- Email: [email protected]
Consumption of mung bean (Vigna radiata L.) attenuates obesity, ameliorates lipid metabolic disorders and modifies the gut microbiota composition in mice fed a high-fat diet
Journal of Functional Foods xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Consumption of mung bean (Vigna radiata L.) attenuates obesity, ameliorates lipid metabolic disorders and modifies the gut microbiota composition in mice fed a high-fat diet ⁎
Dianzhi Hou, Qingyu Zhao, Laraib Yousaf, Jabir Khan, Yong Xue, Qun Shen College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China National Engineering Research Center for Fruit and Vegetable Processing, Beijing 100083, China Key Laboratory of Plant Protein and Grain Processing, China Agricultural University, Beijing 100083, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Mung bean Obesity 16S rRNA Gut microbiota Lipid metabolic disorders
Mung bean is shown having several health benefits, but a little bit of knowledge is known about its effects on high-fat diet (HFD)-induced obesity or its relationship with gut microbiota composition changes. Here, it was observed that consumption of HFD supplemented with cooked mung bean (30%, w/w) for 12 weeks effectively alleviated body weight gain and lipid metabolic disorders, which was accompanied by a decrease in hepatic steatosis and adipocyte size. Furthermore, high-throughput sequencing of 16S rRNA revealed that mung bean supplementation prevented the HFD-induced gut microbiota dysbiosis, which was likely associated with the decreased relative abundance of several HFD-dependent taxa (Ruminiclostridium_9, Mucispirillum, Bilophila, Blautia, Ruminiclostridium, and Odoribacter), and the increased relative abundance of norank_f__Muribaculaceae. Spearman’s correlation analysis indicated that those genera were closely correlated with obesity-related indices. Collectively, the prevention of obesity by mung bean supplementation was at least partially mediated by structural modulation of gut microbiota.
1. Introduction Growing clinical evidence suggested that consumption of calorierich diets has led to increased rates of obesity and overweight, thus becoming major risk factors for many diseases, such as cardiovascular diseases, diabetes, and even some kinds of cancers (Razzoli, Pearson, Crow, & Bartolomucci, 2017). Previous studies have shown that a highfat diet causes severe lipid metabolic disorders (Saad, Santos, & Prada, 2016; Tulipani et al., 2016). Moreover, it has been proved that a highfat diet (HFD) is strongly associated with the disordered profiles of gut microbiota, microbial products, and also some normal bacteria decreased and disappeared, which contribute to the development of obesity (Bianchi, Duque, Saad, & Sivieri, 2019; Greenhill, 2017). For example, in many published studies, the increasing proportion of Firmicutes and decreasing the amount of Bacteroidetes, as well as the elevation of their ratio in major phyla demonstrated an increased risk of obesity (Chang et al., 2015). In order to improve health status and address all of the health problems caused by obesity, many worldwide health organizations call for serious changes in dietary patterns,
prompting the increased consumption of plant-based functional foods (Eichelmann, Schwingshackl, Fedirko, & Aleksandrova, 2016; Hou et al., 2018; Mohamed, 2014). Among dietary approaches, consumption of pulses has been suggested as a safe strategy to attenuate obesity and overweight (Foyer et al., 2016; Rebello, Greenway, & Finley, 2014). The mung bean (Vigna radiata L.) is an important edible traditional legume crop consumed by most Asian countries (Nair, Yang, Easdown, Thavarajah, Thavarajah, & Hughes, 2013). As a nutritional and healthy ingredient, mung bean is commonly prepared into various forms of food, such as soups and congee, or used to enhance the nutritional quality of noodles, confectionery and miscellaneous snacks (Hou et al., 2019). Moreover, in the well-known Chinese pharmacopoeia (Compendium of Materia Medical), mung bean is Chinese traditional medicine, which is recorded to be conducive to the detoxification activities, alleviation of heat stroke, recuperation of mentality, and regulation of gastrointestinal upset. Interestingly, in addition to the ancient description, many other potential health benefits of the mung bean have been reported, such as hypoglycemic and hypolipidemic effects (Inhae et al., 2015; Jang, Kang, Choe, Shin, & Kim, 2014), antihypertensive (Li,
⁎ Corresponding author at: College of Food Science and Nutritional Engineering, China Agricultural University, No.17, Qinghua East Road, Haidian District, Beijing 100083, China. E-mail address: [email protected] (Q. Shen).
https://doi.org/10.1016/j.jff.2019.103687 Received 22 September 2019; Received in revised form 31 October 2019; Accepted 15 November 2019 1756-4646/ © 2019 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Please cite this article as: Dianzhi Hou, et al., Journal of Functional Foods, https://doi.org/10.1016/j.jff.2019.103687
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
2.2. Biochemical analysis
Shi, Liu, & Le, 2006; Xie, Du, Shen, Wu, & Lin, 2019), anticancer (Swee Keong, Hamidah, Nurul Elyani, Boon Kee, Yong, Norlaily Mohd, & Kamariah, 2013), hepatoprotective (Lopes & Martins, 2018), and immunomodulatory properties (Ketha & Gudipati, 2018). Although these studies suggested that mung bean contains more natural phytochemical compounds and special protein that effectively benefit human health, however, mung bean, as a whole food supplemented in high-fat diet, can show health benefits is less known and still needs to figure out. Furthermore, the role of gut microbiota in these experiments has received little attention. More experiments are needed to verify whether the gut microbiota can mediate the metabolic benefits of the mung bean. Therefore, this study was conducted to determine the ability of mung bean-based diet to combat obesity-related metabolic disorder by assessing changes in physiological, histological, and biochemical parameters of mice with HFD-induced obesity. Moreover, our aim was to understand whether mung bean supplementation could modulate the gut microbiota composition of mice fed with HFD, and then find gut microbes responding to the dietary intervention of mung bean, as well as investigate their relationship with the prevention of the obesity-related metabolic disorder. The theoretical evidence presented here provide insights into the interactions between the mung bean and the gut microbiota, especially in terms of their potential health effects in alleviating obesity.
The serum concentration of total cholesterol (TCHO), total triacylglycerol (TG), high-density lipoprotein cholesterol (HDL-C), lowdensity lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were determined by using a 3100 automatic biochemistry analyzer (Hitachi Ltd., Tokyo, Japan). 2.3. Histological analysis The same segments of the liver and epididymal adipose tissues were collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned into 5 μm thick slices, and stained with hematoxylin-eosin (H& E). Frozen liver sections were stained with Oil Red O. All the stained slides were examined through the Zeiss microscope (Axio Observer A1; Carl Zeiss, Oberkochen, Germany). The cell size of adipose was quantified by Image software (National Institutes of Health). 2.4. Fecal sample collection and total genomic DNA extraction Fresh fecal samples were collected from each mouse under sterile conditions by the abdominal massage at the 12-week, and they were immersed in liquid nitrogen immediately and stored at −80 °C until subsequent analysis. Genomic DNA from fecal samples was extracted by a QIAamp-DNA stool mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The concentration of the extracted DNA was quantified by using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). The purity of the extracted DNA was examined by 1% agarose gel electrophoresis.
2. Materials and methods 2.1. Preparation of mung bean, diets, and animal experimental design
2.5. Gut microbiota analysis by 16S rRNA gene sequencing
Mung beans (Vigna radiata L.) were purchased from Dongfangliang Life Technology Co., Ltd. (Shanxi, China). Mung bean seeds were washed and soaked in distilled water at room temperature for 8-h and cooked for 40 mins in a steam chamber (CFXB50A2A-80, Supor Instruments Co., Ltd, Zhejiang, China). Cooked mung bean seeds were then air-dried for 12-h in a drying oven (DVS412C, Yamato Scientific Co., Ltd., Japan) at 50 °C. Finally, the dried mung beans were milled and sieved through an 80-mesh (0.18 mm) to obtain a fine powder, then stored at −20 °C for subsequent usage. Four-week-old C57BL/6J male mice (14–16 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing) and maintained, three mice per cage, in a standard specific-pathogen-free (SPF) facility with controlled conditions (25 ± 2 °C, 55% ± 10% humidity, 12-h light/dark cycle). After one week of acclimation, the mice were randomly divided into 3 groups for a 12-week experiment: (1) normal chow diet (NCD, n = 9) (D12450J, 10% energy derived from fat, 3.85 total kcal/g, Research Diets Inc., New Brunswick, NJ, USA), (2) high fat diet (HFD, n = 9) (D12492, 60% energy derived from fat, 5.24 total kcal/g, Research Diets Inc., New Brunswick, NJ, USA), (3) HFD supplemented with 30% cooked mung bean flour (HFD-CMB, n = 9) (60% energy derived from fat, 5.24 total kcal/g, Shuyishuer Biotech Co., Ltd, Changzhou, China). Importantly, each macronutrient in HFD and HFD-CMB diets contributed equally to the caloric density, thus they were isocaloric diets. The composition and energy densities of the experimental diets were listed in Supplementary Table S1. The level of 30% mung bean supplementation was selected based on the previous studies (Andersson et al., 2017; Nakamura et al., 2016). Mice were having free access to food and water. Food intake was measured twice per week, and body weight was recorded once weekly. All the mice were euthanized after 12 h fasting at the end of 12-week. The blood samples were collected from the orbital vascular plexus, isolated at 3,000 rpm for 10 min at 4 °C to obtain the serum, and then stored at −80 °C until the subsequent analysis. The epididymal white adipose tissues (Epi-WAT), retroperitoneal white adipose tissues (Retro-WAT), and perirenal white adipose tissues (Per-WAT) were weighted.
The V3 and V4 regions of the 16S rRNA gene was amplified in a polymerase chain reaction (PCR) system by using the primers 338F ( 5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The cycling and reaction conditions were described by Wang, Wang, Li, Hu, and Chen (2019). The 2% agarose gel was used to check the PCR products. The PCR products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, USA) according to the manufacturer’s instructions. Purified amplicons were quantified using QuantiFluor-ST (Promega, Madison, WI, USA) and sequenced on an Illumina Miseq platform at Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The sequences obtained were picked into operational taxonomic units by clustering 97% sequence similarity and classified at various taxonomic ranks (phylum, order, class, family and genus). 2.6. Statistical analysis Data are expressed as the mean ± SEM (standard error of the mean). Unpaired Student’s t tests and a one-way ANOVA followed by Tukey’s post hoc test were used to calculate the statistical significance between two groups and more than two groups, respectively. p < 0.05 were considered to be statistically significant. 3. Results 3.1. Mung bean supplementation attenuated body weight gain in HFDinduced mice High fat-diet is always associated with body weight (BW) gain. As expected, a significant increase in BW of mice fed with HFD was observed in comparison with the NCD group over 1 week (Fig. 1A, P < 0.001). However, mung bean supplementation significantly prevented HFD-induced BW gain from week 7 to week 12 (Fig. 1A and B, p < 0.05). Moreover, at the end of the experiment (12 weeks), mung 2
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
Fig. 1. Mung bean supplementation prevented HFD-induced obesity. (A) Changes of body weights in mice fed with NCD, HFD and HFD-CMB. (B) Body weight gain. (C) Adipose tissue weight. (D) Fat mass/body mass. (E) Food intake. (F) Energy intake. Data are shown as means ± SEM (n = 7). # p < 0.05, # #p < 0.01, # # # p < 0.001 versus NCD. *p < 0.05, **p < 0.01, ***p < 0.001 versus HFD.
(p < 0.001, Fig. 2B).
bean supplementation significantly inhibited the accumulation of body fat, including epididymal, retroperitoneal, and perirenal fat (Fig. 1C, p < 0.05), which was aligned with BW gain. Notably, the body fat ratio was also significantly decreased by mung bean supplementation (Fig. 1D, P < 0.001). In addition, there was no statistically difference in the food intake (g/d/mouse) and energy intake (kcal/d/mouse) between the HFD and HFD-CMB groups (Fig. 1E and F), suggesting that the effects of the mung bean on BW gain were not associated with the changes in food consumption.
3.3. Mung bean supplementation prevented HFD-induced liver steatosis and adipose tissue hypertrophy As shown in Fig. 3A and B, the liver tissue of NCD group was in a healthy state, showing that the structure of hepatic lobular was intact, the hepatic sinus has no pathological defects, and the nuclei were having normal morphology. Conversely, compared with the NCD group, HFD feeding caused the widening of the gap junctional channels, cell wall loosened, necrosis or death, and autolysis in hepatocytes. Nevertheless, a neat stacking of hepatic sinuses and a marked reduction of hepatocyte damage were observed in HFD-CMB group, as evidenced by the lower steatosis score (Fig. 3D, p < 0.001). Moreover, Oil Red O staining indicated that there was little lipid accumulation in the hepatocytes of HFD-CMB group (Fig. 3B). All of these results suggested that mung bean supplementation could inhibit lipid storage and prevent the liver steatosis. It is noteworthy that, compared with the NCD group, HFD feeding significantly increased the mean size of epididymal white adipocytes, but this phenomenon was partly reversed by mung bean supplementation (Fig. 3C and E).
3.2. Mung bean supplementation alleviated dyslipidemia and improved AST and ALT levels Obesity is often accompanied by hyperlipidemia. As shown in Fig. 2A, mung bean supplementation was found to significantly reduce the serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) levels of mice in HFD-CMB group in comparison with the HFD group. However, no significant difference in the serum HDL-C level was observed between the HFD-CMB and HFD groups. Notably, mung bean supplementation reduced the levels of serum AST and ALT (p < 0.05), but only significantly in ALT 3
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
Fig. 2. Mung bean supplementation lowered blood serum lipid levels, and AST and ALT. (A) Blood lipid concentration. (B) Activity of serum ALT and AST. Data are shown as means ± SEM (n = 7). # p < 0.05, # #p < 0.01, # # #p < 0.001 versus NCD. *p < 0.05, **p < 0.01, ***p < 0.001 versus HFD.
Fig. 3. Prevention of HFD-induced liver steatosis and WAT hypertrophy by mung bean supplementation. (A) H&E staining of liver tissue (40 × zoom). (B) liver Oil Red O staining (40 × zoom). (C) H&E staining of epididymal white adipose (20 × zoom). (D) steatosis score. (E) Mean adipocyte area. Data are shown as means ± SEM (n = 7). # p < 0.05, # #p < 0.01, # # #p < 0.001 versus NCD. *p < 0.05, **p < 0.01, *** p < 0.001 versus HFD.
4
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
were identified from the phyla to the genus level and were different for each group (Fig. 6A). As shown in Fig. 6B, the NCD group was characterized by the higher proportion of genus norank_f__Muribaculaceae, family Muribaculaceae, order Bacteroidales, class Bacteroidia, phylum Bacteroidetes, while, in the HFD group, the gut microbiota was enriched by a dramatic increase in genus norank_f__Lachnospiraceae, family Lachnospiraceae and Ruminococcaceae, oder Clostridiales, Class Clostridia, Phylum Firmicutes. Remarkably, the mung bean supplementation was also characterized by genus norank_f__Muribaculaceae, family Muribaculaceae, order Bacteroidales, class Bacteroidia, phylum Bacteroidetes in the HFD-CMB group, which was consistent with the NCD group. Taken together, these data suggested that mung bean supplementation could reverse the HFD-induced composition of gut microbiota.
3.4. Mung bean supplementation regulated HFD‑induced gut microbiota dysbiosis In order to evaluate the gut microbiota alterations resulted from HFD-induced obesity as well as the improved effect of mung bean supplementation on gut microbiota, the fecal bacterial composition of mice was detected by the 16S rRNA sequencing based on V3-V4 region. A total of 977,441 raw clean reads were generated from 21 mice (7 samples per group) with 46,544 reads per group on average, and lowquality sequences were removed. The number of clean tags per sample ranged from 35,035 to 59,810. The effective reads were clustered into OTUs (Operational Taxonomic Units) based on 97% similarity. Rarefaction and Shannon-Wiener curves showed that most of the bacterial diversity and richness in different samples have been covered (Supplementary Fig. S1). Interestingly, the ACE and Chao indices were significantly decreased in the fecal microbiota of the HFD group in comparison with that of the NCD group, but they were significantly restored by mung bean supplementation. However, there was no difference in the Shannon and Simpson indices between the three groups. These results suggested that mung bean supplementation could effectively prevent the decrease induced by the HFD in the richness of gut microbiota (Fig. 4A). To better comprehend the shared richness among each group, a Venn diagram analysis was performed at the OTU level (Fig. 4B). More OTUs was found between HFD-CMB and NCD samples (75) than that of HFD-CMB and HFD samples (25). In addition, the further Beta diversity analysis using principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) based on Bray-Curtis showed that there was a palpable clustering in the gut microbiota composition of different groups (Fig. 4C). These results indicated that the composition of gut microbiota changed significantly in response to mung bean supplementation. To identify specific taxa related to mung bean supplementation, relative abundance was assessed at the phylum, family, and genus level. The microbial community structure of three groups was mainly dominated by Firmicutes and Bacteroidetes at phylum level (Fig. 5A). Compared with the HFD group, mung bean supplementation significantly increased the relative abundance of Bacteroidetes and Proteobacteria in HFD-CMB group (Fig. 5B). Interestingly, in our study, mung bean supplementation significantly prevented the increase induced by HFD in the Firmicutes/Bacteroidetes (F/B) ratio (p < 0.05) (Fig. 5C). Furthermore, the genus was represented in a heat map at different levels in the three groups, showing that the dominant bacteria of HFD-CMB group and NCD group were relatively similar, but different from HFD group (Fig. 5D). To better understand the effects of the mung bean on the relative abundance of the key genera in HFD-fed mice, Welch’s t-test was used to analyze the distinguished genera based on mean proportions of 15 key genera within the three groups in the present study (Fig. 5E). In comparison with the NCD group, the relative abundance of norank_f__Muribaculaceae and Faecalibaculum were significantly decreased, but the level of Blautia, norank_f__Lachnospiraceae, unclassified_f__Lachnospiraceae, Ruminiclostridium_9, Ruminiclostridium, Bilophila were significantly increased in the HFD group (Fig. 5E). However, mung bean supplementation significantly enriched the relative abundance of unclassified_f__Lachnospiraceae, norank_f__Muribaculaceae and Lachnospiraceae_NK4A136_group, and lowered the relative abundance of Blautia, Ruminiclostridium, Ruminiclostridium_9, Mucispirillum, Bilophila, and Odoribacter, as compared with the HFD group (Fig. 5E). Notably, some changes of genera in the HFD-CMB group were restored to similar levels as that of the NCD group. Together, these results provided an explanation that the significant changes at genus level mentioned above may contribute to the beneficial effects of the mung bean against obesity. In addition, LEfSe analysis with a logarithmic LDA score threshold of three was performed to identify the specific bacteria characteristic associated with mung bean supplementation. The specific bacterial taxa
3.5. Possible relationships between gut microbiota and obesity-related indices Spearman’s correlation analysis was performed to further explore the correlation between the changes in gut microbiota composition and the obesity-related parameters. As shown in Fig. 7, the HFD enriched norank_f__Lachnospiraceae, unclassified_f__Lachnospiraceae, Ruminiclostridium_9, and Bilophila showed strongly positive correlations with obesity-related indexes. In this study, mung bean supplementation may reduce obesity and modulate the genus by promoting the relative abundance of norank_f__Muribaculaceae and lowering the relative abundance of Ruminiclostridium_9, Mucispirillum, Bilophila, and Odoribacter (Fig. 5E). 4. Discussion Legume consumption has been shown to have beneficial effects on the prevention and management of obesity-related metabolic disorders, because of their low-energy density and high nutritional properties (Marinangeli & Jones, 2012; Rebello et al., 2014; Venn et al., 2010). For mung bean, previous studies have shown that its health benefits are mainly attributed to the bioactive polyphenols, polysaccharides, and protein isolate it contains (Hou et al., 2019). The hypolipidemic and anti-obesity effects of mung bean have been well documented in clinical trials and animal studies (Kohno, Motoyama, Shigihara, Sakamoto, & Sugano, 2017; Yeap et al., 2015). However, most of the studies focused on their effects on host energy expenditure and lipid metabolism (Watanabe et al., 2016; Yao, Zhu, & Ren, 2014), but only a few have reported their impact on the biodiversity and activity of the host intestinal microbiota. To the best of our knowledge, only mung bean protein isolate has its anti-obesity effects abolished in germ-free mice, indicating that the beneficial effects of mung bean protein isolate followed a mechanism that depended on the presence of gut microbiota (Nakatani et al., 2018). Thus, in this study, it was speculated that mung bean supplementation may affect not only community biodiversity and composition but also functional attributes of the gut microbiota in HFDinduced obese mice. Moreover, these changes would have possible positive effects on host health. In the present study, it is important to note that mung bean, as a whole food, were prepared by following traditional cooking practices (soaked and cooked), thus these results may provide may provide valuable reference for the health benefits of mung bean. It was showed that daily consumption of HFD supplemented with mung bean (30%) was sufficient to resist weight gain (Fig. 1A and B) and reduce fat accumulation (Fig. 1C and D) without a significant difference in food intake and energy intake (Fig. 1E and F) as compared with the HFD group. It means that the effect of mung bean on body weight is not due to the reduced food consumption or calorie intake. As well known, HFD affects the serum levels of TC, TG, HDL-C, and LDL-C, which will lead to abnormal lipid metabolism and ultimately to dyslipidemia or non-alcoholic fatty liver. A recent double-blind, placebo-controlled clinical trial of 45 prediabetes patients showed that, after consumption of 2.5 g 5
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
Fig. 4. Effects of NCD, HFD and HFD-CMB on alpha and beta diversity of gut microbiota. (A) richness and diversity indices. (B) Venn diagram showing the OTUs shared by the mice from each group. (C) PCoA and NMDS score plot based on Bray-Curtis. Data are shown as means ± SEM (n = 7). # p < 0.05, ##p < 0.01, ### p < 0.001 versus NCD. *p < 0.05, **p < 0.01, ***p < 0.001 versus HFD.
group. Moreover, ALT and AST are the two biochemical markers normally used for early-stage assessment of liver injury. Previous studies have showed that the aqueous extract and the flavonoid fraction (vitexin and isovitexin) of the mung bean exhibited a stronger hepatoprotective activity by suppressing the accumulation of hepatic lipids in the acetaminophen-induced acute liver injury model than that of other pulses, including adzuki bean, black bean, and rice bean (Liu et al., 2014; Wu, Wang, Lin, & Chang, 2001). In addition, mung bean protein isolate was also found to significantly reduce the plasma ALT and AST concentrations in mice fed with HFD, preventing non-alcoholic fatty
mung bean protein isolates daily for 12 weeks, the triglyceride levels were significantly decreased in the subjects with hyperlipidemia, and these effects were particularly observed in obese subjects (Kohno et al., 2017). In another study, after 4 weeks of oral administration, ethanolic extracts of mung bean (1 g/kg) were found to significantly lower the triacylglycerol and cholesterol levels in KK-Ay mice by decreasing the expression of lipogenic genes (Inhae et al., 2015). Our results (Fig. 2A) are generally in agreement with these results. The mung bean supplementation significantly reduced the serum lipid levels in mice of HFDCMB group, including TC, TG, and LDL-C, in comparison with the HFD
6
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
Fig. 5. Mung bean supplementation modulated the composition of the gut microbiota. (A) Gut microbiota composition at the phylum level. (B) Percent of community abundance on phylum level. (C) F/B ratio. (D) Heatmap analysis at the genus level. (E) Mean proportions of 15 key genus in mice from different groups. Data are shown as means ± SEM (n = 7). # p < 0.05, ##p < 0.01, ###p < 0.001 versus NCD. *p < 0.05, **p < 0.01, ***p < 0.001 versus HFD.
7
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
Fig. 6. Structure and key phylotypes of gut microbiota responding to HFD and mung bean supplementation. (A) Cladogram generated from LEfSe analysis showing the relationship between taxon (the levels represent, from the inner to outer rings, phylum, class, order, family, and genus). (B) LDA scores derived from LEfSe analysis, showing the genus LDA score of > 3 (the length of the bar represents the LDA score).
Turnbaugh, Klein, & Gordon, 2006; Seganfredo et al., 2017). Mung bean supplementation restored the proportion of Bacteroides in the HFD-CMB group to a similar level to that presented in the NCD group (P < 0.05), which reportedly could help reduce the severity of obesity and type 2 diabetes (Moreno-Indias, Cardona, Tinahones, & QueipoOrtuño, 2014; Sharma et al., 2018). It was reported that the host adiposity was associated with an increase in the F/B ratio (Duca & Lam, 2014; Xue, Xie, Huang, Long, Gao, Ou, & Peng, 2016), and its elevation is considered as a hallmark of obesity-driven dysbiosis in many published studies (Murphy, Velazquez, & Herbert, 2015; Stephens, Arhire, & Covasa, 2018). Intriguingly, mung bean supplementation significantly reduced the ratio of F/B in the HFD-CMB group (Fig. 5C). In addition, many studies have demonstrated the bloom of Proteobacteria may play a positive role in leanness and metabolic health (Cho & Blaser, 2012; Moreno-Indias et al., 2014). The relative abundance of Proteobacteria in the gut microbiota community was found to be higher in the HFD-CMB group than that of HFD group. The mechanisms underlying these beneficial effects of mung bean supplementation on obesity are likely related to the alterations of gut microbiota composition, as evidenced by the reduced ratio of F/B in mice of HFD-CMB group. Moreover, the presence of specific species is inextricably linked to the presence of specific biological effects. At the genus level, we observed that the HFD-enriched genera norank_f__Lachnospiraceae, unclassified_f__Lachnospiraceae, Ruminiclostridium_9, Ruminiclostridium, and Bilophila were all positively correlated with obesity and obesity-
liver disease onset and progression (Watanabe et al., 2016). While, in this study, mung bean supplementation significantly decreased the ALT level, indicating mung bean could effectively alleviate HFD-induced liver injury to a certain extent. Those were also supported by the histological analysis, showing that hepatic steatosis and adipocyte size were significantly reduced by mung bean supplementation (Fig. 3). Overall, the interaction between nutrients may be an important explanation for mung bean in ameliorating the obesity-related metabolic disorders. Emerging evidence has revealed that gut microbiota plays an important role in the treatment of obesity (Blaser, 2017; Cox, West, & Cripps, 2015). The composition of gut microbiota can be reshaped by the interaction between dietary components and intestinal microorganisms, thus has a significant impact on host metabolism (Conlon & Bird, 2015; Rowland et al., 2018). Therefore, many dietary supplements have been used to prevent or alleviate metabolic diseases by modulating gut microbiota (Jing et al., 2018; Li et al., 2019). Here, it was found that mung bean supplementation could reverse the HFD-induced gut microbiota dysbiosis, and promote the growth of some specific bacteria. The decreases in the richness of gut microbiota in HFD-induced obese mice were fully prevented by the consumption of mung bean (Fig. 4A), with dysbiosis being one of the hallmarks of obesity (Cotillard et al., 2013). In many studies, HFD can lead to obesity-driven dysbiosis in gut microbiota by increasing the relative abundance of Firmicutes and reducing the relative abundance of Bacteroidetes (Ley,
8
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
Fig. 7. Heatmap of Spearman’s correlation analysis between gut microbiota and obesity-related indexes. The intensity of the colors represented the degree of association (red, positive correlation; green, negative correlation). Significant correlations are marked by *p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
found that the mung bean-mediated metabolic improvements were likely associated with the decreased relative abundance of undesirable bacteria and increased relative abundance of some possible beneficial bacteria in the gut microbiota community. Nevertheless, since a whole food approach was utilized in this study, the complex composition of the mung bean makes it difficult to decide which bioactive component (s) found (non-digestible carbohydrates, protein, and phenolics) are responsible for the beneficial effects on the obesity-related induces, and intestinal phenotype. Thus, the specific effects of individual bioactive components derived from mung bean on the obese phenotype and the microbial community are needed to be further explored.
related physiological markers (Fig. 7). However, mung bean supplementation significantly lowered the relative abundance of Ruminiclostridium_9, Mucispirillum, Bilophila, Blautia, Ruminiclostridium, and Odoribacter, which were enriched in the HFD group (Fig. 5E, p < 0.05). For example, the increased relative abundance of Bilophila-containing clusters is proved to be positively associated with HFD feeding (David et al., 2013; Schneeberger et al., 2015). Zhao et al. reported that a combination of quercetin and resveratrol could dramatically reduce the relative abundance of Bilophila, and then prevent the HFD-induced obesity (Zhao et al., 2017). These results indicated that the effects of mung bean were at least partially due to a decrease in the relative abundance of these obesity-related bacteria. Intriguingly, norank_f__Muribaculaceae had a strongly negative correlation with all of the obesity-related indices in the current study, and mung bean supplementation significantly promoted its relative abundance. Whether the norank_f__Muribaculaceae may play an important role in alleviating obesity and lipids metabolic disorders still needs an accurate trail to confirm the speculation. From the above-mentioned results, it was
5. Conclusions In summary, the results from this study concluded that cooked mung bean, prepared by following traditional human cooking practices at a physiologically relevant intake level, was sufficient to suppress weight gain and fat accumulation and ameliorate the serum lipid and 9
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
ALT levels in HFD-induced obesity mice. Furthermore, the reduction of obesity-related indices was strongly associated with the prevention of HFD-induced gut microbiota dysbiosis. Dietary modulation of gut microbiota by mung bean consumption might contribute to the improvement of the gastrointestinal health and eventually mediate its beneficial effects on the host. Meanwhile, it was unclear that whether cooked mung bean supplementation can still alter gut microbiota under a normal diet. Additional studies of mice consuming normal chow diet and high-fat diet, which both supplemented with cooked mung bean, will be necessary to determine whether some specific bacteria genus is significantly linked to mung bean-based diet. Collectively, the use of the cooked mung bean as a novel method of dietary incorporation or “value-added” ingredient has been beneficial in mitigating the severity of obese phenotype. However, there are still important gaps in our knowledge of the effects of special components in the mung bean on obesity and their mechanisms of anti-obesity, and which components are directly responsible for these beneficial effects on metabolism.
Duca, F. A., & Lam, T. K. T. (2014). Gut microbiota, nutrient sensing and energy balance. Diabetes, Obesity and Metabolism, 16(S1), 68–76. https://doi.org/10.1111/dom. 12340. Eichelmann, F., Schwingshackl, L., Fedirko, V., & Aleksandrova, K. (2016). Effect of plantbased diets on obesity-related inflammatory profiles: A systematic review and metaanalysis of intervention trials. Obesity Reviews, 17(11), 1067–1079. https://doi.org/ 10.1111/obr.12439. Foyer, C. H., Lam, H.-M., Nguyen, H. T., Siddique, K. H. M., Varshney, R. K., Colmer, T. D., ... Considine, M. J. (2016). Neglecting legumes has compromised human health and sustainable food production. Nature Plants, 2(8), 16112. https://doi.org/10. 1038/nplants.2016.112. Greenhill, C. (2017). Gut microbiome and serum metabolome changes. Nature Reviews Endocrinology, 13, 501. https://doi.org/10.1038/nrendo.2017.89. Hou, D., Chen, J., Ren, X., Wang, C., Diao, X., Hu, X., ... Shen, Q. (2018). A whole foxtail millet diet reduces blood pressure in subjects with mild hypertension. Journal of Cereal Science, 84, 13–19. https://doi.org/10.1016/j.jcs.2018.09.003. Hou, D., Yousaf, L., Xue, Y., Hu, J., Wu, J., Hu, X., ... Shen, Q. (2019). Mung Bean (Vigna radiata L.): bioactive polyphenols, polysaccharides, peptides, and health benefits. Nutrients, 11(6), 1238. https://doi.org/10.3390/nu11061238. Inhae, K., Seojin, C., Joung, H. T., Munji, C., Hae-Ri, W., Won, L. B., & Myoungsook, L. (2015). Effects of Mung Bean (Vigna radiata L.) Ethanol Extracts Decrease Proinflammatory Cytokine-Induced Lipogenesis in the KK-Ay Diabese Mouse Model. Journal of Medicinal Food, 18(8), 841–849. https://doi.org/10.1089/jmf.2014.3364. Jang, Y.-H., Kang, M.-J., Choe, E.-O., Shin, M., & Kim, J.-I. (2014). Mung bean coat ameliorates hyperglycemia and the antioxidant status in type 2 diabetic db/db mice. Food Science and Biotechnology, 23(1), 247–252. https://doi.org/10.1007/s10068014-0034-3. Jing, C., Wen, Z., Zou, P., Yuan, Y., Jing, W., Li, Y., & Zhang, C. (2018). Consumption of Black Legumes Glycine soja and Glycine max Lowers Serum Lipids and Alters the Gut Microbiome Profile in Mice Fed a High-Fat Diet. Journal of Agricultural and Food Chemistry, 66(28), 7367–7375. https://doi.org/10.1021/acs.jafc.8b02016. Ketha, K., & Gudipati, M. (2018). Immunomodulatory activity of non starch polysaccharides isolated from green gram (Vigna radiata). Food Research International, 113, 269–276. https://doi.org/10.1016/j.foodres.2018.07.010. Kohno, M., Motoyama, T., Shigihara, Y., Sakamoto, M., & Sugano, H. (2017). Improvement of glucose metabolism via mung bean protein consumption: A clinical trial of GLUCODIA TM isolated mung bean protein in Japan. Functional Foods in Health & Disease, 7, 115–134. https://doi.org/10.31989/ffhd.v7i2.320. Ley, R. E., Turnbaugh, P. J., Klein, S., & Gordon, J. I. (2006). Human gut microbes associated with obesity. Nature, 444(7122), 1022–1023. https://doi.org/10.1038/ 4441022a. Li, G.-H., Shi, Y.-H., Liu, H., & Le, G.-W. (2006). Antihypertensive effect of alcalase generated mung bean protein hydrolysates in spontaneously hypertensive rats. European Food Research and Technology, 222(5), 733–736. https://doi.org/10.1007/ s00217-005-0147-2. Li, S., Yu, W., Guan, X., Huang, K., Liu, J., Liu, D., & Duan, R. (2019). Effects of millet whole grain supplementation on the lipid profile and gut bacteria in rats fed with high-fat diet. Journal of Functional Foods, 59, 49–59. https://doi.org/10.1016/j.jff. 2019.05.030. Liu, T., Yu, X. H., Gao, E. Z., Liu, X. N., Sun, L. J., Li, H. L., ... Yu, Z. G. (2014). Hepatoprotective Effect of Active Constituents Isolated from Mung Beans (Phaseolus radiatus L.) in an Alcohol-Induced Liver Injury Mouse Model. Journal of Food Biochemistry, 38(5), 453–459. https://doi.org/10.1111/jfbc.12083. Lopes, L. A. R., Martins, M. D. C. d. C. e., Farias, L. M. d., Brito, A. K. d. S., Lima, G. D. M., Carvalho, V. B. L. d., . . . Frota, K. D. M. G. (2018). Cholesterol-Lowering and LiverProtective Effects of Cooked and Germinated Mung Beans (Vigna radiata L.). Nutrients, 10(7), 821. doi: https://doi.org/10.3390/nu10070821. Marinangeli, C. P. F., & Jones, P. J. H. (2012). Pulse grain consumption and obesity: Effects on energy expenditure, substrate oxidation, body composition, fat deposition and satiety. British Journal of Nutrition, 108(S1), S46–S51. https://doi.org/10.1017/ S0007114512000773. Mohamed, S. (2014). Functional foods against metabolic syndrome (obesity, diabetes, hypertension and dyslipidemia) and cardiovasular disease. Trends in Food Science & Technology, 35(2), 114–128. https://doi.org/10.1016/j.tifs.2013.11.001. Moreno-Indias, I., Cardona, F., Tinahones, F. J., & Queipo-Ortuño, M. I. (2014). Impact of the gut microbiota on the development of obesity and type 2 diabetes mellitus. Frontiers in Microbiology, 5(190), https://doi.org/10.3389/fmicb.2014.00190. Murphy, E. A., Velazquez, K. T., & Herbert, K. M. (2015). Influence of high-fat diet on gut microbiota: A driving force for chronic disease risk. Current Opinion in Clinical Nutrition and Metabolic Care, 18(5), 515–520. https://doi.org/10.1097/MCO. 0000000000000209. Nair, R. M., Yang, R.-Y., Easdown, W. J., Thavarajah, D., Thavarajah, P., Hughes, J. d. A., & Keatinge, J. (2013). Biofortification of mungbean (Vigna radiata) as a whole food to enhance human health. Journal of the Science of Food and Agriculture, 93(8), 1805-1813. doi: doi:10.1002/jsfa.6110. Nakamura, K., Koyama, M., Ishida, R., Kitahara, T., Nakajima, T., & Aoyama, T. (2016). Characterization of bioactive agents in five types of marketed sprouts and comparison of their antihypertensive, antihyperlipidemic, and antidiabetic effects in fructoseloaded SHRs. Journal of Food Science and Technology, 53(1), 581–590. https://doi. org/10.1007/s13197-015-2048-0. Nakatani, A., Li, X., Miyamoto, J., Igarashi, M., Watanabe, H., Sutou, A., ... Kimura, I. (2018). Dietary mung bean protein reduces high-fat diet-induced weight gain by modulating host bile acid metabolism in a gut microbiota-dependent manner. Biochemical and Biophysical Research Communications, 501(4), 955–961. https://doi. org/10.1016/j.bbrc.2018.05.090. Razzoli, M., Pearson, C., Crow, S., & Bartolomucci, A. (2017). Stress, overeating, and
6. Ethics statement All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The animal experiment was approved by the Institutional Animal Care and Use Committees of China Agricultural University (Beijing, China) with the approval number AW08089102-1-4 and was carried out in accordance with the National Research Council Guidelines. Funding This work was supported by National Key Research and Development Program of China (2017YFD0401202). Declaration of Competing Interest The authors declare that they have no conflict of interest. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.103687. References Andersson, K. E., Chawade, A., Thuresson, N., Rascon, A., Öste, R., Sterner, O., ... Hellstrand, P. (2017). Wholegrain oat diet changes the expression of genes associated with intestinal bile acid transport. Molecular Nutrition & Food Research, 61(7), 1600874. https://doi.org/10.1002/mnfr.201600874. Bianchi, F., Duque, A. L. R. F., Saad, S. M. I., & Sivieri, K. (2019). Gut microbiome approaches to treat obesity in humans. Applied Microbiology and Biotechnology, 103(3), 1081–1094. https://doi.org/10.1007/s00253-018-9570-8. Blaser, M. J. (2017). The theory of disappearing microbiota and the epidemics of chronic diseases. Nature Reviews Immunology, 17, 461. https://doi.org/10.1038/nri.2017.77. Chang, C.-J., Lin, C.-S., Lu, C.-C., Martel, J., Ko, Y.-F., Ojcius, D. M., ... Lai, H.-C. (2015). Ganoderma lucidum reduces obesity in mice by modulating the composition of the gut microbiota. Nature Communications, 6, 7489. https://doi.org/10.1038/ ncomms8489 https://www.nature.com/articles/ncomms8489#supplementaryinformation. Cho, I., & Blaser, M. J. (2012). The human microbiome: At the interface of health and disease. Nature Reviews Genetics, 13, 260. https://doi.org/10.1038/nrg3182. Conlon, M. A., & Bird, A. R. (2015). The impact of diet and lifestyle on Gut Microbiota and human health. Nutrients, 7(1), 17–44. https://doi.org/10.3390/nu7010017. Cotillard, A., Kennedy, S. P., Kong, L. C., Prifti, E., Pons, N., Le Chatelier, E., ... Layec, S. (2013). Dietary intervention impact on gut microbial gene richness. Nature, 500, 585. https://doi.org/10.1038/nature12480 https://www.nature.com/articles/ nature12480#supplementary-information. Cox, A. J., West, N. P., & Cripps, A. W. (2015). Obesity, inflammation, and the gut microbiota. The Lancet Diabetes & Endocrinology, 3(3), 207–215. https://doi.org/10. 1016/S2213-8587(14)70134-2. David, L. A., Maurice, C. F., Carmody, R. N., Gootenberg, D. B., Button, J. E., Wolfe, B. E., ... Turnbaugh, P. J. (2013). Diet rapidly and reproducibly alters the human gut microbiome. Nature, 505, 559. https://doi.org/10.1038/nature12820 https://www. nature.com/articles/nature12820#supplementary-information.
10
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
Zonja, B., ... Andres-Lacueva, C. (2016). Biomarkers of Morbid obesity and prediabetes by metabolomic profiling of human discordant phenotypes. Clinica Chimica Acta, 463, 53–61. https://doi.org/10.1016/j.cca.2016.10.005. Venn, B. J., Perry, T., Green, T. J., Skeaff, C. M., Aitken, W., Moore, N. J., ... Williams, S. (2010). The effect of increasing consumption of pulses and Wholegrains in Obese People: A randomized controlled trial. Journal of the American College of Nutrition, 29(4), 365–372. https://doi.org/10.1080/07315724.2010.10719853. Wang, J., Wang, P., Li, D., Hu, X., & Chen, F. (2019). Beneficial effects of ginger on prevention of obesity through modulation of gut microbiota in mice. European Journal of Nutrition. https://doi.org/10.1007/s00394-019-01938-1. Watanabe, H., Inaba, Y., Kimura, K., Asahara, S.-I., Kido, Y., Matsumoto, M., ... Inoue, H. (2016). Dietary Mung Bean Protein Reduces Hepatic Steatosis, Fibrosis, and Inflammation in Male Mice with Diet-Induced, Nonalcoholic Fatty Liver Disease. The Journal of Nutrition, 147(1), 52–60. https://doi.org/10.3945/jn.116.231662. Wu, S. J., Wang, J. S., Lin, C. C., & Chang, C. H. (2001). Evaluation of hepatoprotective activity of Legumes. Phytomedicine, 8(3), 213–219. https://doi.org/10.1078/09447113-00033. Xie, J., Du, M., Shen, M., Wu, T., & Lin, L. (2019). Physico-chemical properties, antioxidant activities and angiotensin-I converting enzyme inhibitory of protein hydrolysates from Mung bean (Vigna radiate). Food Chemistry, 270, 243–250. https://doi. org/10.1016/j.foodchem.2018.07.103. Xue, B., Xie, J., Huang, J., Long, C., Gao, L., Ou, S., . . . Peng, X. (2016). Plant Polyphenols Altering a Pathway of Energy Metabolism by Inhibiting Fecal Bacteriodetes and Firmicutes In Vitro. Food & Function, 7(3), 10.1039.C1035FO01438G. doi: https:// doi.org/10.1039/C5FO01438G. Yao, Y., Zhu, Y., & Ren, G. (2014). Mung bean protein increases plasma cholesterol by upregulation of hepatic HMG-CoA Reductase, and CYP7A1 in mRNA Levels. Journal of Food and Nutrition Research, 2(11), 770–775. https://doi.org/10.12691/jfnr-2-11-2. Yeap, S. K., Beh, B. K., Ho, W. Y., Mohd Yusof, H., Mohamad, N. E., Ali, N. M., ... Long, K. (2015). In vivo antioxidant and hypolipidemic effects of fermented mung bean on hypercholesterolemic mice. Evidence-based Complementary and Alternative Medicine: eCAM, 2015, 508029. https://doi.org/10.1155/2015/508029. Zhao, L., Zhang, Q., Ma, W., Tian, F., Shen, H., & Zhou, M. (2017). A combination of quercetin and resveratrol reduces obesity in high-fat diet-fed rats by modulation of gut microbiota. Food & Function, 8(12), 4644–4656. https://doi.org/10.1039/ c7fo01383c.
obesity: Insights from human studies and preclinical models. Neuroscience & Biobehavioral Reviews, 76, 154–162. https://doi.org/10.1016/j.neubiorev.2017.01. 026. Rebello, C. J., Greenway, F. L., & Finley, J. W. (2014). A review of the nutritional value of legumes and their effects on obesity and its related co-morbidities. Obesity Reviews, 15(5), 392–407. https://doi.org/10.1111/obr.12144. Rowland, I., Gibson, G., Heinken, A., Scott, K., Swann, J., Thiele, I., & Tuohy, K. (2018). Gut microbiota functions: Metabolism of nutrients and other food components. European Journal of Nutrition, 57(1), 1–24. https://doi.org/10.1007/s00394-0171445-8. Saad, M. J. A., Santos, A., & Prada, P. O. (2016). Linking Gut microbiota and inflammation to obesity and insulin resistance. Physiology, 31(4), 283–293. https://doi. org/10.1152/physiol.00041.2015. Schneeberger, M., Everard, A., Gómez-Valadés, A. G., Matamoros, S., Ramírez, S., Delzenne, N. M., ... Cani, P. D. (2015). Akkermansia muciniphila inversely correlates with the onset of inflammation, altered adipose tissue metabolism and metabolic disorders during obesity in mice. Scientific Reports, 5, 16643. https://doi.org/10. 1038/srep16643. Seganfredo, F. B., Blume, C. A., Moehlecke, M., Giongo, A., Casagrande, D. S., Spolidoro, J. V. N., ... Mottin, C. C. (2017). Weight-loss interventions and gut microbiota changes in overweight and obese patients: A systematic review. Obesity Reviews, 18(8), 832–851. https://doi.org/10.1111/obr.12541. Sharma, V., Smolin, J., Nayak, J., Ayala, J. E., Scott, D. A., Peterson, S. N., & Freeze, H. H. (2018). Mannose Alters Gut Microbiome, Prevents Diet-Induced Obesity, and Improves Host Metabolism. Cell Reports, 24(12), 3087–3098. https://doi.org/10. 1016/j.celrep.2018.08.064. Stephens, R. W., Arhire, L., & Covasa, M. (2018). Gut Microbiota: From microorganisms to metabolic organ influencing obesity. Obesity, 26(5), 801–809. https://doi.org/10. 1002/oby.22179. Swee Keong, Y., Hamidah, M. Y., Nurul Elyani, M., Boon Kee, B., Yong, H. W., Norlaily Mohd, A., . . . Kamariah, L. (2013). In Vivo Immunomodulation and Lipid Peroxidation Activities Contributed to Chemoprevention Effects of Fermented Mung Bean against Breast Cancer. Evidence-Based Complementray and Alternative Medicine,2013,(2013-4-22), 2013(2013), 708464. doi: http://dx.doi.org/10.1155/ 2013/708464. Tulipani, S., Palau-Rodriguez, M., Miñarro Alonso, A., Cardona, F., Marco-Ramell, A.,
11
Contents lists available at ScienceDirect
Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Consumption of mung bean (Vigna radiata L.) attenuates obesity, ameliorates lipid metabolic disorders and modifies the gut microbiota composition in mice fed a high-fat diet ⁎
Dianzhi Hou, Qingyu Zhao, Laraib Yousaf, Jabir Khan, Yong Xue, Qun Shen College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China National Engineering Research Center for Fruit and Vegetable Processing, Beijing 100083, China Key Laboratory of Plant Protein and Grain Processing, China Agricultural University, Beijing 100083, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Mung bean Obesity 16S rRNA Gut microbiota Lipid metabolic disorders
Mung bean is shown having several health benefits, but a little bit of knowledge is known about its effects on high-fat diet (HFD)-induced obesity or its relationship with gut microbiota composition changes. Here, it was observed that consumption of HFD supplemented with cooked mung bean (30%, w/w) for 12 weeks effectively alleviated body weight gain and lipid metabolic disorders, which was accompanied by a decrease in hepatic steatosis and adipocyte size. Furthermore, high-throughput sequencing of 16S rRNA revealed that mung bean supplementation prevented the HFD-induced gut microbiota dysbiosis, which was likely associated with the decreased relative abundance of several HFD-dependent taxa (Ruminiclostridium_9, Mucispirillum, Bilophila, Blautia, Ruminiclostridium, and Odoribacter), and the increased relative abundance of norank_f__Muribaculaceae. Spearman’s correlation analysis indicated that those genera were closely correlated with obesity-related indices. Collectively, the prevention of obesity by mung bean supplementation was at least partially mediated by structural modulation of gut microbiota.
1. Introduction Growing clinical evidence suggested that consumption of calorierich diets has led to increased rates of obesity and overweight, thus becoming major risk factors for many diseases, such as cardiovascular diseases, diabetes, and even some kinds of cancers (Razzoli, Pearson, Crow, & Bartolomucci, 2017). Previous studies have shown that a highfat diet causes severe lipid metabolic disorders (Saad, Santos, & Prada, 2016; Tulipani et al., 2016). Moreover, it has been proved that a highfat diet (HFD) is strongly associated with the disordered profiles of gut microbiota, microbial products, and also some normal bacteria decreased and disappeared, which contribute to the development of obesity (Bianchi, Duque, Saad, & Sivieri, 2019; Greenhill, 2017). For example, in many published studies, the increasing proportion of Firmicutes and decreasing the amount of Bacteroidetes, as well as the elevation of their ratio in major phyla demonstrated an increased risk of obesity (Chang et al., 2015). In order to improve health status and address all of the health problems caused by obesity, many worldwide health organizations call for serious changes in dietary patterns,
prompting the increased consumption of plant-based functional foods (Eichelmann, Schwingshackl, Fedirko, & Aleksandrova, 2016; Hou et al., 2018; Mohamed, 2014). Among dietary approaches, consumption of pulses has been suggested as a safe strategy to attenuate obesity and overweight (Foyer et al., 2016; Rebello, Greenway, & Finley, 2014). The mung bean (Vigna radiata L.) is an important edible traditional legume crop consumed by most Asian countries (Nair, Yang, Easdown, Thavarajah, Thavarajah, & Hughes, 2013). As a nutritional and healthy ingredient, mung bean is commonly prepared into various forms of food, such as soups and congee, or used to enhance the nutritional quality of noodles, confectionery and miscellaneous snacks (Hou et al., 2019). Moreover, in the well-known Chinese pharmacopoeia (Compendium of Materia Medical), mung bean is Chinese traditional medicine, which is recorded to be conducive to the detoxification activities, alleviation of heat stroke, recuperation of mentality, and regulation of gastrointestinal upset. Interestingly, in addition to the ancient description, many other potential health benefits of the mung bean have been reported, such as hypoglycemic and hypolipidemic effects (Inhae et al., 2015; Jang, Kang, Choe, Shin, & Kim, 2014), antihypertensive (Li,
⁎ Corresponding author at: College of Food Science and Nutritional Engineering, China Agricultural University, No.17, Qinghua East Road, Haidian District, Beijing 100083, China. E-mail address: [email protected] (Q. Shen).
https://doi.org/10.1016/j.jff.2019.103687 Received 22 September 2019; Received in revised form 31 October 2019; Accepted 15 November 2019 1756-4646/ © 2019 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Please cite this article as: Dianzhi Hou, et al., Journal of Functional Foods, https://doi.org/10.1016/j.jff.2019.103687
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
2.2. Biochemical analysis
Shi, Liu, & Le, 2006; Xie, Du, Shen, Wu, & Lin, 2019), anticancer (Swee Keong, Hamidah, Nurul Elyani, Boon Kee, Yong, Norlaily Mohd, & Kamariah, 2013), hepatoprotective (Lopes & Martins, 2018), and immunomodulatory properties (Ketha & Gudipati, 2018). Although these studies suggested that mung bean contains more natural phytochemical compounds and special protein that effectively benefit human health, however, mung bean, as a whole food supplemented in high-fat diet, can show health benefits is less known and still needs to figure out. Furthermore, the role of gut microbiota in these experiments has received little attention. More experiments are needed to verify whether the gut microbiota can mediate the metabolic benefits of the mung bean. Therefore, this study was conducted to determine the ability of mung bean-based diet to combat obesity-related metabolic disorder by assessing changes in physiological, histological, and biochemical parameters of mice with HFD-induced obesity. Moreover, our aim was to understand whether mung bean supplementation could modulate the gut microbiota composition of mice fed with HFD, and then find gut microbes responding to the dietary intervention of mung bean, as well as investigate their relationship with the prevention of the obesity-related metabolic disorder. The theoretical evidence presented here provide insights into the interactions between the mung bean and the gut microbiota, especially in terms of their potential health effects in alleviating obesity.
The serum concentration of total cholesterol (TCHO), total triacylglycerol (TG), high-density lipoprotein cholesterol (HDL-C), lowdensity lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were determined by using a 3100 automatic biochemistry analyzer (Hitachi Ltd., Tokyo, Japan). 2.3. Histological analysis The same segments of the liver and epididymal adipose tissues were collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned into 5 μm thick slices, and stained with hematoxylin-eosin (H& E). Frozen liver sections were stained with Oil Red O. All the stained slides were examined through the Zeiss microscope (Axio Observer A1; Carl Zeiss, Oberkochen, Germany). The cell size of adipose was quantified by Image software (National Institutes of Health). 2.4. Fecal sample collection and total genomic DNA extraction Fresh fecal samples were collected from each mouse under sterile conditions by the abdominal massage at the 12-week, and they were immersed in liquid nitrogen immediately and stored at −80 °C until subsequent analysis. Genomic DNA from fecal samples was extracted by a QIAamp-DNA stool mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The concentration of the extracted DNA was quantified by using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). The purity of the extracted DNA was examined by 1% agarose gel electrophoresis.
2. Materials and methods 2.1. Preparation of mung bean, diets, and animal experimental design
2.5. Gut microbiota analysis by 16S rRNA gene sequencing
Mung beans (Vigna radiata L.) were purchased from Dongfangliang Life Technology Co., Ltd. (Shanxi, China). Mung bean seeds were washed and soaked in distilled water at room temperature for 8-h and cooked for 40 mins in a steam chamber (CFXB50A2A-80, Supor Instruments Co., Ltd, Zhejiang, China). Cooked mung bean seeds were then air-dried for 12-h in a drying oven (DVS412C, Yamato Scientific Co., Ltd., Japan) at 50 °C. Finally, the dried mung beans were milled and sieved through an 80-mesh (0.18 mm) to obtain a fine powder, then stored at −20 °C for subsequent usage. Four-week-old C57BL/6J male mice (14–16 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing) and maintained, three mice per cage, in a standard specific-pathogen-free (SPF) facility with controlled conditions (25 ± 2 °C, 55% ± 10% humidity, 12-h light/dark cycle). After one week of acclimation, the mice were randomly divided into 3 groups for a 12-week experiment: (1) normal chow diet (NCD, n = 9) (D12450J, 10% energy derived from fat, 3.85 total kcal/g, Research Diets Inc., New Brunswick, NJ, USA), (2) high fat diet (HFD, n = 9) (D12492, 60% energy derived from fat, 5.24 total kcal/g, Research Diets Inc., New Brunswick, NJ, USA), (3) HFD supplemented with 30% cooked mung bean flour (HFD-CMB, n = 9) (60% energy derived from fat, 5.24 total kcal/g, Shuyishuer Biotech Co., Ltd, Changzhou, China). Importantly, each macronutrient in HFD and HFD-CMB diets contributed equally to the caloric density, thus they were isocaloric diets. The composition and energy densities of the experimental diets were listed in Supplementary Table S1. The level of 30% mung bean supplementation was selected based on the previous studies (Andersson et al., 2017; Nakamura et al., 2016). Mice were having free access to food and water. Food intake was measured twice per week, and body weight was recorded once weekly. All the mice were euthanized after 12 h fasting at the end of 12-week. The blood samples were collected from the orbital vascular plexus, isolated at 3,000 rpm for 10 min at 4 °C to obtain the serum, and then stored at −80 °C until the subsequent analysis. The epididymal white adipose tissues (Epi-WAT), retroperitoneal white adipose tissues (Retro-WAT), and perirenal white adipose tissues (Per-WAT) were weighted.
The V3 and V4 regions of the 16S rRNA gene was amplified in a polymerase chain reaction (PCR) system by using the primers 338F ( 5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The cycling and reaction conditions were described by Wang, Wang, Li, Hu, and Chen (2019). The 2% agarose gel was used to check the PCR products. The PCR products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, USA) according to the manufacturer’s instructions. Purified amplicons were quantified using QuantiFluor-ST (Promega, Madison, WI, USA) and sequenced on an Illumina Miseq platform at Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The sequences obtained were picked into operational taxonomic units by clustering 97% sequence similarity and classified at various taxonomic ranks (phylum, order, class, family and genus). 2.6. Statistical analysis Data are expressed as the mean ± SEM (standard error of the mean). Unpaired Student’s t tests and a one-way ANOVA followed by Tukey’s post hoc test were used to calculate the statistical significance between two groups and more than two groups, respectively. p < 0.05 were considered to be statistically significant. 3. Results 3.1. Mung bean supplementation attenuated body weight gain in HFDinduced mice High fat-diet is always associated with body weight (BW) gain. As expected, a significant increase in BW of mice fed with HFD was observed in comparison with the NCD group over 1 week (Fig. 1A, P < 0.001). However, mung bean supplementation significantly prevented HFD-induced BW gain from week 7 to week 12 (Fig. 1A and B, p < 0.05). Moreover, at the end of the experiment (12 weeks), mung 2
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
Fig. 1. Mung bean supplementation prevented HFD-induced obesity. (A) Changes of body weights in mice fed with NCD, HFD and HFD-CMB. (B) Body weight gain. (C) Adipose tissue weight. (D) Fat mass/body mass. (E) Food intake. (F) Energy intake. Data are shown as means ± SEM (n = 7). # p < 0.05, # #p < 0.01, # # # p < 0.001 versus NCD. *p < 0.05, **p < 0.01, ***p < 0.001 versus HFD.
(p < 0.001, Fig. 2B).
bean supplementation significantly inhibited the accumulation of body fat, including epididymal, retroperitoneal, and perirenal fat (Fig. 1C, p < 0.05), which was aligned with BW gain. Notably, the body fat ratio was also significantly decreased by mung bean supplementation (Fig. 1D, P < 0.001). In addition, there was no statistically difference in the food intake (g/d/mouse) and energy intake (kcal/d/mouse) between the HFD and HFD-CMB groups (Fig. 1E and F), suggesting that the effects of the mung bean on BW gain were not associated with the changes in food consumption.
3.3. Mung bean supplementation prevented HFD-induced liver steatosis and adipose tissue hypertrophy As shown in Fig. 3A and B, the liver tissue of NCD group was in a healthy state, showing that the structure of hepatic lobular was intact, the hepatic sinus has no pathological defects, and the nuclei were having normal morphology. Conversely, compared with the NCD group, HFD feeding caused the widening of the gap junctional channels, cell wall loosened, necrosis or death, and autolysis in hepatocytes. Nevertheless, a neat stacking of hepatic sinuses and a marked reduction of hepatocyte damage were observed in HFD-CMB group, as evidenced by the lower steatosis score (Fig. 3D, p < 0.001). Moreover, Oil Red O staining indicated that there was little lipid accumulation in the hepatocytes of HFD-CMB group (Fig. 3B). All of these results suggested that mung bean supplementation could inhibit lipid storage and prevent the liver steatosis. It is noteworthy that, compared with the NCD group, HFD feeding significantly increased the mean size of epididymal white adipocytes, but this phenomenon was partly reversed by mung bean supplementation (Fig. 3C and E).
3.2. Mung bean supplementation alleviated dyslipidemia and improved AST and ALT levels Obesity is often accompanied by hyperlipidemia. As shown in Fig. 2A, mung bean supplementation was found to significantly reduce the serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) levels of mice in HFD-CMB group in comparison with the HFD group. However, no significant difference in the serum HDL-C level was observed between the HFD-CMB and HFD groups. Notably, mung bean supplementation reduced the levels of serum AST and ALT (p < 0.05), but only significantly in ALT 3
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
Fig. 2. Mung bean supplementation lowered blood serum lipid levels, and AST and ALT. (A) Blood lipid concentration. (B) Activity of serum ALT and AST. Data are shown as means ± SEM (n = 7). # p < 0.05, # #p < 0.01, # # #p < 0.001 versus NCD. *p < 0.05, **p < 0.01, ***p < 0.001 versus HFD.
Fig. 3. Prevention of HFD-induced liver steatosis and WAT hypertrophy by mung bean supplementation. (A) H&E staining of liver tissue (40 × zoom). (B) liver Oil Red O staining (40 × zoom). (C) H&E staining of epididymal white adipose (20 × zoom). (D) steatosis score. (E) Mean adipocyte area. Data are shown as means ± SEM (n = 7). # p < 0.05, # #p < 0.01, # # #p < 0.001 versus NCD. *p < 0.05, **p < 0.01, *** p < 0.001 versus HFD.
4
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
were identified from the phyla to the genus level and were different for each group (Fig. 6A). As shown in Fig. 6B, the NCD group was characterized by the higher proportion of genus norank_f__Muribaculaceae, family Muribaculaceae, order Bacteroidales, class Bacteroidia, phylum Bacteroidetes, while, in the HFD group, the gut microbiota was enriched by a dramatic increase in genus norank_f__Lachnospiraceae, family Lachnospiraceae and Ruminococcaceae, oder Clostridiales, Class Clostridia, Phylum Firmicutes. Remarkably, the mung bean supplementation was also characterized by genus norank_f__Muribaculaceae, family Muribaculaceae, order Bacteroidales, class Bacteroidia, phylum Bacteroidetes in the HFD-CMB group, which was consistent with the NCD group. Taken together, these data suggested that mung bean supplementation could reverse the HFD-induced composition of gut microbiota.
3.4. Mung bean supplementation regulated HFD‑induced gut microbiota dysbiosis In order to evaluate the gut microbiota alterations resulted from HFD-induced obesity as well as the improved effect of mung bean supplementation on gut microbiota, the fecal bacterial composition of mice was detected by the 16S rRNA sequencing based on V3-V4 region. A total of 977,441 raw clean reads were generated from 21 mice (7 samples per group) with 46,544 reads per group on average, and lowquality sequences were removed. The number of clean tags per sample ranged from 35,035 to 59,810. The effective reads were clustered into OTUs (Operational Taxonomic Units) based on 97% similarity. Rarefaction and Shannon-Wiener curves showed that most of the bacterial diversity and richness in different samples have been covered (Supplementary Fig. S1). Interestingly, the ACE and Chao indices were significantly decreased in the fecal microbiota of the HFD group in comparison with that of the NCD group, but they were significantly restored by mung bean supplementation. However, there was no difference in the Shannon and Simpson indices between the three groups. These results suggested that mung bean supplementation could effectively prevent the decrease induced by the HFD in the richness of gut microbiota (Fig. 4A). To better comprehend the shared richness among each group, a Venn diagram analysis was performed at the OTU level (Fig. 4B). More OTUs was found between HFD-CMB and NCD samples (75) than that of HFD-CMB and HFD samples (25). In addition, the further Beta diversity analysis using principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) based on Bray-Curtis showed that there was a palpable clustering in the gut microbiota composition of different groups (Fig. 4C). These results indicated that the composition of gut microbiota changed significantly in response to mung bean supplementation. To identify specific taxa related to mung bean supplementation, relative abundance was assessed at the phylum, family, and genus level. The microbial community structure of three groups was mainly dominated by Firmicutes and Bacteroidetes at phylum level (Fig. 5A). Compared with the HFD group, mung bean supplementation significantly increased the relative abundance of Bacteroidetes and Proteobacteria in HFD-CMB group (Fig. 5B). Interestingly, in our study, mung bean supplementation significantly prevented the increase induced by HFD in the Firmicutes/Bacteroidetes (F/B) ratio (p < 0.05) (Fig. 5C). Furthermore, the genus was represented in a heat map at different levels in the three groups, showing that the dominant bacteria of HFD-CMB group and NCD group were relatively similar, but different from HFD group (Fig. 5D). To better understand the effects of the mung bean on the relative abundance of the key genera in HFD-fed mice, Welch’s t-test was used to analyze the distinguished genera based on mean proportions of 15 key genera within the three groups in the present study (Fig. 5E). In comparison with the NCD group, the relative abundance of norank_f__Muribaculaceae and Faecalibaculum were significantly decreased, but the level of Blautia, norank_f__Lachnospiraceae, unclassified_f__Lachnospiraceae, Ruminiclostridium_9, Ruminiclostridium, Bilophila were significantly increased in the HFD group (Fig. 5E). However, mung bean supplementation significantly enriched the relative abundance of unclassified_f__Lachnospiraceae, norank_f__Muribaculaceae and Lachnospiraceae_NK4A136_group, and lowered the relative abundance of Blautia, Ruminiclostridium, Ruminiclostridium_9, Mucispirillum, Bilophila, and Odoribacter, as compared with the HFD group (Fig. 5E). Notably, some changes of genera in the HFD-CMB group were restored to similar levels as that of the NCD group. Together, these results provided an explanation that the significant changes at genus level mentioned above may contribute to the beneficial effects of the mung bean against obesity. In addition, LEfSe analysis with a logarithmic LDA score threshold of three was performed to identify the specific bacteria characteristic associated with mung bean supplementation. The specific bacterial taxa
3.5. Possible relationships between gut microbiota and obesity-related indices Spearman’s correlation analysis was performed to further explore the correlation between the changes in gut microbiota composition and the obesity-related parameters. As shown in Fig. 7, the HFD enriched norank_f__Lachnospiraceae, unclassified_f__Lachnospiraceae, Ruminiclostridium_9, and Bilophila showed strongly positive correlations with obesity-related indexes. In this study, mung bean supplementation may reduce obesity and modulate the genus by promoting the relative abundance of norank_f__Muribaculaceae and lowering the relative abundance of Ruminiclostridium_9, Mucispirillum, Bilophila, and Odoribacter (Fig. 5E). 4. Discussion Legume consumption has been shown to have beneficial effects on the prevention and management of obesity-related metabolic disorders, because of their low-energy density and high nutritional properties (Marinangeli & Jones, 2012; Rebello et al., 2014; Venn et al., 2010). For mung bean, previous studies have shown that its health benefits are mainly attributed to the bioactive polyphenols, polysaccharides, and protein isolate it contains (Hou et al., 2019). The hypolipidemic and anti-obesity effects of mung bean have been well documented in clinical trials and animal studies (Kohno, Motoyama, Shigihara, Sakamoto, & Sugano, 2017; Yeap et al., 2015). However, most of the studies focused on their effects on host energy expenditure and lipid metabolism (Watanabe et al., 2016; Yao, Zhu, & Ren, 2014), but only a few have reported their impact on the biodiversity and activity of the host intestinal microbiota. To the best of our knowledge, only mung bean protein isolate has its anti-obesity effects abolished in germ-free mice, indicating that the beneficial effects of mung bean protein isolate followed a mechanism that depended on the presence of gut microbiota (Nakatani et al., 2018). Thus, in this study, it was speculated that mung bean supplementation may affect not only community biodiversity and composition but also functional attributes of the gut microbiota in HFDinduced obese mice. Moreover, these changes would have possible positive effects on host health. In the present study, it is important to note that mung bean, as a whole food, were prepared by following traditional cooking practices (soaked and cooked), thus these results may provide may provide valuable reference for the health benefits of mung bean. It was showed that daily consumption of HFD supplemented with mung bean (30%) was sufficient to resist weight gain (Fig. 1A and B) and reduce fat accumulation (Fig. 1C and D) without a significant difference in food intake and energy intake (Fig. 1E and F) as compared with the HFD group. It means that the effect of mung bean on body weight is not due to the reduced food consumption or calorie intake. As well known, HFD affects the serum levels of TC, TG, HDL-C, and LDL-C, which will lead to abnormal lipid metabolism and ultimately to dyslipidemia or non-alcoholic fatty liver. A recent double-blind, placebo-controlled clinical trial of 45 prediabetes patients showed that, after consumption of 2.5 g 5
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
Fig. 4. Effects of NCD, HFD and HFD-CMB on alpha and beta diversity of gut microbiota. (A) richness and diversity indices. (B) Venn diagram showing the OTUs shared by the mice from each group. (C) PCoA and NMDS score plot based on Bray-Curtis. Data are shown as means ± SEM (n = 7). # p < 0.05, ##p < 0.01, ### p < 0.001 versus NCD. *p < 0.05, **p < 0.01, ***p < 0.001 versus HFD.
group. Moreover, ALT and AST are the two biochemical markers normally used for early-stage assessment of liver injury. Previous studies have showed that the aqueous extract and the flavonoid fraction (vitexin and isovitexin) of the mung bean exhibited a stronger hepatoprotective activity by suppressing the accumulation of hepatic lipids in the acetaminophen-induced acute liver injury model than that of other pulses, including adzuki bean, black bean, and rice bean (Liu et al., 2014; Wu, Wang, Lin, & Chang, 2001). In addition, mung bean protein isolate was also found to significantly reduce the plasma ALT and AST concentrations in mice fed with HFD, preventing non-alcoholic fatty
mung bean protein isolates daily for 12 weeks, the triglyceride levels were significantly decreased in the subjects with hyperlipidemia, and these effects were particularly observed in obese subjects (Kohno et al., 2017). In another study, after 4 weeks of oral administration, ethanolic extracts of mung bean (1 g/kg) were found to significantly lower the triacylglycerol and cholesterol levels in KK-Ay mice by decreasing the expression of lipogenic genes (Inhae et al., 2015). Our results (Fig. 2A) are generally in agreement with these results. The mung bean supplementation significantly reduced the serum lipid levels in mice of HFDCMB group, including TC, TG, and LDL-C, in comparison with the HFD
6
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
Fig. 5. Mung bean supplementation modulated the composition of the gut microbiota. (A) Gut microbiota composition at the phylum level. (B) Percent of community abundance on phylum level. (C) F/B ratio. (D) Heatmap analysis at the genus level. (E) Mean proportions of 15 key genus in mice from different groups. Data are shown as means ± SEM (n = 7). # p < 0.05, ##p < 0.01, ###p < 0.001 versus NCD. *p < 0.05, **p < 0.01, ***p < 0.001 versus HFD.
7
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
Fig. 6. Structure and key phylotypes of gut microbiota responding to HFD and mung bean supplementation. (A) Cladogram generated from LEfSe analysis showing the relationship between taxon (the levels represent, from the inner to outer rings, phylum, class, order, family, and genus). (B) LDA scores derived from LEfSe analysis, showing the genus LDA score of > 3 (the length of the bar represents the LDA score).
Turnbaugh, Klein, & Gordon, 2006; Seganfredo et al., 2017). Mung bean supplementation restored the proportion of Bacteroides in the HFD-CMB group to a similar level to that presented in the NCD group (P < 0.05), which reportedly could help reduce the severity of obesity and type 2 diabetes (Moreno-Indias, Cardona, Tinahones, & QueipoOrtuño, 2014; Sharma et al., 2018). It was reported that the host adiposity was associated with an increase in the F/B ratio (Duca & Lam, 2014; Xue, Xie, Huang, Long, Gao, Ou, & Peng, 2016), and its elevation is considered as a hallmark of obesity-driven dysbiosis in many published studies (Murphy, Velazquez, & Herbert, 2015; Stephens, Arhire, & Covasa, 2018). Intriguingly, mung bean supplementation significantly reduced the ratio of F/B in the HFD-CMB group (Fig. 5C). In addition, many studies have demonstrated the bloom of Proteobacteria may play a positive role in leanness and metabolic health (Cho & Blaser, 2012; Moreno-Indias et al., 2014). The relative abundance of Proteobacteria in the gut microbiota community was found to be higher in the HFD-CMB group than that of HFD group. The mechanisms underlying these beneficial effects of mung bean supplementation on obesity are likely related to the alterations of gut microbiota composition, as evidenced by the reduced ratio of F/B in mice of HFD-CMB group. Moreover, the presence of specific species is inextricably linked to the presence of specific biological effects. At the genus level, we observed that the HFD-enriched genera norank_f__Lachnospiraceae, unclassified_f__Lachnospiraceae, Ruminiclostridium_9, Ruminiclostridium, and Bilophila were all positively correlated with obesity and obesity-
liver disease onset and progression (Watanabe et al., 2016). While, in this study, mung bean supplementation significantly decreased the ALT level, indicating mung bean could effectively alleviate HFD-induced liver injury to a certain extent. Those were also supported by the histological analysis, showing that hepatic steatosis and adipocyte size were significantly reduced by mung bean supplementation (Fig. 3). Overall, the interaction between nutrients may be an important explanation for mung bean in ameliorating the obesity-related metabolic disorders. Emerging evidence has revealed that gut microbiota plays an important role in the treatment of obesity (Blaser, 2017; Cox, West, & Cripps, 2015). The composition of gut microbiota can be reshaped by the interaction between dietary components and intestinal microorganisms, thus has a significant impact on host metabolism (Conlon & Bird, 2015; Rowland et al., 2018). Therefore, many dietary supplements have been used to prevent or alleviate metabolic diseases by modulating gut microbiota (Jing et al., 2018; Li et al., 2019). Here, it was found that mung bean supplementation could reverse the HFD-induced gut microbiota dysbiosis, and promote the growth of some specific bacteria. The decreases in the richness of gut microbiota in HFD-induced obese mice were fully prevented by the consumption of mung bean (Fig. 4A), with dysbiosis being one of the hallmarks of obesity (Cotillard et al., 2013). In many studies, HFD can lead to obesity-driven dysbiosis in gut microbiota by increasing the relative abundance of Firmicutes and reducing the relative abundance of Bacteroidetes (Ley,
8
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
Fig. 7. Heatmap of Spearman’s correlation analysis between gut microbiota and obesity-related indexes. The intensity of the colors represented the degree of association (red, positive correlation; green, negative correlation). Significant correlations are marked by *p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
found that the mung bean-mediated metabolic improvements were likely associated with the decreased relative abundance of undesirable bacteria and increased relative abundance of some possible beneficial bacteria in the gut microbiota community. Nevertheless, since a whole food approach was utilized in this study, the complex composition of the mung bean makes it difficult to decide which bioactive component (s) found (non-digestible carbohydrates, protein, and phenolics) are responsible for the beneficial effects on the obesity-related induces, and intestinal phenotype. Thus, the specific effects of individual bioactive components derived from mung bean on the obese phenotype and the microbial community are needed to be further explored.
related physiological markers (Fig. 7). However, mung bean supplementation significantly lowered the relative abundance of Ruminiclostridium_9, Mucispirillum, Bilophila, Blautia, Ruminiclostridium, and Odoribacter, which were enriched in the HFD group (Fig. 5E, p < 0.05). For example, the increased relative abundance of Bilophila-containing clusters is proved to be positively associated with HFD feeding (David et al., 2013; Schneeberger et al., 2015). Zhao et al. reported that a combination of quercetin and resveratrol could dramatically reduce the relative abundance of Bilophila, and then prevent the HFD-induced obesity (Zhao et al., 2017). These results indicated that the effects of mung bean were at least partially due to a decrease in the relative abundance of these obesity-related bacteria. Intriguingly, norank_f__Muribaculaceae had a strongly negative correlation with all of the obesity-related indices in the current study, and mung bean supplementation significantly promoted its relative abundance. Whether the norank_f__Muribaculaceae may play an important role in alleviating obesity and lipids metabolic disorders still needs an accurate trail to confirm the speculation. From the above-mentioned results, it was
5. Conclusions In summary, the results from this study concluded that cooked mung bean, prepared by following traditional human cooking practices at a physiologically relevant intake level, was sufficient to suppress weight gain and fat accumulation and ameliorate the serum lipid and 9
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
ALT levels in HFD-induced obesity mice. Furthermore, the reduction of obesity-related indices was strongly associated with the prevention of HFD-induced gut microbiota dysbiosis. Dietary modulation of gut microbiota by mung bean consumption might contribute to the improvement of the gastrointestinal health and eventually mediate its beneficial effects on the host. Meanwhile, it was unclear that whether cooked mung bean supplementation can still alter gut microbiota under a normal diet. Additional studies of mice consuming normal chow diet and high-fat diet, which both supplemented with cooked mung bean, will be necessary to determine whether some specific bacteria genus is significantly linked to mung bean-based diet. Collectively, the use of the cooked mung bean as a novel method of dietary incorporation or “value-added” ingredient has been beneficial in mitigating the severity of obese phenotype. However, there are still important gaps in our knowledge of the effects of special components in the mung bean on obesity and their mechanisms of anti-obesity, and which components are directly responsible for these beneficial effects on metabolism.
Duca, F. A., & Lam, T. K. T. (2014). Gut microbiota, nutrient sensing and energy balance. Diabetes, Obesity and Metabolism, 16(S1), 68–76. https://doi.org/10.1111/dom. 12340. Eichelmann, F., Schwingshackl, L., Fedirko, V., & Aleksandrova, K. (2016). Effect of plantbased diets on obesity-related inflammatory profiles: A systematic review and metaanalysis of intervention trials. Obesity Reviews, 17(11), 1067–1079. https://doi.org/ 10.1111/obr.12439. Foyer, C. H., Lam, H.-M., Nguyen, H. T., Siddique, K. H. M., Varshney, R. K., Colmer, T. D., ... Considine, M. J. (2016). Neglecting legumes has compromised human health and sustainable food production. Nature Plants, 2(8), 16112. https://doi.org/10. 1038/nplants.2016.112. Greenhill, C. (2017). Gut microbiome and serum metabolome changes. Nature Reviews Endocrinology, 13, 501. https://doi.org/10.1038/nrendo.2017.89. Hou, D., Chen, J., Ren, X., Wang, C., Diao, X., Hu, X., ... Shen, Q. (2018). A whole foxtail millet diet reduces blood pressure in subjects with mild hypertension. Journal of Cereal Science, 84, 13–19. https://doi.org/10.1016/j.jcs.2018.09.003. Hou, D., Yousaf, L., Xue, Y., Hu, J., Wu, J., Hu, X., ... Shen, Q. (2019). Mung Bean (Vigna radiata L.): bioactive polyphenols, polysaccharides, peptides, and health benefits. Nutrients, 11(6), 1238. https://doi.org/10.3390/nu11061238. Inhae, K., Seojin, C., Joung, H. T., Munji, C., Hae-Ri, W., Won, L. B., & Myoungsook, L. (2015). Effects of Mung Bean (Vigna radiata L.) Ethanol Extracts Decrease Proinflammatory Cytokine-Induced Lipogenesis in the KK-Ay Diabese Mouse Model. Journal of Medicinal Food, 18(8), 841–849. https://doi.org/10.1089/jmf.2014.3364. Jang, Y.-H., Kang, M.-J., Choe, E.-O., Shin, M., & Kim, J.-I. (2014). Mung bean coat ameliorates hyperglycemia and the antioxidant status in type 2 diabetic db/db mice. Food Science and Biotechnology, 23(1), 247–252. https://doi.org/10.1007/s10068014-0034-3. Jing, C., Wen, Z., Zou, P., Yuan, Y., Jing, W., Li, Y., & Zhang, C. (2018). Consumption of Black Legumes Glycine soja and Glycine max Lowers Serum Lipids and Alters the Gut Microbiome Profile in Mice Fed a High-Fat Diet. Journal of Agricultural and Food Chemistry, 66(28), 7367–7375. https://doi.org/10.1021/acs.jafc.8b02016. Ketha, K., & Gudipati, M. (2018). Immunomodulatory activity of non starch polysaccharides isolated from green gram (Vigna radiata). Food Research International, 113, 269–276. https://doi.org/10.1016/j.foodres.2018.07.010. Kohno, M., Motoyama, T., Shigihara, Y., Sakamoto, M., & Sugano, H. (2017). Improvement of glucose metabolism via mung bean protein consumption: A clinical trial of GLUCODIA TM isolated mung bean protein in Japan. Functional Foods in Health & Disease, 7, 115–134. https://doi.org/10.31989/ffhd.v7i2.320. Ley, R. E., Turnbaugh, P. J., Klein, S., & Gordon, J. I. (2006). Human gut microbes associated with obesity. Nature, 444(7122), 1022–1023. https://doi.org/10.1038/ 4441022a. Li, G.-H., Shi, Y.-H., Liu, H., & Le, G.-W. (2006). Antihypertensive effect of alcalase generated mung bean protein hydrolysates in spontaneously hypertensive rats. European Food Research and Technology, 222(5), 733–736. https://doi.org/10.1007/ s00217-005-0147-2. Li, S., Yu, W., Guan, X., Huang, K., Liu, J., Liu, D., & Duan, R. (2019). Effects of millet whole grain supplementation on the lipid profile and gut bacteria in rats fed with high-fat diet. Journal of Functional Foods, 59, 49–59. https://doi.org/10.1016/j.jff. 2019.05.030. Liu, T., Yu, X. H., Gao, E. Z., Liu, X. N., Sun, L. J., Li, H. L., ... Yu, Z. G. (2014). Hepatoprotective Effect of Active Constituents Isolated from Mung Beans (Phaseolus radiatus L.) in an Alcohol-Induced Liver Injury Mouse Model. Journal of Food Biochemistry, 38(5), 453–459. https://doi.org/10.1111/jfbc.12083. Lopes, L. A. R., Martins, M. D. C. d. C. e., Farias, L. M. d., Brito, A. K. d. S., Lima, G. D. M., Carvalho, V. B. L. d., . . . Frota, K. D. M. G. (2018). Cholesterol-Lowering and LiverProtective Effects of Cooked and Germinated Mung Beans (Vigna radiata L.). Nutrients, 10(7), 821. doi: https://doi.org/10.3390/nu10070821. Marinangeli, C. P. F., & Jones, P. J. H. (2012). Pulse grain consumption and obesity: Effects on energy expenditure, substrate oxidation, body composition, fat deposition and satiety. British Journal of Nutrition, 108(S1), S46–S51. https://doi.org/10.1017/ S0007114512000773. Mohamed, S. (2014). Functional foods against metabolic syndrome (obesity, diabetes, hypertension and dyslipidemia) and cardiovasular disease. Trends in Food Science & Technology, 35(2), 114–128. https://doi.org/10.1016/j.tifs.2013.11.001. Moreno-Indias, I., Cardona, F., Tinahones, F. J., & Queipo-Ortuño, M. I. (2014). Impact of the gut microbiota on the development of obesity and type 2 diabetes mellitus. Frontiers in Microbiology, 5(190), https://doi.org/10.3389/fmicb.2014.00190. Murphy, E. A., Velazquez, K. T., & Herbert, K. M. (2015). Influence of high-fat diet on gut microbiota: A driving force for chronic disease risk. Current Opinion in Clinical Nutrition and Metabolic Care, 18(5), 515–520. https://doi.org/10.1097/MCO. 0000000000000209. Nair, R. M., Yang, R.-Y., Easdown, W. J., Thavarajah, D., Thavarajah, P., Hughes, J. d. A., & Keatinge, J. (2013). Biofortification of mungbean (Vigna radiata) as a whole food to enhance human health. Journal of the Science of Food and Agriculture, 93(8), 1805-1813. doi: doi:10.1002/jsfa.6110. Nakamura, K., Koyama, M., Ishida, R., Kitahara, T., Nakajima, T., & Aoyama, T. (2016). Characterization of bioactive agents in five types of marketed sprouts and comparison of their antihypertensive, antihyperlipidemic, and antidiabetic effects in fructoseloaded SHRs. Journal of Food Science and Technology, 53(1), 581–590. https://doi. org/10.1007/s13197-015-2048-0. Nakatani, A., Li, X., Miyamoto, J., Igarashi, M., Watanabe, H., Sutou, A., ... Kimura, I. (2018). Dietary mung bean protein reduces high-fat diet-induced weight gain by modulating host bile acid metabolism in a gut microbiota-dependent manner. Biochemical and Biophysical Research Communications, 501(4), 955–961. https://doi. org/10.1016/j.bbrc.2018.05.090. Razzoli, M., Pearson, C., Crow, S., & Bartolomucci, A. (2017). Stress, overeating, and
6. Ethics statement All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The animal experiment was approved by the Institutional Animal Care and Use Committees of China Agricultural University (Beijing, China) with the approval number AW08089102-1-4 and was carried out in accordance with the National Research Council Guidelines. Funding This work was supported by National Key Research and Development Program of China (2017YFD0401202). Declaration of Competing Interest The authors declare that they have no conflict of interest. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.103687. References Andersson, K. E., Chawade, A., Thuresson, N., Rascon, A., Öste, R., Sterner, O., ... Hellstrand, P. (2017). Wholegrain oat diet changes the expression of genes associated with intestinal bile acid transport. Molecular Nutrition & Food Research, 61(7), 1600874. https://doi.org/10.1002/mnfr.201600874. Bianchi, F., Duque, A. L. R. F., Saad, S. M. I., & Sivieri, K. (2019). Gut microbiome approaches to treat obesity in humans. Applied Microbiology and Biotechnology, 103(3), 1081–1094. https://doi.org/10.1007/s00253-018-9570-8. Blaser, M. J. (2017). The theory of disappearing microbiota and the epidemics of chronic diseases. Nature Reviews Immunology, 17, 461. https://doi.org/10.1038/nri.2017.77. Chang, C.-J., Lin, C.-S., Lu, C.-C., Martel, J., Ko, Y.-F., Ojcius, D. M., ... Lai, H.-C. (2015). Ganoderma lucidum reduces obesity in mice by modulating the composition of the gut microbiota. Nature Communications, 6, 7489. https://doi.org/10.1038/ ncomms8489 https://www.nature.com/articles/ncomms8489#supplementaryinformation. Cho, I., & Blaser, M. J. (2012). The human microbiome: At the interface of health and disease. Nature Reviews Genetics, 13, 260. https://doi.org/10.1038/nrg3182. Conlon, M. A., & Bird, A. R. (2015). The impact of diet and lifestyle on Gut Microbiota and human health. Nutrients, 7(1), 17–44. https://doi.org/10.3390/nu7010017. Cotillard, A., Kennedy, S. P., Kong, L. C., Prifti, E., Pons, N., Le Chatelier, E., ... Layec, S. (2013). Dietary intervention impact on gut microbial gene richness. Nature, 500, 585. https://doi.org/10.1038/nature12480 https://www.nature.com/articles/ nature12480#supplementary-information. Cox, A. J., West, N. P., & Cripps, A. W. (2015). Obesity, inflammation, and the gut microbiota. The Lancet Diabetes & Endocrinology, 3(3), 207–215. https://doi.org/10. 1016/S2213-8587(14)70134-2. David, L. A., Maurice, C. F., Carmody, R. N., Gootenberg, D. B., Button, J. E., Wolfe, B. E., ... