[advances in food research] advances in food research volume 13 volume 13 || astringency of fruits...

ASTRINGENCY OF FRUITS AND FRUIT PRODUCTS IN RELATION TO PHENOLIC CONTENT

BY M. A. ,JOSLYN AND .JUDITH I,. GOLDSTEIN

Department of Nutritional Sciences, C 7 ? i ivrrsit .v of Calif ornin. B o k e l e y, Cnlifornin

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The Sensation of Astringency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

111. Protein Precipitation and Protein Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Analytical Methods for Tannin and Astringency Asmy . . . . . . . . . . . . . . . . . V. Astringency in Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A. Factors Influencing Bstringmcy in Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Theories Proposed to Account for the Loss in

Astringency in Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

179 180 185 191 195 205

205 208 209

I . INTRODUCTION Significant progress has been made in recent years in the methodology

of organoleptic evaluation of quality in foods (Kramer and Twigg, 1962). Sensory attributes such as color and texture can be evaluated by both organoleptic and objective physical methods (Mackinney and Little, 1962 ; Matz, 1962; Soc. Chem. Ind., 1960). Even though the objective evaluation of flavor and odor still depends largely on sensory perception (Little, 1958; Campbell Soup Co., 1961; Crocker, 1945), the methodology of taste cliff erence testing has been greatly refined. Less progress has been made in basic psychometric, physiological, and chemical evaluations of the processes actually involved in the psychological and physiological sensory reception, generation, and transmission of stimuli; in developing the molecular and macromolecular mechanisms of perception ; and in evaluating the factors influencing these reactions (Soc. Chem. Ind., 1957 ; Zotterman, 1962). Thus, while differences in the degree of sweetness of foods can be detected with a high degree of precision, our knowledge of the basic reactions involved is too incomplete to allow the prediction of whether or not a particular chemical compound will be sweet. Knowledge of the basic psychology and physiology of sensory perc-ptions is particularly lacking in the quality called astringency. It has long been known that the accepta- bility and palatability of fruits and fruit products, as well as the stability

179

180 11. A . JOSLYN AND JUDITH L. GOLDSTEIN

of a t least certain fruit products, is dependent on the type and concentration of astringents present (Bate-Smith, 1954a, 1954b, 1958). The palatability of wine depends on the balance between the sugar, acid, and “tannin” contents. The concentration of astringent materials present is as important as the sugar-acid ratio in determining the quality of fruit juices such as apple, cherry, and grape juice.

The nature of the constituents responsible for the sensation of astringency and the physiology of the astringency sensation and the factors influencing it are known only partially. The naturally occurring phenolic compounds have long been associated with astringency. Many of these compounds were first recognized in tanning liquors, from which their name “tannin” was derived. The chemistry of vegetable tannins has been reviewed in the classic publications of Nierenstein (1927, 1934), Freuden- berg (1932, 19331, and Russell (1935), and in the more recent publications of Howes (1954), Schmidt (1955), Gustavson (1956), White (1958), Ollis (1961), and Jurd (1962). Available information on the sensation of astringency and the factors that affect astringency in fruits and fruit products is critically reviewed here.

Interest in flavonoid compounds in foods, as \?-ell as in plant phenolics in general, has continued to increase since the first review, by Bate-Smith (1954b). Largely as a result of the interest aroused and investigations in this field by Bate-Smith and his collaborators, particularly Swain, of the Low Temperature Research Station a t Cambridge, England, a Plant Phenolics Group was founded in England. The inaugural meeting of this interdisciplinary group was held in the Department of Botany, Cniversity of Cambridge, England, on April 9, 1957. That meeting was followed by a symposium on “The Oxidation of Plant Phenolics,” reported hy Bate- Smith (1957). Since then the group has met annually, and the proceedings of several syniposia have heen published (Fairbairn, 1959; Pridhani, 1960, 1963; Ollis, 1961). A similar group of scientists interested in plant phenolics was organized in the United States in 1962. In addition to the proceedings referred to previously, three important reference texts have been published dealing with various aspects of plant phenolics (Geissman, 1962; Gore et al., 1962; Hayaishi, 1962).

II. THE SENSATION OF ASTRINGENCY

The word “astringent” is derived froin the Latin ad (to) and stringere (bind). Astringency is thus defined as a “binding” reaction. In pharmacology, astringents are recognized as drugs that precipitate proteins. Be- cause of this effect, they are used to stop heniorrliage and diarrhea and to inhibit secretions a t mucous surfaces and in various glands (Goodman nnd Gilman, 1955; Claus, 1956; Sollmann, 1957). Astringents in medicine

PHENOLS AND FRUIT ASTRINGENCY 181

are defined as any substances that cause the constriction of organic tissue; tlie definition given in Encyclopaedia Britannica (1957) is “a group of agents which tend to shrink the mucous membranes and raw surfaces and dry up secretions.”

Ruemele (1937) separated astringents into two classes: the true astringents, compounds that react with skin proteins; and the pseudo- astringents, which, although astringent, do not react with skin proteins. The pseudo-astringents include epinephrine, ephedrine, cold water, and dextrins.

The true astringents are subdivided into the following four groups (Greene, 1921; McDonough, 1935; Lesser, 1939) :

1) Salts of multivalent metallic cations such as aluminum, chromium, zinc, lead, calcium, magnesium (borax and boric acid are included with these)

2) Vegetable tannins, e.g., gallotannic acid 3) Dehydrating agents, e.g., ethyl alcohol, acetone, glycerine 4) Mineral acids (including halogenated acetic acid) Astringents such as aluminum or zinc salts used cosmetically to pre-

vent sweating of underarm regions, alcohol used in after-shave lotions to close skin pores, or the zinc astringents used topically in mouth washes or in eye drops, although sirnilar in drying up secretions and shrinking tissues, probably differ in their physiological effects. These therapeutic astringents have little in common with the plant phenolics, which also have astringent properties. Whether the drying up of mucous secretions occurs through contraction or closure of salivary ducts, or through actual precipitation of the mucins secreted, or both, is not known. The precipitation of tissue proteins is accompanied by the shrinkage of tissue due to loss of water and a decrease in the permeability of this tissue to water and solutes. A similar change occurs in the conversion of collagen, a hydrophilic colloid gel, into the relatively non-hydrophilic leather. Astringency was defined by Moncrief (1946) as a “contracting or drying taste.” In the United States, astringency is considered to be the dry, puckery sensation perceived in the mouth after ingestion of unripe fruits. For this reason Bate-Smith (1954a) suggested that astringency is a sensation of touch instead of taste, “since dryness or puckeriness has to do with feeling not with taste.” Bate-Smith (1954b) stated further that “astringency is, stxictly, concerned with the sense of touch and is due to the coagulation of the proteins of the saliva and mucous epithelium by conibination with the astringent body.”

In the flavor-profile method, proposed for specifying flavors and odors by Cairncross and Sjostroni (1949), astringency was recognized as a coin- ponent of flavor, which was defined to include all the sensations of taste,

182 M. A. JOSLYN AXD J U D I T H L. GOLDSTEIN

sinell, and feeling. Astringency was recognized, however, to be not a tastc sensation but was considered to he a feeling factor (Little, 1963). The chemical feeling factors that stimulate the end organs of feeling in tlic mouth and throat inay actually be related to pain sensations. When liigli concentrations of astringents are tasted, the severe dryness that results is almost painful. The immediate reaction is to rinse the mouth with water, but this is of no avail, since the sensation of dryness and tightness per- sists.

