Miracle Fruit Research

After extensive testing on both lab animals and humans, Miracle Fruit has been found to be absolutely safe for human
consumption. There are reports showing that lab animals given a Miracle Fruit diet ended up healthier at the end of testing
than the control animals that ate none!

Improvement of insulin resistance by miracle fruit (sYNSEPALUM DULCIFICUM) in fructose-richchow-fed rats. - In PTR. Phytotherapy Research
ISSN 0951-418X ; 2006, vol. 20, no11, pp. 987-992 [6 page(s) (article)] (25 ref.). The authors CHEN Chang-Chih (1) ; LIU
I.-Min (2) ; CHENG Juei-Tang (3).
The researchers concluded that the in vivo insulin sensitivity was markedly raised by miracle fruit. In conclusion, the results
suggest that miracle fruit may be used as an adjuvant for treating diabetic patients with insulin resistance because this fruit
has the ability to improve insulin sensitivity.
(1) Department of Emergency Medicine, Mackay Memorial Hospital and College of Oral Medicine,Taipei Medical
University, Taipei City, 10401, TAIWAN, PROVINCE DE CHINE
(2) Department of Pharmacy, Tajen University, Yen-Pou, Ping Tung Shien, 90701, TAIWAN, PROVINCE DE CHINE
(3) Department of Pharmacology. College of Medicine, National Cheng Kung University, Tainan City, 70101, TAIWAN,
PROVINCE DE CHINE

At Florida State University in Tallahassee

A professor of biophysics. Dr. Lloyd Beidler, began his studies of miracle fruit in the late 1950's. Together with Dr. Kenzo
Kurihara/ he successfully isolated the active principle, publishing their results in "Science", Vol. 161, September 20, 1968.
Research performed independently in the Netherlands under the sponsorship of the Unilever Company culminated in the
same year. It was found that a glycoprotein causes the taste-modification effect, a giant, 'macro-molecule' with a molecular
weight of 44,000. The size of this molecule made it difficult, if not impossible, to synthesize it. This was one of the reasons I.
M. C. had determined that the miracle fruit had no real commercial potential; vast plantations would have been necessary to
supply enough fruit for the large scale production they envisioned.
Drs. Beidler and Kurihara had access to a sufficient number of fresh berries, which were grown in a greenhouse at the
university. The miracle fruits were stored in the deep freeze until needed; 300 at a time were used to make a potent solution
through standard scientific procedures. Through their thorough tests, they discovered that the taste-modifying activity was
destroyed by heat, or when exposed to organic solvents, and was greatly reduced by exposure to pH above 12.0 or below
2.5 at room temperature. Situations with a pH of 3.7 and temperature of 4C caused the activity to remain stable for one
month. It was also concluded that the protein was basic, and contains no other protein within the active component. It does
have bound to it two sugar molecules; the active principle therefore contains a small amount of sugar6.7%, which was
determined not to be an impurity. This is what makes the active principle of S. dulcificum a glycoprotein. Glycoproteins are
known to be completely innocent of any toxicity and are readily metabolized by the body.

Other research, by Dr. Linda Bartoshuk

... on behalf of the U. S. Army, was begun in 1966 at their laboratories in Natick, Massachusetts.
Dr. Bartoshuk's specialty was the psychology of taste; her interest was in the miracle fruit's military potential. Since the fruit
could make such barely palatable foods as those consumed in West Africa into culinary delights, it seemed logical that Army
food could also stand similar improvement.
After years of exhaustive research, she read a paper in 1970 at the Army Research Conference in West Point, that was
very positive in its support of miracle fruit. Thorough analysis concluded that no toxic heavy metals were present. Huge
quantities of miracle fruit concentrate- 3,000 times ordinary human consumption were proven to cause absolutely no ill
effects. (In fact, the health of their laboratory animals was improved by miracle fruit consumption!)
Foods such as vegetables, meats and others that were not usually sour were not affected, although in some cases the
flavor of vegetables did improve. It was believed that some foods had flavors which were previously masked, and were
beautifully brought out by the miracle fruit's principle.
This effect would last for at least an hoursome variation seemed to depend upon how long the fruit was held in the
mouth before eating other foods (possibly due to how well the glycoprotein coated the taste buds). Until saliva eventually

hydrolyzed the glycoprotein, acidic foods would continue to taste sweet as the sweet receptors continued to "fire" by
exposure to sour foods. This has been known to last 18 hours in rare cases. No aftertaste was ever reported; although other
flavors were slightly enhanced, such as the degree of saltiness.
It should be noted here that it was also proven beyond doubt that the central nervous system is not affected by the miracle
fruit, which was a concern of the FDA, which was also fearful that children could be harmed by the dulling of their natural
taste defenses, allowing them to consume harmful substances. Small children are most frequently poisoned by aspirin;
miraculin was tested and proven NOT to mask its characteristically bitter taste. Organic acids- especially citric acidare the
substances that are modified to the greatest degree. Battery acid will NOT become a tasty drink.
Dr. Linda Bartoshuk now works at the Center for Smell and Taste at the University of Florida.

"Cortical representation of taste-modifying action of miracle fruit in humans."

Chizuko Yamamoto(a), Hajime Nagai(b), Kayo Takahashi(a), Seiji Nakagawa(c), Masahiko Yamaguchi(c), Mitsuo Tonoike(c,1), and
Takashi Yamamoto(a),
Here we present the abstract of their work:
Red berries of a tropical plant called miracle fruit, Richadella dulcifica, reduce the sour and aversive taste of acids and
add sweet and palatable taste. To elucidate the brain mechanism of this unique action of miracle fruit, we recorded tasteelicited magnetic fields of the human cerebral cortex. The initial taste responses were localized in the fronto-parietal
opercular/insular cortex reported as the primary taste area. The mean latency of the response to citric acid after chewing
miracle fruit was essentially the same as that for sucrose and was 250300 ms longer than that for citric acid. Since it is
known that stimulation with acids after the action of miracle fruit induces both sweetness and sourness responses in the
primate taste nerves, the present results suggest that the sourness component of citric acid is greatly diminished at the level
of sub cortical relays, and mostly sweetness information reaches the cortical primary taste area. We propose the idea that
the qualitative aspect of taste is processed in the primary taste area and the affective aspect is represented by the pattern of
activation among the different cortical areas.
(a) Department of Behavioral Physiology, Graduate School of Human Sciences, Osaka University, 1-2 Yamadaoka, Suita,
Osaka 565-0871, Japan
(b) Research and Development, Product Quality and Regulatory Affairs, Cerebos Pacific Limited, 18 Cross Street 12-01-08
China Square Central, 048423, Singapore
(c) Institute for Human Science and Biomedical Engineering, National Institute of Advanced Industrial Science and
Technology (AIST), 1-8-31, Midorigaoka, Ikeda, Osaka 563-8577, Japan

"Analyses of gustatory-related brain magnetic fields induced by taste sensation".

Hajime Nagai(a, b), Chizuko Yamamoto(b, c), Kayo Takahashi(c), Seiji Nakagawa(b), Masahiko Yamaguchi(b), Yoshie Kurihara(d),
Mitsuo Tonoike(b), and Takashi Yamamoto(c)
Here we present the abstract of their work:
Recent studies on noninvasive recordings from human brain have shown the existence of taste-elicited activation
areas in the cerebral cortex. While functional MRI (f-MRI) and positron CT (PET) are often used for these studies, the
magneto encephalogram (MEG) is the most commonly used instrument for these noninvasive measurements. One
advantage of the MEG measurements is the ability to measure rapid taste-elicited time-course data.
In this current study, we used brain magnetic fields to quantitate the stimulus latencies evoked by different taste stimuli that
use different peripheral transduction mechanisms. Recent work has shown that taste stimuli that presumably act through
different transduction processes show different MEG-measured latencies. Here, we measured the latencies due to citric acid
and sucrose and compared these with the latency due to the action of the taste-modifying substance contained in miracle
fruit during stimulation by citric acid. Miracle fruit has the property of changing the sour taste of acids to sweet taste. The use
of this taste-modifying substance allows us to compare the latency of two very different sweet-taste-evoking substances.
(a) Institute for New Product Development, Suntory Research Center, 1-1-1 Wakayamadai, Shimamoto, Mishima, Osaka
618-8503, Japan
(b) Life Electronics Research Center, Electromechanical Laboratory, Osaka 563-8577, Japan
(c) Faculty of Human Sciences, Osaka University, Osaka 565-0871, Japan d Biological Function Division, National Food
Research Institute, Japan

"Determination of disulfide array and subunit structure of taste-modifying protein, miraculin."

Hiroshi Igeta(a), Yoichiro Tamura(a), Kazuyasu Nakaya(b), Yasuharu Nakamura(b)and Yoshie Kurihara(a),
Here we present the abstract of their work:
The taste-modifying protein, miraculin (Theerasilp, S. et al. (1989) J. Biol. Chem. 264, 66556659) has seven cysteine
residues in a molecule composed of 191 amino acid residues. The formation of three intrachain disulfide bridges at Cys-47Cys-92, Cys-148-Cys-159 and Cys-152-Cys-155 and one interchain disulfide bridge at Cys-138 was determined by amino
acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. The presence of an interchain
disulfide bridge was also supported by the fact that the cystine peptide containing Cys-138 showed a negative color test for
the free sulfhydryl group and a positive test after reduction with dithiothreitol. The molecular mass of nondashreduced
miraculin (43 kDa) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was nearly twice the
calculated molecular mass based on the amino acid sequence and the carbohydrate content of reduced miraculin (25 kDa).
The molecular mass of native miraculin determined by low-angle laser light scattering was 90 kDa. Application of a crude
extract of miraculin to a Sephadex G-75 column indicated that the taste-modifying activity appears at 52 kDa. It was
concluded that native miraculin in pure form is a tetramer of the 25 kDa-peptide and native miraculin in crude state or
denatured, nondashreduced miraculin in pure form is a dimer of the peptide. Both tetramer miraculin and native dimer
miraculin in crude state had the taste-modifying activity.
(a) Department of Chemistry, Faculty of Education, Yokohama National University, Yokohama, Japan b School of
Pharmaceutical Sciences, Showa University, Tokyo, Japan

"Sweet proteins - potential replacement for artificial low calorie sweeteners".

Ravi Kant
... Published an article
The Abstract of his paper says: Exponential growth in the number of patients suffering from diseases caused by the
consumption of sugar has become a threat to mankinds health. Artificial low calorie sweeteners available in the market
may have severe side effects. It takes to figure out the long term side effects and by the time these are established, they are
replaced by a new low calorie sweetener. Saccharine has been used for centuries to sweeten foods and beverages without
calories or carbohydrates. It was also used on a large scale during the sugar shortage of the two world wars but was
abandoned as soon as it was linked with development of bladder cancer. Naturally occurring sweet and taste modifying
proteins are being seen as potential replacements for the currently available artificial low calorie sweeteners. Interaction
aspects of sweet proteins and the human sweet taste receptor are being investigated.
Published in the Nutritional Journal 2005, 4:5. His article is available at http://www.nutritionj.com/content/4/1/5.

New technologies for taste modifying proteins - "Proteins pack muscle to modify taste"
The article mentions the following:
Miracle fruit or Synsepalum dulcificum fruit are ellipsoid, red and have a palatable pulp containing the seed. They have
the ability to alter the perception of taste in a way that makes sour foods like lemon or vinegar taste sweet. This alteration of
taste perception persists for many minutes. Miraculin may be used as a food additive to alter the taste of food, such as by
masking sourness in some foods, or may be taken separately, before consuming a meal.
While fresh berries of S. dulcificum are very potent, damaged fruit or crude extracts become quickly denatured, and so the
aim of protein purification is to separate miraculin from factors in a crude extract that cause protein breakdown, followed by
preservation of miraculin. For purification, the fruits of S. dulcificum are homogenized in 1% PEG 20,000 in water. Dissolution
of miraculin is achieved by adjusting the pH to 7 with saturated sodium carbonate solution. The suspension is filtered through
glass and added to an equal volume of acetone with stirring. The precipitate is harvested by centrifugation at 1200g for 10
minutes and is washed with 2 volumes of 50% v/v acetone. The washed precipitate is dissolved in 0.1 M potassium
phosphate buffer pH 7.0.

Future Trends
Bulk sweeteners and flavorsome ingredients such as sugars are essential in foods and food processing. However, foods
can be substantially improved by the use of intense sweeteners and flavor enhancers. Recent advances are removing the
barrier of scarcity that has limited the use of TMPs (Taste Modifying Proteins), and new advances will further exploit
structural and biochemical studies of TMPs.
The production of TMPs for the food industry has been limited by the expense of protein purification and by the limited
availability of fruit to process. The protein nature of these ingredients and their amenability to genetic engineering and
biotechnology suggest a solution to these limits.

Molecular biology will be used in the future to manipulate the quality rather than the quantity of TMPs available. Properties
that may be manipulated in the future are taste intensity, aftertaste and taste profile, which have already been modulated in
biochemical experiments. Once the molecular basis of these changes in taste have been elucidated, then protein
engineering may be used to alter the taste properties of TMPs and allow the production of tailor-made molecules on an
industrial scale.
The preceding article was condensed and edited from its original, "New technologies for taste modifying proteins,
"published in the July 1998 Trends in Food Science & Technology, Elsevier Science Ltd.
Michael Witty can be reached by phone at +44-1223-330219; fax: +44-1223-333953; e-mail:mw132@mole.bio.cam.ac.uk

Complete amino acid sequence and structure characterization of the taste-modifying protein,
miraculin
S Theerasilp, H Hitotsuya, S Nakajo, K Nakaya, Y Nakamura and Y Kurihara. Department of Chemistry, Faculty of
Education, Yokohama National University, Japan.
The taste-modifying protein, miraculin, has the unusual property of modifying sour taste into sweet taste. The complete
amino acid sequence of miraculin purified from miracle fruits by a newly developed method (Theerasilp, S., and Kurihara, Y.
(1988) J. Biol. Chem. 263, 11536- 11539) was determined by an automatic Edman degradation method. Miraculin was a
single polypeptide with 191 amino acid residues. The calculated molecular weight based on the amino acid sequence and
the carbohydrate content (13.9%) was 24,600. Asn-42 and Asn-186 were linked N-glycosidically to carbohydrate chains. High
homology was found between the amino acid sequences of miraculin and soybean trypsin inhibitor.
J. Biol. Chem., Vol. 264, Issue 12, 6655-6659, 04, 1989

Complete purification and characterization of the taste-modifying protein, miraculin, from miracle
fruit
S Theerasilp and Y Kurihara. Department of Chemistry, Faculty of Education, Yokohama National University, Japan.
The taste-modifying protein, miraculin, has the unusual property of modifying a sour taste into a sweet taste. Previous
attempts to isolate miraculin from deeply colored alkaline extracts of the miracle fruit were unsuccessful. We found that
miraculin is extracted with 0.5 M NaCl solution. The extracted solution is colorless and shows the strong sweet-inducing
activity. Miraculin was purified from the extracted solution by ammonium sulfate fractionation, CM-Sepharose ion-exchange
chromatography, and concanavalin A-Sepharose affinity chromatography. The purified miraculin thus obtained gave a single
sharp peak in reverse phase high performance liquid chromatography, indicating that it is highly pure. The sample also gave
a single band having molecular weight 28,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This value was
much lower than the values reported previously (40,000-48,000). The amino acid composition of the purified miraculin was
determined. Sequence analysis of the purified miraculin indicated that it is composed of a pure single polypeptide and
identified 20 amino-terminal amino acids. The purified miraculin contained as much as 13.9% of sugars, which consisted of
glucosamine, mannose, galactose, xylose, and fucose in a molar ratio of 3.03:3.00:0.69:0.96:2.12.
J. Biol. Chem., Vol. 263, Issue 23, 11536-11539, 08, 1988

Structural study of asparagine-linked oligosaccharide moiety of taste- modifying protein,

miraculin
N Takahashi, H Hitotsuya, H Hanzawa, Y Arata and Y Kurihara. Department of Biochemistry, Nagoya City University
College of Nursing, Japan.
The structures of the N-linked oligosaccharides of miraculin, which is a taste modifying glycoprotein isolated from miracle
fruits, berries of Richadella dulcifica, are reported. Asparagine-linked oligosaccharides were released from the protein by
glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a
fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by
high performance liquid chromatography (HPLC) on an ODS-silica column. More than five kinds of oligosaccharide fractions
were separated by the one chromatographic run. The structure of each oligosaccharide thus isolated was analyzed by a
combination of sequential exoglycosidase digestion and another kind of HPLC with an amidesilica column. Furthermore, high
resolution proton nuclear magnetic resonance (1H NMR) measurements were carried out. It was found that 1) five
oligosaccharides obtained are a series of compounds with xylose- containing common structural core, Xyl beta 1----2 (Man
alpha 1----6) Man beta 1----4-GlcNAc beta 1----4 (Fuca1----3)GlcNAc, 2) a variety of oligosaccharide structures are significant

for two glycosylation sites, Asn-42 and Asn-186, and 3) two new oligosaccharides, B and D, with unusual structures
containing monoantennary complex-type were characterized. (formula; see text)
J. Biol. Chem., Vol. 265, Issue 14, 7793-7798, May, 1990<

Cloning and sequencing of a cDNA encoding a taste-modifying protein, miraculin

Yutaka Masuda(a), Satoru Nirasawa(b), Kazuyasu Nakaya(a) and Yoshie Kurihara(b). Available online 27 December 1999.
References and further reading may be available for this article. To view references and further reading you must purchase
this article.
Abstract:
A cDNA clone encoding a taste-modifying protein, miraculin (MIR), was isolated and sequenced. The encoded precursor to
MIR was composed of 220 amino acid (aa) residues, including a possible signal sequence of 29 aa. Northern blot analysis
showed that the mRNA encoding MIR was already expressed in fruits of Richadella dulcifica at 3 weeks after pollination and
was present specifically in the pulp.
Author Keywords: Sweetness-inducing protein; Richadella dulcifica; miracle fruit; Northern blot
Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, DNA complementary to RNA; kb, kilobase(s) or 1000 bp; MIR,
miraculin; MIR, gene (DNA) encoding MIR; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction; Rd,
Richadella dulcifica; SDS, sodium dodecyl sulfate
(a) Laboratory of Biological Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo 142, Japan. Tel. (813) 3...
(b) Department of Chemistry, Faculty of Education, Yokohama National University, Yokohama 240, Japan
Received 4 January 1995; accepted 20 February 1995; Received by A. Nakazawa
_________________________________________________________________________________________________
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Miraculin, the Sweetness-inducing Protein from Miracle Fruit

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

J. N. BROUWER, H. VAN DER WEL, A. FRANCKE & G. J. HENNING. Unilever Research Laboratory, Vlaardingen, The
Netherlands.
The berries of Richardella dulcifica (Schum. and Thonn.) Baehni, formerly designated as Synsepalum dulcificum(1), a
shrub indigenous to tropical West Africa, have long been known for their taste-changing properties(2). These berries, called
miraculous berries or miracle fruit, have the property of modifying the taste of sour foods and dilute mineral and organic acids
into a sweet taste after the fruit pulp has been chewed. This modifying effect lasts for some time, usually for 12 h.
Baehni, C. , Boissiera, 11, 1 (1965).
Daniell, W. F. , Pharm. J., 11, 445 (1852).
Inglett, G. E. , Dowling, B. , Albrecht, J. J. , and Hoglan, F. A. , J. Agric. Food Chem., 13, 284 (1965).
Badran, A. M. , and Jones, D. E. , Nature, 206, 622 (1965). Article
Goldstein, J. L. , and Swain, T. , Phytochemistry, 4, 185 (1965).
Hultin, H. O. , and Levine, A. S. , J. Food Sci., 30, 917 (1965).
Reisfeld, R. A. , Lewis, U. J. , and Williams, D. E. , Nature, 195, 281 (1962). | Article | PubMed | ISI |
ChemPort |
Scott, T. A. , and Melvin, E. H. , Anal. Chem., 25, 1656 (1953). | Article | ISI | ChemPort |
Garin, S. , and Hood, D. B. , J. Biol. Chem., 131, 211 (1939).
Beidler, L. M. , J. Food Sci., 31, 275 (1966); Sci. News Lett., 88, 329 (1965).
Nature 220, 373 - 374 (26 October 1968); doi:10.1038/220373a0

ON THE GUSTATORY EFFECTS OF MIRACULIN AND GYMNEMIC ACID IN THE MONKEY

G. HELLEKANT, E. C. HAGSTROM*, Y. KASAHARA**, and Y. ZOTTERMAN. Department of Physiology Veterinrhgskolan,
104 05 Stockholm 50, Sweden
The summated response from the chorda tympani proper nerve of 9 monkeys was recorded during stimulation with
solutions of acetic and citric acids, sodium chloride, quinine sulfate, sucrose, glucose and fructose before and after
application of extracts of Synsepalum dulcificum-miraculin- and Gymnema sylvestre-gymnemic acid-on the tongue. It was
observed that (a) miraculin enhanced the response to all acids used (b) miraculin had no significant effect on the response of
the other taste stimuli (c) its effect lasts for more than h and was not removed by rubbing of the tongue (d) gymnemic acid
had no significant effect on the response to any of the stimuli used if miraculin had not been applied beforehand (e)

gymnemic acid applied after miraculin diminished the response to acid, then miraculin enhanced the response to acid again.
It was concluded that these electrophysiological findings in monkey parallel the psychophysical observations in man with
regard to the effect of miraculin and gymnemic acid on the response to acids, but that they differ with regard to the effect of
gymnemic acid on the response to sugars.
* On leave from Dept. of Psychology, University of New Hampshire, U.S.A.** On leave from Dept. of Oral Physiology,
Osaka University, Japan.Chemical Senses 1: 137-145, 1974

Determination of disulfide array and subunit structure of taste-modifying protein, miraculin.

Igeta H, Tamura Y, Nakaya K, Nakamura Y, Kurihara Y. Department of Chemistry, Faculty of Education, Yokohama National
University, Japan.
The taste-modifying protein, miraculin (Theerasilp, S. et al. (1989) J. Biol. Chem. 264, 6655-6659) has seven cysteine
residues in a molecule composed of 191 amino acid residues. The formation of three intrachain disulfide bridges at Cys-47Cys-92, Cys-148-Cys-159 and Cys-152-Cys-155 and one interchain disulfide bridge at Cys-138 was determined by amino
acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. The presence of an interchain
disulfide bridge was also supported by the fact that the cystine peptide containing Cys-138 showed a negative color test for
the free sulfhydryl group and a positive test after reduction with dithiothreitol. The molecular mass of non-reduced miraculin
(43 kDa) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was nearly twice the calculated
molecular mass based on the amino acid sequence and the carbohydrate content of reduced miraculin (25 kDa). The
molecular mass of native miraculin determined by low-angle laser light scattering was 90 kDa. Application of a crude extract
of miraculin to a Sephadex G-75 column indicated that the taste-modifying activity appears at 52 kDa. It was concluded that
native miraculin in pure form is a tetramer of the 25 kDa-peptide and native miraculin in crude state or denatured, nonreduced miraculin in pure form is a dimer of the peptide. Both tetramer miraculin and native dimer miraculin in crude state
had the taste-modifying activity.
Biochim Biophys Acta. 1991 Sep 20; 1070(3):303-7.

The sweetness-inducing effect of miraculin; behavioural and neurophysiological experiments in

the rhesus monkey Macaca mulatta.
J. N. Brouwer*, D. Glaser , C. Hard af Segerstad, G. Hellekant, Y. Ninomiya and H. van der Wel*. Department of Veterinary
Science, University of Wisconsin, Madison, WI 53706 U.S.A.
Wisconsin Regional Primate Center, Madison, WI 53706 U.S.A.Unilever Research Laboratory, Vlaardingen, The
NetherlandsAnthropologisches Institut der Universitat Zurich, Switzerland
1. The gustatory effects of miraculin, the sweetness-inducing protein from the miracle fruit Synsepalum dulcificum, was
studied in the rhesus monkey, Macaca mulatta.2. The intake of five acids was recorded in two-bottle preference tests, one
bottle containing acid and the other tap water, before and after miraculin treatment. All the acids tasted more pleasant after
miraculin.3. The electrical activity of the chorda tympani nerve to stimulation of the tongue with a variety of sweeteners,
acids, sodium chloride and quinine hydrochloride was recorded in anaesthetized animals.4. Pre-treatment of the tongue with
03-5 mg miraculin doubled the summated nerve response to the acids and diminished the response to sucrose by about
10%. The enhancement lasted for at least an hour and the diminution up to 20 min.5. After miraculin treatment the
Spearman's rank correlation coefficient between the order of increased intake of acids and the order of enhancement of the
summated nerve response was 099.6. A solution of 01 mg miraculin per ml. elicited a weak nerve response. No preference
over water for this concentration of miraculin was recorded in the two-bottle tests.7. The activity of twenty-nine single taste
fibres, selected for their responsiveness to sweetness or acids or both, was recorded after miraculin treatment. Effects were
obtained in nine fibres which were similar but more pronounced than those observed in the summated recordings. Before
miraculin, these fibres responded better and to a larger variety of sweeteners (81%) than the other fibres (40%). After
miraculin, acids elicited on the average 23 times more activity than before, while the response to sweeteners was depressed.
In twenty fibres no effect of miraculin was observed. These fibres responded to fewer of the sweeteners and were more
stimulated by the non-sweet stimuli than the first group.8. The results suggest that miraculin acts on those structures in the
taste cell membrane that are involved in perception of the sweet taste, making them sensitive to acids. The new quality of
sweetness after miraculin treatment is signalled by taste fibres which normally respond to sweet substances but which, under
the influence of miraculin, are responding to acids. It is likely that the quality of a taste stimulus is conveyed by the identity of
the taste fibres.* On leave from the Department of Oral Physiology, Gifu College of Dentistry, Gifu, Japan.

J Physiol Vol 337 pp 221-240

Copyright 1983 byThe Physiological Society

Taste in chimpanzees. III: Labeled-line coding in sweet taste.

Hellekant G, Ninomiya Y, Danilova V(1)
In peripheral taste the coding mechanism remains an enigma. Among coding theories the "across-fiber pattern" argues that
activity across fibers codes for taste, whereas the "labeled line" claims that activity in a particular set of fibers underlies a
taste quality. We showed previously that chimpanzee chorda tympani taste fibers grouped according to human taste qualities
into an S-cluster, responding predominantly to sweet stimuli, a Q-cluster, sensitive to bitter tastants, and an N-cluster,
stimulated by salts. The analysis showed that information in the S-line suffices to distinguish stimuli of one taste quality from
the others. However, one condition for the labeled line remained: that blockage of activity in a particular line must cause
blockage of one taste quality, but of no other, or its onset give rise to the sensation of a taste quality. Here we studied this
requirement with gymnemic acids and miraculin. In humans and chimpanzees, gymnemic acids suppress the sweet taste of
all sweeteners whereas miraculin adds a sweet taste quality to sour stimuli. Gymnemic acids also abolish miraculin-induced
sweet taste. We found that gymnemic acids practically abolished the response to every sweetener in the chimpanzee Scluster. Equally important, they had no effect on the responses of the Q- and N-clusters. After miraculin, the S-cluster fibers
responded to acids as well as to sweeteners, although they had not responded to acids before miraculin. Gymnemic acids
abolished this miraculin-induced response to acids and responses to sweeteners in the S-fibers. These results link the sweet
taste quality to activity in fibers of the S-cluster. Thus the S-cluster fibers satisfy the definition of the labeled-line theory: "that
activity in a particular fiber type represents a specific taste quality."
(1) The University of Wisconsin and Wisconsin Regional Primate Center, Madison 53706, USA.GH@ahabs.wisc.edu
Unpleasant sweet taste: a symptom of SIADH caused by lung cancer
Y Nakazato, K Imai, T Abe, N Tamura, K Shimazu

Abstract:
A 56 year old woman with large cell lung carcinoma complained of an unpleasant sweet taste (dysgeusia). She developed
hyponatraemia caused by the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Dysgeusia disappeared
when serum sodium normalised and recurred when hyponatraemia relapsed. Dysgeusia was the initial and only symptom of
SIADH in this case.
Keywords: SIADH; dysgeusia; lung cancer; miraculin
Department of Neurology, Saitama Medical School, Saitama, Japan
Correspondence t
Dr Yoshihiko Nakazato
Department of Neurology, Saitama Medical School, 38 Morohongo Moroyama, Iruma-gun, Saitama 350-0495, Japan;
nakachan@saitama-med.ac.jp
Journal of Neurology, Neurosurgery, and Psychiatry 2006;77:405-406; doi:10.1136/jnnp.2005.073726
Copyright 2006 by the BMJ Publishing Group Ltd.

A quantitative enzyme immunoassay for miraculin in Richadella dulcifica (miracle fruit).

Shigeo Nakajo, Sarroch Theerasilp(1), Kazuyasu Nakaya, Yasuharu Nakamura and Yoshie Kurihara(1)
We have developed an enzyme-linked immunoabsorbent assay for miraculin, a glycoprotein which is capable of modifying
sour taste into sweet. Antiserum against purified miraculin was raised in rabbits and the anti-miraculin in IgG was purified
using a Protein A-Cellulofine column and then conjugated with horseradish peroxidase. The established enzyme
immunoassay method was able to accurately quantitate pg amounts of miraculin in crude extracts of miracle fruit. The
amounts of miraculin in miracle fruit increased dramatically seven weeks after pollination and at eight weeks reached 102
g/mg proteins in the fruit.
Chemical Senses 13: 663-669, 1988(1) Department of Chemistry, Faculty of Education, Yokohama National University
Tokiwadai, Hodogaya-ku, Yokohama, Japan Laboratory of Biological Chemistry, School of Pharmaceutical Sciences, Showa
University 1-5-8 Hatanodai, Sinagawa-ku, Tokyo

Purification of miraculin glycoprotein using tandem hydrophobic interaction chromatography

Document Type and Number: United States Patent 5886155

Abstract:
A method for purifying a plant protein comprises: (a) preparing a crude plant extract; (b) passing the crude plant extract with
a first buffer solution through a guard column comprising a hydrophobic interaction chromatography medium of
hydrophobicity sufficiently high to prevent tannins and polyphenols from eluting from the column in the presence of the buffer
solution, but sufficiently low that a protein fraction elutes with the first buffer solution; (c) passing the protein fraction through
a capture column coupled in series to the guard column, the capture column comprising a hydrophobic interaction
chromatography medium of hydrophobicity sufficiently high to prevent the protein from eluting in the presence of the buffer
solution; and (d) eluting the protein from the capture column as a purified fraction. Preferably the plant is miracle fruit, the
protein is Miraculin, and the method comprises ion exchange chromatography and gel filtration chromatography of the
purified fraction of the protein. Foods and beverages may be sweetened with the purified Miraculin.

Genetically stable expression of functional miraculin, a new type of alternative sweetener, in

