Metabolites of biologically active compounds formed during digestion that then reach the blood and target organs often have different biological activity compared to primary compounds [1
]. These phenolic compounds, which occur in the human diet in the largest quantities, do not always show the highest biological activity after consumption; this may be due to their limited absorption in the digestive tract, intensive metabolism to derivatives with lower activity, or their rapid elimination (degradation). For example, the antioxidant activity (AOX) of quercetin glycosides is half that of aglycon [2
Individual groups of polyphenols differ significantly in bioavailability, which is associated with their structure, molecular weight, polarity, and form. The metabolic reaction of most dietary polyphenols has several similarities as follows: (1) glycosides of these compounds undergo hydrolysis prior to absorption; (2) mainly glucuronates and sulfates of native particles are present in the plasma; (3) in the polyphenols containing hydroxyl groups in the ortho position, methylation may occur; (4) and aglycons are either absent in the bloodstream or present in small quantities (except for catechins derived from green tea) [3
]. The highest number of metabolic transformations and absorption of polyphenols occurs in the small intestine, but the course of this digestive stage differs for individual flavonoid groups [5
]. Polyphenols that have not been absorbed in the small intestine, and those that have been secreted through bile or directly from enterocytes due to metabolic processes, reach the large intestine. In this part of the digestive tract, intestinal microbiota resides, which has extremely diverse enzymatic activity. Because of bacterial enzymes, a large number of reactions occur in the large intestine, including deconjugation, dehydroxylation, demethylation, isomerization, decarboxylation, hydrolysis of various chemical bonds, and cleavage of aromatic rings, resulting in a mixture of simple phenolic acids [9
Edible fruits differ in the content of phenolic compounds in their tissues. Some of them are very rich in antioxidants, e.g., chokeberry, blackcurrant, and wild rose. Others, such as apples, do not contain significant amounts of phenolic compounds; however, due to the frequency or amount in which they are consumed, they are an important source of these ingredients from a nutritional point of view.
Apples are one of the most common fruits in the world due to their taste and the ability to obtain a wide range of processed products (juices, purees, wines, and confectionery additives). Fruits contain polyphenols from various groups, including procyanidins, phenolic acids, dihydrochalcones, flavan-3-ols, flavonols, and anthocyanins, but their concentration is not high and strongly depends on which part of the plant they are taken from, fruit variety, climatic conditions, and many other factors [10
]. In addition, the polyphenol content decreases when processing apples [14
]. Apples are one of the most commonly used raw materials in fruit winemaking, due to their availability, price, and chemical composition, which allow to obtain a high-quality product with good sensory qualities.
Blackcurrant, which belongs to the gooseberry family, is grown mainly in Europe, and is primarily used for the production of nectars, syrups, jams, and wines, as well as ingredients for juices and non-alcoholic beverages. Blackcurrant contains a number of phenolic compounds and is characterized by high antioxidant activity; this increases the possibility of its use in the functional food sector [15
]. Blackcurrants are a good source of vitamins (A, C, E, and folic acid), provitamins (carotene and lutein), mineral compounds (calcium and selenium), and phytosterols; however, their health-promoting properties are primarily associated with the presence of phenolic compounds (anthocyanins, flavonols, flavan-3-ols, proanthocyanidins, soluble tannins, and phenolic acids) as the main bioactive components of fruits [15
]. Regardless of how the fruit is processed, there is always a reduction in polyphenols of up to 80% in the final product [17
]; this is associated with a high content of pectins in the skin cell wall making it difficult to extract these compounds. Despite this, among the berry fruit juices, blackcurrant juice has the highest content of phenolic compounds and the best ability to quench free radicals [18
]. Blackcurrants are a valued raw material for wine due to their characteristic strong aroma and taste, but the high acidity of this fruit makes it necessary to deacidify the musts by dilution or by the biological method [19
The processing of fruit into juices and wines is very popular; however, during enzymatic treatment, pressing, clarification, and other technological processes, as well as fermentation itself, there is a transformation of phenolic compounds, which significantly affects the final content and profile of polyphenols in beverages. In addition, the different chemical composition of beverages is associated with the different bioavailability of these valuable ingredients during digestion. The bioavailability of polyphenolic compounds from lyophilized currant juice has already been described previously [20
], but the authors focused mainly on whether the anti-inflammatory effects of blackcurrant fruits could be modulated by metabolic transformations. The bioavailability of polyphenols from apple juice was also analyzed [21
]. However, there are still no publications comparing the bioavailability of polyphenolic compounds from fruit musts and the wines obtained from them. Therefore, for this study, musts were made from apples and blackcurrants, and then they were subjected to ethanol fermentation to obtain wines. The aim of the study was to assess the content and profile of phenolic compounds and antioxidant activity at various stages of digestion of wines and musts in a simulated human digestive tract.
