Assessing the Effects of Ginger Extract on Polyphenol Profiles and The

nutrients Article Assessing the Effects of Ginger Extract on Polyphenol Profiles and the Subsequent Impact on the Fecal Microbiota by Simulating Digestion and Fermentation In Vitro Jing Wang 1,2,3,4, Yong Chen 3,5 , Xiaosong Hu 2, Fengqin Feng 1,3, Luyun Cai 1,3,4 and Fang Chen 2,* 1 Ningbo Research Institute, Zhejiang University, Ningbo 310027, China; [email protected] (J.W.); [email protected] (F.F.); [email protected] (L.C.) 2 College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China; [email protected] 3 College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310027, China; [email protected] 4 School of Biological and Chemical Engineering, NingboTech University, Ningbo 310027, China 5 College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China * Correspondence: [email protected]; Tel./Fax: +86-10-62737645-18 Received: 27 September 2020; Accepted: 16 October 2020; Published: 19 October 2020 Abstract: The beneficial effects of ginger polyphenols have been extensively reported. However, their metabolic characteristics and health effects on gut microbiota are poor understood. The purpose of this study was to investigate the digestion stability of ginger polyphenols and their prebiotic effects on gut microbiota by simulating digestion and fermentation in vitro. Following simulated digestion in vitro, 85% of the polyphenols were still detectable, and the main polyphenol constituents identified in ginger extract are 6-, 8-, and 10-gingerols and 6-shogaol in the digestive fluids. After batch fermentation, the changes in microbial populations were measured by 16S rRNA gene Illumina MiSeq sequencing. In mixed-culture fermentation with fecal inoculate, digested ginger extract (GE) significantly modulated the fecal microbiota structure and promoted the growth of some beneficial bacterial populations, such as Bifidobacterium and Enterococcus. Furthermore, incubation with GE could elevate the levels of short-chain fatty acids (SCFAs) accompanied by a decrease in the pH value. Additionally, the quantitative PCR results showed that 6-gingerol (6G), as the main polyphenol in GE, increased the abundance of Bifidobacterium significantly. Therefore, 6G is expected to be a potential prebiotic that improves human health by promoting gut health. Keywords: in vitro fermentation; ginger extract; gut microbiota; 6-gingerol; short-chain fatty acids 1. Introduction Ginger is the rhizome of Zingiber officinale Roscoe (Zingiberaceae) and is commonly consumed as spice or flavoring agent. Nowadays, ginger is widely used in making beverages and foods all over the world [1]. As a medicinal and edible plant, ginger has strong pharmacological activity and has been used as a Chinese herbal medicine for more than 2500 years. In recent years, the health-related applications of ginger have been widely studied, especially in various diseases, such as cancer [2], nausea and vomiting [3,4], gastrointestinal disease [5], osteoarthritis [6], and metabolic syndromes [7]. The nutritional and pharmacological activities of ginger mainly come from its bioactive polyphenols, especially the pungent principles gingerols and shogaols [8]. However, just a few studies have Nutrients 2020, 12, 3194; doi:10.3390/nu12103194 www.mdpi.com/journal/nutrients Nutrients 2020, 12, 3194 2 of 13 examined the digestion stability, bioavailability and metabolic characteristics of ginger and its bioactive phytochemicals [9,10]. The phenolics in ginger extract (GE) showed lower solubility in different pH buffers and remained stable in simulated gastric and intestinal fluids, indicating the suitability of these compounds during oral administration [11]. Additionally, similar stability of 6-shogaol, one of the major polyphenol compounds in ginger, was observed in simulated gastrointestinal fluids within 2 h [12]. As far as we know, most phenolic compounds are difficult to absorb in the small intestine, and then enter the colon to interact with the colonized gut microbiota [8]. The presence of these compounds in the colon leads to complex interactions between polyphenolic compounds and gut microbiota, including modulation of the gut microbiota by polyphenols and biotransformation of polyphenols by the gut microbiota [13]. Our previous research has shown that ginger can play an anti-obesity role by modulating the composition of gut microbiota [14]. However, the interactions between ginger polyphenols and the gut microbiota are still unclear. In vitro simulated digestion and fermentation models are widely applied to predict bioavailability of foods or nutrients because these models are relatively inexpensive and without ethical concerns; moreover, the experimental conditions are controllable, the sampling is simple and the results are repeatable [15]. Several studies have focused on the potential applications of in vitro models. The biostability and catabolism of phenolic compounds in pomegranate have been reported