Letting wine polyphenols functional: Estimation of wine polyphenols bioaccessibility under different drinking amount and drinking patterns

Food Research International 127 (2020) 108704

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Letting wine polyphenols functional: Estimation of wine polyphenols bioaccessibility under different drinking amount and drinking patterns

T

Xiangyu Suna,b,1, Xianghan Chenga,1, Jingzheng Zhanga, Yanlun Jua, Zhiluo Quea, Xiaojun Liaob, ⁎ ⁎⁎ Fei Laob, Yulin Fanga, , Tingting Maa, a

College of Enology, College of Food Science and Engineering, Viti-viniculture Engineering Technology Center of State Forestry and Grassland Administration, Shaanxi Engineering Research Center for Viti-Viniculture, Heyang Viti-viniculture Station, Northwest A&F University, Yangling 712100, China b Beijing Key Laboratory for Food Nonthermal Processing, Key Lab of Fruit and Vegetable Processing of Ministry of Agriculture, National Engineering Research Center for Fruit and Vegetable Processing, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China

ARTICLE INFO

ABSTRACT

Keywords: Wine Polyphenols Bioaccessibility Drinking amount Drinking pattern In vitro digestion

Effects of drinking amount and patterns of wine on the digestive characteristics and bioaccessibility of wine polyphenols under in vitro gastrointestinal digestion were investigated. Wine polyphenols released well during mouth and stomach digestion, and the release rates in the “serum-available” fraction, “colon-available” fraction, and after the colon were much lower. Red wine showed a higher biological activity than white wine, but white wine had a better bioaccessibility than red wine, especially under binge drinking. The bioaccessibility of most polyphenols decreased as the drinking amount increased, indicating that drinking larger volumes of wine did not increase the bioaccessibility of polyphenols. Additionally, the relevant biological activities did not increase as the drinking amount increased. Drinking after a meal showed significantly better results than drinking before a meal in most of the tests. Hence, in order to let wine polyphenols play its functional for human health, there still need a moderate consumption amount of wine and drinking after meal is better.

Chemical compounds studied in this article: (+)-catechin (PubChem CID: 9064) (−)-epicatechin (PubChem CID: 72276) (−)-epigallocatechin (PubChem CID: 72277) (−)-epicatechin gallate (PubChem CID: 107905) (−)-epigallocatechin gallate (PubChem CID: 65064) Gallic acid (PubChem CID: 370) Protocatechuic acid (PubChem CID: 72) Caffeic acid (PubChem CID: 689043) Ferulic acid (PubChem CID: 1794427) Chlorogenic acid (PubChem CID: 1794427)

1. Introduction Since the “French Paradox” was reported in 1992 (Renaud & De Lorgeril, 1992), wine has been considered to have certain health benefits for humans. In fact, wine has been found to have many effects on health, such as in maintaining redox balance (Marhuenda et al., 2016), in modulating fecal microbiota and reducing markers of metabolic syndrome in obese patients (Moreno-Indias et al., 2016), in decreasing the relative risks of developing coronary heart disease and cancer (Mazué et al., 2014), anti-inflammatory activity (Nunes et al., 2013), antihypertensive activity (Pino-García, Rivero-Pérez, González-

SanJosé, Croft, & Muñiz, 2017), and in protecting cognitive function (Boban et al., 2016). Wine is a complex matrix containing many classes of compounds, such as alcohols, sugars, acids, tannins, minerals, proteins, and other secondary metabolites, such as phenolic compounds, organic acids, and volatile compounds (Artero, Artero, Tarín, & Cano, 2015). Among these bioactive compounds, phenolic compounds are considered to be one of the biggest contributors to the benefits attributed to wine (Sun et al., 2015) mainly because dietary polyphenol intake has been associated with numerous health benefits for humans (Xiang, Zhang, Apea-Bah, and Beta, 2019), such as antioxidant (Sun et al., 2015; Xiang, Li, Ndolo, and Beta, 2019), antibacterial, antiviral,

Corresponding author. Corresponding author. E-mail addresses: [email protected] (J. Zhang), [email protected] (Y. Fang), [email protected] (T. Ma). 1 These authors contributed equally to this work. ⁎

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https://doi.org/10.1016/j.foodres.2019.108704 Received 25 February 2019; Received in revised form 17 September 2019; Accepted 21 September 2019 Available online 31 October 2019 0963-9969/ © 2019 Elsevier Ltd. All rights reserved.

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antifungal, antiproliferative, anti-inflammatory, anti-allergic, anti-hypertensive and antithrombotic activities as well as the prevention of dental diseases and positive effects on human microbiota composition and functionality (Fraga, Croft, Kennedy, & Tomás-Barberán, 2019). However, all these health benefits are based on the consumption of a moderate amount of wine (Artero et al., 2015). As with the intake of bioactive grape phytochemicals, large amounts of ethanol will also be ingested into the body. Excessive ethanol intake is related to illness such as different types of cancers, particularly those of the digestive tract, liver and breast, and even early death (Boban et al., 2016; Garaycoechea et al., 2018). In reality, it is estimated that harmful alcohol use is the third largest cause of early death and illness in the European Union (EU, 2006), and it is a significant contributor to the global burden of disease as it is the third most important risk factor globally and the most important risk factor in middle-income countries (WHO, 2010, 2014). In fact, alcohol consumption is a fashion around the world. Governments generally set guidelines for amounts of alcohol consumption. Now, in USA (U.S. Department of Health and Human Services and U.S. Department of Agriculture, 2015), UK (House of commons science and technology committee, 2012), Canada (Canadian Centre on Substance Abuse, 2013), and China (National health and family planning commission of China, 2016) all had set their drinking advice. Meanwhile, consumers have a certain understanding of the harm of alcohol from beer and other alcoholic beverages with high alcohol content. However, there was an undesirable trend in wine consumption, as consumers thought of wine as safe and healthy, mainly due to its low alcohol content, its high content of bioactive compounds such as polyphenols and the publicity of wine health benefits such as the “French Paradox”; thus, there was no need to control the amount of wine consumed. Under these conditions, even though bioactive compounds such as polyphenols are present in wine, this level of wine consumption could still cause harm to humans. Except that, a previous study showed that drinking wine with meals is associated with more beneficial effects (Boban et al., 2016); however, the wine drinking patterns are not usually associated with meals. Except for excessive alcohol intake, as previously mentioned, another problem remains; the bioavailability of polyphenols. The possible health benefits of polyphenols in the human body are substantially determined by their bioavailability (Minekus et al., 2014). The abundant polyphenols in the consumed wine are not necessarily those that will result in the highest tissue concentrations or those with biological effects, owing to the considerable differences in the bioavailabilities of polyphenols (Alminger et al., 2014). Although human nutritional studies are still considered the “gold standard” for addressing diet-related questions, these studies are time consuming, costly, and restricted by ethical concerns (Alminger et al., 2014; Minekus et al., 2014). Meanwhile, In recent years, an increasing number of studies have used in vitro digestion models which was showed well correlated results with in vivo and clinical studies (Celep, Charehsaz, Akyüz, Acar, & Yesilada, 2015) to investigate the gastrointestinal behavior of foods and bioavailabilities of nutrients (Celep et al., 2015; Corrêa et al., 2017; Gumienna, Lasik, & Czarnecki, 2011; Lingua, Wunderlin, & Baroni, 2018), mainly due to the advantage of being faster, less expensive, less labor intensive, not having ethical restrictions, and allowing a relatively large number of samples to be measured in parallel for screening purposes. Therefore, in order to let wine polyphenols play its functional for human health, in this study, the effects of drinking patterns of wine, including drinking before or after a meal, on the digestive characteristics and bioaccessibility of wine polyphenols under simulated in vitro gastrointestinal digestion were first investigated. In addition, the effects of the amount of wine consumed on the digestive characteristics and bioaccessibility of wine polyphenols during in vitro digestion are also discussed. These results are expected to provide a scientifically understand the “pro and con” for drinking.

2. Materials and methods 2.1. Samples and chemicals The “Xueyuanpai” red wine (Cabernet Sauvignon) (Alcohol: 13.5%) and white wine (Chardonnay) (Alcohol: 12%) were kindly supplied by Wine Technology Development Center of China Agricultural University. All standards were from Sigma-Aldrich (St. Louis, MO, USA), including five flavan-3-ol standards [(+)-catechin (CAT), (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG)] and eleven phenolic standards, including six hydroxybenzoic acids (gallic acid, protocatechuic acid, phydroxy benzoic acid, gentisic acid, vanillic acid and syringic acid) and five hydroxycinnamic acids (caffeic acid, p-coumaric acid, ferulic acid, chlorogenic acid and sinapic acid). Juvitana Infant formula were purchased from Enter Asia (Hong Kong, China). Methanol and acetonitrile were of HPLC grade (Spectrum Chemical Co., Irvine, CA, USA), and all of the remaining reagents were of analytical grade. 2.2. In vitro gastrointestinal (GI) digestion The in vitro GI digestion assay was performed according to the procedures described previously (Alminger et al., 2014; Minekus et al., 2014) with slight modifications. Four sequential steps, mouth, stomach, small intestine and colon digestion, were included in this model to mimic in vivo GI digestion. For each digestion step, the sample groups were independent such that the mouth step had twelve parallel sets for each treatment, while there were nine in the stomach, six in the small intestine and three in the colon. The flow chart of the digestion experiment was shown as ***Fig S1. 2.2.1. Mouth digestion For each sample, a 100 mL sample was added to an amber glass flask containing 100 mL of an artificial saliva solution (Alminger et al., 2014; Liu, Ma, Zhang, Gao, & McClements, 2017; Ma et al., 2019a) (50 mM NaCl, 10 mM NaH2PO4, and 40 mM NaHCO3), after which the pH was adjusted to 6.7–6.9 using 1 M NaOH, followed by the addition of 1 mL of a fresh α-amylase preparation containing 25 U. Next, the mixtures were shaken at 100 g for 1 min in a shaking incubator at 37 °C to simulate agitation in the mouth. After 2 h, three samples for each treatment were immediately snap frozen in liquid nitrogen to stop the reaction, and the samples were stored until further treatment. The other samples were transfer for the following steps. 2.2.2. Stomach digestion From this step, there were two drinking pattern groups: drinking before a meal and drinking after a meal. The simulated gastric fluid for before a meal consisted of 2 g of NaCl, 450 units of pepsin per mL, enough HCl to adjust the pH to 1.2, and enough distilled water to dilute the mixture to 1 L. The simulated gastric fluid for after a meal consisted of 2 g of NaCl, 450 units of pepsin per mL, 45 g Juvitana Infant formula which was cut into 5-mm cubic pieces, enough HCl to adjust the pH to 5, and enough distilled water to dilute the mixture to 1 L (Ministry of agriculture notice No. 869-2-2007). The Juvitana infant formula added here were a food matrix with defined composition as 20% turkey meat, 25% boiled corn paste, 10% boiled potato paste, 5% rice flour, 0.1% NaCl and 39.9% water (mixture contained 3% protein, 10% carbohydrate and 1% fat), free of preservatives, antioxidants, artificial flavors and aromas, and was used to simulate standard meal (food matrix) here. There were three groups based on the amount of wine consumed: low wine drinking (with a wine volume/simulated gastric fluid volume ratio of 0.5:1), moderate wine drinking (with a wine volume/simulated gastric fluid volume ratio of 1:1), and binge drinking (with a wine volume/simulated gastric fluid volume ratio of 3:1). The wine volumes in the three treatment groups were the same. The volume in each group was made constant by adding physiological saline and using HCl to 2

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adjust the pH to 1.2 or 5 for the different treatments. Each mixture of wine and simulated gastric fluid was transferred to a tube with a silver paper cap and placed in a water bath at 37 °C in an incubated shaker with continuous swirling at 100 g for 2 h to simulate gastric digestion. During the processing, the pH of the reaction system was monitored and maintained stable by 1 M HCl or NaOH and using an automatic titration unit (Metrohm, Riverview, FL, USA). Then, three samples for each treatment were immediately snap frozen in liquid nitrogen to stop the reaction, and stored until further treatment. The other samples were transfer for the following steps.

0.45 μm pore filters, fractionated into microtubes, and stored at −80 °C until analysis. 2.3. Determination of the polyphenols 2.3.1. Determination of the total phenolics (TP), total flavonoids (TFO), total flavan-3-ols (TFA), and total anthocyanins (TA) The TP content was determined according to the Folin-Ciocalteu colorimetric method (Sun, Ma, Han, Huang, & Zhan, 2017; Xiang, ApeaBah, Ndolo, Katundu, and Beta, 2019). The results are expressed as mg gallic acid equivalents (GAE)/L wine (mg GAE/L wine). The TFO content was determined according to a previously described protocol using aluminium trichloride colorimetric method (Ma et al., 2014; Zhang et al., 2019a). The results are expressed as mg catechin equivalents (CTE)/L wine (mg CTE/L wine). The TFA content was estimated using a slightly modified version of the DMACA (p-dimethyl-aminocinnamaldehyde) method (Ma et al., 2014). The results are expressed as mg CTE/L wine. The TA content was estimated using the pH differential method (Sun et al., 2017). The results are expressed as mg cyanidin-3glucoside (CGE)/L wine (mg CGE/L wine). All spectrophotometric measurements were performed using a UV–Vis double beam Hitichi U3010 spectrometer (Hitichi, Kyoto, Japan).

2.2.3. Small intestine digestion The simulated intestinal fluid consisted of 7 g of KH2PO4, 4 mg/mL pancreatin, 25 mg/mL bile salts, enough NaOH to adjust the pH to 7.5, and enough distilled water to dilute the mixture to 1 L (Ministry of agriculture notice No. 869-2-2007). After stomach digestion, the test mixture was transferred to a glass beaker, and half of the simulated gastric fluid volume of simulated intestinal fluid was added. The volume of each group was made consistent by adding physiological saline. NaOH was used to adjust the pH to 7.5. A full, bubble-free, closed dialysis bag (molecular weight cut-off 12 kDa) containing sufficient NaHCO3 at pH 7.5 was added, and the beaker was sealed with parafilm. The glass beaker was placed in a water bath at 37 °C in the dark in an incubated shaker with continuous swirling at 100g for 2 h to simulate small intestine digestion. The pH of the reaction system was then monitored and maintained by titrating the reaction mixture with 0.25 M sodium hydroxide solution using an automatic titration unit (Metrohm, Riverview, FL, USA). After this time, the solution inside the dialysis bag was separated and stored, which representing the fraction available for absorption into the circulatory system by passive diffusion, and hence could simulate the “serum-available” fraction in vivo of polyphenol (Alminger et al., 2014; Minekus et al., 2014). On the other hand, the solution outside of the dialysis bag, the nondialyzable fraction, was separated and stored; this is the material that remained in the gastrointestinal tract and would reach the colon (simulate the “colonavailable” fraction in vivo (Alminger et al., 2014; Minekus et al., 2014)). After 2 h, the “serum-available” and “colon-available” fraction of the three sample tubes for each treatment were taken and acidified with formic acid to pH = 2 to neutralize the NaHCO3 and stored until further treatment. The other samples were transfer for the following steps.

2.3.2. Detection of phenolic acids A Waters Alliance 2695 HPLC system with a Waters 2996 PDA (photodiode array) detector (Waters Corp., Milford, Massachusetts, USA) was used to simultaneously separate and analyze the phenolic acids. The system was run at 1.0 mL/min using a 100RP-18e column (250 mm × 4.0 mm, inner diameter 5 μm) from Merck LiChrospher (Darmstadt, Germany) and an RP-18 (10 mm × 4 mm) guard column that was also from Merck. The column temperature was set at 30 °C, and the injection volume was 10 μL. The detection wavelengths were 280 nm and 320 nm. External standards were used to identify the target analytes. Mobile phase A was methanol, acetic acid and water (10:2:88), and mobile phase B was methanol, acetic acid and water (90:2:8). The gradient elution was as follows: 0 to 25 min, phase B from 0 to 15%; 25 to 45 min, phase B from 15 to 50%; and 45 to 53 min, phase B from 50 to 0%. All samples were filtered through a 0.45-μm organic Millipore filter before injection (Merck Millipore, 290 Concord Road, Billerica, MA, USA) (Zhang et al., 2019b). The HPLC method was validated as described in ****Fig S2 A. We can see that a good separation was achieved for the 11 phenolic acids under the chromatographic conditions, and the R2 values were greater than 0.9992 ***(Table S1), which indicated that the eleven standards showed good linear relationships in the concentration range tested.

2.2.4. Colonic digestion The fecal inoculums were obtained from fresh feces collected from the entire large intestines of male SD rats (70-day-old animals, average 300 g) immediately after euthanasia (Corrêa et al., 2017). A fecal pool was made from 5 animals. All the procedures of the animal experiment were in accordance with the State Code of Practice for the Care and Use of Animals for Scientific Purposes. After collection, the material was immediately homogenized with the culture medium at a ratio of 1:10 (w/v). The fermentation medium was prepared according to a previous method (Fu et al., 2018), and the material was sterilized at 121 °C for 30 min before use. Ten milliliters of the solution outside of the dialysis bag from the small intestine digestion step was thoroughly mixed with 45 mL of fecal inoculum and 45 mL of fermentation medium using a vortex mixer. The pH was adjusted to 8.0 by the addition of 2 M NaHCO3. The experimental samples were transferred to different anaerobic sealed tubes in an anaerobic incubator, and the samples were incubated at 37 °C and shaken at 100 g for 18 h. Then, the fermentation products were collected and plunged into ice water for 5 min to stop fermentation. These fermentation products were centrifuged at 8000g for 15 min to separate the supernatants for further analysis.

2.3.3. Detection of flavan-3-ols A Waters Alliance 2695 HPLC system with a Waters 2996 PDA detector was used to simultaneously separate and analyze the flavan-3-ols. The system was run at 1.0 mL/min using a 100RP-18e column (250 mm × 4.0 mm, inner diameter 5 μm) and an RP-18 (10 mm × 4 mm) guard column from Merck LiChrospher. The detection wavelength was 280 nm, the column temperature was 30 °C, and the injection volume was 10 μL. External standards were used to identify the target analytes. Mobile phase A was water. Mobile phase B was acetic acid and water (10:90). The flow rate was 1 mL/min. The gradient elution was as follows: 0 to 20 min, phase B from 7.5 to 65%; 20 to 30 min, phase B from 65 to 80%; 30 to 48 min, phase B from 80 to 90%; 48 to 55 min, phase B at 90%; and 55 to 63 min, phase B at 7.5%. Samples were filtered through a 0.45-μm Millipore membrane before injection. The HPLC method validation is described ***in Fig S2 B. We can clearly see that the 5 flavan-3-ols were well separated under these chromatographic conditions, and the R2 values were greater than 0.9993 ***(Table S2), which indicated that the five standards showed good linear relationships in the concentration range tested.

2.2.5. Sample preparation for analysis Aliquots from the mouth, stomach, intestine (the “serum-available” and “colon-available”) and colonic digestions were centrifuged at 13,000g for 10 min. All samples were immediately filtered through 3

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2.4. Analysis of the antioxidant capacity

version 7.05, Hangzhou, China).

Four different methods, DPPH, ABTS, ORAC and FRAP, were used in this study. The DPPH scavenging activity and ABTS assays were based on previously described methods (Sun et al., 2017) with slight modifications. The oxygen radical absorbance capacity (ORAC) assay and the fluorescence recovery after photobleaching (FRAP) assays were performed essentially as described previously (Ma et al., 2017) with some modifications. The results are expressed as μM Trolox/L wine.

3. Results and discussion 3.1. The digestive characteristics of wine polyphenols 3.1.1. TP, TFO, TFA and TA The TP, TFO, TFA and TA contents are shown in Fig. 1. Overall, during mouth digestion, there was a good release of TP (Fig. 1A1–A4), which with no significant difference with wine. A previous report also demonstrated that there was only little change in wine polyphenols during the mouth digestion (Lingua et al., 2018), mainly due to the duration in mouth is very short for liquid food (Minekus et al., 2014). During stomach digestion, there was a good release of TP, with release rates of 88.59% to 95.86% for red wine and 76.04% to 85.49% for white wine, which was in accordance with previous reports (Celep et al., 2015; Gumienna et al., 2011; Lingua et al., 2018). After stomach digestion, food stuffs are transported to the small intestine and are then been absorbed (“serum-available” fraction, the bioaccessibility of bioactive compounds), while others are transferred to the colon (“colon-available” fraction). In the “serum-available” fraction, the TP showed an average release rate of 40–50%, while the average release rate in the colon fraction was 20%, which means that approximately half of the polyphenols in the wines were absorbed in small intestine digestion. Then, the wine stuffs were transported to the colon, which contains a diverse ecosystem of microorganisms. The polyphenols could serve as substrates for the community of microorganisms in the colon and influence the ecosystem of microorganism or continue on to be absorb into the serum, which happens with only a small proportion of the polyphenols (Celep et al., 2015). After colon fermentation, an average of 10% of the polyphenols were left in the colon. There were no differences in stomach digestion with different consumption volumes. However, in the small intestine digestion process, as the amount of wine consumed increased, the TP in the “serum-available” decreased with both red and white wine, and the TP in the colon-available fraction increased. This might due to the total TP amount increased, but transportation rate was constant, and resulting in a lower content in serum and a higher content in colon, which still need further verification in the future. This indicated that drinking to much wine did not result in increased polyphenol absorption. Meanwhile, the release rates in serum for white wine were significantly higher than for red wine (for example, the release rates were 42% and 34.92% in binge drinking before a meal). This might be due to the differences in polyphenol contents between red and white wines, as red wine always shows a polyphenol content several times higher than that of white wine. This indicated that even when drinking a large amount of white wine, the human body could still absorb the polyphenols better than when drinking red wine. In the colon digestion process, the TP tended to increase with increasing drinking amount. Of the two drinking patterns, drinking before a meal resulted in a lower TP content than drinking after a meal in the stomach digestion, which might be due to the difference in digestive enzyme activities under different pH conditions in the stomachs of the two drinking patterns (Alminger et al., 2014). Additionally, drinking after a meal showed a significantly higher serum TP value than drinking before a meal in all three wine drinking amounts and a lower colon-available TP value, which means that the polyphenols were better absorbed when drinking wine after a meal. TFO also showed a good release during mouth and stomach digestion (Fig. 1 B1–B4). In fact, there was no significant difference with wine during mouth digestion. Meanwhile, a higher TFO content was observed in the stomach following red wine consumption (with release rates of 106.71–123.07%). This might be due to the hydrolysis of the proanthocyanidins in red wines, which are hydrolyzed to their monomeric units after simulated stomach digestion due to the strong acidic conditions (Fernández & Labra, 2013). The release rate of TFO in white wine was much lower than in red wine but still higher than the release