3. Materials and Methods
The research material consisted of apples of the Rubin variety, originating from the pomological orchard of the University of Agriculture in Krakow, Poland, and blackcurrant fruit from plantations in the Krakow area.
The apples and currants were washed, the apples were additionally crushed (with a laboratory crusher, Robix, Veszprem, Hungary), and then the must was pressed out of the fruit (wooden manual basket press, dimensions: φ = 25 cm, h = 36 cm). The musts were subjected to correction of the extract (up to 20 °Blg) and acidity (for apple must 4.5 g/L–5.0 g/L, for currant must 7 g/L), and then inoculated with rehydrated Erbslöh Oenoferm® InterDry F3 yeast (Marxam, Krakow, Poland) in the amount of 0.3 g/L. Fermentation of apple must was carried out at 20 °C ± 2 °C for thirty days, and currant must for fifty days, and then the wines were aged at 5 °C (cold room with adjustable temperature) for two months. Full anaerobic conditions were ensured; the wines were aged in bottles sealed with rubber stoppers and secured with parafilm. The access of light to the wines was also completely eliminated (darkened, closed cold store room).
The musts and wines obtained from them served as the material for analysis and were used in the simulated digestive process. All tests and analyses were performed in a minimum of three physical replicates.
3.1. Determination of Extract Content, Alcohol, Total Acidity, and Volatile Acidity
Determinations of total extract, ethyl alcohol, and acidity were carried out in accordance with the methods recommended by the International Organization of Vine and Wine (OIV) [56
]. The total extract and ethanol content were determined using distillation methods with pycnometric density determination. Briefly, the wine sample (100 mL) was distilled, the distillate was made up to 100 mL with distilled water, and its density was measured using a pycnometer. The alcohol content in the wine was read from the tables. Then, the distillation residue was quantitatively transferred to a volumetric flask, made up to 100 mL with distilled water, and the extract content was measured with an Abbe refractometer (PZO S.A., Warsaw, Poland). Total acidity was determined by potentiometric titration with 0.1 M NaOH. The results were expressed in g of malic acid/L. The volatile acidity analysis was performed by steam distillation of the wine followed by titration of the distillate with 0.1 M NaOH. The results were expressed in g of acetic acid/L.
3.2. Procedure of In Vitro Digestion
Musts and extracts (0.5 mL) were acidified to pH = 2 with HCl (0.5 M, POCh, Gliwice, Poland) into screw-capped vials. Then were added 0.75 mL of pepsin solution (62.6 mg of pepsin, EC 22.214.171.124, with activity 3440 U/mg, Sigma-Aldrich, St. Louis, MO, USA) dissolved in 20 mL of 0.1 M HCl (POCh) and redistilled water (obtained by ultrafiltration, Simplicity Millipore, Temecula, CA, USA) to a total volume of 3 mL. After thorough mixing, the test tubes were incubated in a water bath at 37 °C for 2 h. At this stage (digestion stage I, stomach), some samples were centrifuged (880× g, 10 min) and the supernatants forwarded for further analysis.
NaHCO3 (POCh) in an amount providing pH = 7.0 was added to the remaining samples, followed by 0.375 mL of pancreatin and bile solution. The solution was prepared by mixing 66.7 mg of pancreatin, (EC 232-468-9, with activity 8 × USP, Sigma-Aldrich) and 833.3 mg of bile (EC 232-369-0, Sigma-Aldrich), which was then dissolved in 10 mL of 0.1 M NaHCO3. The sample volume was adjusted to 5 mL with redistilled water. Vials were incubated in a water bath (37 °C, 4 h) and centrifuged (1380× g, 10 min). Then, some samples were centrifuged and the obtained supernatants were retained for further analysis (digestion stage II, small intestine).
