INTERACTIONS OF POLYPHENOLS WITH PROTEINS IN PLANTS AND PLANT PRODUCTS*

R.L.M. SYNGE

{Norwich)

ABSTRACT

The plant polyphenols axe a very heterogeneous group, some universally and others widely distributed among plants, and often present in surprisingly high concentrations. There have been diverse speculations about their significance for the life of the plant. For present purposes, they are conveniently discussed as tannins and non-tannins.

Tannin-protein reactions are important in the preparation and enjoyment of tea, wine and beer. Tannins interfere with the digestion of proteins in poultry, pigs and human beings, but sometimes may influence that process favourably in ruminants. Non-tannin polyphenols can undergo oxidation to semiquinones and quinones. These can undergo further oxidative polymerization, as well as coupling to proteins, by a wide variety of chemical reactions. Some of these reactions may serve to protect the plant against infections, parasites or predators. Such reactions, under the name 'enzymic browning', cause difficulties in food processing. More detailed study is required of possible damage to the nutritive value of proteins; this is particularly important for poultry and pig rations.

It has become increasingly clear that these reactions of plant polyphenols contribute substantially to producing the organic matter of soils. Indeed, it is possible that this function has determined the evolutionary history of some of these polyphenols, and that they have no immediate function in the living plant. As the nature and quantity of potyphenols differ greatly between plant species, agricultural scientists should conside~ more seriously the effects of diffeient crop plants mad of different agricultural practices on soil organic matter.

Plant polyphenots are, in themselves, a fascinating group of substances and have at tracted some of the most distinguished o f the Nobel-Prize-winning organic chemists, including Emil Fischer, Willst~itter, Robinson (much helped by the botanical and gardening interests of his first wife) and Todd, besides many other excellent chemists. The polyphenols have often shown themselves tr ickier as Ph.D. fodder for research students than have those other favourite groups o f natural products - the terpenoids and the alka- loids. The continuing fascination o f polyphenols for chemists involved with

* Wechselwirkungen zwischen Polypheno~n und EiweiNtoffen in Pflanzen und pflanzlichen Produkten Vortrag gehalten auf der 24. Tagung der Nobelpreis~ger in Lindau (Bodensee) l.-5. Jufi 1974.

Qual. Plant. - P1. Fds. hum. Nutr. XXIV, 3/4: 337-350, 1975.

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plant constituents is shown by the fact that both the British and North American PhytochemicaI Societies originally styled themselves 'plant phenolics groups' - and, earlier this year, a new 'plant phenolics group' has established itself in France.

The plant polyphenols are a highly diverse group, the chemically known members of which can be counted in hundreds, if not in thousands (Harborne, 1964; Rib6reau-Gayon, 1972; Karrer, 1958). Yet our knowledge of their distribution, particularly in quantitative terms, is still fragmentary. The late Professor A.I. Virtanen used to express the hope that phyto- chemists would some time try to account quantitatively in chemical terms for all the recognizable constituents of a single piece of plant material. He himself, perhaps imitating the aged Peer Gynt, aimed at doing that for the onion, but was quickly diverted into tracing the innumerable sulphur com- pounds which contribute to the wonderful flavour of that vegetable. The so-called Weende system of analysis, which is still to some extent in use among agricultural chemists, has a much shorter way with plant materials - w e have ether-soluble material, ash, fibre, 'crude protein' (that is, N × 6.25) and finally (by difference) the so-called 'nitrogen-free extract- ives'. Many people are under the impression that these are mostly starch and other carbohydrates which can be brought into solution. For some plants, that is indeed true, but for many plants a more important place is taken by polyphenols. Consider, for example, the tea plant (Camellia sinensis), in which as much as 40% of the dry matter of the leaves may consist of polyphenolic materials. Many other plants contain comparable amounts of polyphenols, and biologists have been at a loss to explain the function of these lavishly accumulated materials. Few of them are known to have well- defined biological effects, apart from the obvious function of anthocyanins and other flavonoid pigments in making flowers and fruits visually attractive to insects and birds. I will touch again on this problem in the course of this lecture.

Today, I am lecturing about the interactions of polyphenols with plant proteins, and I propose to lay particular emphasis on aspects of this topic that are of practical and economic importance. For my purpose, it is con- venient to divide the polyphenols into tannins and non-tannins.

TANNINS

The tannins (Haslam, 1966), although chemically the more complicated group, can be dealt with rather more shortly. Their traditional use is in the tanning of leather, and they are loosely defined as polyphenols having the property of precipitating proteins - to possess this property they must have

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a substantial, but not excessive, molecular weight and a good number of phenolic groups in the molecule. The important tannins of tea are formed by oxidative condensation of smaller, non-tannin, polyphenols. In the preparation of black tea, enormous care is taken in encouraging the fight enzymes to act to the right extent. It is theaftavin, and definitely not caffeine, that makes what the English call °a good cup of tea' or, in North of England idiom, 'makes spoon stand up in cup'. This astringency, this 'curl- ing up of the tongue', is a characteristic which you Germans may not appreciate in tea, but you probably do appreciate it in red wine; it is a taste which certainly both Germans and English share in beer.

It is generally agreed that tannins interact with proteins by multiple hydrogen bonding of phenolic groups to 'peptide' or 'amide' bonds (Loomis & Battaile, 1966; Van Sumere et al., 1975) - but as there is much contro- versy about exactly which of the various possible tautomers are interacting, I do not like to give any detailed representation of this bond. It is also clear that this sort of bonding is reversible. The reaction may be slow, and the subsequent precipitation may be slower. European beers tend to contain an excess of polyphenols (derived both from the hops and the malt), and these tend slowly to precipitate the malt proteins, resulting in a cloudiness or 'haze' in the beer. Brewers, accordingly, often remove some of the poly- phenols by treating beers with substances rich in amide bonds, such as poly(vinylpyrrolidone) (PVP) or nylon. This achieves one purpose, in allow- hag their beers to remain clear, while travelling further and staying longer undrunk in stores and public houses --but it is generally held that such beers may have lost a lot of their 'tang'. It is notable, in England, how some of the smaller breweries, which have resisted monopolization, are able to conduct equally or more profitable business by cutting down on advertizing and transport costs, supplying beer to local pubs only. There it is quickly drunk by 'regulars' who esteem the 'tang', which they find missing from many of the most widely advertized and widely sold brews. Polyphenol content is thus probably the chief technical desideratum of that new popular movement in Britain, the Campaign for Real Ale.

In the U.S.A., much of their beer, which is noted for its insipidity, is made from rice (Pomeranz, 1972), which contributes an excess of protein. Extra- neous tannins are added by brewers to remove this. Prof. R.D. Haworth has an amusing story about how he was called in some years ago to consult about problems arising because the U.S. Government had forbidden the im- portation of Chinese gallotannin, on account of their bad political relations with China. Turkish gallotannin had been substituted, with unsatisfactory results. 'If they'd read Emil Fischer's and Freudenberg's papers, they could have expected the results to be bad', Prof. Haworth said. 'I advised them to mix their beer with an equal volume of English beer and centrifuge the mix-

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ture, but that idea didn't seem to appeal to them'. Eventually, a solution satisfactory to all parties, except the Chinese, was achieved.

Tannins are a problem for biochemists, too, when they try to isolate enzymes or other proteins from plant material. As soon as the organization of the tissue is destroyed, tannins get loose from the vacuoles of the cells and precipitate the proteins. PVP has been taken over by plant biochemists from the brewers, and has proved very valuable for preventing this (Loomis & Battaile, 1966).

It is notable that tannins are absent from most of the staple vegetable foods which we eat (Bate-Smith, 1972). In many cases, the tannins may have been bred out in the course of deriving the domesticated plant from its wild precursor, and this may have made the domesticated plants more difficult to protect from predators and parasites. One thing is quite clear - besides their unpleasant taste, tannins make it more difficult to digest the proteins of foods. For ourselves, our pigs and our poultry (all monogastric animals) that is a disadvantage, but perhaps it is not so for our ruminant animals - the cattle, sheep and goats. They certainly eat with relish plant material which is rich in tannins. It is now agreed that the micro-organisms of the fore-stomach (the rumen) of these animals attack proteins of the diet, degrading them in a wasteful way to ammonia if they are too soluble. Experiments on adding tannins to protein-rich concentrates for cattle have been done, particularly in France; it is probably that benefit was obtained, and there were few obvious bad effects (Hatfield, 1973). Attention has recently become diverted from tannins by the successful use, in Australia, of formaldehyde treatment of protein concentrates for the same purpose (Hatfield, 1973).

NON-TANNINS

The non-tannin polyphenols do not bond nearly as strongly to proteins, being smaller molecules. However, they are much more widely distributed

among plants. As examples, I can give first 3-(3',4'-dihydroxyphenyl)- L-alanine (L-DOPA) which has been known as a major constituent of broad beans (Vicia faba) for more than 60 years (Karrer, 1958). Although it is not abundantly present in most plants, it is readily formed, by enzymic hydro- xylation, from the universally occurring amino acid tyrosine. DOPA is often encountered as an intermediate in biosynthetic processes in the plant, e.g. in biosynthesis of betalains (Centrospermae) and of alkaloids. DOPA often cyclizes to give indolic compounds; in animals, at least, the further poly- merization of these to melanins has been rather thoroughly studied. My second example is chlorogenic acid, an ester of caffeic acid which is very widely distributed in plants (Harborne, 1964; Rib~reau-Gayon, 1972;

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Karrer, 1958; Van Sumere et al., 1975). Indeed, substituted cinnamic acids occur, free or linked by ester or amide bonds, in a very wide range of plant substances. Neukom and colleagues showed some years ago that they are present in cereal glycoproteins, esterified to a carbohydrate moiety (Painter & Nenkom, 1968); more recently, Van Sumere and colleagues have shown that, in amide linkage, they occur as the N-terminal group of some of the important barley proteins (Van Sumere et al., 1975). Low-molecular plant cinnamamides have also been shown to have important antifungal properties (Stoessl, 1970). A few low-molecular polyphenols may have direct toxicity for animals (Singleton & Kratzer, 1973) or perhaps be beneficial (Fr6hlich & Mayr, 1973).

Today, though, I want to dwell not so much on the occurrences of these polyphenols as on what happens when they undergo enzymic or spontan- eous oxidation. As most of them are o-diphenols, there results an o-semi- quinone radical or an o-quinone molecule. These are among the most chemically reactive of all organic compounds, and are particularly liable to undergo oxidative polymerization with formation of new C-C bonds or of ether bridges between molecules. Pierpoint (1971)has dealt admirably with the many biologically important processes in which o-quinones seem to be involved. Horspool (1969) has reviewed their varied chemical reactions. Pierpoint has himself shown that oxidizing chlorogenic acid can couple with the protein moiety of a plant virus; inactivation of the virus did not in this case happen, though it has been widely postulated (Pierpoint, i973). Indeed, quinone formation and polymerization in individual plant cells when they are attacked by infective agents, predators or parasites - t h e 'necrotizing reaction' - is probably an important means of self-defence for the plant. In food processing, these so-called 'enzymic browning' reactions are usually regarded with disfavour on aesthetic grounds - people do not like a brown colour in their apples or potatoes- and it is a common practice to inhibit such reactions with bisulphite or with ascorbic acid, which rapidly reduce any quinone which may be formed.

I want now to discuss in more chemical detail the reactions of quinones with proteins. Of the various potentially reactive groups of the amino acid sidechains, the most abundant are the primary e-amino groups of lysine residues. Few, if any, model coupling compounds involving lysine have been described, but a quinone derivative involving the amino group of the simplest amino acid, glycine, was prepared by Emil Fischer long ago (Fischer & Schrader, 1910).

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O tt C

/ HC

I1 C2HsOOC.CH2.NH.C

\ C II 0

\ C.NH.CH2 .COOC2 Hs ti CH

/

Nuclear coupling, rather than imine formation, occurs preferentially, and it is worth noting that the resulting bond is a vinylogous amide group, which can be hydrolysed to give back the amine, though usually in poor yield and with formation of ammonia (Cranwelt & Haworth, 1971). Treatment of o-benzoquinone with excess amine can lead further to quinone imines, which my colleague Dr. Roger Davies has shown to be major products among the fifty-odd separable coloured compounds formed by reacting o-benzoquinone with alkylamines (Davies, 1975).

NR O 0 II ]1

C C I I / \ / \ C CH RNH.C CH

I fi II II HC C.NRH HC C.NHR

% / \ / C C

tl NHR NR

Quinones couple in similar fashion, but much more avidly, with the thiol groups of cysteine residues in proteins. With free cysteine, more com- plicated reactions both of thiol and amino groups occur. R.A. Nicolaus and G. Prota and their colleagues in Naples have shown such products to be responsible for the red pigments of feathers and hair (Thomson, 1974). It does not, either, seem to be excluded that quinones can simply oxidize thiols to sulphenic, sulphinic or sulphonic acids. Sulphinic acids so formed would couple readily with quinones to give sulphones of the parent diphenol - it is a standard procedure to 'trap' quinones thus with benzenesulphinic acid.

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Quite recently, it has been shown that thio-ether groups, as in methionine, can couple with quinones (Vithayathil & Murthy, 1972; Bosshard, 1972). With methionine, too, direct oxidation to sulphoxides and sulphone can occur, and some of these products might couple with oxidizing polyphenols. It is something of a public scandal that the constitution of another oxida- tion product, 'dehydromethionine', described by T.F. Lavine as long ago as 1945 (Lavine, 1945, 1949), when he postulated the little-known 'isothiazo- lidinium' structure, has never yet been confirmed by synthesis and study of relevant analogues (Gensch & Higuchi, 1967; M~Shrle, 1967). Methionine is usually, from the point of view of nutrition, the critically deficient amino acid in plant proteins. All these reactions could conceivably occur to methionine residues linked in proteins, and their nutritional consequences have not been properly assessed, which is not surprising when the organic chemistry of methionine is in such a bad state.

One can conceive of indole groups of tryptophan undergoing coupling with quinones (Horspool, 1969); tryptophan is also very readily oxidized. Even coupling of quinones to peptide bonds cannot be excluded; it has been suggested that that could interfere with digestibility (Horigome, 1973).

In view of all these chemical facts, many of them well established, which concern especially the nutritionally critical amino acids lysine, cyst(e)ine and methionine, it is quite extraordinary that in not a single text book of nutrition have I ever seen mentioned the possibility of nutritional damage to proteins by quinones etc., whereas damage by heating, by fats and by reducing sugars is discussed quite extensively. The first direct evidence that these reactions can damage proteins was brought in 1968 by Horigome & Kandatsu (t968). They exposed casein to various polyphenols undergoing enzymic oxidation and established that the resulting brown products were nutritionally impoverished.

Although methionine is often the critically deficient amino acid in plant proteins, cereal proteins are particularly deficient in lysine, and those proteins that are used to supplement the basal cereal diets of pigs and poultry, such as soya-bean meal and fishrneal, are esteemed chiefly for their contribution of lysine. The virtual disappearance of Peruvian fishmeal from the world market was followed, in 1973, by President Nixon's prohibition of the export of soya products from the U.S.A. These events, coupled with the special economic situation in the U.K., have greatly stimulated interest among us in the use of leaf protein, which is rich in lysine, for pig and poultry rations (Pirie, 1971). The greatest interest is in the process some- times called 'fodder fractionation'. With intensive fertilization of grassland, high yields of dry rhatter/hectare are inevitably accompanied by an increase in protein (% of dry matter), which often rises far above the 14% or so which is desirable for the lactating cow. If the fresh herbage is ground up

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and some of the resulting protein-rich slurry is pressed out from the fibrous residue, this slurry can be added wet to cereals, to give a mash on which pigs do very well, while the fibrous residue still contains enough protein to give good milk yields when fed to dairy cattle. Encouraging results along these lines are being got in Britain, at the National Institute for Research in Dairying near Reading and at the Rowett Research Institute near Aberdeen, and such processes are already in commercial use in California.