The sensation of astringency-one of extreme dryness and puckeriness --unlike a true taste sensation, is not confined to a particular region of the inouth or tongue but is perceived as a diffuse stimulus. The perccp- tion of astringency in the mouth is not instantaneous but requires appreciable time for development. Astringency and dryness are considered by Little (1963) as delayed effects, in contradistinction to long-lasting effects such as bitterness. Actually, astringency may persist and carry over, as noted by Hinreiner et al . (1955).

Astringency differs from taste also in that the threshold for perception is much greater. In fact, Sollmann (19211, over 40 years ago, reported that the astringency of substances could be detected better by tasting powdered preparations rather than solutions. The minimum concentration of added tannic acid that could be detected in peach purees was 15 to 25 iiig per 100 g (Guadagni and Nimmo, 1953). Differences in astringency between peaches containing 69 and 111 nig were detectable by 12 of 14 judges, but those between peaches containing 151 and 181 mg hy only 6 out of 12 judges. An increase in 50 to 80 mg of tannin per 100 g of peacli puree was readily detectable by taste. Marshall (1954) reported that a “tannin” content of less than 0.1 g per 100 ml does not impart a detectablc astringency to cherry juice, and this is also true for apple juice. Hinreincr et al. (1955) reported that 100 mg of added grape seed tannin could be detected by taste per 100 ml white wine, and 150 mg in red wine, although as littlc as 20 mg per 100 ml could be detected in water (Berg et al., 1955).

A mucous membrane covers all the exposed surfaccs of the mouth, epiglottis, and digestive tract. The epithelial layer of the mouth surfaces is moistened by the secretions of the salivary glands. The location of the t h e e pairs of salivary glands in the mouth is shown schematically in Fig. I (Massler and Schour, 1946). The parotid is the largest of these glands, situated below the ears, at each side of the mouth. I ts ducts opcn into the hard-palate region, adjacent to the anterior mandibles, and i t secretes a thin, watery fluid. The other two glands, sublingual and sub- maxillary, secrete a more viscous fluid; their ducts open just beneath the tongue in the front of tlie mouth. Their secretion contains inucin and amylase enzymes.


FIG. 1 . Major salivary glands and their location in the mouth (Masslpr and Schour, 1946).

The physiology of astringency is still not well defined (Winton and Bayliss, 1955), either as regards the inolecular structure of the substances responsible, the constituents of the mucous membranes actually involved, or the mechanism of the action taking place. Secretions from the parotid and other salivary glands may be inhibited by the astringent, or the ducts may be constricted. It is not known whether the primary reaction is precipitation of mucins or constriction of the ducts. The tongue as well as the palate becomes dry and rough, owing to constriction of surface tissues.

Two main types of glycoproteins are known to occur in the epithelial mucous, fucomucin and sialomucin (Levene, 1925 ; Wolstenholme and O’Connor, 1958; Jakowski, 1963). The inucopolysacchnrides of the con- nective tissue are known to differ in cornposition and structure from those present in the epithelial iiiucous (Hoffman and Meyer, 1962). Hashinioto and Pignxtn (1962) have summarized data available on the chemical composition of the mucins responsible for the viscous characteristics of the mucous secretions of higher animals. The mucins of the epithelial tissue and of the salivary secretions contain acctylated glucosamine and

184 M. A . JOSLTN A S D JUDITH L. GOLDSTEIN

galactosamines, as well as sialic acid and proteins. Jenkins (1960) r t - ported that mucin is the protein present in highest concentration in human saliva (which averages about 0.3% protein). The parotid saliva does not contain sialic acid containing niucopolysaccharides, but is a rich source of enzymes (acid phosphatase, esterases, aldolase, cholinesterase, glycuro- nidase, lipase, and lipozyme’l .

The possibility has been proposed that a preferential reaction occurs between the astringent and mucoprotein. Bate-Smith (1954a) suggested that the salivary mucopolysaccharides may have a special affinity for astringent substances, but this has not been confirmed. Burton and Reed (Faraday Society, 1954) reviewed the information available on the role of mucoid material in tanning. In practice it has been known that the interfibrillary material present in hides and skins interferes with tanning and dyeing, but the actual nature of this material and the best methods for removal are not known. Histological, electron microscope, and pliysi- ological investigations of skin have indicated that mucoid material is present in association with protein fibers and that this largely determines tissue cohesion and stability. Both hyaluronic acid and chondroitin sul- fate are known to occur; the latter is more firmly bound in the skin and is responsible for water-repellent properties. Mucolytic enzymes are much more efficient in removing mucoid material than lime or sulfide, and leave the fiber structure in a well-opened-up condition, ready for tanning, without the usual subsequent treatment with tryptic enzymes. Whether or not niucoid material in hides reacts with tannins, however, is not known.

That possibility is supported by the fact that linear polyamides in the form of fibers or fine powders are able to adsorb polar substances such as phenols from solution, with the formation of hydrogen bonds, first es- tablished by Carelli e t al. (1955) and confirmed by Grassmann e t al. (1956, 1957) and others. The sorption of phenolics by polyamides is restricted to the amorphous region, where a solution process of the polar substances into the polymer takes place. Henimcrling (1933) investigated the effect of astringents on mucous membranes by applying tannin or aluminum acetate daily for four weeks to rabbit oral niucosa, and reported characteristic vasoconstriction and hyperkeratosis. Treatment of the oral inucosa with these astringents prevented inflaniniation otherwise caused by soap.

The mouth and salivary glands are innervated by both sympathetic (adrenergic) and parasympathetic (cholinergic ) nerves. The adrenergic nerves of the sympathetic system secrete epinephrine and norepinephrine ; both of these cause the constriction of blood vessels. Contraction of blood vessels and muscles is known to be stimulated by secretions from the nerres of the sympathetic nervous system and the adrenal gland. Epine-

PHEXOLS AND FRCIT ASTRINGENCY

OH I

185

H-6- OH I

H- C-H I

NH I

Epinephrine (Adrenalin)

CH3

H-C-OH I

H-C-H I

H-N-H

Norepinephrine (Sympathin E)

Frc. 2. Strncture of epinephrine and norepinephrine.

phrine and norepinephrine (whose structures are shown in Fig. 2) are closely related to phenolic compounds present in the tanning extracts. Similar cornpounds may be present in plant tissues. A number of closely related ainines have been isolated from bananas (Marshal, 1959), notably tramine, dopamine, serotonin, and norepinephrine.