2.5. Evaluation of the inhibitory effects of α-amylase and α-glucosidase 2.5.1. α-Amylase inhibition assay The methods used for the enzymatic inhibition assays performed in this study were adapted from those developed in a previous study (Ma et al., 2015) with some modifications. Fifty microliters of the samples or acarbose solutions of different concentrations were added to 100 µL of 5 U/mL α-amylase solution (in 0.2 M sodium phosphate buffer at pH 6.6), and the mixtures were each blended in a timely manner. Then, 100 µL of 1% soluble starch solution (dissolved in sodium phosphate buffer and boiled for 15 min) was added to each tube, and the mixtures were incubated at 37 °C for 10 min. The reactions were then terminated by the addition of 0.5 mL of DNS reagent (1% 3,5-dinitrosalicylic acid and 12% sodium potassium tartrate in 0.4 M NaOH). The mixtures were then incubated in a boiling water bath for 10 min. The mixtures were diluted with 3.75 mL of distilled water in an ice bath, and then they were allowed to come to room temperature. The absorbance at 540 nm was measured with a microplate reader. The control contained 100 µL of the buffer solution in place of the α-amylase solution. The blank contained 100 µL of the buffer solution instead of the soluble starch solution. The inhibitory activity (I) was calculated using the following equation:

I(%) = [1

(A sample

Abackground)/A control] × 100

(1)

2.5.2. α-Glucosidase inhibition assay The α-glucosidase inhibitory activity was measured as described in a previous report (Ma et al., 2019b) with slight modifications. Briefly, 0.1 mL of the samples or acarbose solutions of different concentrations were added to 0.1 mL of 4 U/mL α-glucosidase solution (in 0.2 M sodium phosphate buffer pH 6.8), and the mixtures were each blended in a timely manner. Then, 0.1 mL of 6 mmol/L 4-nitrophenyl-α-d-glucopyranoside (pNPG) solution (in 0.2 M sodium phosphate buffer at pH 6.8) was added to each tube, and the mixtures were incubated at 37 °C for 30 min. The reactions were then terminated by the addition of 3 mL of 1 mol/L Na2CO3. The absorbance at 400 nm was then measured with a microplate reader. The control contained 0.1 mL of buffer solution in place of the α-glucosidase solution, and the background contained 0.1 mL of buffer solution instead of the pNPG solution. The inhibitory activity (I) was calculated using Equation (1). 2.6. Bacterial count After colonic digestion, a previously described method (Gumienna et al., 2011) was used for counting the number of Entrobacteriaceae (using MacConkey selective medium), Lactobacillus (using MRS medium-agar), Enteroccocus (using agar with kanamycin, esculin and sodium azide) and Bifidobacterium (using Garch’s medium). The number of live bacterial cells was assessed by Koch’s plate method. 2.7. Statistical analysis The experimental results are expressed as the means ± standard deviations (SD) of three replicate fermentations for each treatment. Correlations were calculated using a linear regression. Statistical analyses were performed using data processing system software (DPS, 4

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Fig. 1. The total phenolics (TP), total flavonoids (TFO), total flavan-3-ols (TFA), and total anthocyanins (TA) of wine after in vitro GI digestion. (A1) TP of red wine before a meal; (A2) TP of red wine after a meal; (A3) TP of white wine before a meal; (A4) TP of white wine after a meal; (B1) TFO of red wine before a meal; (B2) TFO of red wine after a meal; (B3) TFO of white wine before a meal; (B4) TFO of white wine after a meal; (C1) TFA of red wine before a meal; (C2) TFA of red wine after a meal; (C3) TFA of white wine before a meal; (C4) TFA of white wine after a meal; (D1) TA of red wine before a meal; and (D2) TA of red wine after a meal. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

groups, the serum TFA contents in the drinking after a meal group were significantly higher than those of the drinking before a meal group with red wines, and the colon-available TFA contents were lower, indicating that the TFA was better absorbed when drinking wine after a meal. As shown in Fig. 1D1–D2, TA showed no significant difference with wine in the mouth step. In the stomach digestion step, the trends in the TA contents were obviously different between the two different drinking patterns. In the drinking before a meal group, the stomach TA contents were significantly higher than in the wine and increased with increasing drinking amount. However, in the drinking after a meal group, the stomach TA contents were significantly lower than in the wine. This was mainly due to the different pH conditions in the stomach. Gastric pH before a meal in healthy human subjects is in the range of 1.0–2.5. After a meal, the stomach pH sharply increases to approximately 4.5–6.2 (Alminger et al., 2014). The stability of anthocyanins in the digestive process appeared to be related to their structure. A lower pH could result in most of the anthocyanins being in the flavylium cation conformation (Yang et al., 2018), which resulted in the apparent increase in the stomach TA content in the drinking before a meal group. In addition, the pH in the drinking after a meal group could promote the cleavage of the C-ring of the anthocyanin structure (Lingua et al., 2018; Yang et al., 2018), which would result in a decrease in the TA content. The TA contents after small intestine and colon digestion were only approximately 40% and 10%, respectively, which were mainly due to the pH conditions and the effect of digestive enzymes. This is consistent with the report by Lingua et al. (2018) but somewhat different from the report by Yang et al. (2018), mainly because Yang et al. did not consider the digestive enzymes in their in vitro GI digestion model.

rate of TP. This might be due to the content of proanthocyanidins in white wines being obviously lower than in red wines; hence, the increase in the TFO release rate was lower than what was observed for red wines (Cáceres-Mella et al., 2013). The trends in the TFO contents in the small intestine and colon digestion were similar to those of TP. Considering the drinking amount, the TFO content in serum decreased in both red and white wine with increasing amount of wine consumed except for a moderate amount of white wine consumed before a meal. In addition, the colon-available TFO and the after colon TFO contents increased along with the drinking amount for both red and white wines. Of the different drinking pattern groups, under binge drinking conditions, a higher serum TFO value was observed compared to what was seen for drinking after a meal, indicating that drinking after a meal could mitigate the decrease caused by excessive drinking. In addition, in the low volume group, the TFO content in the serum in the drinking after a meal group was much higher than with the drinking before a meal group (85.05% and 58.63%), and the TFO in the colon-available fraction (18.58% and 6.25%) was what was left over after the other digestion steps, which indicated a better bioaccessibility of TFO. There was also no significant difference with wine for the TFA during mouth digestion, which was same with TP and TFO (Fig. 1C1–C4). And the TFA contents significantly increased during the stomach digestion with both red and white wines, and the release rate reached 144.84% in red wines. The reason might be the same as that of TFO; the proanthocyanidins were hydrolyzed to their monomeric units (Fernández & Labra, 2013). Lingua et al. (2018) reported a similar result. There was no significant differences in serum TFA contents among the three different drinking amounts, but the colon-available TFA and the after colon TFA still increased with increasing drinking amount for both red and white wines. For the two different drinking pattern 5

6

flavan-3-ol standards

hydroxycinnamic acids

White wine hydroxybenzoic acids

flavan-3-ol standards

hydroxycinnamic acids

Red wine hydroxybenzoic acids

Individual phenolic acids

gallic acid Pr-acid Ph-acid gentisic acid vanillic acid syringic acid THBA caffeic acid Pc-acid ferulic acid chlorogenic acid sinapic acid THCA TPC CAT EC EGC ECG EGCG TFA

caffeic acid Pc-acid ferulic acid chlorogenic acid sinapic acid THCA TPC CAT EC EGC ECG EGCG TFA

gallic acid Pr-acid Ph-acid gentisic acid vanillic acid syringic acid THBA

0.87 ± 0.11 a 6.78 ± 0.23 a 2.19 ± 0.22 a 6.00 ± 0.80 a 0.12 ± 0.01 a 2.11 ± 0.11 a 18.07 ± 1.48 a 3.50 ± 0.47c 0.38 ± 0.05 a 0.35 ± 0.05 a 1.64 ± 0.23 a 7.65 ± 0.45 a 13.52 ± 1.25 a 31.59 ± 2.73 a 4.55 ± 0.60 a 2.02 ± 0.27 a 1.22 ± 0.12 a 0.36 ± 0.05 a 0.11 ± 0.01 a 8.26 ± 1.05 a

30.10 ± 1.09c 12.31 ± 0.87 a 1.86 ± 0.17 a 5.74 ± 0.23 a 18.57 ± 0.78 a 68.58 ± 3.14 a 233.99 ± 6.07 a 42.85 ± 0.78c 6.26 ± 0.23b 2.97 ± 0.11 d 43.55 ± 2.01b 0.56 ± 0.10c 96.19 ± 3.23b

90.79 ± 0.57a 26.78 ± 0.59 a 7.19 ± 0.13 a 19.03 ± 0.48 a 16.62 ± 0.87b 5.00 ± 0.29 a 165.41 ± 2.93a

Wine

Table 1 The individual phenolic acids in wine after in vitro GI digestion with low wine consumption.

