Interactions between Salivary Proteins and Apple Polyphenols and the Fate of Complexes during Gastric Digestion.

Beneficial polyphenols in apples can reach the stomach as complexes formed with salivary proteins. The present study aimed at documenting the interactions between salivary proteins and cider apple polyphenols and the fate of complexes during gastric digestion. A polyphenolic extract was mixed with human saliva, and interactions were characterized by analyzing proteins and polyphenols in the insoluble and soluble fractions of the mixtures, before and after in vitro gastric digestion. Results confirmed that proline-rich proteins can efficiently precipitate polyphenols and suggested that two zinc-binding proteins can also form insoluble complexes with polyphenols. The classes of polyphenols involved in such complexes depended on the polyphenol-to-protein ratio. In vitro gastric digestion led to extensive proteolysis of salivary proteins, and we formulate the hypothesis that the resulting peptides can interact with and precipitate some procyanidins. Saliva may therefore partly modulate the bioaccessibility of at least procyanidins in the gastric compartment.

Native and fermented waxy cassava starch as a novel gluten-free and clean label ingredient for baking and expanded product development.

Amylose-free and wild-type cassava starches were fermented for up to 30 days and oven- or sun-dried. The specific volume (ν) after baking was measured in native and fermented starches. The average ν (across treatments) for waxy starch was 3.5 times higher than that in wild-type starches (17.6 vs. 4.8 cm3 g-1). The best wild-type starch (obtained after fermentation and sun-drying) had considerably poorer breadmaking potential than native waxy cassava (8.4 vs. 16.4 cm3 g-1, respectively). The best results were generally obtained through the synergistic combination of fermentation (for about 10-14 days) and sun-drying. Fermentation reduced viscosities and the weight average molar mass led to denser macromolecules and increased branching degree, which are linked to a high loaf volume. The absence of amylose, however, was shown to be a main determinant as well. Native waxy starch (neutral in taste, gluten-free, and considerably less expensive than the current alternatives to cassava) could become a new ingredient for the formulation of clean label-baked or fried expanded products.

Preparative isolation of apple flavan-3-ols monomers and oligomers using pH-zone-refining centrifugal partition chromatography combined with reversed-phase liquid chromatography.

Flavan-3-ols (catechin monomers and procyanidins) are the main class of polyphenols in apples and are found in high concentrations in cider apple varieties. They are known to be involved in bitterness and astringency in apple-based beverages, and also contribute to polyphenol nutritional intake.Therefore, highly purified flavan-3-ol fractions isolated from raw materials are needed to study their various properties. For this purpose, a gentle strategy combining pH-zone-refining centrifugal partition chromatography (pH-ZRCPC) and preparative reversed-phase liquid chromatography (Prep-RPLC) was developed to recover one hundred milligrams of a high purity apple flavan-3-ol fraction. First, pH-ZRCPC fractionation in descending mode was optimized to remove hydroxycinnamic acid derivatives using a biphasic mixture composed of ethyl acetate/n-butanol/water (3/2/5, v/v). Trifluoroacetic acid and sodium hydroxide were used as retainer and eluter, in the upper and lower phases, respectively. Secondly, Prep-RPLC separation was carried out in isocratic mode at 20% ACN to remove dihydrochalcones. Finally, from one gram of a crude polyphenol extract, four hundred and nine milligrams of a highly purified fraction of flavan-3-ols with an average degree of polymerization close to 3.1 was obtained with 73% recovery.

Heat-unstable apple pathogenesis-related proteins alone or interacting with polyphenols contribute to haze formation in clear apple juice.

Physico-chemical instability is a damaging defect that can occur in clear bottled beverages leading to the formation of haze. In a previous study, we showed the presence of proteins in haze gathered from apple juices. For the first time, proteomics was used to sequence and identify four pathogenesis-related proteins (PRPs) from the haze of a commercial apple juice. Then, a study involving purified PRPs and polyphenols from apple juice was conducted in model solution to understand the mechanisms by which they are involved in haze formation. Visual assessment revealed that apple juice pathogenesis-related proteins are able to form haze alone when thermally denatured. These proteins were also able to interact with apple juice procyanidins to form complexes that can be precipitated using ultracentrifugation, even without prior heating. These interactions were greater when the degree of polymerization of tannins increased.

Preparative fractionation of 5'-O-caffeoylquinic acid oxidation products using centrifugal partition chromatography and their investigation by mass spectrometry.

When apples are processed into juices or ciders, a great variety of neoformed molecules are generated by enzymatic oxidation of polyphenols. These phenolic oxidation products could be responsible of specific organoleptic properties in apple juices and ciders. 5'-O-Caffeoylquinic acid (CQA) is the major hydroxycinnamic acid in apple and the preferential substrate of apple polyphenoloxidase (PPO). Its main oxidation products were synthetized and purified at the multi-milligrams scale to decipher their structures using mass spectrometry. CQA oxidation products were first synthetized in model solution by oxidizing CQA, in the presence of oxygen and PPO. Then, a specific method involving centrifugal partition chromatography (CPC) was developed to fractionate the main oxidation products corresponding to CQA dehydrodimers (MW 706 Da). For this purpose, CPC was performed in Elution-Extrusion Countercurrent Chromatography (EECCC) mode using a two-phase solvent system precisely selected according to the partition coefficient of the targeted compounds. After a last purification step using semi-preparative reversed phase HPLC, ten CQA dehydrodimers resulting from oxidative coupling were successfully purified, with a UV 280 nm chromatographic purity superior to 85%. Hypothetical structures were formulated for all CQA dehydrodimers based on their UV-vis, MS and MSn spectra. According to this study, centrifugal partition chromatography in combinaison with semi-preparative HPLC was a promising tool to fractionate or purify phenolic oxidation products.

Self-aggregation of oxidized procyanidins contributes to the formation of heat-reversible haze in apple-based liqueur wine.

The ability of tannins to self-associate or form complexes with other macromolecules has important nutritional implications but can also result in defects in beverages. In addition, oxidation may be involved in the aggregation properties of tannins. In order to assess the impact of tannin oxidation on their self-association, oligomeric procyanidins were oxidized in a model solution and their aggregation kinetics were studied using light scattering. Under the conditions tested, only oxidized procyanidins were involved in haze formation. An increase in the level of oxidation and the degree of polymerization of procyanidins enhanced aggregation. Procyanidin oxidation products were depolymerized and the evolution of their markers was monitored throughout the aggregation process using liquid chromatography coupled with mass spectrometry. This revealed the involvement of intramolecular coupling in reversible haze formation. The haze formed in a model solution was partially reversible at high temperature. This property was similar in pommeau, an apple-based beverage. This work highlighted the involvement of oxidized tannins in reversible haze.