The dry, puckery sensation that is perceived on ingestion of astringents also occurs with certain drugs, such as atropine, and is experienced by persons suffering from diabetes insipidus and in conditions of severe thirst or alcoholism (Wolf, 1958). The feeling of dryness under all these conditions may be related to the accumulation of catechol ainines in the cyto- plasm of the responsive tissues. Normally, these catechol amines are methylated on liberation, and rendered inactive (Axelrod, 1960). The rnethylation of tlicse compounds is catalyzed by an O-niethyltransferase, which requires S-adcnylniethionine as the methyl donor, methylation occurring in thc meta position. Axelrod and Laroche (1959) reported that, in viuo, pyrogallol acted as a competitive inhibitor. Even earlier, DeEds e t al. (1957) discovered tha t 3,4-dihydroxyphenyl conipounds such as caffeic acid undergo 0-methylation as well as dehydroxylation in the animal body. Trihydroxyphenyl compounds such as gallic acid also undergo 0-inethylation (Booth et al., 1959). Phenolic compounds such as tannins may thus contribute to astringency both directly, by reaction with niucins, anti indirectly, by interfering with the normal biological deactiva- tion of catechol amines. Goldstein (1963) recently observed that, after ingestion of an astringent, rinsing the inoutli with methionine relieved the dryness of astringency whereas rinsing with water had no effect.

111. PROTEIN PRECIPITATION AND PROTEIN BINDING

Gustavson (1949) defined astringents as substances causing the sensation of puckering on the tongue, hut pointed out that a numerical

186 31. A. JOSLPN AXD J U D I T H L. GOLDSTEIN

classification of the various tannins and vegetable-tanning materials according to their degree of astringency is not possible, because of the large number of factors involved. Astringency produced by tannins appears to be a function of molecular size, the pH of solution, degree of purity, electrical charge, the nature of anions and neutral salts present, temperature, and tannin content. It has been assumed that the affinity of the tannins for collagen, under comparable experimental conditions, is a measure of astringency. Quebracho tannins [ MW (molecular weight) 24201 and tannic acid (MW 3100-3400) are classified as astringent tannins, whereas gambier (MW 520) and myrobalan tannins (MW 1900), possessing a lower degree of affinity for collagen, belong to the nonastringent group. Hilbert ( 1938) reported that astringency varies not only from one tanning material to another but also with the method used in extracting a given tannin. Extracts prepared by low-temperature leach- ing are less astringent than extracts prepared by autoclaving, possibly because autoclaving results in extraction of higher polymers.

Astringents in general possess tanning properties, that is, they have the property of cross linking and precipitating proteins from aqueous solution and of converting hide collagen into leather. The cross links formed between the protein micelles are believed to involve hydrogen bonds as well as ionic bonds. Multivalent cations, vegetable tannins, polymeric phenolsulfonic acids, and aldehydes have been used widely as protein precipitants or tanning agents. The chemistry of tanning with vegetable tannin extracts has been investigated as a colloid chemical process by Wilson, Thomas, and others (see Wilson, 1928) and, more recently, from the standpoint of macromolecular chemistry, by a number of investigators (McLaughlin and Theis, 1945 ; Gustavson, 1949, 1956 ; Flaherty et al., 1958).

Recent X-ray diffraction and electron microscope studies have indicated that collagen probably possesses a helical structure similar to that of keratin. Significant advances have been made in our knowledge of the chemistry of vegetable tannins, particularly ellagitannins (Schmidt, 1956), and, more recently, the chemistry of gallotannins has been eluci- dated fully (Haslam e t al., 1961a,b). The actual reactions involved between hide collagen and tannins, however, are still not resolved, largely Lecause the reaction has been followed between chemically undefined hide constituents and chemically undefined tannins. Various surface-chemistry techniques, including surface pressure, surface potential, and viscosity, have been used in following the reaction sequence, but X-ray diffraction and light-scattering techniques have not yet been applied. The data available, however, indicate that leather has a rather compact cross- linked structure as a result of the multipoint association between the

PHENOLS AND FRUIT ASTRlNCENCY 187

multifunctional polyhydroxybenzene derivatives and a number of protein chains.

A variety of physicocheniical methods have been used in investigating the chemistry of leather tanning. These includes studies on the diffusion of tannins into hide by measurement of the rate of penetration of tannin into hide by microscopic examination of dichromate-stained sections and of changes in density measured pycnometrically. As tannin diffuses into the skin i t displaces water, and the apparent density increases. Arm- strong (Faraday Society, 1954) measured changes in the apparent density of sections of one skin immersed in solutions of mimosa tannins and tannic acid, and found this to vary with the square root of time. The tanning process was simpler with mimosa tannin than with tannic acid. The amount of tannic acid diffusing into skin reached a limiting value as the concentration of tannic acid increased. Ellis and Pankhurst (Fara- day Society, 1954) investigated the interaction of tannins with collagen by following changes in surface pressure, surface potential, and surface viscosity a t the interface of collagen and underlying solution of tannins. They reported that tannins were characterized by the development of a highly viscous film accompanied by changes in surface potential and/or surface pressure, which varied characteristically with the type of tannins used. Catechol tannins were found to produce a marked condensation of the monolayer, indicating multipoint association between the multifunctional tannin molecules and a number of protein chains, producing a compact crose-linked structure. The surface pressure was not dependent on pH in the range of 3-6, but the surface potential increased linearly with increase in pH, and the surface viscosity decreased exponentially. Mimosa tannin was electrophoretically inert a t pH 3 but became increasingly negatively charged as pH increased. At pH 3, where the tannin is electrophoretically neutral and where tanning is not accompanied by any change in surface potential, the chief mechanism responsible for the tanning process is hydrogen bond formation between phenolic hydroxyl groups and carboxyl, amino, and keto-imide groups of protein. At higher pH values, both hydrogen bond formation and electrovalent combination between cationic collagen and anionic tannins are involved.

Vegetable tannins are usually classified into hydrolyzable tannins and condensed tannins. The hydrolyzable tannins are subdivided into : gallotannins, yielding, on acid hydrolysis, glucose and gallic acid, with smaller quantities of shikimic or quinic acids; and ellagitannins, which yield glucose and ellagic acid, together with gallic acid. Armitage e t al. (1961) showed that gallotannin is an octa- or nonagalloylated glucose, consisting of a pcntagalloylglucose nucleus to which three or four additional galloyl groups are attached by depside links. The galloyl nuclei are attached to

188 &I. A. JOSLYN A S D JUDITH L. GOLDSTEIN

glucose by ester linkages, and additional galloyl groups are attached by depside bonds (ester linkages between polyhydroxybenzoic acid nuclei). These ester linkages are readily hydrolyzed by hot mineral acid, giving the component glucose and gallic acids.

The condensed tannins, on the other hand, contain little if any carbo- hydrate material, and are converted into insoluble amorphous phloba- phenes by the action of mineral acids. Although the monomeric flavonols present in nonhydrolyzable tannins are known, the molecular structure of the naturally occurring polymers resulting from the condensation, oxidative or nonoxidative, of flavonoid compounds such as catechin, is unknown (Freudenberg and Weinges, 1962; Hergert, 1962; Hathway and Seakins, 1957; Swain, 1960; Hillis, 1956; ROUX, 1957, 1958).

The condensed tannins as a class are more widely distributed in tlie higher plants than the hydrolyzable tannins, and are tlie main tannin constituent in fruit tissues. These tannins have not been isolated in pure conditions; most of the preparations investigated are known to be mixtures of varying molecular size and may contain more than one molecular species. Until recently they were considered to be polymers of catechin, but in 1953 Bate-Smith and Swain (1953) proposed that leucoanthocyanins are responsible for astringency and the characteristic phlobapliene formation. Subsequently, Bate-Smith (1954a,b), Swain and Bate-Smith (1956, 1962), and Swain (1962) presented additional data to confirm this proposal, as did Hillis (1956) and Roux (1957, 1958).