0.82 ± 0.08 a 6.23 ± 0.17 a 2.11 ± 0.12ab 5.78 ± 0.79 a 0.11 ± 0.01 a 1.98 ± 0.14 a 17.03 ± 1.31 a 3.77 ± 0.67c 0.33 ± 0.09 a 0.34 ± 0.08 a 1.51 ± 0.32 a 7.22 ± 0.56 a 13.17 ± 1.72 a 30.20 ± 3.03 a 4.31 ± 0.43 a 2.09 ± 0.19 a 1.18 ± 0.15 a 0.41 ± 0.04 a 0.11 ± 0.02 a 8.10 ± 0.83 a

32.12 ± 0.98c 11.09 ± 0.99 a 1.88 ± 0.21 a 5.32 ± 0.37 a 17.09 ± 1.01a 67.50 ± 3.56 a 231.71 ± 5.58a 44.12 ± 1.02c 7.19 ± 0.48b 3.31 ± 0.28 d 45.09 ± 2.87b 0.99 ± 0.12c 100.7 ± 4.77b

91.02 ± 0.29 a 27.02 ± 0.37 a 6.99 ± 0.09 a 17.09 ± 0.38 a 17.22 ± 0.66 a 4.87 ± 0.23 a 164.21 ± 2.02a

mouth

Before meal

0.78 ± 0.04b 5.28 ± 0.22b 1.98 ± 0.09b 4.87 ± 0.10c 0.08 ± 0.02c 0.98 ± 0.09b 13.97 ± 0.56b 4.98 ± 0.33b 0.18 ± 0.02b 0.37 ± 0.02 a 0.76 ± 0.03b 5.39 ± 0.12b 11.68 ± 0.52b 25.65 ± 1.08b 3.89 ± 0.22b 1.98 ± 0.11b 1.00 ± 0.06b 0.38 ± 0.02 a 0.09 ± 0.02 a 7.34 ± 0.43b

38.99 ± 0.99b 6.51 ± 0.24c 1.78 ± 0.09 a 2.87 ± 0.25b 10.28 ± 0.65b 60.43 ± 2.22b 215.09 ± 5.95b 76.29 ± 1.20b 13.87 ± 0.44 a 5.27 ± 0.29b 77.28 ± 1.22 a 2.76 ± 0.03 a 175.47 ± 3.18 a

87.12 ± 0.99b 24.01 ± 0.39b 6.29 ± 0.72b 15.38 ± 0.78b 17.83 ± 0.37 ab 4.03 ± 0.48b 154.66 ± 3.73b

stomach

0.28 ± 0.03 d 2.98 ± 0.17c 0.78 ± 0.06 e 3.87 ± 0.11 e 0.03 ± 0.01 d 0.39 ± 0.02c 8.33 ± 0.40c 1.87 ± 0.21 d 0.09 ± 0.02c 0.31 ± 0.03 a 0.48 ± 0.03c 3.98 ± 0.14c 6.73 ± 0.43c 15.06 ± 0.83c 1.01 ± 0.09 cd 0.67 ± 0.04c 0.45 ± 0.02 d 0.18 ± 0.01b 0.04 ± 0.01b 2.35 ± 0.17c

13.09 ± 0.22 e 3.87 ± 0.09 e 1.22 ± 0.12b 1.76 ± 0.09c 4.89 ± 0.33 d 24.83 ± 0.85 d 128.29 ± 3.07d 10.82 ± 0.76 ef 4.87 ± 0.33c 2.98 ± 0.18 d 30.38 ± 1.22 d 1.04 ± 0.09b 50.09 ± 2.74 d

67.09 ± 1.21 d 10.87 ± 0.23 e 2.29 ± 0.09 d 10.27 ± 0.24 d 9.28 ± 0.26 d 3.66 ± 0.19c 103.46 ± 2.22d

serum

0.02 e 0.09 d 0.03f 0.04f 0.00f 0.01 d 0.19 d 0.18 e 0.01 d 0.03b 0.07c 0.18 e 0.47 e 0.66 d 0.08 d 0.02 d 0.01 e 0.01c ± 0.12 d

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

(continued on next page)

0.11 2.04 0.38 0.76 0.01 0.10 3.40 1.46 0.04 0.22 0.44 1.22 3.38 6.78 0.99 0.23 0.10 0.09 nd 1.41

8.98 ± 0.22 g 1.88 ± 0.19 g 0.91 ± 0.14c 1.34 ± 0.09 d 3.98 ± 0.22 e 17.09 ± 0.86 e 58.55 ± 2.86 e 10.29 ± 0.82f 1.87 ± 0.38 d 0.76 ± 0.09 e 18.28 ± 0.90 e 0.21 ± 0.04 d 31.41 ± 2.33 e

19.28 ± 0.98 g 11.09 ± 0.28 d 2.89 ± 0.09c 3.09 ± 0.23 e 4.09 ± 0.33 e 1.02 ± 0.09 d 41.46 ± 2.00f

colon

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nd 0.47 0.04 nd nd nd 0.51 0.23 nd 0.07 0.21 0.34 0.85 1.36 0.23 nd nd nd nd 0.23

0.01c 0.04 d 0.02 h 0.27 g 0.48 e 0.03 e

± 0.03 e

± ± ± ± ± ±

± 0.21f ± 0.2 g

± 0.20 e ± 0.01 h

2.44 ± 0.09 i 0.36 ± 0.04 i 0.04 ± 0.01 d 0.47 ± 0.09 e 1.08 ± 0.05 h 4.39 ± 0.28 g 12.19 ± 0.97 g 4.09 ± 0.33 g 0.33 ± 0.01f nd 5.87 ± 0.49 g nd 9.99 ± 0.83f

± 0.09 g ± 0.04 e ± 0.69 h

± 0.28 i ± 0.19 g ± 0.09 e

0.87 ± 0.11 a 6.78 ± 0.23 a 2.19 ± 0.22 a 6.00 ± 0.80 a 0.12 ± 0.01 a 2.11 ± 0.11 a 18.07 ± 1.48 a 3.50 ± 0.47c 0.38 ± 0.05 a 0.35 ± 0.05 a 1.64 ± 0.23 a 7.65 ± 0.45 a 13.52 ± 1.25 a 31.59 ± 2.73 a 4.55 ± 0.60 a 2.02 ± 0.27 a 1.22 ± 0.12 a 0.36 ± 0.05 a 0.11 ± 0.01 a 8.26 ± 1.05 a

30.10 ± 1.09c 12.31 ± 0.87 a 1.86 ± 0.17 a 5.74 ± 0.23 a 18.57 ± 0.78 a 68.58 ± 3.14 a 233.99 ± 6.07 a 42.85 ± 0.78c 6.26 ± 0.23b 2.97 ± 0.11 d 43.55 ± 2.01b 0.56 ± 0.10c 96.19 ± 3.23b

90.79 ± 0.57a 26.78 ± 0.59 a 7.19 ± 0.13 a 19.03 ± 0.48 a 16.62 ± 0.87b 5.00 ± 0.29 a 165.41 ± 2.93a

mouth

after colon

3.01 2.09 1.22 nd 1.02 0.46 7.80

After meal

Before meal

0.81 ± 0.03 a 5.22 ± 0.18b 2.03 ± 0.22 a 5.01 ± 0.89b 0.09 ± 0.02b 0.82 ± 0.11b 13.98 ± 1.45b 5.21 ± 0.87 a 0.19 ± 0.02b 0.33 ± 0.02 a 0.79 ± 0.05b 5.03 ± 0.14b 11.55 ± 1.10b 25.53 ± 2.55b 4.01 ± 0.34 ab 1.88 ± 0.23b 0.89 ± 0.04c 0.33 ± 0.03 a 0.10 ± 0.01 a 7.21 ± 0.65b

40.28 ± 1.02 a 7.89 ± 0.45b 1.82 ± 0.23 a 3.02 ± 0.22b 11.48 ± 0.76b 64.49 ± 2.68b 207.18 ± 7.50c 80.27 ± 2.09 a 14.37 ± 0.98 a 6.02 ± 0.47 a 76.29 ± 1.28 a 2.87 ± 0.09 a 179.82 ± 4.91a

71.09 ± 1.09c 27.09 ± 0.88 a 6.09 ± 0.46b 16.09 ± 0.99b 18.04 ± 1.23 a 4.29 ± 0.17b 142.69 ± 4.82c

stomach

0.33 ± 0.03c 2.78 ± 0.12c 0.89 ± 0.03 d 4.02 ± 0.33 d 0.03 ± 0.01 d 0.37 ± 0.02c 8.42 ± 0.54c 1.99 ± 0.12 d 0.11 ± 0.01c 0.30 ± 0.02 a 0.44 ± 0.03c 3.05 ± 0.12 d 5.89 ± 0.30 d 14.31 ± 0.84c 1.22 ± 0.09c 0.55 ± 0.03c 0.39 ± 0.03 d 0.18 ± 0.02b 0.04 ± 0.01b 2.38 ± 0.18c

15.02 ± 0.98 d 4.88 ± 0.05 d 1.34 ± 0.09b 1.78 ± 0.11c 5.98 ± 0.23c 29.00 ± 1.46c 125.83 ± 5.83d 12.98 ± 0.98 d 4.37 ± 0.34c 3.22 ± 0.03c 38.47 ± 2.01c 1.21 ± 0.18b 60.25 ± 3.54c

55.28 ± 1.28 e 13.22 ± 0.98c 2.98 ± 0.22c 11.29 ± 0.78c 10.28 ± 0.88c 3.78 ± 0.23c 96.83 ± 4.37 e

serum

0.14 2.08 1.11 0.55 0.02 0.09 3.99 1.22 0.05 0.23 0.49 0.99 2.98 6.97 0.99 0.22 0.11 0.08 0.01 1.41

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 e 0.19 d 0.09c 0.04 g 0.00 e 0.01 d 0.36 d 0.09f 0.01 d 0.02b 0.02c 0.07 g 0.21f 0.57 d 0.07 d 0.02 d 0.02 e 0.01c 0.00c 0.12 d

9.91 ± 0.91f 2.09 ± 0.17 g 0.87 ± 0.18c 1.39 ± 0.22 d 3.28 ± 0.19f 17.54 ± 1.67 e 58.65 ± 3.62 e 11.09 ± 0.92 e 2.03 ± 0.11 d 0.46 ± 0.19f 16.28 ± 0.23f 0.19 ± 0.04 d 30.05 ± 1.49 e

22.47 ± 0.87f 9.09 ± 0.49f 2.33 ± 0.09 d 2.99 ± 0.22 e 3.22 ± 0.19f 1.01 ± 0.09 d 41.11 ± 1.95f

colon

nd 0.49 0.19 nd nd nd 0.68 0.29 nd 0.02 0.22 0.41 0.94 1.62 0.29 nd nd nd nd 0.29

0.02 d 0.01 d 0.02f 0.08 g 0.13 e 0.03 e

± 0.03 e

± ± ± ± ± ±

± 0.05 e ± 0.03 g

± 0.03 e ± 0.02 g

3.92 ± 0.33 h 1.11 ± 0.09 h 0.04 ± 0.01 d 0.39 ± 0.03 e 2.41 ± 0.19 g 7.87 ± 0.65f 20.86 ± 1.69f 3.98 ± 0.18 g 0.34 ± 0.02 e nd 5.88 ± 0.34 g nd 10.20 ± 0.54f

9.01 ± 0.59 h 1.94 ± 0.21 h 1.08 ± 0.18 e nd 0.67 ± 0.04 h 0.29 ± 0.02f 12.99 ± 1.04 g

after colon

Note: Pr-acid, protocatechuic acid; Ph-acid, p-hydroxy benzoic acid; Pc-acid, p-coumaric acid; THBA, total hydroxybenzoic acid; THCA, total hydroxycinnamic acid; TPC, total phenolic acids; TFA, total flavan-3-ols acids; CAT, (+)-catechin (CAT); EC, (−)-epicatechin; EGC, (−)-epigallocatechin; ECG, (−)-epicatechin gallate; EGCG, (−)-epigallocatechin gallate (EGCG). Different letters in the same line indicate significant differences (Duncan’s test: P < 0.05, performed by DPS software (version 7.55, China).