My colleagues in Norwich and I became interested in such leaf-protein concentrates because some of them fall far short of the nutritional value to be expected from their amino acid composition. We suspected reaction of lysine e-NH2 groups with quinones. We were able to show that various proportions of these groups had become inaccessible to deamination by nitrous acid, and that this inaccessibility correlated with poor nutritional performance (Allison et al., 1973). Furthermore, the higher the proportion of lysine that was inaccessible to deamination, the lower was the total lysine yielded by the protein on acid hydrolysis. As all bulk preparations of leaf proteins have, if handled gently, a very constant amino acid composition, this fits in well with the supposition of direct coupling of -NH2 groups to aromatic nuclei, for Cranwell & Haworth (1971) found, as I have already mentioned, that only a proportion of amino acid residues so coupled can be regenerated under the usual conditions of acid hydrolysis of proteins.

We are hoping to be able to demonstrate such involvement of lysine residues by criteria acceptable to chemists, and have had some encouraging results from the catalytic hydrogenation of model compounds to cyclo- hexane derivatives. In particular, the Fischer compound (Fischer & Schrader, 1910), which on acid hydrolysis gives a little ammonia, a lot of glycine and a mass of brown insoluble matter, hydrogenates smoothly to give derivatives of a new bis-imino acid which is stable to acid hydrolysis (Davies et al., 1975).

Effects of the kind which I have been discussing are by no means confined to leaf proteins. Seed endosperms are often relatively free of polyphenol s, but nutritional damage to plant proteins from 'enzymic browning' is suggested from studies of sunflower seeds (Girault et al., 1970; Sabir et al., 1974a, b), sorghum (Oswalt, 1973) and a number of tropical articles of diet. A closely similar phenomenon has been fully studied with cottonseed, which contains the toxic polyphenol gossypol. Here, it is the free or potential aldehyde groups which couple with lysine to give Schiff bases. Thus, on heating under suitable conditions, the gossypol ceases to be toxic, but the protein loses a lot of its nutritional value. Hence, removal of gossypol by solvent extraction or its inactivation by reaction with added amines have been the means by which cottonseed has become established as a valuable and new protein concentrate, appealing to a wide market, whereas in former

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times it was thrown away or used as a fertilizer on the fields (Singleton & Kratzer, 1973).

ORGANIC MATTER OF SOILS

In conclusion, I want to say a little about the involvement of plant proteins in the formation of the organic matter of the soil. There is still controversy about whether this originates mainly from lignin, from microbial residues or from plant polyphenols, and indeed these theories are not mutually ex- clusive. But, with gradually advancing application of the newer physical and chemical methods of study to these humic materials, a balance of opinion is developing in favour of them being to a great extent products of oxidative polymerization of polyphenols (Haworth, 1971). One can picture large 'core' molecules of chaotic condensed-aromatic structure, including a proportion of cyclic NS and O atoms. Such a 'core' would have, at its periphery, phenolic and quinonoid groups available to couple with proteins. 14C-Dating experiments have shown that the 'hydrolysable' amino acid-rich moiety of humic acid may have an average age measured in decades to centuries, whereas the insoluble unhydrolysable 'core' may have an age measured in centuries to millennia (Campbell et al., 1967; Jenkinson, 197t-3). It seems reasonable to picture such aromatic cores as gradually 'weathering away', with renewed formation of quinonoid groups and renewed coupling to protein. Two powerful arguments can be adduced for this view. First, Perry & Adams (1971) exposed humic acid to 14C-labelled glycylglycine. Radioactivity was taken up by the humic acid. When this humic acid was subjected to acid hydrolysis, eight times more 14C remained insoluble when the N-terminal glycine residue was labelled than when the C-terminal residue was labelled. This is in complete agreement with the model experiments of CranweU & Haworth (t971) on the coupling products of amino acids and peptides with quinones. The second argument emerged from studies of the amino acid compositions of a wide range of humus materials, including ancient products from brown coals, shales, buried soils, etc. In general, the more the humus material has been weathered (especially by ploughing and by tropical climates) and the lower its N content and yield of total amino acids after acid hydrolysis, the greater is the proportion of these amino acids contributed by lysine (Stevenson, 1956a, b; Caries et aL, 1958; Caries & Decau, 1960a, b; Scharpenseel & Krausse, 1962; Yamashita & Akiya, 1963; Wang et al., 1967; Decau, 1969). This suggests that the quantitatively most important point of attachment of proteins to the resis- tant aromatic core is through the e-NH2 groups of Iysine residues, which are thus the last amino acid residues to be eroded away as the humus dis- appears.