Rosenheim (1920) first reported the presence of colorless precursors of anthocyanins in grape leaves, and named these leucoanthocyanins. Freudenberg and Weinges (1960 \ proposed the name proanthocyanidins, t o include anthocyanidin precursors a t the same oxidation level as the anthocyanidins and those a t lower oxidation levels. The real leucoanthocyanidins, according to Freudenberg (1962), are considered to he the flav-3-en-3-01 and flav-2-en-3-01, as well as their keto, open-chain, and hydrated forms. The naturally occurring flavan-3,4-diols are considered by Freudenberg ( 1962) to be water adducts of leucoanthocyanidins. Oligomeric and polymeric proanthocyanidins are belicved to be formed from these diols by mechanisms similar to those found with catechins. The literature has described several naturally occurring dinieric proanthocyanidins that, on being heated with dilute acids, arc converted into catechin or epicatechin and anthocyanidin (either cyanidin or delphinidin). This conversion occurs more rapidly and at lower concentrations of acid than the conversion of monomeric flavan-3,4-diols into anthocyanidins and aniorphous polymers, or the conversion of catechin into cyanidin (Freudenberg, 1962).

PHENOLS AND FRCIT ASTRINGENCY 189

The formation of condensed “catechin” tannins by acid-catalyzed polymerization of catechin has been favored by Freudenberg, Schmidt, and others (see Geissnian, 1962), whereas Hergert (1962) favors the original suggestion of Bate-Smith and Swain (1953) that a large majority of condensed tannins are derived by polymerization of flavan-3-01s and flavan-3,4-diols, thc so-called leucoanthocyanins. Catechins, and the anthocyanidin corresponding to the leucoanthocyanin present, as well as phlobaphencs, are frequently found in acid hydrolysates of condensed tannins. The anthocyanidin may be derived either from the polymeric catechin, tlie copolymer of catechin and leucoanthocyanin, or the polymeric Irucoanthocyanin. The hydrolyzable tannins, because they are derivatives of polyhydroxybenzoic acid, are more acid (pK 4.5 to 5.0) than the condensed tannins (pK 5 to 6 ) , which contain less phenolic hydroxyl groups per benzene ring, and no free carboxyl groups. The structures catechin, gallocaterhin, leucocyanidin, and cyanidin are shown in Fig.

HO Ho+oH

OH OH \ \

HO HO

OH

Catechin Gallo c ate chin

HO&OH \ - H O f l O H \ -

’ OH OH HO OH HO

Leucocyanidin Cyanidin

FIG. 3. Structure of catechin and rrlated phenolics.

of 3.

Catechin, having a molecular weight of 290, does not precipitate gelatin or tan hide but, according to Bate-Smith (1954~~1, is astringent, whereas the closely related clilorogenic acid is not a t all astringent. Ellis and Pankhurst (1954), however, pointed out that catechin is not astringcnt

190 3C. .4. JOSLYK’ AKD .JUDITH L. GOLDSTEIN

although its polyiners are. The extent of polymerization required to pro- duct astringency is not known, and neither is the change in astringency with polymerization. It is likely that even dimers may be astringent. The astringent leucoanthocyanins isolated from cacao and from apple and other fruits apparently are polymers, whereas those obtained from woody tissues are monomers.

Although the property of astringency is generally ascribed to tannins, it is known that many phenolics classed as tannins will not, in fact, tan leather or precipitate proteins.

The main protein-binding reaction of the tannins is thought to be between the hydrogen of the hydroxyl group in the tannin and thc oxygen of the keto-imide bond in the protein, -CO*NH-, i.e., the peptide bond. This has been confirmed by the demonstration that tannins are adsorbed by polyamide, whose only reactive group is the keto-imide group (Gustavson, 1949,1954,1956). Grassmann et al. (1956) first demonstrated the binding capacity of the keto-imide group by the precipitation of water-soluble urea-formaldehyde condensation products with tannins. The urea-formaldehyde chain has the structure: -XH * CO * NH CH2. NH . CO NH . CH2--. The 6-amino group in lysine molecules in the protein is thought to be involved in the ionic bonds formed at an acid pH with the carboxyl groups of the tannin.

For precipitation to occur, botli the tannin and the protein must have tlic correct steric structure and molecular weight. The aromatic liydroxyl groups in the tannins are stabilized by resonance, forming quinoncs. An interesting example of steric effect due to the phenolic hydroxides was found by Tu and Lollar (1950). They found that 3,5-dihydroxy-9-phenyl- xanthene acted as a protein precipitant but that the m-substituted 2,7- dihydroxy derivative did not. However, the hydroxide group is not ob- ligatory in this reaction, since the syntan (Otto, 1953) Tanigan supra LL, which contains the activc -CH= group in the aromatic ring but has no liydroxyl group, acts as a perfect tannin. The molecular weight of the tannins that cause protein precipitation appears to be around 1000-3000, although these figures are only a mean value.

The hydrogen ion concentration is of great importance in the protein- binding reactions, partly because it affects the degree of swelling of the protein, thereby facilitating diffusion of the tannins. It has been reported that tannins precipitatc only hydratcd polyamide powdcr (Gustavson, 1954).

pH is also important in that i t affects the degree of ionization of the protein and tannin, thereby increasing the likelihood of ionic reactions. In general, proteins are precipitated on either side of their isoelectric point. The degree of binding again decreases a t pH 2 and below, and a t pH 8 and above.

PHENOLS AND FRUIT ASTRIKGENCT 191

IV. ANALYTICAL METHODS FOR TANNIN AND ASTRINGENCY ASSAY

A variety of procedures have been used for the determination of tannin content. These are described in older publications by Nierenstein (1927) and Freudenberg (1932), and in more recent publications by Joslyn (1950), Schmidt (1955) , Snell and Snell (1953), and Swain and Goldstein (1963). The methods available are based on: the reduction by poly- hydroxybenzenes of weakly acidic pernianganate, or by alkaline phos- pliotungstate-pliosphoniolybdate; the formation of colored ferric com- plexes; the formation of colored products of reaction with various electrophilic reagents such as diazotized p-nitroaniline, vanillin-sulfuric acid, nitrous acid; or precipitation from solution by a variety of reagents including salts of aluminum, zinc, or lead, proteins like gelatin, and alkaloids. h’one of these methods is specific, even the classic protein precipitation or hide powder binding. Tannins will precipitate or be adsorbed by substances other than proteins, e.g., alkaloids, starch, agar, or polyvinyl- pyrollidone (Goldstein, 1962) . Davis et al. (1945) reported quantitative precipitation of tannins by azo dyes containing free amino acids. Phenols, aromatic acids, and catechins did not precipitate the dyes they selected.

In earlier published data on the astringency of fruits the method used most widely was the Loewenthal-Proctor permanganate reduction procedure, essentially as described by Neubauer (1872). In this procedure the permanganate-reducing material present in an aqueous extract of the fruit tissue is determined before and after precipitation of the “tannins” with gelatin in the presence of kaolin, or before and after adsorption of “tannins” on boneblack (Joslyn, 1950). The total permanganate-reducing material present was usually reported as “total astringency,” and the non- precipitable or nonabsorbable material as “astringent non-tannins.” The unreliability of this procedure was recognized early by Caldwell (1925), who recognized that the precipitation of tannins by gelatin was variable and not quantitative, that there was variable adsorption of coloring matter by the filter paper or the gelatin-kaolin-tannin precipitate, and that error was involved in using the equivalent weight of tannic acid as a factor in converting results. Bate-Smith (1954b), Sniit e t al. (1955) ~ and others subsequently reported the unspecificity of the pernianganate titration. Permanganate a t room temperature in weakly acid solution will oxidize any o- or p-dihydroxybenzene derivative but not monohydroxy or metahydroxy dcri\Tatives unless their molecule is otherwise open to attack. Nevertheless, changes in astringency as detected by taste were reported to be correlated with changes in tannin content as determined by permanganate titration during the growth and ripening of fruits such as apples, cherries, and grapes.

192 31. A . JOSLYN A S D JUDITH L. GOLDSTEIN

Of the other available procedures for the deterniination of tannin, tlie most widely used is the colorimetric method based on the reduction of phosphomolybdic-phosphotungstic acids by phenols to niolybdenuni blue in alkaline solution (Snell and Snell, 1953). This phenolic reagent, intro- duced by Folin and Denis (1912, 1915), was adapted to the determination of tannins in wines and whiskey by Rosenblatt and Peluso (1941) and Pro (19521, and has been used for the determination of tannins in fruit hy Guadagni e t aZ. (1949), Sherman e t al. (1953), Swain and Hillis (19591, and Craft (1961 I . The Folin-Denis reagent is even less specific than permanganate, and reacts with monohydroxybenzene as well as dihydroxybenzene compounds. Many other reducing substances also gire moly1)- denum blue with this reagent, including sulfides, sulfurous acid, hydrogen peroxide, aromatic amines, unsaturated aliphatic compounds, and glucose, as indicated by Snell and Snell (1953). Of particular importance in the analysis of fruit and plant tissue is reduction of the Folin-Denis phenol reagent by ascorbic acid (Sherman e t aZ., 1953).

Reeve (1951) proposed a modification of the nitroso reaction for tannins in plant tissues, first used by Vinson (19101 and in modified form by Hoepfner (19321, Vorsatz (19421, and Adams and Merrill (1949), as a histochemical test for polyphenols in fresh plant tissue; and Reeve (1959) later modified this for quantitative measurement of catechol tannins in peacli tissue based on determination of reflectance with a Photovolt pho- tometer and a 550-A filter.

To improve the spccificity of the clieniical methotls for measuring “active” or astringent tannin content of fruit juices or fruit tissue extracts, several modifications of the more commonly used procedures have been proposed. Lloyd (1916) very early observed that the degree of as- tringcncy in persimmons depends on the degree of adsorption of tannins by a cellulose component. He explained a decrease in astringency during ripening by an increase in the concentration of this component, and thus a decrease in tlie available tannin that could be adsorbed by the mucous membranes. He proposed as a measure of astringency the use of moistened filter paper as an adsorbent. The amount of tannin taken up hy the filter paper from a cut surface of the fruit, after developriicnt with a tannin reagent, was reported to be as accurate as tasting in determining astringency.

Barnell and Barnell (1945 reported tha t the degree of astringency of banana pulp and skin as determined organoleptically was not closely correlated with tannin content as determined by the usual methods. Thc total tannin content of the pulp of some varieties of bananas may actually increase on ripening. They dcvelopcd a procedure for estimating the so-called active tannins on tlie basis of an early observation that tannin

PHEPI’OLS AND FRUIT ASTRINGENCY 193

precipitated or inactivated diastase. Active tannin content was deter- inined froin the difference between tlie time rcquired for the hydrolysis of soluble starch by a standard diastase preparation under standard conditions in the presence and absence of an aqueous extract of banana tissue. The “active tannin” content so determined decreased progressively during ripening, and correlated well with astringency. Subsequently, Swain and Goldstein (1962a) reported the inhibition of emulsin activity by tannic acid, but this was not applied to the detcrrnination of astringency.

Enzyine inhibition by tannins and related phenolics has long been known (Williams, 1963). This includes inhibition of pectic enzymes by phenolics, such as the inhibition of coinmercial pectinase by astringent persiiurnons reported by Nakayania and Chichester (1963). This inhibition niay be due to inactivation of enzyme or combination with substrate. Thus, the inhibition of diastase is due to combination with starch while that of ernulsin is due to precipitation of enzyme protein.

Guadagni e t al. (1949) determined the total phenolic content of alco- holic extracts of peach fruit tissue by the Rosenblatt and Peluso (1941) procedure and also measured the percentage of the total phenolase-oxi- dized phenols in a slurry of peach tissue by the naturally occurring pol- yphenol oxidase. This was found to vary from 65 to 80% in the range of 32-194 nig% of total tannin. They suggested that the oxidizable tannin content not only was a measure of the tendency of fruit to brown but also could be used as an index of astringency.

Swain and Hillis (1959) deterniined the total phenolic content of plum tissue extracts by a iiiodification of the Folin-Denis procedure, and also determined the vanillin-reactive flavanols containing phloroglucinol resi- dues by photometric rncasurernent of the concentration of the red car- boniuin ion formed with vanillin in 70F sulfuric acid, and the leucoanthocyanin content by a modification of the procedure used by Piginan e t nl. (1953), which also was modified later by Manson (1960).

Goldstein and Swain (1963a) reported that the ratio of vanillin to Folin-Denis values for phenolic content decreased with molecular size and could be used as a measure of degree of polymerization of flavans.

The leucoanthocyanin content as determined by yield of anthocyanidin formed on heating with acidified amyl alcohol, propanol, or butanol solution is also known to vary with degree of polymerization. Manson (1960) reported that the yield of cyanidin obtained by heating a propanol solution of the monomeric flavan diol was considerably greater than tha t obtained from leucoanthocyanins as extracted froin spruce bark, presumably because the latter were polymeric. Roux and Paulus (1962) were the first to actually determine the effect of degree of polymerization of leucoanthocyanins on yield of anthocyanidin. For leucofisetinidins isolated from the

194 hl. A. JOSLYN AND J U D I T H L. GOLDSTEIN

heartwood of the black-wattle tree, they found that the yield of fisetinidin chloride under the conditions of acid treatment used by Pigman e t al. (1953) decreased from 23.5270 for the monomeric flavan diol to 6.75% for trimeric, 4.78% for the pentameric, and 4.9570 for the decameric tannin. See also Joslyn and Goldstein (1963).

Roux and Bill (1959) briefly reviewed the mechanism of the conversion of flavan-3,4-diols into anthocyanidins, which was believed to be dehydration of the 3,4-diol group followed by oxidation or disproportiona- tion. They found that the rate of formation and extent of conversion were pronioted by exclusion of moisture from the reaction mixture. Instead of yields of 3% in aqueous hydrochloric acid solutions or of 20% in propanol- hydrochloric acid mixtures containing 3N HCI, yields of 374272 were obtained by using anhydrous .02S hydrochloric acid in propanol under pressure.