flavan-3-ol standards

hydroxycinnamic acids

White wine hydroxybenzoic acids

flavan-3-ol standards

hydroxycinnamic acids

Red wine hydroxybenzoic acids

Individual phenolic acids

Table 1 (continued)

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8

flavan-3-ol standards

hydroxycinnamic acids

White wine hydroxybenzoic acids

flavan-3-ol standards

hydroxycinnamic acids

Red wine hydroxybenzoic acids

Individual phenolic acids

gallic acid Pr-acid Ph-acid gentisic acid vanillic acid syringic acid THBA caffeic acid Pc-acid ferulic acid chlorogenic acid sinapic acid THCA TPC CAT EC EGC ECG EGCG TFA

gallic acid Pr-acid Ph-acid gentisic acid vanillic acid syringic acid THBA caffeic acid Pc-acid ferulic acid chlorogenic acid sinapic acid THCA TPC CAT EC EGC ECG EGCG TFA 0.87 ± 0.11 a 6.78 ± 0.23 a 2.19 ± 0.22 a 6.00 ± 0.80 a 0.12 ± 0.01 a 2.11 ± 0.11 a 18.07 ± 1.48 a 3.50 ± 0.47b 0.38 ± 0.05 a 0.35 ± 0.05 ab 1.64 ± 0.23 a 7.65 ± 0.45 a 13.52 ± 1.25 a 31.59 ± 2.73 a 4.55 ± 0.60 a 2.02 ± 0.27 a 1.22 ± 0.12 a 0.36 ± 0.05 a 0.11 ± 0.01 a 8.26 ± 1.05 a

90.79 ± 0.57b 26.78 ± 0.59c 7.19 ± 0.13 a 19.03 ± 0.48 a 16.62 ± 0.87b 5.00 ± 0.29 a 165.41 ± 2.93a 30.10 ± 1.09b 12.31 ± 0.87 a 1.86 ± 0.17 ab 5.74 ± 0.23 a 18.57 ± 0.78 a 68.58 ± 3.14 a 233.99 ± 6.07a 42.85 ± 0.78b 6.26 ± 0.23b 2.97 ± 0.11 d 43.55 ± 2.01c 0.56 ± 0.10f 96.19 ± 3.23c

Wine

Table 2 The individual phenolic acids in wine after in vitro GI digestion with moderate wine consumption.

0.82 ± 0.08 a 6.23 ± 0.17 a 2.11 ± 0.12ab 5.78 ± 0.79 a 0.11 ± 0.01 a 1.98 ± 0.14 a 17.03 ± 1.31 a 3.77 ± 0.67c 0.33 ± 0.09 a 0.34 ± 0.08 a 1.51 ± 0.32 a 7.22 ± 0.56 a 13.17 ± 1.72 a 30.20 ± 3.03 a 4.31 ± 0.43 a 2.09 ± 0.19 a 1.18 ± 0.15 a 0.41 ± 0.04 a 0.11 ± 0.02 a 8.10 ± 0.83 a

91.02 ± 0.29 a 27.02 ± 0.37 a 6.99 ± 0.09 a 17.09 ± 0.38 a 17.22 ± 0.66 a 4.87 ± 0.23 a 164.21 ± 2.02a 32.12 ± 0.98c 11.09 ± 0.99 a 1.88 ± 0.21 a 5.32 ± 0.37 a 17.09 ± 1.01a 67.50 ± 3.56 a 231.71 ± 5.58a 44.12 ± 1.02c 7.19 ± 0.48b 3.31 ± 0.28 d 45.09 ± 2.87b 0.99 ± 0.12c 100.7 ± 4.77b

Mouth

Before meal

0.88 ± 0.07 a 5.98 ± 0.34b 2.03 ± 0.23b 4.65 ± 0.33b 0.09 ± 0.02b 0.89 ± 0.09b 14.52 ± 1.08b 5.43 ± 0.34 a 0.19 ± 0.03c 0.39 ± 0.02 a 0.81 ± 0.11b 4.99 ± 0.41b 11.81 ± 0.91b 26.33 ± 1.99b 3.78 ± 0.27b 1.89 ± 0.11b 1.02 ± 0.09b 0.33 ± 0.02 a 0.10 ± 0.01 a 7.12 ± 0.50 a

92.09 ± 2.09 a 28.22 ± 0.98b 6.99 ± 0.45 ab 16.24 ± 0.89b 17.02 ± 0.67 ab 3.99 ± 0.22b 164.55 ± 5.3 a 40.38 ± 0.98 a 7.35 ± 0.34c 1.69 ± 0.18b 2.99 ± 0.09b 12.03 ± 0.99b 64.44 ± 2.58 a 228.99 ± 7.88 a 78.26 ± 1.29 a 14.02 ± 0.98 a 5.76 ± 0.29b 80.21 ± 3.09 a 2.65 ± 0.19b 180.9 ± 5.84 a

Stomach

0.31 ± 0.03b 2.76 ± 0.11c 0.99 ± 0.03 d 3.78 ± 0.23c 0.04 ± 0.01c 0.41 ± 0.02c 8.29 ± 0.43c 2.01 ± 0.18 d 0.11 ± 0.02 d 0.33 ± 0.02b 0.55 ± 0.05c 3.67 ± 0.14c 6.67 ± 0.41c 14.96 ± 0.84c 0.99 ± 0.03c 0.78 ± 0.09c 0.49 ± 0.04c 0.15 ± 0.01c 0.05 ± 0.01b 2.46 ± 0.18b

60.28 ± 1.22 d 12.38 ± 0.98 e 2.97 ± 0.34 d 9.84 ± 0.47 e 8.99 ± 0.76c 2.89 ± 0.23c 97.35 ± 4.00c 15.29 ± 0.98c 3.82 ± 0.05 e 1.29 ± 0.11c 1.55 ± 0.12 d 5.01 ± 0.34 d 26.96 ± 1.60c 124.31 ± 5.6c 13.29 ± 0.98c 5.33 ± 0.34c 3.20 ± 0.09c 38.27 ± 1.09 d 1.21 ± 0.19c 61.30 ± 2.69 d

Serum

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 d 0.22 d 0.21 e 0.09 d 0.01 d 0.01 d 0.55 d 0.18 e 0.01f 0.02c 0.03 d 0.09 d 0.33 d 0.88 d 0.1c 0.02 e 0.01 d 0.01 d 0.01c 0.15c

(continued on next page)

0.09 2.47 0.78 0.58 0.01 0.14 4.07 1.38 0.04 0.28 0.47 1.23 3.40 7.47 1.04 0.19 0.11 0.07 0.02 1.43

22.11 ± 1.29f 11.27 ± 0.78 e 3.08 ± 0.37c 3.67 ± 0.13f 5.02 ± 0.22 d 1.21 ± 0.09 d 46.36 ± 2.88d 10.29 ± 0.98d 2.01 ± 0.09f 1.10 ± 0.07 d 1.56 ± 0.15 d 4.89 ± 0.34 d 19.85 ± 1.63d 66.21 ± 4.51d 12.38 ± 0.56d 1.09 ± 0.08 e 0.87 ± 0.03 e 19.37 ± 0.77f 0.44 ± 0.09f 34.15 ± 1.53f

Colon

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nd 0.48 0.11 nd nd nd 0.59 0.26 nd 0.09 0.19 0.39 0.93 1.52 0.19 nd nd nd nd 0.19

0.02 0.02 0.04 0.11 0.16 0.02

d e e e e d

± 0.02 d

± ± ± ± ± ±

± 0.05f ± 0.03 g

± 0.03 e ± 0.02 g

0.82 ± 0.08 a 6.23 ± 0.17 a 2.11 ± 0.12ab 5.78 ± 0.79 a 0.11 ± 0.01 a 1.98 ± 0.14 a 17.03 ± 1.31 a 3.77 ± 0.67c 0.33 ± 0.09 a 0.34 ± 0.08 a 1.51 ± 0.32 a 7.22 ± 0.56 a 13.17 ± 1.72 a 30.20 ± 3.03 a 4.31 ± 0.43 a 2.09 ± 0.19 a 1.18 ± 0.15 a 0.41 ± 0.04 a 0.11 ± 0.02 a 8.10 ± 0.83 a

91.02 ± 0.29 a 27.02 ± 0.37 a 6.99 ± 0.09 a 17.09 ± 0.38 a 17.22 ± 0.66 a 4.87 ± 0.23 a 164.21 ± 2.02a 32.12 ± 0.98c 11.09 ± 0.99 a 1.88 ± 0.21 a 5.32 ± 0.37 a 17.09 ± 1.01a 67.50 ± 3.56 a 231.71 ± 5.58a 44.12 ± 1.02c 7.19 ± 0.48b 3.31 ± 0.28 d 45.09 ± 2.87b 0.99 ± 0.12c 100.7 ± 4.77b

Mouth

After colon

7.28 ± 0.38 h 2.09 ± 0.21 g 1.56 ± 0.14 e nd 1.98 ± 0.12f 0.48 ± 0.04f 13.39 ± 0.89f 4.92 ± 0.19 e 0.44 ± 0.09 h 0.03 ± 0.02f 0.33 ± 0.03f 2.01 ± 0.14f 7.73 ± 0.47f 21.12 ± 1.36f 3.99 ± 0.37 e 0.10 ± 0.02f 0.21 ± 0.02f 5.29 ± 0.66 h 0.04 ± 0.01 h 9.63 ± 1.08 h

After meal

Before meal

0.82 ± 0.07 a 6.04 ± 0.56 a 2.09 ± 0.18b 4.78 ± 0.34b 0.07 ± 0.02b 0.89 ± 0.07b 14.69 ± 1.24b 5.67 ± 0.34 a 0.22 ± 0.02b 0.39 ± 0.03 a 0.76 ± 0.04b 5.34 ± 0.43b 12.38 ± 0.86 a 27.07 ± 2.10b 3.98 ± 0.32b 1.89 ± 0.12b 1.03 ± 0.11b 0.38 ± 0.07 a 0.07 ± 0.02b 7.35 ± 0.64 a

89.01 ± 2.09c 30.12 ± 1.01 a 6.76 ± 0.45b 15.28 ± 0.87c 18.02 ± 0.59 a 4.21 ± 0.44b 163.4 ± 5.45 a 42.39 ± 1.95 a 8.33 ± 0.76b 1.99 ± 0.18 a 3.09 ± 0.29b 12.87 ± 0.19b 68.67 ± 3.37 a 232.07 ± 8.82 a 79.28 ± 1.46 a 14.98 ± 0.87 a 6.24 ± 0.45 a 68.29 ± 2.77b 3.01 ± 0.39 a 171.8 ± 5.94b

