Sweet, antisweet and sweetness-inducing substances

Review Recent progress in studies of antisweel substance;,, sweet proteins and sweetness-inducing proteins is reviewed. Gymnemic acid, ziziphin and hodulcin, which are triterpene glycosides, suppress the sweet taste sensation in humans, and gurmarin, which is a peptide, suppresses the sweet taste response in the rat. Five sweet proteins, monellin, thaumatin, pentadin, curculin and mabinlin, are known. There are two sweetness-inducing proteins. Miraculin has the ability to modify a sour taste into a sweet taste. Curc~din, which is itself a sweet protein, induces a sweet taste in response to water, as

Sweet, antisweet and sweetness-inducing substances Yoshie Kuriharaand Satoru Nirasawa

well as having a taste-modifying activity similar to that of

homologues and determined their structures to be glucuronides of a triterpene whose OH group at C-21 in the genin is esterified with either 2-methylbutyric acid or 2-methylcrotonic acid 2. Hydrolysis of the acyl group led to a complete loss of the antisweet activity. In recent years, the numbers of diabetic and overweight Recently, a number of gymnemic acid homologues people have greatly increased worldwide. For the pre- were isolated by Arihara's group 3. The structural differvention of obesity or the therapy of diabetes, it is import- ence between these homologues is in the number and ant to limit the ingestion o f sugars. There are many type of substituent acyl groups on the gymnemagenin, low- or no-calorie artificial sweeteners, but most cannot which has six hydroxyl groups. In general, the antisweet be used as food additives because of harmful side effects. activity of the gymnemic acid homologues decreased In general, proteins cannot stimulate taste receptors with decreasing numbers of acyl groups on the genin, because of sterie hindrance, but exceptionally a few pro- suggesting that hydrophobicity plays an important role teins, such as monellin, thaumatin, pentadin, curculin in the generation of the antisweet activity. and mabinlin, elicit a sweet taste. Two proteins, miracThe leaves of G. sylvestre have been used for the therulin and curculin, are known to modify the taste func- apy or prevention of diabetes in India. According to tion, having the ability to modify a sour taste into a Yoshioka ~, gymnemic acid has the ability to suppress sweet taste. Curculin also has the ability to induce a glucose absorption in the small intestine. He examined sweet taste in response to water. Both the sweet proteins the effect of gymnemic acid on blood glucose levels in and the sweetness-inducing protein curculin elicit a sweet the rat as a function of time after the oral administration taste at very low concentrations. Most of these proteins of sucrose. Sugar ingestion without gymnemic acid are derived from plants and have been used to add a ingestion brought about an overshoot increase in the sweet taste to foods or drinks by native people and can blood glucose level; concurrent ingestion of gymnemic be safely ingested. Hence these proteins may have acids suppressed the oversrloot increase. These results suggested that gymnemic acids may be useful for the potential as low-calorie sweeteners. In this paper, we review recent progress in the study therapy of diabetes. of the sweet proteins and sweetness-inducing proteins. In addition, we briefly discuss antisweet substances, Ziziphin which are useful as tools for elucidating the receptor The leaves of the plant Zizyphusjujuba MILL contain mechanisms of sweet taste. The sources and activities of a sweetness-inhibiting substance known as ziziphin5. all three classes of substances are summarized in Table 1. We have purified ziziphin and determined tts structure to be a glycoside of the triterpene jujubogenin 6. Ziziphin inhibited the swe:t taste of all the sweeteners examined Antisweetsubstances Gymnemic acid (D-glucose, D-fru-tose, stevioside, sodium saccharin and The leaves of Gymnema sylvestre R. Br. contain an aspartame), while it had no suppressive effect on the active component, gymnemic acid~ which has the ability salty taste of NaC1, the sour taste of HCI, nor the bitter to suppress sweet taste in humans. If one chews the taste of quinine. Thus, the taste-suppressing activity of leaves, sugar powder tastes like sand and sugar sol- ziziphin is highly specific for sweetness. ution tastes like plain tap water. About 25 years ago, Reichstein and his co-workers purified gymnemic acid Hodulcin The leaves of Hovenia dulcis THUNB contain a and determined its chemical structure t. The sample they named hodulcin. isolated, however, contained many homologues, which sweetness-inhibiting substance could be separated by high-performance liquid chroma- Kennedy et aL 7"8 partially purified hodu!cin and estimated that its aglycone is jujubogenin, the same as that tography (HPLC). We have since purified two of the of ziziphin, but that ziziphin and hodulcin differ in their YoshieKuriharaand SatoruNirasawaare at the Departmenlof Chemistry, substituent saccharides. Hodulcin has the ability to supFacultyof Education,YokohamaNationalUniversity,Yokohama240, Japan. press the sweetness of sucrose without reducing the

miraculin.

Trends in Food Science& TechnologyFebruary1994 [Vol. 51

~,~,,4.~,,~.,,~.,s......... ~,,,,~.,.,~-.,.,.~.~,,4so:"~,o

3,

Tabhl, 1. Sourcesand activitiesof anlisweet substances,sweet proteinsand sweetness-inducingproteins Substlance

Source

Growing district

Activity

Gym ~emic acid Ziziplhin Hodtl,tcin Gurmari,

India North China China. Japan India

Suppression of sweetness Suppression of sweetness Suppression of sweetness Suppression of sweetness

West Africa

Sweet taste

Thaurnatm Pent~ldin Mabinlin CurolJlin

Leaves of Gymnema syIvestre Leaves of Ziz~,phusjujuba Lecves of Hovenia dulcis Leaves of Gr,mnemasylvestre Fruitsof Dioscoreophyllim cumminsii Fruits of Thaumatococcusdanielli Fruits of Pentadiplandrabrazzeana Seeds of Capparis masaikai Fruits oi Curculigc, iatifolla

West Africa Africa South China Malaysla

Miraculin

Fruits of Richadella dulcifica

West Africa

Sweet taste Sweet taste Sweet taste Sweet tasle Sour taste -~ sweet taste Water * sweet taste Sour taste -~ sweet taste

Monellin

tastes of NaCI, citric acid and qtfinine sulfate. Recently, Yoshiikawa et al. ~ purificd five new dammarane glycosides having antisweet activity l rein the leaves of H. dulci.+. Three analogues were glycosides ofjujubogenin, as Kennedy et al. had estimated.

and gel filtration. The molecular mass of the subunit of pentadin was estimated to be around 12000Da. The sweetness intensity of pentadin was about 500 times that of sucrose on a mass basis. Data on the heat stability of pentadin have not been reported.

Gurmarin

Mabinlin

Oymnentic acids, extracted from tile leaves of G. syh'estre, suppress the sweet taste sensation i~TM, humans (see above), but do not suppress the responses to sweeteners in the rat. However, lmoto et al. "~ have found that tlhe hot water extract from the leaves of G. syh,estre also contains a separate substance that does suppress the rat taste neural response to sucrose when the extract is applied to the tongue surface. They purified the active principle, named it gunnarin, and determined its structure u~. Gurmarin is a peptide consisting of 35 amino acids and having a molecular mass of 4209 Da. It took several hours to recover the suppressed sweet response after gumlarin ingestion in the lat j°, while the suppressive effeclt of gymnemic acid on sweet taste in humans only lasted for ~10min. Gurmarin does not suppress sweet taste sensation in humans.

Plants of Caplmris masaikai L6vl. grow in the south of Yunnan in China and bear fruits the size of tennis balls. The seeds of mature fruits have a sweet taste and the sweet principle has been named mabinlin 23. Recently, we purified five homologues of mabinlin z4. The molecular masses of the mabinlin homologues were ~ 12 000 Da. For each homologue, the maximum sweetness induced by an 8XI0-4M mabinlin solution in water was equivalent to that of a 0.3 M sucrose solution; thus mabinlin is 375 times sweeter than sucrose. Of the five homologues, mabinlin 11 was the most heat stable. Its sweet activity was unchanged by heating in solution at 80°C for over 48h. On the other hand, the sweet activity of one of the other homologues, mabinlin I-1, was completely lost after a 1 h incubation at 80°C. Circular dichroism spectroscopy showed that the a-helical structure of mabinlin 1-1 was completely destroyed by the I h incubation at 80°C, in parallel with the loss of sweet activity, whereas that of mabinlin 11 was unchanged under the same conditions 25. The properties of mabinlin !I are summarized in Table 3.

Sweet proteins

Fi'¢e sweet proteins are known: thaumatin ~-~,monellin ~3, pentadin u, curculin and mabinlin. Curculin also has the ability to induce sweetness and is discussed in more detail below, under the heading "Sweetness-inducing proteins'. There are already several good articles and reviews on monellin and thaumatin ~s-2~ and hence this review deals only briefly with these proteins. Table 2 summarizes the properties of monellin and thaumatin 1. Monellin and thaumatin 1 are 3000 and 1600 times sweeter than sucrose on a mass basis, respectively. Both are basic proteins. The sweet activity of thaumatin I is not destroyed by boiling its solution for over I h (Ref. 22). Natural monellin is composed of A and B chains and its sweet activity is destroyed by boiling; however, the sweet activity of a single-chain monellin synthesized by crosslinking the two chains is retained on boiiing t'~.

38

Table 2. Properties of monellin and thaumatin I

Properly Sweet taste' Number of amino acids ~ Molecular mass (Da): [ Monomer i A chain B chain : [soelectric point

Penlladin

" Datatakenfrom Ref. 15

The fruits of the plant Pentadiplandra brazceana Bai~lon contain a sweet protein named pentadin, van tier Well et a/. ~ partially purified pentadin by uhrafiluation

b Datataken (romReL16

Monellin '~

Thaumatinl u

3000

1600

94

207

11 086 5251 5835

22 209

9.3

12.0

Sweetness,.'.'ascomparedwith that of sucroseon a massbasis

Trends in Food Science & Technology February 1994 [Vol. 5[

We have determined the amino acid sequences of mabinlin II (Ref. 24) and mabinlin I-1 (Ref. 25). Both proteins are composed of an A chain and a B chain. There is a high degree of similarity between the two proteins, Both proteins have eight cysteine residues and the same disulfide structures :~. The A and B chains are connected by two disulfide bridges, and there are two intrachain disulfide bonds within the B chain (Fig. la). The difference in the heat stabilities of the two proieins seems to arise from a difference in part of the amino acid sequence. It seems that a local core structure present in mabinlin I1 but absent in mabinlin I- I contributes to the heat stability o+r the a-helix. Sweetness-inducing proteins Miraculin The plant Richadella dzdciJic~t bears fruits called miracle fruit, which have

r. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Table 3. Properlies of mabinlin II, miraculin and turtulin Property i Molecular mass (Da): i Monomer Dimer ~'Carbohydrate content l~oelectric pbint ! Maximum s~,eetnessof protein solution'~ Maximum sweetness induced by b.! ',~ citric acid d ! :Maximum sweetnes~ induced by warerd

Mabinlin IP

Miraculln b

Curc,.lin ~

12 400 NA

24600 43000

lZ 4(}0 27 800 NA

NA

I {.9%

] 1.3

9.1

7. i

375

NA

9000

NA

400 000

9000

NA

NA

9000

I itj Data taken f¢om Ref.24 ~'Data taken from Rei. 21'} ~ Data taken from Ref. 27 The sweetnessof each protein solution in ,.'cateris comparedto that oi a sucrosesolution,on a molar basis;see lexl for detai';~, i NA Not applicable

! i i

1 s la 33 A chain - - . . ' ~ 7 ~ ' ~ S S

I 121

I 110

:he unusual property of being able to (a) change a sour taste into a sweet taste. s s For example, lemon elicits a sweet taste after chewing the fruit. A pew extraction method to purify the active component, miraculin, has recently been developed in our laboratory-+6. 1 The properties of miraculin are sumi st S' ~ S , S S marized in Tnble 3. Pure miraculin (b) itself has no taste. All the acids tested S--S $ S I I elicited a sweet taste if tasted after I l, l l ,,, S--S miraculin had been held in the mouth 1 for a few minutes. The sweetness in2:, ~ , ...... ,,,,, duced by 0. I M citric acid after tasting S S S S a 1 p,M miraculin solution is equiv(c) i i alent to that of a 0.4 M sucrose solS S S S ution; thus, the sweetness of a mirt i I 1 114 aculin solution induced by 0.1 M citric acid solution is 400000 times that of a sucrose solution. The sweetness- Fig. 1 inducing activity of miraculin lasts for Structures of: la), mabinlin II (data taken from Refs 24 and 23); (b), miraculin (data taken from Refs 29 2 h after the application of miraculin and 30); (c), curculin (data taken fro;n Ref. 27). to the mouth:". Miraculin in the fruit is present as a homodimer; but in the pure state, two has 51.1% homology, h is not known why miraculin miraculin dimers aggregate to form a tetramer. Both the shows a high degree of homology with soybean trypsin dimer and the tetramer have taste-modlfying activity:L inhibitor. We have attempted to clone the miraculin gene with The primary structure of miraculin has been determined. The miraculin monomer is composed of 191 amino acid the goal of determining the effects of site-specific muresidues3~'; there are three intrachain disulfide bridges in tations on the sweetness of the protein, and thus elucidateach monomer, and an interchain disulfide bridge forms ing the active site of miraculin. On the basis of the the dimer +-'~(Fig. lb). There are two substituent sugar determined amino acid sequence of mimculin, we have chains, which are connected to the 42nd and 186th constructed a DNA duplex using an automated solid-phase asparagine residues~L Surprisingly, the amino acid phosphite approach (Horiuchi, S. et al., ur,published), sequence of miraculin has a high degree of homology The 601-base pair DNA duplex obtained was inserted with ~hat of soybean trypsin inhibitor; the N-terminal into an expression vector, and. antil:vady-binding studies sequence has 36.4% homology and C-terminal sequence (Western blotting analysis) showed that miraculin Trends in Food Science & Technology February 1994 [Vol. 51

3'-)

pmduc>tion was induced in E, colt. However, this miraculin showed no sweemess4nducing activity, possibly becausle the disulfide bridges were not correctly formed it',, E. ,',Idi.

The sweetness of a 4.2X10-~M curculin solution is equivalent to that of a 0.38 M sucrose solution; thus, curculin is about 9000 times sweeter than sucrose• The sweetness of curculin decreases with time, and is finally abolished a few minutes after the application of curculin Curculiin :o the mouth; subsequent application of water to the Cureuligo la~ifolio is grown under gum trees in mouth then re-elicits a sweet taste. The sweetness inMalay:fia. The fruits have a sweet taste; in addition, duced by water after a 4.2×10-sM curculin solution was after c,hewing the fruits, water or black tea without held in the mouth was equivalent to that of a 0.38 M added :sugar elicits a sweet taste. Sour substance';., such sucrose solution. Curculin also has the ability to modify as citriic acid or ascerbic acid also induce strong sweet- a sour tas,'e into a sweet taste. The sweetness induced by ness. siimilar to the case of miraculin. We have purified a 0.1 M citric acid solution after a 4.2×10-5M curculin the actiivc principle of the fruits, using a similar method solution was held in the mouth was also equivalent to to that employed for the purification of miraculin, and that of a 0.38 M sucrose solution2°. We have investigated n~mled it curculin-'L Using the purified sample, we have the possibility that certain components in saliva supdetermined the amino acid sequence of curculin. press the sweetness of curculin; 1 mM Ca 2÷ or Mg 2÷ solThe properties of curculin are summarizzd in Table 3. utions (corresponding to the approximate concentrations Curculiin is a homodimer: the curculin monomer is com- found in saliva) both completely suppress the swee,'ness -'°. posed of 114 amino acid residues 27. There are 4 cysteine A model for the taste-modifying action of curculin is re~idutl:s, which form two interchain disulfide bridges given in Fig. 3. The active site of curculin binds to the and one intrachain disulfide bridge per monomer sweet taste receptor site, which induces a sweet taste. In (Fig, Ic/. Recently, we succeeded in cD,stallizing cur- the presence of divalent cations in saliva, the active site culin (Fig. 2). The c~stals diffract X-rays to at least a of curculin does not stimulate the sweet receptor site resolution of 3.0,'~ and are suitable for X-ray crystal- and hence the sweet taste sensation is abolished. ElimIographic studies ~:. ination of saliva by tasting water leads to regeneration of the sweet taste. S~milar to the case of miraculin, curculin elicits a sweet taste in response to acids. In the presence of acids, divalent cations do not suppress the sweetness induced by curculin, probably because divalent cations do not bind to the receptor site at acidic pH. Therefore, the sweetness lasts longer in the presence of acids. The activity of curculin in solution is stable below 50°C, and curcuhn powder can be stored at room temperature without loss of activity for at least one year~3. In order to elucidate the active site of curc':lin, we are currently undertaking molecular cloning experiments with the goal of determining the effects of site-specific mutations on the sweetness of the protein. We have obtained two cDNA inserts encoding preprocurculin, which is composed of 158 amino acid residues, including a signal sequence of 22 residues and a carboxy• t4 termir, a! extension peptide of 22 residues . However, curcnlin expressed in E. colt after incorporation of the eDNA inserts had no sweet activity. Similar to the case with miraculin, it seems that the disulfide bridges were not correctly formed in E. colt. Antibodies raised against curculin antibody crossreact with miraculin, and antibodies raised against miraculin antibody crossreact with curculin3s. These results suggest that there is a structure common to both proteins. However. there is no high degree of similarity in the amino acid sequences of the proteins. In addition, there is no higk degree of similarity between the amino acid Fig. 2 sequences of mabinlin, monellin, thaumatin, miraculin and curculin. The fact that these proteins all have a ClfTsials of curcu!in, crystallized using the hanging drop vapour sweet ta.,;te seems to have no evolutionary significance. diffusi,l:,n method using polyethylene gl$,col a00 as a precipitant. The crvsl;al5 belong Io the orlholhombic space group P2~2~2~ with unit Conclusions cell dimensions: a = 271 A, b = I05 ,~, c= 48.7 ,~. A,.erage clystal Of the known antisweet substances, three (gymnemic length: 0,4-3 mm. (Photograph cuurtes;' ot Harada, S. el al?:i acid. ziziphin and hodulcin) are glycosides of triterpene. ;o

Trends i~ Food Science & Technology February 1994 IVol. 51

These substances all have the ability to suppress sweet taste senCurculin/ sation in humans, and they have been used mainly to elucidate the \\ mechanisms of sweet sensation by psychophysical means. Another Sv,,,eet taste receptor site antisweet substance (gurmarin) is a peptide. It does not suppress Sweet sweet taste sensation in humans, _H~ Ca 2. or ,'v'ig2. but does suppress the sweet /,,/.H~ i - ~ in saliva response in rats. Hence, gurmarin has been used to elucidate ;he mechanisms of sweet sensation by electrophysiological means. Five sweet proteins and two sweetness-inducing proteins, some of which may have potential for use as sweeteners in foods, are known. Table 3 shows a summary of the properties of the three most recently Non-sweet characterized sweet/sweetnessSweet inducing proteins, mabinlin II, miracutin and curculin. Only mimcu- Fig. 3 lin contains sugars. Mabinlin and A schematic model of the mechanism oi the sweetness- nducing acfi,, itv (,'f curculin; see !ext for details curculin elicit a sweet taste on their (adapted from Ref, 20). own, while acids elicit a sweet taste Yoshika,.:a, K., Amimolo, K., Arihara, S. and Matsuura, K. t198ql after miraculin and curculin are held in the mouth, and Tetrahedron Left. ~0. 1103-1 I06 water elicits a sweet taste after curculin is held in the mouth. Mabinlin is the most heat stable, but its sweetness 4 Yoshiaka, S. It 986) I, Yonatec, t..led. Soc, ~7, 142-1-34 Kennedy, L.M. and Halpem. B.P. ~I q801 Phvsinl. Beha;,. 24, 135-143 is not as strong as that of curculin. Of miraculin, mabinlin 5 Kurihara, Y., Ookubo. K., Tasaki. H.. kodama H.. Akivama, Y., and curculin, curculin seems to have the most potential Yagi, A. and Halpem, B. 11988! Tetrahedran 44.61-l~b for development as a low-calorie sweetener since it has 7 Kennedy, LM., Saul, L.R., Sefe~ka, R. and Sle,,en% D.A. (i9881 (.hem, a strong sweet taste and is relatively stable. In addition, Senses 13,529-543 curculin has the ability to change a sour taste into a ,~weet a Kenned',', L.M.. Bourassa, D,M. and Rogers. ME. l lqq ~I in S~.eel-Tx~te taste, a property not found in thaumatin or monellin. The Chenroreception lMathlouthi, M.. Kanters, ].A. and ['hrch, C.G.. edsl, plant that produces curculin can be easily cultured in the pp. ]i7-tS1, Ekevier tropics. A culture project has started and hence a large 9 Yoshikawa, K., ]umura, S. Yamada, K. and Anhara, S. It 9t)2, Chem. .°harm. Bull 40, 2287-2291 amount of curculin will be available in the near future. The active sites of the sweet proteins and the ~0 Irnoto. T.. Miyasaka. A., Ishima, R, and Akasaka, K. i]9911Comp. Bloc hem, Phy~ial. I OOA, ]0q- ~ 14 sweetness-inducing proteins have not yet been claritied. Ongoing biotechnological studies on these proteins will 11 Kamei, K., Takano. R., Mi;,asaka, A,, Imoto, T and Hara, S. I]"~21 L Biochem. 11 I. ]09-I 12 explore the structure of their active sites. These studies ,.'an der Wel, H. and Loeve. K. d97 !b htr. I. Bioc hem. H, 221-225 may also make !t possible to produce mc, re heat-stable t2 ,".1ofris,i.A. and Cagan, R.H, 119721Bio(hm~. Biqnh~x .~cta 201,114-t22 miraculin and curculiu. 13 •,:an der Wel, H., Laesnn. G., Hladik. C..'x.L HeH,,,Lml. (1. and Claser. D.

