Phenolic compounds, which are present in the epidermis zone, parenchyma zone, core zone, and seeds of fruit (Malus domestica var. Kermerrien), were extracted from freeze-dried material by three successive solvent extractions. The dry methanol extract and the dry aqueous acetone extracts were analyzed using reversed-phase HPLC coupled with diode array detection following thiolysis to quantify phenolic compounds according to their classes (hydroxycinnamic acid derivatives, flavan-3-ols, flavonols, and dihydrochalcones). The method is suitable for the determination of the relative proportions of the different classes of polyphenols and provided information on the constitutive units and the average degree of polymerization of oligomeric and polymeric procyanidin structures. Results showed that procyanidins are the predominant phenolic constituents in cider apple fruits, much of them corresponding to highly polymerized structures. Keywords: Apple; phenolics; procyanidins; cider; HPLC; thiolysis

The cortex of 14 French apple varieties (12 cider and 2 juice varieties), one English cider variety, and one dessert apple (i.e., Golden Delicious) were studied for their polyphenol composition. Total polyphenols were assayed by the Folin-Ciocalteu method, and the precise polyphenolic composition (monomeric catechins, proanthocyanidins, hydroxycinnamic acids, and dihydrochalcones) was obtained by HPLC following thiolysis. ESI-MS and ESI-MS/MS analyses showed that chlorogenic acid and p-coumaroylquinic acid were methylated under the conditions of thiolysis. Depending on the variety, the global polyphenol concentration varied from 1 to 7 g per kilogram of fresh cortex. Cider varieties globally showed a higher polyphenol concentration than the dessert apple Golden Delicious, bitter varieties being the more concentrated. The proportion of the polyphenol classes varied greatly from one cultivar to another. For all varieties, procyanidins were always the predominant class. They were mainly constituted of (-)-epicatechin units with a small proportion of (+)-catechin as a terminal unit. The average degree of polymerization ranged between 4.2 and 7.5 depending upon the variety with an exception for the sharp varieties Guillevic and Avrolles which showed significant concentrations of procyanidins with DPn of 40 and 50, respectively.

Five French cider apple varieties were compared on the basis of their detailed polyphenol profile in the cortex and in the juices. Among the factors studied, variety was the most important variability factor in fruits, whereas polyphenol profiles showed an overall stability from one year to another, and a limited decrease of polyphenol concentration was observed during the starch regression period of fruit maturation. In juices, procyanidins remained the preponderant polyphenol class with concentrations up to 2.4 g/L even in centrifuged juices. Compared to the fruits, the average degree of polymerization of procyanidins was significantly reduced in the juice. Centrifugation of the crude juice had only minor effects on the polyphenol composition. For one variety, highly polymerized procyanidins with average degrees of polymerization of 25 were shown to be soluble in the centrifuged juice at a concentration of close to 1.2 g/L. Oxygenation of the juices during processing resulted in a significant decrease of all classes of native polyphenols. Catechins and procyanidins were particularly affected by oxidation, whereas caffeoylquinic acid was partly preserved. The transfer of polyphenols after pressing was maximal for dihydrochalcones and minimal for procyanidins with extraction yield values close to 80 and 30%, respectively.

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The rate of consumption of dissolved oxygen by apple polyphenol oxidase in cider apple juices did not correlate with polyphenol oxidase activity in the fruits and decreased faster than could be explained by the decrease of its polyphenolic substrates. The kinetics parameters of a crude polyphenol oxidase extract, prepared from apple (Braeburn cultivar), were determined using caffeoylquinic acid as a substrate. Three apple procyanidin fractions of n 80, 10.5, and 4 were purified from the parenchyma of cider apples of various cultivars. Procyanidins, caffeoylquinic acid, (-)-epicatechin, and a mixture of caffeoylquinic acid and (-)-epicatechin were oxidized by reaction with caffeoylquinic acid o-quinone in order to form oxidation products. All the fractions were evaluated for their inhibitory effect on PPO activity. Native procyanidins inhibited polyphenol oxidase activity, the inhibition intensity increasing with n. The polyphenol oxidase activity decreased by 50% for 0.026 g/L of the fraction of n 80, 0.17 g/L of the fraction of n 10.5, and 1 g/L of the fraction of n 4. The inhibitory effect of oxidized procyanidins was twice that of native procyanidins. Oxidation products of caffeoylquinic acid and (-)-epicatechin also inhibited polyphenol oxidase.

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Procyanidins (i.e. condensed tannins) are polyphenols commonly found in fruits. During juice and cider making, apple polyphenol oxidase catalyzes the oxidation of caffeoylquinic acid (CQA) into its corresponding o-quinone which further reacts with procyanidins and other polyphenols, leading to the formation of numerous oxidation products. However, the structure and the reaction pathways of these neoformed phenolic compounds are still largely unknown. Experiments were carried out on a model system to gain insights into the chemical processes occurring during the initial steps of fruit processing. Procyanidin B2 was oxidized by caffeoylquinic acid o-quinone (CQAoq) in an apple juice model solution. The reaction products were monitored using high performance liquid chromatography (HPLC) coupled to ultraviolet (UV)-visible and electrospray tandem mass spectrometry (ESI-MS/MS) in the negative mode. Oxidative conversion of procyanidin B2 ([M-H](-) at m/z 577) into procyanidin A2 at m/z 575 was unambiguously confirmed. In addition, several classes of products were characterized by their deprotonated molecules ([M-H](-)) and their MS/MS fragmentation patterns: hetero-dimers (m/z 929) and homo-dimers (m/z 1153 and 705) resulting from dimerization involving procyanidin and CQA molecules; intramolecular addition products at m/z 575, 573, 927, 1151 and 703. Interestingly, no extensive polymerization was observed. Analysis of a cider apple juice enabled comparison with the results obtained on a biosynthetic model solution. However, procyanidin A2 did not accumulate but seemed to be an intermediate in the formation of an end-product at m/z 573 for which two structural hypotheses are given. These structural modifications of native polyphenols as a consequence of oxidation probably have an impact on the organoleptic and nutritional properties of apple juices and other apple-derived foods.