using simulated gastrointestinal digestion and colonic fermentation models in vitro [16], and the importance of fruit seeds in the prevention of oxidative stress-related diseases has been evaluated using a similar approach [17]. In vitro approaches were also used to assess the polyphenol availability of açai and its subsequent health impact on the fecal microbiota [18]. Following in vitro digestion, fermentation of the nondigestible compounds in apple modulates bacterial populations in feces from obese mice with an increasing trend in the production of butyric acid [19]. However, to our knowledge, this study is the first to assess the effect of GE-containing phenolic compounds on changes in fecal bacteria in an in vitro model of fecal microbiota-mediated fermentation. This study aimed to evaluate the stability of GE, which is rich in polyphenols and its beneficial effects on gut microbial using an in vitro model. Furthermore, the effect of 6G on promoting the amounts of specific genera was determined by quantitative real-time polymerase chain reaction (qPCR). 2. Materials and Methods 2.1. Plant Materials and Chemicals After lyophilization, dried ginger (Laiwu, Shandong Province, China) was milled and stored in a moisture-controlled cabinet until use. The 6-, 8-, and 10-gingerols and 6-shogaol were purchased from Chromadex (Irvine, CA, USA). Unless otherwise noted, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Preparation of Ginger Extract Dried ginger powder (200 g) was steeped in ethyl acetate (w:v, 1:5), stirred (4 ◦C) for 2 h and centrifuged (12,000 g, 4 C) for 20 min. After extraction three times, the supernatants extracted continuously were × ◦ combined, and the solvent was evaporated under reduced pressure at 40 ◦C using a rotary evaporator (IKA, Staufen, Germany). After lyophilization, the fractions were stored at 80 C until use. − ◦ 2.3. Simulated Digestion Model In Vitro Simulated gastrointestinal and small-intestinal digestion of GE was carried out by a SPH-100D thermostat shaker (Shiping Instrument Corp., Shanghai, China) according to previous methods with minor modifications [20]. In brief, 0.5 g of dried GE was dissolved in 40 mL of Tris-maleate buffer (0.1 mol/L, pH 6.9) at 37 ◦C (water bath). For in vitro gastrointestinal digestion, the pH was adjusted to 2.0 using HCl (0.1 mol/L) solution; 16.5 µL of pepsin ( 250 units/mg; 160 mg/mL solution in 0.1 mol/L ≥ HCl) were then added, and the sample was incubated at 37 ◦C for 2 h for complete gastric digestion. Nutrients 2020, 12, 3194 3 of 13 In the simulated intestinal phase, 125 µL of pancreatin-bile extract (4 mg of pancreatin (350 FIP-U/g protease, 6000 FIP-U/g lipase, 7500 FIP-U/g amylase) and 25 mg of bile extract in 0.1 M NaHCO3) were added to the gastric mixture. After adjusting the pH to 7.5 using 0.1 M NaHCO3, digestion continued at 37 ◦C for another 2 h. Under the same conditions, culture media without GE was set as the control group. All experiments were carried out in the absence of light and oxygen by covering with foil and flushing with N2 for 10 min before digestion. Samples obtained from each digestion step were lyophilized and stored at 20 C for analysis. − ◦ 2.4. Simulated Fecal Fermentation In Vitro Samples collected from the digestion described above were lyophilized for fecal fermentation in vitro. The culture medium was prepared according to a previously described method [21]. In brief, the sterile medium containing peptone (2 g/L), yeast extract (2 g/L), hemin (0.05 g/L), NaCl (0.1 g/L), MgSO4 7H2O (0.01 g/L), CaCl2 6H2O (0.01 g/L), bile salts (0.5 g/L), NaHCO3 (2 g/L), L-cysteine (0.5 g/L), K2HPO4 (0.04 g/L), KH2PO4 (0.04 g/L), Tween 80 (2 mL/L), vitamin K1 (10 mL/L), resazurin (1 mg/L) and distilled water. The medium was adjusted to pH 7.0 and continuously sparged with O2-free N2 overnight. The inoculum was prepared from fresh feces collected from male mice (C57BL/6 species) that were fed standard diets (D12450B; Research Diets) for 10 weeks and had not received antibiotics at any time. All experimental procedures were conducted with approval from the Biomedical Ethical Committee of Peking University (Beijing, China) with the approval number LA2018288. Freshly collected feces are immediately diluted with 10 mmol/L PBS (pH = 6.8) (w/v, 1:10). Next, the diluted feces were filtered with four layers of medical gauze. Then, the diluted feces were mixed with the culture medium (1:3) and the lyophilized fraction of the simulated digested GE from the process described above or the lyophilized fraction of digesta lacking GE (as a control). The mixture was distributed in disposable tubs (10 mL/tub/incubation time) and fermented at 37 ◦C in a LAI-3T anaerobic incubator (Longyue Instrument Corp., Shanghai, China) filled with gas mixture (H2 5%, CO2 10%,N2 85%). All incubations were performed in triplicate. During the 24-h fermentation process, samples were collected at 0, 6, 12, and 24 h for analysis.

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