transgenic tomato plants
Hyeon-Jin Sun, Hiroshi Kataoka, Megumu Yano and Hiroshi Ezura*
Abstract:
Miraculin is a taste-modifying protein isolated from the red berries of Richadella dulcifica, a shrub native to West Africa.
Miraculin by itself is not sweet, but it is able to turn a sour taste into a sweet taste. This unique property has led to increasing
interest in this protein. In this article, we report the high-yield production of miraculin in transgenic tomato plants. High and
genetically stable expression of miraculin was confirmed by Western blot analysis and enzyme-linked immunosorbent assay.
Recombinant miraculin accumulated to high levels in leaves and fruits, up to 102.5 and 90.7 g/g fresh weight, respectively.
Purified recombinant miraculin expressed in transgenic tomato plants showed strong sweetness-inducing activity, similar to
that of native miraculin. These results demonstrate that recombinant miraculin was correctly processed in transgenic tomato
plants, and that this production system could be a good alternative to production from the native plant.
KEYWORDS: low-calorie sweetener miracle fruit miraculin sweetness-inducing activity taste-modifying
protein transgenic tomatoCorrespondence (fax +81 29 853 7263; e-mail:ezura@gene.tsukuba.ac.jp
Copyright 2007 The Authors
Journal compilation 2007 Blackwell Publishing Ltd
* Graduate School of Life and Environmental Sciences, Gene Research Center, University of Tsukuba, Tennodai 1-1-1,
Tsukuba, Ibaraki 305-8572, Japan

New technologies for taste modifying proteins

Michael Witty
Department of Plant Sciences
University of Cambridge
Cambridge CB2 3EA, UK
(tel: +44-1233-333938; fax: +44-1233-333953; e-mail:m.bwit@seqnet.dl.ac.uk)
Available online 3 December 1998. References and further reading may be available for this article. To view references
and further reading you must purchase this article.
Abstract:
Taste-modifying proteins are a natural alternative to artificial sweeteners and flavour enhancers and have been in use in
some cultures for centuries. Use has been limited by the stability and availability of these proteins, but recently advances in
biotechnology have been made that will increase their availability. These include production in transgenic organisms and
protein engineering to increase stability. Taste-modifying proteins will be available for much wider use in the food industry,
and will reduce dependence on synthetic alternatives.
Article Outline:
1. Thaumatin purification
2. Purification of monellin
3. Purification on miraculin4. New technologies
5. Future trends
References
Received 27 April 2007; revised 27 June 2007; accepted 29 June 2007.

On the sense of taste in two Malagasy Primates (Microcebus murinus and Eulemur mongoz)
G. Hellekant, C.M. Hladik(1), V. Dennys(1), B. Simmen(1), T.W. Roberts, D. Glaser(2), G. DuBois(3)and D.E. Walters(4)
University of Wisconsin, Department of Animal Health and Biomedical Sciences, and Wisconsin Regional Primate Center
Madison, WI 53706, USA
The relationship between phylogeny and taste is of growing interest. In this study we present recordings from the chorda
tympani proper (CT) nerve of two lemuriforme primates, the lesser mouse lemur (Microcebus murinus) and the mongoose
lemur (Eulemur mongoz), to an array of taste stimuli which included the sweeteners acesulfame-K, alitame, aspartame, Dglucose, dulcin, monellin, neohesperidin dihydrochalcone (NHDHC), saccharin, sodium superaspartame, stevioside,
sucralose (TGS), sucrose, suosan, thaumatin and xylitol, as well as the nonsweet stimuli NaC1, citric acid, tannin and
quinine hydrochloride. In M.murinus the effects of the taste modifiers gymnemic acid and miraculin on the CT response were
recorded. Conditioned taste aversion (CTA) experiments in M.murinus and two-bottle preference (TBP) tests in E.mongoz
were also conducted. We found that all of the above tastants except thaumatin elicited a CT response in both species. The
CTA technique showed that M.murinus generalized from sucrose to monellin but not to thaumatin. The intake of aspartame,
ranging in concentration from 0.1 to 30 mM was measured in E.mongoz with TBP tests. At no concentration did we see a
preference, but there was a significant rejection of 10 and 30 mM aspartame (P 0.025). Miraculin had no effects on the CT
response to acids, and gymnemic acid did not selectively suppress the CT response to sucrose or that of any other
sweeteners. The absence of ability to taste thaumatin in these species supports the dichotomy between catarrhine and noncatarrhine species. The difference in results with thaumatin and monellin indicate that their sweet moieties are not identical. It
also points to a phylogenetic difference in taste within the prosimian group. Further, the results with aspartame indicate that
the perception of sweetness from aspartame is limited to catarrhine species. Finally, neither miraculin nor gymnemic acid
exhibit the same taste modifying effects in lemuriformes as they do in hominoidea. Thus the results with gymnemic acid and
miraculin corroborate those obtained earlier in other prosimians.
Miracle fruit is an evergreen shrub that grows in West Africa. The berry of the miracle fruit plant is used as
medicine.
People take miracle fruit to treat diabetes and correct chemotherapy-related taste disturbances.
In foods, miracle fruit is used as a low-calorie sugar-free sweetener.
How does it work?

Miracle fruit contains a chemical that affects taste receptors in the tongue. This chemical makes the tongue
register sour tastes as sweet tastes. The chemical itself has no taste at all.