Although phenolic assays of the above types can be applied to juices, n ines, and similar fruit products without special precautions other than protection against the loss of phenolics by oxidation during sampling, storage, and analysis, their application to fruit tissues involves the prob- lcin of extraction. Guadagni e t al. (1949), on the basis of phenolic content of extracts. proposed blending 50 g of ground peach tissue in 300 ml of 70% ethanol. They found that this extract had higher phenolic content than extract prepared by using 9570 ethanol, 0 . 3 7 oxalic acid, or water alonc. They gave no data on the completeness of extraction of the phenolics present. Hillis and Swain (1959) prepared extracts of plum tissue by first extracting with hot absolute inethanol until the residue was free of inethanol-soluble phenols and then extracting the marcs so obtained with 50% methanol. They reported that the concentration of leucoanthocyanins c xtractable with absolute methanol decreased as plums ripened, whercas the concentration of leucoanthocyanins extractable with 50% niethanol increased. Craft (1961) extracted the polyphenols of Elbertn peaches hy blending stcanied peaches with sufficient 957% ethanol to obtain :L final concentration of 70%, but determined leucosnthocyanins in both the fil- trate and slurry. He found that appreciable amounts of leucoanthocyanin were retained by the ethanol-extracted pulp.

Russell (1935) stressed the use of inert solvents in extracting tannins, and preferred acetone. Methanolysis is known to occur during the extraction of gallotannins (Haslam et al., 1961a,b). Duthie (1938) reported 407( acetone to be a better extracting agent for cacao tannins than hot water. Concentrated acetone solutions, however, are not superior to ethanol or methanol, and interfere with vanillin determination and also are lcss efficient in extracting proanthocyanidins from dried astringent tissues cuch as sulfited persimmons.

Astringent drugs are discussed in general by Greene (19211, Mc-

PHENOLS AND FRUIT ASTRINGEKCP 195

Donough (1935), Ruernele (1937), and Lesser (1939). A variety of procedures have been proposed for bioassay and determination of astringent materials. Sollmann (1921) early compared various criteria of astringency and found them to give fairly concordant results. Hc reported (Sollmann, 1920) that the simplest and most satisfactory appeared to be the precipitation of protein from aqueous solution. It has long been known that substances which precipitate proteins, such as the salts of heavy metals or tannic acid, are conspicuous among the astringents used in medicine and among the substances that are astringent in the mouth. Although tlic chemistry of the process of cross linking and precipitation of tannins has been investigated for many years, particularly by M'ood (1908), Trunkel (1910), and Wilson (1919, 1920), and considerable information is available on the changes involved and the factors influencing this (Gustavson, 1949, 1956), our knowledge of this complex reaction is still incomplete. Inhibition of hemolyses of erythrocytes was proposed as a method of nieasuririg astringency by Sollmann (1921) and Buckendahl (19331, and was investigated more completely by Wilbrandt and Wips (1947). Lux and Christian (1951) reported that the permeability of frog skin to Na+ and I- increased after treatment with aluminum salts. Koniiyama (19261, and more recently Wilbrandt e t al. (1952), reported that astringents show two typcs of action on rattail tendon, a tanninlike action in which the bridges between the protein fibers become more numerous and the expan- sibility of the protein decreases and a urea-like action in which the bridges between the protein strands become dissolved and the elasticity increases. Theis (1930) proposed dilatometric measurement of the increase in volume of a well-hydrated tissue on treatment with astringent solution such as alum or zinc chloride. Preliminary data obtained in our laboratory indicate an appreciable increase in the volume of gelatin solution when tannic acid solution is added.

None of these methods for measuring astringency, however, have been applied to the investigation of astringency in fruit tissues or to changes in astringency during growth and ripening.

Wilson (1920) defined tanning and astringency on the basis of mutual colloidal electrolyte interaction between positively charged collagen and negatively charged tannin particles. Mezey (1925), although supporting the concept that astringency is a function of the potential difference between tannin solutions and collagen, pointed out that measurement of this property is not practical as a routine control procedure.

V. ASTRINGENCY IN FRUITS

Variety, maturity, and climate are known to influence the astringency of fruit. Caldwell (1925) reported fairly extensive data on the effect of seasonal conditions on the astringency of American grape juices, and also

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on apple juices (Caldwell, 1928). Caldwell (1934) reported additional data on the astringency of eight varieties of apples, five varieties of cherries, and three varieties of strawberries, including data on changes in concentration during growth and ripening. As pointed out by Hulme (1958), data on changes in the astringent constituents of apple and pear fruits are limited. Thc more extensive investigations of changes in the astringency of perry pears during growth and ripening are those of Jac- quin and Tavernier (1954) and Jacquin and Cherel (1957). The changes in astringency of grapes with variety and maturity have been investigated quite extensively, and the older available data have been summarized by Caldwell ( 1928, 1934) and, more recently, by Amerine (1954). Among the deciduous fruits, peaches have been investigated more completely than other fruits-by Blake and Davidson (1941), Blake (1942), Gua- dagni e t al. (1949), Peynaud (1950), Johnson et al. (1951), Guadagni and Nirnmo (1953), Reeve (1959), and Craft (1961). The distribution of tannins within the banana fruit and the changes in their conditions and amount during ripening were investigated by Barnell and Barnell (194.3) , von Loesccke (1950), \Tinton and n’inton (19421, and others. The tannins of the persimmon fruit and their changes during ripening were investigated even earlier, by Lloyd (1911, 1916). I to and Oshima (1962) recently re-

0 10 2 0 30 4 0 5 0 0 10 2 0 30 4 0 5 0

M A Y 5 - JUNE 2 2 M A Y 6 - JUNE 2 2

FIG. 6. Changes in tannin content during growth of cherries (Caldwell, 1934). A t left, Napoleon sweet c*herry ; right, Montmorency red sour cherry.

198 M. A . JOSLYN AND JUDITH L. GOLDSTEIN

portcd tlie isolation of a leucoanthocyanin from Japancsc persimmon fruit t isue.

The changes in tannin content of applcs, as reported by Caldwell (1934), are sliown in Fig. 4 for Baldwin apples (typical of table apples) and in Fig. 5 for Launette, a French cider apple. The tannin content, expressed as percent by weight of tissue, is considerably higher for the Launette than the Baldwin variety but increases in both to a maximum and then decreases, the maximum being reached sooner with the Baldwin than the Launette. The tannin content per fruit, however, increases over a longer portion of the growing period, and then decreases more slowly than tlie percentage of tannin. Similar data are shown for cherries in Fig. 6 and for strawberries in Fig. 7. Changes in tannin content during gromtli arc sliown for Gaume peaches in Fig. 8 and for Elberta peaches in Fig. 9. Changes in the phenolic content of pears are shown in Fig. 10.

It is well known that certain fruits are astringent when they are un-

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V E R Y HARD WHITE- 4 0 % 7 5 % PALE F I R M F U L L H A R D GREEN NlNG PALE PALE RED R I P E R I P E

FIG. 7 . Changes in tannin content during growth of Howard 17 strawberries (Caltl- well, 1934).


ripe. Ih r ing ripening on the tree or in storage, this astringency disappears. This loss in astringency may be accompanied by a change in the tannin fraction. The actual change in tannins, determined chemically, may be very small indeed and may either increase or decrease. In bananas there usually is no correlation between total tannin content and astringency, but thc “active” tannin content decreases, as shown in Fig. 11. In astringent fruits the main phenolic compounds found are flavanols. Both the flavanols

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FIG. 8. Changes in tannin content during growth of Gaume peaches (Rceve, 1959).

