Philippe Sanoner

Characterization of procyanidin B2 oxidation products in an apple juice model solution and confirmation of their presence in apple juice by high-performance liquid chromatography coupled to electrospray ion trap mass spectrometry

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.

Stabilisation of the colour of anthocyanins in solutions by admixture with phytocomponents from apple.

Summary The colour of anthocyanins can be enhanced by planar chemical complex formation, or stabilised by the formation of adducts. We investigated these properties using fruit extracts. Delphinidine and its glucoside, myrtilline, at 0.1 mM were mixed with 10 mM caffeoylquinic acid (CQA; chlorogenic acid) or 6 mM ferulic acid (FeA). In a second experiment CQA, ferulic acid, and a CQA-rich apple extract were added in variable concentrations to a blackcurrant pigment preparation and incubated at 37°C in 0.02 M citrate buffer at pH 1.5, pH 3.0, or pH 4.5. The colour change was followed by measuring the absorbance at 540 nm and the composition of the solutions was assayed by HPLC using diode array detection. Deglycosylation of anthocyanins leads to a loss of colour and chemical stability. It is therefore of major importance that the enzyme preparations used during juice extraction be devoid of glycosidases. Addition of phenolic acids to an anthocyanin solution increases and stabilises the colour, which is accompanied by an acceleration of the loss of the native anthocyanins and the formation of coloured adducts, presumably based on the pyranoanthocyanin structure. The glycosyl residue in anthocyanins is necessary for colour stabilisation, which appears to be due to the formation of new pigments. Caffeoylquinic acid appeared to be more efficient than ferulic acid, and a CQA-rich extract from apple also resulted in stabilisation of the colour of a blackcurrant extract.