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Humus is esteemed by agricultural scientists for its water-holding proper- ties, its ion-exchanging properties, its ability to chelate trace minerals and by its forming a 'bank' of only slowly mineralizing nitrogen, sulphur and phosphorus, which thus gradually become available to growing plants over the course of several years. Farmers in the eastern part of England are learning to their cost the consequences of ruthless cropping year after year with little else than sugar-beet and barley, followed by burning of the straw. These practices have denuded the soils of humus. This year, the dryness of the topsoil was such that germination failed. In other recent years the March winds have often caused dust storms, blowing away both topsoil and the seeds sown in it.

I want to put it to you that the evolution in plants of massive contents of polyphenols was not directed mainly by the physiological need for them of the plant when it was alive, but so that, when the plant or its subaereal parts died off, a soil should become established more suitable for the growth of subsequent generations o f plants. In this process, the coupling of proteins with polymerizing polyphenols (e.g. in the senescent lea0 could be an important step in getting scarce nitrogen and sulphur into a form not readily attacked by mineralizing micro.organisms or leached away. As different plants contain very different assemblages of polyphenols, I think that agricultural chemists concerned about improving soil quality could usefully study in much greater detail the contributions made by natural plant com- munities and by different kinds of crops to the development o f the organic matter of the soil.

ZUSAMMENFASSUNG

Die Polyphenole sind eine sehr heterogene Gruppe yon Stoffen, die sich in iiber- raschend groiq0er Menge h~iufig in pflanztichen Substanzen finden. Ober ihre Bedeutung fiir die Pflanze sind schon die verschiedensten Spekulationen angestettt worden. Fiir unsere Darstellung soUen sie lediglich in Tannine und Nicht-Tannine unterteilt werden.

Die Tannin-Eiweif~Reaktionen spielen bei der Herstellung und beim Genut~ yon Tee, Wein und Bier eine grofSe Rolle. Tannine stiSren die EiweifSverdauung bei Gefliigel, Schweinen und beim Mensehen, fiSrdern sie jedoch unter Umstgnden bei Wiederk/iuern.

Potyphenole, die keine Tannine sind, k6nnen zu Semichinonen und Chinonen oxidiert werden. Das Ftihrt zu weRerer oxidativer Polymerisation trod einer fiber die verschiedensten Reaktionen erfolgenden Ankopplung an Eiweit?~e. Diese Reaktionen tragen vieUeicht zum Schutz der Pflanze gegen Infektionen, Sch~idtingsbefall und Ver- zehr dutch Tiere bei. Unter dern Begriff 'Enzymbr/iunung' verursachen sie Schwierig- keiten bei der Konservierung. Bisher ist noch nicht genau genug bekannt, inwieweit solche Reaktionen den Nghrwert yon Eiweit~en, besonders in der Schweine- und Ge- fliigetfiitterung, beeintr~ichtigen kOnnen.

Es wird immer deutlicher erkennbar, daf5 durch diese Reaktionen die pflanzlichen Polyphenote wesentlich zum Aufbau organischer Materie im Boden beitragen. Viel- leicht sind viete dieser Verbindungen nut zu diesem Zweck entstanden und brauchen in

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der lebenden Pflanze iiberhaupt keine funktionelle Rolle zu spielen. Da sich Menge und Art der Polyphenole je nach der Nutzpflanze stark voneinander unterscheiden, w~ire die Agrarwissenschaft gut beraten, wenn sic landwirtschaftliclie Praktiken unter dem Ge- sichtspunkt betrachtete, wetchen Beitrag jede Kulturpflanze zur Erhaltung oder Steigerung des Gehalts an organischer Materie im Boden leistet.

