I. Tests like heavy metals, catalysts, residual solvents, microbial limit will be added on the current specification. The solubility of ANS9801 in water is 0.099g/dl at 25°C for 30 minutes. This is far greater than the necessary solubility required to obtain a sweetness level matching a 10% sucrose solution. A co-crystal of ANS9801 and Aspartame was prepared to improve the dissolution rate. A s shown in Figure 8, in the case of the mixture initial dissolution rate of ANS9801 is slower than that of Aspartame, however, in mixed crystal (ANS9801:Aspartame = 0.022:1 mol/mol), the dissolution rate of ANS9801 is the same as that of Aspartame. 3

2

2


471

Table I. The advanced analysis results of ANS9801


Specification Parameter Identification Description IR absorption spectrum Purity Assay

N-[N-[3-(3-hydroxy-4methoxyphenyl)propyla-aspartyl]-L-phenylalanine Total other related substances Water Residue on ignition Lead Specific rotation [a]20D

Specification Valut

White to yellow powder Same as Reference standard Not less than 97.0% and not more than 102.0% on anhydrous basis Not more than 1.0% Not more than 1.5% Between 2.5% and 5.0% Not more than 0.2% Not more than 1 ppm Between-45° a n d - 3 8 °

Figure 8. Dissolution rate of co-crystal ofANS9801 and Aspartame(APM) (Temp:25degree, Concentration:PSE=10).


472

Functionality


Functionality as a Flavor Enhancer Functionality of ANS9801 in various foods was evaluated. The ANS9801 has flavor enhancement effects in foods such as lemon tea, orange juice and strawberry yogurt at very low concentrations. According to the definition of the U.S. F D A , a flavor enhancers is a substances added to supplement, enhance, or modify the original taste and/or aroma of food, without imparting a characteristic taste or aroma of its own. For example, in die case of strawberry yogurt, the difference threshold value, or minimum concentration to be able to detect the change in specific taste intensity under 10 wt% sugar solutions, is 0.005mg/dL Other examples of evaluation of vanilla, lemon, orange and strawberry flavor are summarized in Table II. In strawberry yogurt, the aroma becomes fresher with more characteristic strawberry taste and flavor.

Table II. Flavor enhancer effects of ANS9801

PSE

Item

(%)

Vanilla flavored water

10

Lemon flavored water

10

Lemon tea

10

100% orange juice

Strawberry yogurt

10

10

Addition at below difference threshold value (^equivalent cone, to sugar

Effects of ANS9801

Aroma

enhancing the overallflavor,improving the palatability

Taste

enhancing the mouthfulness, full-body

Aroma

impressing die topflavor,enhancing the overall flavor

Taste

adjusting the balance between sweetness and flavor, improving the palatability

Aroma

more natural lemonflavor,real lemon flavor

Taste

adjusting the balance between sweetness and sourness, mouthfulness, no astringency of tea, clear after sweetness

O.OOSmg/lOOml (0.10%)

Aroma

morefreshflavor,suitable time-intensity balance of sweetness and flavor extension

Taste

enhancing the mouthfiillness/improving the balance of overall taste


Aroma

more freshflavor,more characteristic and pleasant flavor

Taste

more pleasant, more characteristic taste like strawberry

0.005mg/100ml (0.10%)


0 009mg/100ml (0.18%)

Functionality as a Sweetener The taste profile and the effectiveness of ANS9801 as a sweetener was examined through a series of sensory evaluations at various concentrations in water and compared to sucrose or aspartame-sweetened solutions.


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Sweetness Potency The sweetness potency of ANS9801 was compared to Aspartame in water using a qualified sensory panelists, where the panelists measured the sweetness profile of ANS9801 using the sucrose equivalency scale. The panelists were screened and specially trained to correctly identify the level of sweetness (i.e., strong, slightly strong, moderate, slightly weak, weak and trace level sweetness) and familiarized with the sucrose equivalency line scale (31 points). The perceived sweetness intensity of ANS9801 and Aspartame increased with increasing concentrations of each as shown in table 3. The relative sweetness potency o f ANS9801 compared to Aspartame (i.e., ratio of equi-sweet concentrations) varied from approximately 119 to 70 times the sweetness potency of Aspartame over a wide range of sweet intensities (4% to 14% SE).