Under anaerobic conditions, intestinal bacteria of documented human origin (Bifidobacterium catenulatum, Escherichia coli, Ruminococcus gauvreauii, Enterococcus caccae, Lactobacillus sp.; Leibniz Institut DSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany), an inoculum containing 106 bacterial cells (with equal participation of individual species) was prepared in 1 mL of the mixture. After being centrifuged (4000× g–5000× g, 20 min), the supernatant was discarded leaving the bacterial pellet. Under anaerobic conditions, samples after previous digestion (supernatant from the small intestine stage) were added to the bacterial pellet, mixed, saturated with inert gas (N2), and incubated at 37 °C for 16 h (interaction of intestinal microbial enzymes). The samples were then centrifuged and the supernatant obtained was retained for further analysis (digestion stage III, large intestine).
3.3. Antioxidant Activity (AOX)
Antioxidant activity (AOX) was determined according to the method described by Tarko et al. [57
], using an active cation radical of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic) acid (ABTS, Sigma-Aldrich). AOX was calculated on the basis of a calibration curve, prepared each time for synthetic vitamin E ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, Trolox, Sigma-Aldrich) and expressed as mg of Trolox/100 mL.
3.4. Total Polyphenol Content (TPC)
Total polyphenol content (TPC) in musts, along with all obtained fractions of simulated digestion, was determined by the method described previously in detail by Tarko et al. [57
], i.e., by the Folin–Ciocalteu method. The results were expressed as mg of (+)-catechin/100 mL.
3.5. Analysis of Phenolic Compound Profiles
Prior to analysis, the samples were filtered through a nylon syringe filter (0.45 µm, Chemland, Stargard Szczecinski, Poland). Analysis of the polyphenol profile was carried out using high-performance liquid chromatography (HPLC, Shimadzu, Kyoto, Japan) equipped with a DAD detector. A Synergi Fusion RP-80A column 150 mm × 4.6 mm (4 µm) (Phenomenex, Torrance, CA, USA), thermostated at 30 °C temperature, was used for all analyses. Acetonitrile (POCh) and 2.5% aqueous solution of acetic acid (POCh) were used as a mobile phase. The detailed gradient program and detection wavelengths were described in detail by Tarko et al. [58
For quantitative analyses, calibration curves were prepared for the following standards: ferulic acid, caffeic acid, chlorogenic acid, gallic acid, hippuric acid, p-coumaric acid, protocatechuic acid, ellagic acid, (+)-catechin, quercetin, resveratrol, kaempferol (Sigma-Aldrich), phloridzin, (−)-epicatechin, procyanidins B1 and B2, cyanidin-3-O-galactoside, cyanidin-3-O-sambubioside, cyanidin-3-O-arabinoside cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, pelargonidin-3-O-glucoside, and peonidin-3-glucoside (Extrasynthese, Genay, France). Compounds were identified by comparing the retention time of individual peaks and UV spectra with those of standards (using the spectrum library for the standards). Polyphenols not detected in any experimental variants were not included in the tables.
3.6. Determination of Sugar and Glycerol Concentration (HPLC)
The concentration of sugars and glycerol was determined by the HPLC method. The analysis of the sugar profile was conducted with the Shimadzu (Kyoto, Japan) NEXERA XR apparatus with an RF-20A refractometric detector. The separation was conducted with an Asahipak NH2P-50 4.6 mm × 250 mm Shodex column (Showa Denko Europe, München, Germany) thermostated at 30 °C. The mobile phase consisted of an acetonitrile aqueous solution (70%), and the isocratic elution program (0.8 mL/min) lasted 16 min.
Quantitative determinations were made using calibration curves prepared for appropriate standards, i.e., glucose, fructose, sucrose, and glycerol (POCh, Gliwice, Poland).
3.7. Statistical Analysis
A minimum of three repetitions of the analysis was conducted, and the results are shown as the arithmetic mean with standard deviation (±SD). Statistical analysis was performed using InStat v. 3.01 (GraphPad Software Inc., San Diego, CA, USA). A single-factor analysis of variance (ANOVA) with post hoc Tukey’s test was applied to determine the significance of differences between means. The Kolmogorov–Smirnov test was carried out to assess the normality of the distribution.