R~St~

Les potyph6nols sont un groupe tr~s h6t6rog~ne de substances, qui se trouvent parfois en des quantit6s surprenantes dans les tissus v6g6taux. Les speculations les plus diverses ont d6j/t 6t6 faites sur leur signification pour le v6g6tal. Notre expos6 les divisera en tamfins et non tannins.

La r6action tannin - prot6ine joue dans la pr6paration et la consommation de th6, de vin, de blare un grand r61e. Les tannins g~nent la digestion des prot6ines chez la volaitle, les porcins, rhomme, mais la facilitent patrols chez les ruminants.

Les polyph6nols - q u i ne sont pas des tannins- peuvent ~tre oxyd6s en semi- quinones et en quinones. Le fait conduit ~ des polym6risations oxydatives, et ~ une liaison avec les prot6ines par les r6actions tes plus diverses. Ces r6actions contfibuent peut ~tre ~ la protection des plantes contre les infections, les parasites et contre la consommation par les animaux. Comme de 'brunissement enzymatique' ils conduisent des difficult6s chez les conserves. Pour l'instant, nous ne savons pas avec pr6cision suf- fisante sices r6actions peuvent diminuer la valeur nutritive des prot6ines, notamment dans ralimentation des porcins et de h volaille.

ll est de plus en plus clair que ces r6actions des polyph6nols v6g6taux jouent un r61e important darts la constitution de ta matiSre organique darts le sol.

Peut ~tre beaucoup de ees polyph6nols ont ils pris naissance uniquement dans ce but, et n'ont pas besoin de jouer un r61e fonctionnel dans la plante vivante.

La nature et la quantit6 des potyph6nols varient consid6rablement avec l'esp~ee des plantes cultiv6es. La science agronomique serait peut ~tre bien inspir6e, d'envisager le point de rue suivant: quelle contribution chaque plante cultiv6e apporte-t-elle au maintien ou h l'augmentation du taux de mati~re organique du sol?

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Bosshard, H. (1972). Ueber die Anlagerung yon Thio~thern an Chinone und Chino- nimine in stark ~uren Medien. Hetv. chim. Acta 55: 32.

Campbell, C.A., Paul, E,A., Rennie, D.A. and MeCallum, K.J. (1967). Applicability of the Carbon-Dating Method of Analysis to Soil Humus Studies. Soil. Sci. 104:21 Z

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Cranwell, P.A. & Haworth, R.D. (1971). Humic Acid-IV: The reaction of o~-Amino Acid Esters with Quinones. Tetrahedron 27: 1831.

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Fischer, E. & Schrader, H. (1910). Verbindungen yon Chinon mit Aminos~iureestern. Ber. dtsch, chem. Ges. 43: 525.

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Gensch, K.-H. & Higuchi, T. (1967). Kinetic Investigation of Reversible Reaction between Methionine and Iodine. J. phar~ Sci. 56:177.

Girault, A., Baudet, J. & Mossg, J. (1970). Etude des Protdines de la Gralne de Toume- sol en vue de t'Amdlioration de leur Teneur en Lysine. In 'Improving Plant Protein by Nuclear Techniques. Proceedings of a Symposium held in Vienna 8-12 June, 1970', p. 275. International Atomic Energy Agency, Wien.

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Itaslam, E. (1966). Chemistry of Vegetable Tannins. Academic Press, London. Hatfield, E.E. (1973). Treating Dietary Proteins with Tannins or Aldehydes. In 'Effect

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Haworth, R.D. (1971). The Chemical Nature of Humic Acid. Soil Sci. 111: 71. Hofigome, T. (1973). [Nutritive Value of N-Acetylcasein and Brown-cotoured

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Author's address: Prof. Dr. R.L.M. Synge Agricultural Research Council's Food Research Institute Colney Lane Norwich NR4 7UA England

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