J/

14

Acknowledgements We wish to express heartfelt gratitude to Professor Beidler of the Florida State University lbr constant encouragement and suggestion. We also express our thanks to Professor Asoh in our University who cultured the plants used in the present study. We also express our thanks to Professor Arai. Department of Agricultural Chemistry. University of Tokyo !or experiments on the molecular cloning of curculin,

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Review

Biogenic amines and their production by microorganisms in food

usually produced by the decarboxylation of amino acids. Figure 1 shows the major pathways for the formation of di- and polyamines. Several biogenic amines (serotonin. histamine and tyramine) play important roles in many human and animal physiological functions. In plants, the diamine putrescine and the polyamines spermidine and spermine are implicated in a number of physiological processes, such as cell division, flowering, fruit development, response to stress and senescence "3. Biogenic amines in foods are of concern in relation to both lbod spoilage and food safety. They are generated either as the result of endogenous amino acid decarboxylase activity in raw food materials, or by the growth of decarboxylase-positive microorganisms under conditions favourable to enzyme activity ~. As the microbial spoilage of food may be accompanied by the This paper pre5enls an overview of the origin and importance increased production of decarboxylases, the presence of of hiogenuic anfines iound in foods, with special reference to biogenic amines might serve as a useful indicator of food spoilage. biogenic amines resulting from the metabolic activities of Monoamine oxidases and diamine oxidases play a food-associated microo~ganisms. Compared with foods of major role in amine degradation in the human body. animal origin le.g. milk and meat productsl, relativeb, little Biogenic amines do not usually represent any health iniormation is available on the levels of biogenic amines in hazard to individuals unless large amounts are ingested, or the natural mechanism for the catabolism of the •,egetable, foods and in novel and prepacked convenience amines is inhibited or genetically deficient. Typical ioods. symptoms may be observed in certain individuals, and include nausea, sweating, headache and hyper- or hypotension s. The most frequent foodborne intoxications caused by Biogenic amines are low molecular weight organic biogenic amines involve histamine. Histamine poisonbases that po~.,sess biological activity, They can be ing is also referred to as "scombroid fish poisoning" lormed and degraded as a result of normal metabolic becm~se of the association of this illness with the conactivity in animals, plants and microorganisms, and are sumption of scombroid fish, such as tuna, mackerel and sardines". Another phenomenon is the "cheese reaction' Anna Hal.~szand AgnesBar.~lhaw al Ihe Cenlr,/lFI)I)(IRe~,ejrti~Institute. caused by high levels of tyramine in cheese r. Determination of the exact toxicity threshold of bio}'~t.rrIljrl(]111(iut ]:~ H-]~~(~~u(lJ[;ot,I. HungJr'~ LiviaSimon-Sarkadii~ at th(. Dt,F,artr~wntrJl B~f~(ht'm;qr~,rod FoodleOm(flo~ ]t'(hnit,¢.LSni~t,r~il~ genic amines in individuals is extremely difficult. The toxic dose is strongly dependent on the efficiency of Ir~ l~d,!l)v!,! H-i l] BUdallU,l. [qunK~r~.. Wilhelm Holzapfel ~torwH)onding ,RJ¢:;~ i. d) tilt,ln.t)l~Jh,el }(~gl(,,,t',Irl(ito,fitoio;~ lt'(h'ra]Rt'~(.,irth C(')IIF(, the detoxilicatiou mechanisms of different individuals. T,)F",L:IFH,;I,'~ [r~14*'~-t"qr 20 I)-7()111 Kdrl~ruh(.,, (it,mldr1\ Upper limits of IOOmg histamine/kg in foods, 2rag

Anna Hal~isz,Agnes Bar~ith, Livia Simon-Sarkadiand Wilhelm Holzapfel

42

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Trends i!'~ Food gcience & T hn.'1og'/February 1994 [Vo]. 5l