Stomach

0.34 ± 0.09b 2.98 ± 0.18c 0.97 ± 0.03 d 3.87 ± 0.19c 0.02 ± 0.01 d 0.38 ± 0.03c 8.56 ± 0.53c 2.19 ± 0.19c 0.12 ± 0.02 d 0.28 ± 0.03c 0.48 ± 0.03 d 3.43 ± 0.19c 6.50 ± 0.46c 15.06 ± 0.99c 1.08 ± 0.12c 0.57 ± 0.04 d 0.43 ± 0.02c 0.20 ± 0.02b 0.05 ± 0.01 2.33 ± 0.21b

64.29 ± 1.22 14.37 ± 0.98 d 2.79 ± 0.25 10.22 ± 0.65 d 9.77 ± 0.38c 2.98 ± 0.19c 104.42 ± 3.67b 14.92 ± 0.46c 5.28 ± 0.38 d 1.39 ± 0.09c 1.87 ± 0.11c 6.94 ± 0.45c 30.40 ± 1.49b 134.82 ± 5.16b 14.09 ± 0.67c 3.98 ± 0.34 d 3.04 ± 0.18c 29.37 ± 1.22 e 1.01 ± 0.03 d 51.49 ± 2.44 e

Serum

0.22 2.39 1.21 0.48 0.01 0.11 4.42 1.46 0.07 0.28 0.51 1.09 3.41 7.83 0.98 0.19 0.09 0.09 0.02 1.37

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03c 0.19 d 0.02c 0.03 d 0.01 d 0.01 d 0.29 d 0.09 e 0.02 e 0.02c 0.03c 0.09 d 0.25 d 0.54 d 0.09c 0.02 e 0.01 d 0.02 d 0.01c 0.15c

25.29 ± 0.98 e 12.98 ± 0.82 e 3.21 ± 0.23c 2.70 ± 0.19 g 2.98 ± 0.22 e 1.09 ± 0.09 e 48.25 ± 2.53d 11.02 ± 0.87d 2.01 ± 0.21f 1.01 ± 0.09 d 1.47 ± 0.12 d 3.99 ± 0.14 e 19.50 ± 1.43d 67.75 ± 3.96d 13.09 ± 0.57c 3.98 ± 0.29 d 0.99 ± 0.09 e 18.28 ± 1.2f 0.89 ± 0.05 e 37.23 ± 2.2f

Colon

0.03 0.53 0.18 nd nd nd 0.74 0.33 nd 0.04 0.23 0.34 0.94 1.68 0.18 nd nd nd nd 0.18

0.01 0.01 0.02 0.06 0.12 0.02

e e e e e d

± 0.02 d

± ± ± ± ± ±

± 0.06 e ± 0.02f

± 0.01 e ± 0.03 e ± 0.02f

10.02 ± 0.87 g 3.09 ± 0.33f 1.69 ± 0.09 e nd 1.11 ± 0.12 g 0.49 ± 0.03f 16.40 ± 1.44e 5.22 ± 0.23 e 1.02 ± 0.09 g 0.11 ± 0.03 e 0.48 ± 0.03 e 1.98 ± 0.12f 8.81 ± 0.50 e 25.21 ± 1.94e 4.23 ± 0.19 e 1.02 ± 0.09 e 0.23 ± 0.02f 6.37 ± 0.12 g 0.10 ± 0.02 g 11.95 ± 0.44 g

After colon

Note: Pr-acid, protocatechuic acid; Ph-acid, p-hydroxy benzoic acid; Pc-acid, p-coumaric acid; THBA, total hydroxybenzoic acid; THCA, total hydroxycinnamic acid; TPC, total phenolic acids; TFA, total flavan-3-ols acids; CAT, (+)-catechin (CAT); EC, (−)-epicatechin; EGC, (−)-epigallocatechin; ECG, (−)-epicatechin gallate; EGCG, (−)-epigallocatechin gallate (EGCG). Different letters in the same line indicate significant differences (Duncan’s test: P < 0.05, performed by DPS software (version 7.55, China).

flavan-3-ol standards

hydroxycinnamic acids

White wine hydroxybenzoic acids

flavan-3-ol standards

hydroxycinnamic acids

Red wine hydroxybenzoic acids

Individual phenolic acids

Table 2 (continued)

X. Sun, et al.

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3.1.2. Individual phenolic acids The polyphenol composition after in vitro GI digestion is shown in Tables 1–3. The contents of eleven individual phenolic acids were monitored. Most phenolic acids showed trend similar to that of TP. For example, gallic acid is the main hydroxybenzoic acid in red wine. No significant difference were observed for most phenolic acids with wine in the mouth step. After stomach digestion, the content of gallic acid in the low and moderate drinking amounts groups significantly decreased. This was consistent with the report by Lingua et al. (2018) on red wine but unlike blueberry wine (Celep et al., 2015), mainly due to the difference in raw materials. Considering the drinking amount, the gallic acid content increased with increasing drinking amount in the stomach digestion step. However, the gallic acid content in the serum was only two thirds that in the wines and decreased with increasing drinking amount; in contrast, the gallic acid content in the colon-available fraction increased with increasing drinking amount, which might due to the same reason with TP that the total gallic acid amount increased, but transportation rate was constant, and resulting in a lower content in serum and a higher content in colon. As one of the main hydroxycinnamic acids in wine, caffeic acid showed a different trend than other individual phenolic acids. After stomach digestion, the caffeic acid content significantly increased for both red and white wines. However, the chlorogenic acid content also showed a different trend, as it decreased a much higher rate (nearly 50%) than other phenolic acids after stomach digestion, which might be due to hydrolysis to caffeic acid and quinic acid (Gumienna et al., 2011), which was mainly due to the exist of digestive enzyme; however, it still need further verification in the future. Furthermore, after colon fermentation, gentisic acid in red wine was not detected in some groups, and gallic acid, gentisic acid, vanillic acid, syringic acid and caffeic acid were not detected in any of the white wine groups. In a previous report (Lingua et al., 2018), only 14 and 13 out of 35 phenolic compounds were detected in the serumavailable fraction and colon-available fraction, respectively, (this study did not contain a colon digestion process). In another study (Gumienna et al., 2011), all the phenolic compounds were detected in the small intestine and after colon fermentation. This discrepancy might be mainly due to differences in experimental materials and the in vitro GI digestion conditions. Overall, the total hydroxybenzoic acid and the total phenolic acid contents showed similar trends; they increased in the stomach digestion and decreased in the serum fraction with increasing wine drinking amount for red wine.

3.2. Influence of different drinking amounts and drinking patterns on the antioxidant capacity The positive effects of wine on human health are mainly attributed to the antioxidant substances that they contain and their associated antioxidant activities (Ma et al., 2017). Hence, the antioxidant activities under different drinking amounts and drinking patterns were evaluated in this study (Fig. 2), and based on previous reports (Ma et al., 2014; Sun et al., 2017), four methods were used. No significant difference were observed with wine in the mouth step. After stomach digestion, the four methods all showed a higher antioxidant capacity in red wines than in white wines except the DPPH value in the drinking after a meal group (Fig. 2A1–A2, B1–B2, C1–C2, D1–D2), which might due to the differences in the methods (Ma et al., 2019a). The white and red wines showed opposite trends; the antioxidant capacity in stomach digestion was significantly lower for white wines than in red wines (Fig. 2A3–A4, B3–B4, C3–C4, D3–D4). This result was consistent with the TP value (Fig. 1A1–A4), and previous reports have also demonstrated a strong correlation between antioxidant capacity and TP values (Celep et al., 2015; Lingua et al., 2018; Ma et al., 2014). In the small intestine digestion, the antioxidant capacities in the serum-available fraction and in the colon-available fraction sharply dropped, with average values of 40–60% and 10–20% for the wine antioxidant capacities, respectively. After colon fermentation, the antioxidant capacity continued to decrease, which was consistent with the polyphenol results. Considering the drinking amount, the antioxidant capacity in serum decreased in both red and white wine with increasing wine consumption, while the antioxidant capacity in the colon-available fraction increased with increasing wine consumption. Under the two different drinking patterns, a higher antioxidant capacity in the serum-available fraction and a lower antioxidant capacity in the colon-available fraction were observed in the drinking after a meal group under binge drinking conditions, which was due to the difference in the polyphenol contents (Fig. 1). Under binge drinking conditions, a higher serum antioxidant capacity was observed in the drinking after a meal group, which was also in line with the polyphenol values (Fig. 1). 3.3. Influence of different drinking amounts and drinking patterns on the inhibitory effects of α-amylase and α-glucosidase 3.3.1. α-Amylase inhibition assay By controlling starch breakdown and intestinal glucose absorption, foods rich in polyphenols could offer an attractive strategy for managing postprandial hyperglycemia for type two diabetes (BurgosEdwards, Jiménez-Aspee, Thomas-Valdés, Schmeda-Hirschmann, & Theoduloz, 2017; Kwon, Apostolidis, & Shetty, 2008; Ma et al., 2015). Hence, the inhibitory effects of α-amylase and α-glucosidase under different drinking amounts and drinking patterns after in vitro GI digestion were investigated (Fig. 3). Red wine and white wine showed inhibition ratios for α-amylase of 32.18% and a 7.67%, respectively (Fig. 3A1–A4), which were consistent with a previous report (Kwon et al., 2008). No significant difference were observed with wine in the mouth step. After stomach digestion, the α-amylase inhibition dropped for both red and white wines (approximately 30% and 5%, respectively). The α-amylase inhibition activities of the serum-available fraction and in the colon-available fraction and after colon fermentation continued to decrease, indicating that α-amylase inhibition was associated with wine polyphenols. Considering the drinking amount, the low and moderate drinking amounts groups showed similar trends in αamylase inhibition. In addition, under binge drinking conditions, the αamylase inhibition capacity in the serum decreased, and the α-amylase inhibition capacity in the colon-available fraction increased (for example, by 6.87% and 8.98% for red wine in the drinking before a meal group). Of the two different drinking patterns, drinking after a meal resulted in higher α-amylase inhibition than drinking before a meal in the stomach and serum fractions, which was also due to the difference

3.1.3. Individual flavan-3-ols Five individual flavan-3-ols were analyzed in this study (Tables 1–3). No significant difference were observed for most individual flavan-3-ols with wine in the mouth step. The contents of most of the five individual flavan-3-ols significantly increased after stomach digestion in red wines, while they tended to decrease with white wines. This could explain the changes observed in the TFA contents (Fig. 1C1–C4). This effect was mainly due to the hydrolysis of the proanthocyanidins to their monomeric units (Fernández & Labra, 2013). However, the contents of all five individual flavan-3-ols sharply dropped in the serum-available fractions and colon-available fractions, which is similar to what was observed for the TFA content. In summary, the individual phenolic compounds showed different stabilities in this study, and these results are similar to those reported for wines, fruit wines (Celep et al., 2015; Gumienna et al., 2011; Lingua et al., 2018; Yang et al., 2018) and some fruits (Corrêa et al., 2017; Fernández & Labra, 2013). Throughout the digestive process, foods are physically and chemically broken down due to churning motions, hydrolytic enzymes and chemical reactions. The complex conditions in the digestive tract, such as various pH conditions and digestive enzymes, will all influence the stability of the phenolic compounds and consequently change their phenolic profiles (Lingua et al., 2018). 10

11

flavan-3-ol standards

hydroxycinnamic acids

White wine hydroxybenzoic acids

flavan-3-ol standards

hydroxycinnamic acids

Red wine hydroxybenzoic acids

Individual phenolic acids

gallic acid Pr-acid Ph-acid gentisic acid vanillic acid syringic acid THBA caffeic acid Pc-acid ferulic acid chlorogenic acid sinapic acid THCA TPC CAT EC EGC ECG EGCG TFA

gallic acid Pr-acid Ph-acid gentisic acid vanillic acid syringic acid THBA caffeic acid Pc-acid ferulic acid chlorogenic acid sinapic acid THCA TPC CAT EC EGC ECG EGCG TFA 0.87 ± 0.11 a 6.78 ± 0.23 a 2.19 ± 0.22 a 6.00 ± 0.80 a 0.12 ± 0.01 a 2.11 ± 0.11 a 18.07 ± 1.48 a 3.50 ± 0.47b 0.38 ± 0.05 a 0.35 ± 0.05b 1.64 ± 0.23 a 7.65 ± 0.45 a 13.52 ± 1.25 a 31.59 ± 2.73 a 4.55 ± 0.60 a 2.02 ± 0.27 a 1.22 ± 0.12 a 0.36 ± 0.05 a 0.11 ± 0.01 a 8.26 ± 1.05 a

90.79 ± 0.57b 26.78 ± 0.59b 7.19 ± 0.13 a 19.03 ± 0.48 a 16.62 ± 0.87 a 5.00 ± 0.29 a 165.41 ± 2.93b 30.10 ± 1.09b 12.31 ± 0.87 a 1.86 ± 0.17c 5.74 ± 0.23 a 18.57 ± 0.78 a 68.58 ± 3.14 a 233.99 ± 6.07b 42.85 ± 0.78b 6.26 ± 0.23b 2.97 ± 0.11c 43.55 ± 2.01b 0.56 ± 0.10 d 96.19 ± 3.23b

Wine

Table 3 The individual phenolic acids in wine after in vitro GI digestion under binge drinking conditions.

0.82 ± 0.08 a 6.23 ± 0.17 a 2.11 ± 0.12ab 5.78 ± 0.79 a 0.11 ± 0.01 a 1.98 ± 0.14 a 17.03 ± 1.31 a 3.77 ± 0.67c 0.33 ± 0.09 a 0.34 ± 0.08 a 1.51 ± 0.32 a 7.22 ± 0.56 a 13.17 ± 1.72 a 30.20 ± 3.03 a 4.31 ± 0.43 a 2.09 ± 0.19 a 1.18 ± 0.15 a 0.41 ± 0.04 a 0.11 ± 0.02 a 8.10 ± 0.83 a

91.02 ± 0.29 a 27.02 ± 0.37 a 6.99 ± 0.09 a 17.09 ± 0.38 a 17.22 ± 0.66 a 4.87 ± 0.23 a 164.21 ± 2.02a 32.12 ± 0.98c 11.09 ± 0.99 a 1.88 ± 0.21 a 5.32 ± 0.37 a 17.09 ± 1.01a 67.50 ± 3.56 a 231.71 ± 5.58a 44.12 ± 1.02c 7.19 ± 0.48b 3.31 ± 0.28 d 45.09 ± 2.87b 0.99 ± 0.12c 100.7 ± 4.77b

Mouth

Before meal

0.86 ± 0.04 a 6.18 ± 0.45 a 2.21 ± 0.12 a 4.88 ± 0.34b 0.11 ± 0.02 a 1.02 ± 0.09b 15.26 ± 1.06b 5.87 ± 0.45 a 0.17 ± 0.02b 0.41 ± 0.03 a 0.77 ± 0.10c 6.02 ± 0.05b 13.24 ± 0.65 a 28.50 ± 1.71b 3.90 ± 0.14c 2.01 ± 0.09 a 0.89 ± 0.09b 0.4 ± 0.02 a 0.08 ± 0.02 ab 7.28 ± 0.36 ab

110.23 ± 3.98a 32.09 ± 1.28 a 7.21 ± 0.45 a 17.02 ± 1.67b 16.87 ± 0.87 a 4.32 ± 0.44b 187.74 ± 8.69 a 42.09 ± 2.09 a 8.01 ± 0.67b 1.91 ± 0.23b 3.01 ± 0.09b 12.78 ± 0.56b 67.80 ± 3.64 a 255.54 ± 2.33 a 80.28 ± 1.98 a 15.22 ± 0.77 a 5.58 ± 0.34 a 75.28 ± 1.29 a 2.56 ± 0.22 a 178.92 ± 4.60 a

Stomach

0.33 ± 0.03b 3.04 ± 0.12b 1.02 ± 0.11b 4.01 ± 0.34c 0.04 ± 0.02b 0.44 ± 0.02c 8.88 ± 0.64c 1.99 ± 0.09c 0.09 ± 0.03c 0.37 ± 0.03b 0.49 ± 0.04 e 4.01 ± 0.22c 6.95 ± 0.41b 15.83 ± 1.05c 1.03 ± 0.09 d 0.56 ± 0.05c 0.58 ± 0.04c 0.19 ± 0.02b 0.03 ± 0.01c 2.39 ± 0.21c

55.98 ± 2.09 d 13.99 ± 1.48 d 3.09 ± 0.34b 8.29 ± 0.56 d 8.77 ± 0.48b 2.98 ± 0.29 d 93.10 ± 5.24 d 13.87 ± 0.98 d 4.28 ± 0.39 d 1.38 ± 0.12 d 1.56 ± 0.09 e 4.99 ± 0.34 d 26.08 ± 1.92b 119.18 ± 7.16d 10.98 ± 0.87 d 4.29 ± 0.43c 3.01 ± 0.09 bc 33.47 ± 1.02c 1.32 ± 0.04b 53.07 ± 2.45c

Serum

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 d 0.19c 0.09b 0.02 d 0.01b 0.01 d 0.34 d 0.09 d 0.01 e 0.02c 0.03 e 0.11 d 0.26c 0.60 d 0.09 d 0.02 d 0.02 d 0.01c 0.01c 0.15 d

(continued on next page)

0.11 2.55 1.23 0.78 0.02 0.11 4.80 1.49 0.03 0.22 0.43 1.34 3.51 8.31 1.21 0.22 0.09 0.11 0.02 1.65

38.21 ± 2.10e 12.38 ± 1.20d 2.81 ± 0.29c 4.99 ± 0.39 e 4.98 ± 0.22 d 1.28 ± 0.19 e 64.65 ± 4.39 e 14.88 ± 0.89d 3.02 ± 0.29 e 1.27 ± 0.09 1.78 ± 0.12 d 5.01 ± 0.33 d 25.96 ± 1.72b 90.61 ± 6.11 e 14.98 ± 0.19c 1.23 ± 0.15 d 1.46 ± 0.22 d 28.09 ± 1.09d 0.98 ± 0.11c 46.74 ± 1.76d

Colon

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nd 0.39 0.24 nd nd nd 0.63 0.31 nd 0.10 0.18 0.37 0.96 1.59 0.19 nd nd nd nd 0.19

0.02 d 0.02f 0.03 e 0.09 e 0.14f 0.03 e

± 0.03 e

± ± ± ± ± ±

± 0.05 e ± 0.02 e

± 0.02 d ± 0.03c

0.82 ± 0.08 a 6.23 ± 0.17 a 2.11 ± 0.12ab 5.78 ± 0.79 a 0.11 ± 0.01 a 1.98 ± 0.14 a 17.03 ± 1.31 a 3.77 ± 0.67c 0.33 ± 0.09 a 0.34 ± 0.08 a 1.51 ± 0.32 a 7.22 ± 0.56 a 13.17 ± 1.72 a 30.20 ± 3.03 a 4.31 ± 0.43 a 2.09 ± 0.19 a 1.18 ± 0.15 a 0.41 ± 0.04 a 0.11 ± 0.02 a 8.10 ± 0.83 a

91.02 ± 0.29 a 27.02 ± 0.37 a 6.99 ± 0.09 a 17.09 ± 0.38 a 17.22 ± 0.66 a 4.87 ± 0.23 a 164.21 ± 2.02a 32.12 ± 0.98c 11.09 ± 0.99 a 1.88 ± 0.21 a 5.32 ± 0.37 a 17.09 ± 1.01a 67.50 ± 3.56 a 231.71 ± 5.58a 44.12 ± 1.02c 7.19 ± 0.48b 3.31 ± 0.28 d 45.09 ± 2.87b 0.99 ± 0.12c 100.7 ± 4.77b

Mouth

After colon

12.03 ± 0.38 g 3.28 ± 0.29 e 1.88 ± 0.19 d 0.47 ± 0.03 g 1.23 ± 0.11f 0.99 ± 0.02f 19.88 ± 1.02 g 6.98 ± 0.23 e 1.28 ± 0.18 g 0.10 ± 0.02 e 0.49 ± 0.03 g 1.47 ± 0.11 g 10.32 ± 0.57c 30.20 ± 1.59 g 5.04 ± 0.19 e 0.05 ± 0.01 e 0.47 ± 0.03 e 10.29 ± 0.78e 0.18 ± 0.02 e 16.03 ± 1.03e

After meal

Before meal

0.89 ± 0.09 a 6.65 ± 0.38 a 2.18 ± 0.29 a 5.28 ± 0.23 ab 0.10 ± 0.02 a 1.09 ± 0.08b 16.19 ± 1.09 ab 6.22 ± 0.44 a 0.19 ± 0.03b 0.38 ± 0.03 ab 0.83 ± 0.07b 6.33 ± 0.05b 13.95 ± 0.62 a 30.14 ± 1.71 a 4.02 ± 0.12b 1.09 ± 0.09b 1.21 ± 0.09 a 0.35 ± 0.04 a 0.11 ± 0.02 a 6.78 ± 0.36b

118.22 ± 4.78 a 30.19 ± 2.09 a 7.36 ± 0.45 a 18.22 ± 0.98 ab 17.02 ± 1.02 a 4.49 ± 0.34b 195.50 ± 9.66 a 41.98 ± 3.49 a 8.20 ± 0.87b 2.08 ± 0.33 a 3.11 ± 0.28b 13.67 ± 0.98b 69.04 ± 5.95 a 264.54 ± 15.6a 83.09 ± 2.39 a 16.28 ± 1.87 a 6.37 ± 0.79 a 77.38 ± 2.98 a 3.08 ± 0.38 a 186.20 ± 8.41 a