Table III· Sweetness potency of ANS9801 ANS9801 Relative Potencies Sweet Intensity Relative to Aspartame Relative to Sucrose (% Sucrose Equivalency) (Aspartame/ANS9801) (Sucrose/ANS9801) 3 4 5 6 7 8 9 10 11 12 13 14

120 119 118 116 114 112 109 105 100 94 85 70

47778 44074 40370 36637 32963 29259 25556 21852 18148 10741 10741 7037

Taste Profile Descriptive analysis of taste profile of ANS9801 in water was carried out using the QDA® methodology (19). To identify the flavor profile of ANS9801 in water, it was compared to Aspartame at different concentrations. Flavor profile was evaluated at 500 ppm and 1600 ppm for Aspartame and 5 ppm and 16ppm for ANS9801. Results are summarized in figure 9. ANS9801 has



474 dominant sweet flavor, while the perceived intensities for bitter flavor and sour flavor are very weak. The sensory profile of ANS9801 is similar to that of Aspartame. Aftertaste attributes of ANS9801 is shown in Figure 10. A t lower concentration, ANS9801 has a little bit higher after taste compare with the same sweetness Aspartame solution. However at higher concentration, these differences are negligible. ANS9801 has very clean sweet taste similar to Aspartame and as previously stated its sweetness potency is twenty thousands times sweeter than sucrose and 100 times than that of Aspartame. Due to its high potency, very small amount of ANS9801 will be effective to add sweetness with various foods. Moreover, ANS9801 can be used in combination with sugar or other high intensity sweeteners. The amount o f ANS9801 used for some foods in case all sweetness comes solely from it is shown in Table IV.

Overall Flavor 50T

Artificial Flavor Λ

Sweet Flavor

Natural Flavor

y

Bitter Flavor

Sour Flavor ANS9801-5ppm — - Aspartame-500ppm

ANS9801-16ppm Aspartame-1600ppm

Figure 9. Flavor profile ofANS9801 at higher concentrations

Stability ANS9801 in dry form such as table top sweetener or powdered soft drink mix is very stable and keeps its functionality under the usual storage conditions (25°C /60%RH, Figure 11).


475


Overall Aftertaste

\

/

\

Sour Aftertaste

Bitter Aftertaste

- - - ANS9801-5ppm — - Aspartame-500ppm

— - ANS9801-16ppm — Aspartame-1600ppm

Figure 10. Aftertaste attributes ofANS9801.

Table I V . Calculated use level of ANS9801 in various foods Item Table top sweetener Powder soft drink Carbonated soft drink Hot packed beverage Chewing gum Fruit yogurt Yellow cake

concentration ofANS9801 SOOppm in 1.3g sachet (in drink:2~4ppm) 110~600ppm (in drink:2~7ppm) 4~7ppm 2.5~3.5ppm 35~50ppm 4~7ppm 10~14ppm



476 Figure 12 shows the stability of ANS9801 in model carbonated soft drink. The stability was measured using a typical ingredient, pH=3.2 that means typical pH of CSD under storage conditions of 25°C /60% relative humidity. After 20 weeks, ANS9801 remained about 60%. However, the satisfactory sweetness level remained throughout the experiment. A t 0, 8, 15 and 20 weeks, sensory evaluation was carried out to check the sweetness acceptability. About 50% of panelist accepted the sweetness of test sample as just about right even at week 20. This result means physico-chemical stability and functional stability is different. High intensity sweeteners have relatively higher sweetness potency in a lower concentration. This phenomenon can be explained by the concentration-response relationships of HIS, the equation of which asymptotically approaching maximal response and fits to the Beidler equation (20).

Usage Example A usage example of ANS9801 in combination with other sweeteners is shown in Figure 13. In lime flavored water sweetened with high fructose corn syrup, 30% of H F C S can be replaced by ANS9801 without any change of taste character.

Conclusion In the discussion above, the procedure to discover new sweet molecules and the approach taken to develop a new sweetener has been described. A novel non-nutritive sweetener, N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl-a-aspartyl]-L-Phenylalanine 1-methyl ester, monohydrate (Laboratory code; ANS9801) was discovered by applying the lead optimization and structure-activity relationships studies based on the sweet taste recognition models of Dr. Ariyoshi or Dr. Goodman. After evaluations of the potentiality as a sweetener, ANS9801 was chosen as a candidate for the development. ANS9801 is made from vanillin and Aspartame. It has very clean sweet taste similar to Aspartame and its sweetness potency is twenty thousands times of sucrose and 100 times of Aspartame. Also ANS9801 has flavor enhancement effects in foods such as lemon tea, orange juice and strawberry yogurt at very low concentrations. Since its high intensity, reduction o f sweeteners cost by combination with other nutritive or non-nutritive sweetener is a possible utilization of ANS9801. In the sweetener market, nutritive sweetener has a 90% share as sweetness equivalent. Currently, the population with obesity and diabetics are increasing in the world including developing countries, and they are major risk factor for other health problems. In this situation, high intensity sweeteners can contribute toward calorie management and open opportunities for expanded usage.


477


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• • •

10 Hi

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80 70 bO SO HO 30

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20

0£

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0 10

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It

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months

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ao

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70

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bO

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50 MO 30 20 10

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10

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2fa

months

Figure / / . Stability ofANS980! in dry form. A; Tabletop form B; Powdered soft drink mix stored at 25°C/60%RH


478

Stability


• using a typical ingredient • pH3.2, typical pH o f CSD • Storage conditions 2 5 ° C / 6 0 % R H - 20 weeks

Sweetness Acceptability • Sensory evaluation at each stability time point 100 a πτιττπι

DDNotfluie Enough • Just About Right • SlightVToo Sweet Π Π uch Too Sweet

weeks

Figure 12. Stability in carbonated soft drink and sweetness acceptability of ANS9801.

Figure 13. High Fructose Corn Syrup (HFCS) substitution byANS9801 in lime flavored water at PSE 5 and 8%, pH3.2, tested by triangle method with 15 expert panels.


479

Acknowledgments Y . A . wishes to thank late Dr. Goodman for many helpful discussions during the course of S A R study.


References 1.

Mazur, R. H.; Schlatter, J. M.; Goldkamp, A. H. J. A m . Chem. Soc. 1969, 91, 2684-2691. 2. Glowaky, R. C.; Hendrick, M. E.; Smiles, R. E.; Terres, A. In: Sweeteners: Discovery, Molecular Design, and Chemoreception; Walters, D. E., Orthoefer, F. T.; DuBois, G. E, Eds; ACS Symposium Series 450, American Chemical Society: Washington, DC, 1991; p57. 3. Ariyoshi, Y.; Agric. Biol. Chem. 1976, 40, 983-992. 4. D'Angelo, L. L.; Sweeny, J. G.; US 5,286,509 (Feb. 15, 1994). 5. Nofre, C.; Tinti, J.-M.; FR 2697844 (May. 13, 1994). 6. Yamazaki, T.; Benedetti, E.; Kent, D.; Goodman, M. Angew. Chem. Int. Ed. Engl. 1994, 33, 1437-1451. 7. Goodman, M.; Del Valle, J. R.; Amino, Y.; Benedetti, E.; Pure Appl. Chem. 2002, 74, 1109-1116. 8. Nakamura, R.; Takahashi, M.; Amino, Y.; Sakamoto, K.; Abstract of Papers, 2nd IUPAC International Symposium on sweeteners, Hiroshima; IUPAC, 2001; P-02, p117. 9. Ariyoshi, Y.; Kohmura, M.; Hasegawa, Y.; Ota, M.; Nio, N. In: Sweeteners: Discovery, Molecular Design, and Chemoreception; Walters, D. E., Orthoefer, F. T.; DuBois, G. E, Eds; ACS Symposium Series 450, American Chemical Society: Washington, D C , 1991; p41. 10. Ando, T.; Ota, M.; Kashiwagi, T.; Nagashima, N.; Ariyoshi, Y.; Chadha, R. K.; Yamazaki, T.; Goodman, M. J. Am. Chem. Soc. 1993, 115, 397-402. 11. Goodman, M; Zhu, Q.; Kent, D. R.; Amino, Y.; Iacovino, R.; Benedetti, E.; Santini, A. J. Peptide Science, 1997, 3, 231-241. 12. Mattern, R-H.; Amino, Y.; Benedetti, E.; Goodman, M. J. Peptide Res. 1997, 50, 289-299. 13. Mattern, R-H.; Amino, Y.; Benedetti, E.; Goodman, M. Biopolymers, 1999, 49, 525-539. 14. De Coupa, Α.; Goodman, M.; Amino, Y.; Saviano, M.; Benedetti, Ε. ChemBioChem, 2006, 7, 377-387. 15. Tinti, J.-M.; Nofre, C. In: Sweeteners: Discovery, Molecular Design, and Chemoreception; Walters, D. E., Orthoefer, F. T.; DuBois, G. E, Eds; ACS Symposium Series 450, American Chemical Society: Washington, D C , 1991; p206.



480 16. Amino, Y.; Yuzawa, K.; Takemoto, T.; Nakamura, R. WO 9952937 A1 (Oct. 21, 1999). 17. Amino, Y.; Yuzawa, Κ.; Takemoto, T.; Nakamura, R. WO 2000000508 A1 (Jan. 6, 2000). 18. Amino, Y.; Yuzawa, K.; Takemoto, T.; Nakamura, R. WO 2000017230 A1 (Mar. 30, 2000). 19. H. Stone and J. L. Sidel; Food Technology, 1998, 52(8), 48-52. 20. DuBois, G, E.; Walters, D. E.; Schiffman, S. S.; Warwick, Z. S.; Booth, B. J.; Pecore, S. D.; Gibes, K.; Carr, B. T.; Brands, L. M. In: Sweeteners: Discovery, Molecular Design, and Chemoreception; Walters, D. E., Orthoefer, F. T.; DuBois, G. E, Eds; ACS Symposium Series 450, American Chemical Society: Washington, DC, 1991; p261.


Development of New, Low Calorie Sweetener: New Aspartame

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