200 I\.[. A. JOSLYN AXD J U D I T H L. GOLDSTEIN

(the catcchinn) and thc flavan diols (the leucoanthocyanins) occur. The other major constituents of the phenolic fraction consist of the flavanols, kaempferol, quercetin, myricetin, chlorogenic acid, and the cinnarnic acids such as caffeic acid. Only members of the condensed tannin class occur in fruits.

In all histocheniical investigations of thc occurrence of tannins, it has been found that they are confined to certain cells, the tannin cells or idio- blasts. Thus, Lloyd (1911, 1912) showed that the tannins in thc persimmon fruit were confined to certain cells. Tokugawa (1919) confirmed this

0 10 2 0 30 4 0 5 0 6 0

D A Y S G R O W T H June 2 3 - A u g 18

Fro. 9. Changes in tannin content during growth of Elberta peaches (Craft, 1961).

observation. Tlic appc’arance of typical tannin cells in unripe Hacliiya persirninon is shown in Fig. 12, in comparison with that of ripe persimmon in Fig. 13. Barncll and Barnell (1945) sliowed that the same is true for the banana and that tlie tannins are confined to the cells of the latex ducts and to certain isolated tannin cells. Recve (1959) again showed tha t the phenolic compounds in the peach wcre restricted to ccrtain cells. If tannins arc rcsponsible for the astringency in unripe fruits, then any hypothesis suggesting a mechanism for the reduction of astringency must take into accoiint the localization of these compounds in certain cells.

It is of intercst that in the most astringent fruits, bananas and persiiii- inons, the anthocyanogens or leuconntliocpanins prcsent are those yielding

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FIG. 10. Changcs In tannin content duiing giowtli of Muscachet pears (Jacyuin and Clieiel, 1957).

202 121. A. JOSLTN A S U JL’DITH L. (nOLDsTEIN

delphinidin on acid hydrolysis. In the green carob pod, also, leucodelphini- din occurs in tannin cells present in astringent meristematic tissue whereas leucocyanidin occurs in seed testa, which are not astringent.

Although some data are available on changes in extractable tannin content, expressed on a weight basis, very little data are available on total tannin content per cell. Most of the latter are limited to peach fruit, but data such as obtained histochemically by Reeve (1959) on catechol tannin per cell include all phenolics containing aromatic orthodihydroxyls. “Tan- nin” content as determined by chemical analysis has rarely been corrc- lated with actual degree of astringency. The meager quantitative data available in this field are limited largely to peaches (Guadagni and Ximnio, 1953).

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FIG. 11. Changes in tannin contcxnt during ripening of banan:rs ( h m c . 1 1 ~ ~ t l Bar- nell, 1945). G, green; C , colorcd ; E K , eating ripc ; OR, overripe.

PHESOLS A N D F R U I T ASTRINGENCY 203

FIG. 12. T'yp1cal tannin cell in unnpc Hachiy:~ prrsimmon S o t e that the cells are turgid and suriounded by smooth membrane

204 11. A. JOSLYW Ah-D J U D I T H L. GOLDSTEIN

FIG. 13. Typical tannin cells in rip^ Hnrhiyn pr1siInnIon. Note that the c ~ l l merw hrnnc is folded.

PHENOLS AND FRUIT .ISTRINt;ENCP 205

A. FACTORS I S F L U E N C I N G ASTRIKGENCY I N FRUITS

Apart from environmental factors such as locality, season, stage of developmmt, and mineral nutrition, which are known to affect the level of phenolic compounds in fruits, and hence astringency, various secondary treatments can decrease the degree of astringency in fruits, for example, treatment with carbon dioxide (Gore, 19121, ethylene (Chace and Church, 1927) ; precipitation of the tannin with acetic acid, formaldehyde, or n-propyl aldehyde (Kakeshita, 1930a,b) ; or oxidation with ozone (Jordt, 1930). Astringency may also be reduced by air drying, particularly in persimmons. If the fruit is chilled, astringency may increase. Barnell and Barnell (1945) found this in the banana and suggested tha t the permeability of the tannin cells had been altered so that the tannins could diffuse into the surrounding tissue. Once this happened, the tannins did not undergo the postharvest changes that lead to the normal loss in astringency.

1%. THEORIES PROPOSED TO ACCOI-NT FOR T H E LOSS I N

ASTRINGENCY I N FRUITS Investigations on the chemistry and physiology of loss in astringency

in Japanese persimmons, begun over 40 years ago by various investigators (Tokugawa, 1919; Koniatsu and Ucda, 1923; Kakeshita, 1930a,b; and others), have been reviewed recently (Xakaniara, 1961 ; Ito and Oshima, 1962; Ito, 1962; Sato et nl., 1962; and others). The methods of removal of astringency practiced in $Japan include treatment with warm water, treatment with carbon dioxide, treatment with ethyl alcohol or acetaldehyde, freezing, drying, and coating with resin films. It has long been known in Japan that, when sun dried, unsulfured or lightly sulfured fruit would lose astringency but heavily sulfured fruit If-ould retain astringency (Ito, 1963).

Matsui and Murata (1960) reported data on the effect of sulfuring on the quality of two varieties of Japanese dried persimmons. When per- sirnmons were sulfured by exposure to fumes produced by burning 1.5- 50.0 g of sulfur per cubic meter, the sulfur dioxide absorbed by the fresh fruit varictl from 15-16 to 320-389 ppm, and after drying for 5 days a t ambient temperatures of 32-34°C aniounted to 5-34 ppm. At all levels of sulfur dioxide content tested, the dried fruit was not astringent. The usual procedure is t o use 3.5 g of sulfur per cubic meter and obtain nonastringent dried fruit containing 7-7.5 ppni of sulfur dioxide. We find that sulfuring to levels of 200-1000 ppni on R tirictl-weight basis produces astringent fruit.

The effect of sulfur dioxide on phenolics of the persimmon fruit has

206 \I. A. JOkiLYh- h X D J U D I T H L. GOLDHTEIS

not been investigated. It is interesting in this connection, however, that Forsyth and Roberts (1960) reported tha t although cacao leucocyanidin yielded cyanidin and epicatechin on hydrolysis with hydrochloric acid, when hydrolyzed with sulfurous acid it yielded epicatechin and a colorless sulfur derivative tha t could be partly converted into cyanidin and an unknown purple pigment.

No data are available on changes in extractable phenolics during treatment with carbon dioxide, a t atmospheric pressure or above, or on treatment with alcohol or acetaldchydc (usually added a t the rate of 1 ml of ethanol per kilogram of fruit). This practice is described in a recently put)- lished booklet on persimmon culture in Japan (Sobagima, 1959).

It is interesting that astringency of persimmon fruit can he removed by holding fruit for 15-24 hours in warm water a t 40°C and also by freezing the whole fruit and storing it for 10 to 90 days, the period dc- prnding on the varicty. Xakaiiiura (1961) investigated the latter process in some dctnil and reported data on changes in soluble tannin content of 13 varieties of fruit stored at -12" to -75°C. Both as regards loss in astringency and loss in solublc tannin, storage a t -25°C was most effec- tive; changes n-ere slower a t -75°C and least a t -12°C. At -25"(', loss of astringency required from 15 to over 90 days of storage, depending on variety, and this was paralleled by the time required to reduce soluble tannin content from its initial value (0.5-0.73$) to 0.05./;.

The various theories proposed to explain loss in astringency iiiay he classified into the following three groups:

1. Binding

I n histochmiical investigations of the tannin cells, Lloyd (1911, 1912), Tokugawa (19191, Barnell and Hurnell (19451, and Reeve (19591 found that the solubility of the tannins changed. I n unripe fruit the tannin cells rupture easily and the tannins diffuse out into the surrounding tissue or, if the fruit is being eaten, out into the mouth. When the fruit ripens, i t loses its astringency, the tannin cells shrink, and the tannin congeals. Lloyd (1911, 1912) suggested that the tannins in the pcrsiminori com- bined with a iiiucilaginous or gelatinous substance of a protein nature. In 1916 hc modified this view and suggested tha t the tannin conibincd with cellulose. Tokugawa (1919) confirmed this cellular binding hypothesis. He postulated that the t m n i n in persimmons coinbincs with a jellylike carrier. Barnell and Barncll suggested tha t the tannins in the hanana coinhined with an aldchydc, anti bclieved that acetaldehyde might be involved. Kuniagai and Tazaki 11922) found no changc in the total p11c- nolic content of the persiniiiion on r1I)cning. However, those worker> found a change in the solubility of thebe compounds. The solubility of the tan-


nins in ethanol increased the solubility in hot water. Hillis and Swain (1959) found that there was a change in the solubility of the leucoanthocyanin fraction in the plum fruit on ripening. A decrease in the 100% methanol-soluble phenolic fraction occurred with an increase in the 5070 methanol-extractable material. This observation could support a binding or polynierization hypothesis for the loss of astringency in fruits. Gold- stein (1962), Swain antl Goldstein (1962b), and we have found this to occur with other f r u i t b a n a n a s , peaches, and persimmons-and also with carob tissues. On ripening, the phenolics extractable with 100% methanol and the ratio of vanillin to Folin-Denis values for phenolic content decreased. This would suggest tha t decreased extractability accompanying loss in astringency was due to polymerization, but no actual determination of changes in molecular weight of extracted phenolics is available. Neither are data on changes in actual dcgrce of extractability with maturity, such as could be obtained by a coinbination of Reeve’s (1951) procedure with that of Hillis and Swain (1959).

2. Change in Molecular Size of the Tannins

Swain (1960) and Swain and Goldstein (1962) suggested that the oligomeric but not the inonorneric or polymeric leucosnthocyanins are astringent. When the fruit ripens, the leucoanthocyanins present polyme- rize through C-C or C-0 bonds. Once this polymerization occurs, as a result of both decrease in solubility and increzse in molecular size, they would no longer react with the proteins or mucopolysaccharides in the mouth. The evidence usctl to support this hypothesis was the change in solubility of the tannins. iiicntioned aboye. The widespread occurrence of leucoanthocyanins in fruits and the fact that flavans are known to poly- merize very casily was cited in support of this hypothesis (Freudenberg and Weinges, 1962; Hergtrt, 1962; Hathway and Seakins, 1957; Hillis and Swain, 1959; Swain, 1960). The tliird piece of evidence (Swain antl Goldstein, 1962b) used wab the change in the chemical reactivity of the tannin-reacting material (Goldstein and Swain, 1963a,c). They found that a greater decrease occurred in the vanillin-reacting material. This reagent is a measure of the unsubstituted sites in a phloroglucinol or resorcinol ring, and these groups arc1 characteristic of the condensed tannins. Siegel- man (1960), working with persimmons, suggested that tannins depolynier- ize on loss of astringency, and favored the idea tha t astringency is caused by a macromolecule, i.e., a high degree of molecular polymerization is necessary.

Craft (1961) rcportcd data on changes in both cxtructable and slurry leucoantliocyanin content of Elberta peaches during maturation. The extractable leucoanthocyanin content decreased by some 40-50% during

208 11. .S. JOSLYK A S D .JUDITH L. GOLDSTEIN

ripening, but tliis decrease was not acconipanied hy an increahe in iion- extractable leucoantliocyanin contcnt. The latter was sinall and variable. While astringency also decreased during ripening, Craft (1961 ) could not correlate this with decrease in concentration of total phenolics (chiefly leucoantliocyanin, followed in decreasing order by chlorogenic acid, catechin, and flavonols) or in Icucoantliocyanin content. The cxtractahle lcucoanthocyanins coniposed 3040% of total phenolics calculatcd on the same basis.

Nakayaina and Cliichester (1963) recently reported data indicating a rapid and extensive decrease in anthocyanogen (lcucoantliocyanin ) content during the ripening of persimnions. They found tha t the soluble an- tliocyanogcn content first incrcascd to ti iiiaxiiiiuin and then tlecrcascd during ripening.

3. A Change in the Hydroxylatzon Pattern of the Phenolic Compound

Tlie most widely occurring leucoanthocyanin, leucocyanin iRohinson and Robinson, 1933; Bate-Smith, 1961), has the cntechol grouping of hydroxyl groups in the p-ring of the flavonoid nucleus. I t o and Osliinia (1962) , however, found leucodclpliinidin in the persimmon, and Siiiiinons (1959) has dciiionstrated the saii1~ phenolic in tlie hanana. It may be of some significance that coiiipounds rvitli n pyrogallol group are present in these astringent fruits. This grouj) is also ~ c . r y cliaracteristic of tannins helonging to hytlrolyztiblr~ tannin> Actually, tlic cyanidin-yielding tannins, such as cacao and grape tannin, are iiot as astringent as banana iiritl

persimmon tannins. I t is possiblc that a change in liydroxylation pattern, c.g., conversion of catccliol groulw into pyrogallol groups, could occur during ripening or storage and hc responsible for loss in astringency, but this is unlikely. At any rate, such a change has not yet been o h s e r v d

Although iiiany workcrs liar-c niadt. extensive analysis of the plienolic constituents in fruits, :is yet tlicre is very little evidence to substantintc any singlc theory to explain the cause of tlie disappearance of astringency in fruits. A relatively large cliangc in astringency, detcrniined organo- Icptically, is usually accoiiipanied hy a sriiall change in the tannin content, determined cheiiiically. Tlie tannin content has been shon-n to increase (Goldstein 1962), decrease (Barnell and Barncll, 1945; Craft, 1961) or remain thc saiiie (Tokugawa, 1919; Johnson et al., 1951) on loss of astringency. I t must be r(wimiljc~ c d , however, that thc disappearance of astringency may not bc tlic saiiic’ for all fruits.

AkChSO~TLEDGMENTS

This rcvicn w a s 1)rcparcd t i > Imrt of a project on chemistry and pliyhi- ology of fruit Icuco:trithory:iniri~ and related phenolic compo~l~tlh s : u ~ -

PHENOLS AND FRUIT ASTRINGENCI 209

ported in part by a Reecarch Grant (EF 00080) froin the U. S. Public Health Service.

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