Stomach

0.38 ± 0.11b 3.04 ± 0.19b 1.21 ± 0.09b 4.22 ± 0.45c 0.03 ± 0.01b 0.39 ± 0.02c 9.27 ± 0.87c 2.03 ± 0.12c 0.13 ± 0.02 bc 0.37 ± 0.02b 0.51 ± 0.03 d 3.98 ± 0.13c 7.02 ± 0.33b 16.29 ± 1.20c 1.11 ± 0.09 d 0.47 ± 0.02c 0.55 ± 0.02c 0.17 ± 0.01b 0.06 ± 0.02b 2.36 ± 0.16c

68.49 ± 2.39c 14.09 ± 0.98 cd 3.23 ± 0.39b 9.02 ± 0.89c 9.87 ± 0.77b 3.29 ± 0.34c 107.99 ± 5.76c 16.76 ± 1.22c 4.90 ± 0.45c 1.29 ± 0.09 d 1.67 ± 0.22 de 6.99 ± 0.39c 31.61 ± 2.37b 139.60 ± 8.13c 9.98 ± 0.33 d 4.23 ± 0.41c 3.33 ± 0.19b 33.27 ± 2.33c 0.99 ± 0.05c 51.80 ± 3.31c

Serum

0.24 2.78 1.45 0.49 0.02 0.10 5.08 1.57 0.06 0.21 0.56 1.24 3.64 8.72 1.02 0.24 0.13 0.11 0.03 1.53

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.02c 0.19c 0.22b 0.03 d 0.01b 0.02 d 0.49 d 0.11 d 0.02 d 0.02c 0.03 d 0.09 d 0.27c 0.76 d 0.09 d 0.02 d 0.02 d 0.03c 0.01c 0.17 d

18.09 ± 0.98f 15.29 ± 1.20c 3.29 ± 0.23b 5.38 ± 0.43 e 5.39 ± 0.30c 1.01 ± 0.09 ef 48.45 ± 3.23f 13.87 ± 0.98d 3.09 ± 0.29 e 1.22 ± 0.09 d 2.01 ± 0.14c 4.51 ± 0.15 e 24.70 ± 1.65b 73.15 ± 4.88f 15.28 ± 0.18c 4.99 ± 0.19c 1.27 ± 0.11 d 30.28 ± 2.09c 1.23 ± 0.13b 53.05 ± 2.70c

Colon

0.09 0.48 0.25 nd nd nd 0.82 0.29 nd 0.06 0.94 0.43 1.72 2.54 0.15 nd nd nd nd 0.19

0.02d 0.05b 0.03 e 0.11 d 0.17 e 0.03 e

± 0.03 e

± ± ± ± ± ±

± 0.06 e ± 0.01 e

± 0.02 d ± 0.02 d ± 0.02c

7.49 ± 0.34 h 3.09 ± 0.19 e 1.98 ± 0.11 d 1.02 ± 0.09f 1.87 ± 0.11 e 0.46 ± 0.03 g 15.91 ± 0.87 h 4.98 ± 0.19f 1.98 ± 0.11f 0.08 ± 0.02 e 0.89 ± 0.19f 2.04 ± 0.12f 9.97 ± 0.63c 25.88 ± 1.50 g 3.98 ± 0.03f 1.23 ± 0.09 d 0.57 ± 0.09 e 11.20 ± 0.98e 0.19 ± 0.02 e 17.17 ± 1.21e

After colon

Note: Pr-acid, protocatechuic acid; Ph-acid, p-hydroxy benzoic acid; Pc-acid, p-coumaric acid; THBA, total hydroxybenzoic acid; THCA, total hydroxycinnamic acid; TPC, total phenolic acids; TFA, total flavan-3-ols acids; CAT, (+)-catechin (CAT); EC, (−)-epicatechin; EGC, (−)-epigallocatechin; ECG, (−)-epicatechin gallate; EGCG, (−)-epigallocatechin gallate (EGCG). Different letters in the same line indicate significant differences (Duncan’s test: P < 0.05, performed by DPS software (version 7.55, China).

flavan-3-ol standards

hydroxycinnamic acids

White wine hydroxybenzoic acids

flavan-3-ol standards

hydroxycinnamic acids

Red wine hydroxybenzoic acids

Individual phenolic acids

Table 3 (continued)

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Fig. 2. The antioxidant capacity of wine after in vitro GI digestion. (A1) DPPH of red wine before a meal; (A2) DPPH of red wine after a meal; (A3) DPPH of white wine before a meal; (A4) DPPH of white wine after a meal; (B1) ABTS of red wine before a meal; (B2) ABTS of red wine after a meal; (B3) ABTS of white wine before a meal; (B4) ABTS of white wine after a meal; (C1) ORAC of red wine before a meal; (C2) ORAC of red wine after a meal; (C3) ORAC of white wine before a meal; (C4) ORAC of white wine after a meal; (D1) FRAP of red wine before a meal; (D2) FRAP of red wine after a meal; (D3) FRAP of white wine before a meal; and (D4) FRAP of white meal after wine. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

in the polyphenol contents (Fig. 1).

Enteroccocus and Entrobacteriaceae, was investigated (Fig. 4). First, under in vitro colonic digestion, the survival rates of all four groups of bacteria were quite high but were lower than in the inoculum. After the colon digestion process, the concentrations of the tested bacteria were found to be between 104 and 107 cfu/ml, which was much higher than those reported previously (Gumienna et al., 2011). In addition, a clinical study by Queipo-Ortuño et al. (2012) reported that the consumption of red wine significantly increased the number of gut microorganisms, including Enterococcus and Bifidobacterium, and the difference might be due to the experimental method (in vitro compared to in vivo). More specifically, Bifidobacterium and Entrobacteriaceae were significantly more inhibited than Lactobacillus and Enteroccocus. Red wine showed a higher inhibitory effect on all four microorganisms than did white wine, mainly due to the differences in their polyphenol contents. Considering the drinking amount, the inhibition capacity of red wine, especially for Bifidobacterium and Enteroccocus, decreased with increasing drinking amount, while no significant difference was observed in white wines. Under the two different drinking patterns, drinking after a meal resulted in higher inhibition of Lactobacillus and Enteroccocus than drinking before a meal for both red and white wine, while no significant differences were observed for Bifidobacterium and Entrobacteriaceae. From the above, the drinking amount and drinking patterns of wine showed a significant influence on the digestive characteristics and bioaccessibility of wine polyphenols and the biological activity of wine polyphenols, such as their antioxidant capacity, the inhibitory effects of α-amylase and α-glucosidase and the effects on gut microbiota. However, all these results were observed under in vitro GI digestion. Although there are an increasing number of studies using in vitro digestion models to investigate the gastrointestinal behavior of foods (Alminger et al., 2014; Minekus et al., 2014), it is obvious that the in vitro simulation method does not account for every step of GI digestion, and most importantly, it does not fully mimic the active transportation

3.3.2. α-Glucosidase inhibition assay Red wine and white wine both showed much higher inhibition ratios for α-glucosidase (87.18% and 26.16%, respectively) than for αamylase (Fig. 3B1–B4), which was in accordance with the report by Kwon et al. (2008) on wines. Ma et al. (2019b) reported a similar result for polyphenol extracts from kiwifruit and their products Sphallerocarpus gracilis stems and leaves. No significant difference were observed with wine in the mouth step. After stomach digestion, the αglucosidase inhibition capacity was still very high and even increased in red wines. In the serum-available fraction, the α-glucosidase inhibition capacity was approximately half of that in the stomach, while in the colon-available fraction and after colon fermentation, the capacity was lower. Considering the drinking amount, the α-glucosidase inhibition capacity decreased with increasing drinking amount in the serumavailable fraction and increased in the colon-available fraction, which was in line with the changing trend with TP. In the binge drinking before a meal group, the inhibition capacity in the colon-available fraction (37.81%) was higher than in the serum-available fraction (31.87%), which was also resulted from the TP changing. No similar trends were observed in the two different drinking patterns. 3.4. Influence of different drinking amounts and drinking patterns on selected intestinal microbiota After stomach digestion, food stuffs are transported to the small intestine, and then after being absorbed into the serum, they are transferred to the colon. Previous reports have shown that polyphenols can influence the microorganism ecosystem (Celep et al., 2015; Gumienna et al., 2011; Moreno-Indias et al., 2016; Shi et al., 2016; Queipo-Ortuño et al., 2012). Hence, the influence of wine polyphenols on four important intestinal microbes, Bifidobacterium, Lactobacillus, 13

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Fig. 3. The inhibitory effects of α-amylase and α-glucosidase of wine after in vitro GI digestion. (A1) α-amylase of red wine before a meal; (A2) α-amylase of red wine after a meal; (A3) α-amylase of white wine before a meal; (A4) α-amylase of white wine after a meal; (B1) α-glucosidase of red wine before a meal; (B2) α-glucosidase of red wine after a meal; (B3) α-glucosidase of white wine before a meal; and (B4) α-glucosidase of white wine after a meal. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

processes. For example, in this study, the effects on gut microorganisms were not the same as those observed in a clinical study (Queipo-Ortuño et al., 2012). The in vitro results in this study are a prediction and reference for human nutritional studies. Hence, future studies in vivo and in the clinic are still needed. From this study, it can be observed that although wine is considered to be very good for humans, wine must be consumed in moderation. As the drinking amount increases, the bioaccessibility of most polyphenols decreases, indicating that drinking larger volumes of wine does not result in increased polyphenol bioaccessibility. Additionally, the relevant biological activities did not increase as much as the drinking amount increased. However, the alcohol absorption did increase as the drinking amount increased, which can cause many illnesses, such as different types of cancers (Garaycoechea et al., 2018), and this may occur in a dose-dependent manner. As shown by the two different drinking pattern groups, drinking after a meal showed significantly better results than drinking before a meal in most

of the tests based on the bioaccessibility of polyphenols. Boban et al. (2016) also reported that drinking wine after meals resulted in more beneficial effects. Hence, there needs to be increased publicity and education to correct the false understandings of consumers regarding binge wine consumption, especially in China, due to the “GANBEI” custom in social situations. 4. Conclusions In conclusion, under in vitro GI digestion, a good release of wine polyphenols was observed during stomach digestion, while the release rate in the “serum-available” fraction, “colon-available” fraction, and after colon fraction decreased. The relevant biological activities showed trends similar to those of the polyphenol contents. Red wine showed a higher biological activity than white wine, but white wine had a better bioaccessibility than red wine, especially under binge drinking 14

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Fig. 4. Changes in selected intestinal microbes after in vitro GI digestion. (A) Bifidobacterium; (B) Lactobacillus; (C) Enteroccocus; and (D) Entrobacteriaceae.

conditions, mainly due to the differences in the polyphenol contents in red and white wines, as red wine always show polyphenol contents several times higher than those in white wines. Considering the drinking amount, the bioaccessibility of most polyphenols decreases with increasing drinking amount, while drinking after a meal showed significantly better results than drinking before a meal in most of the tests of the bioaccessibility of polyphenols and biological activities. In order to let wine polyphenols play its functional for human health, wine consumption must be treated the same as the consumption of other alcoholic beverages, and thus the consumption amount must be controlled.

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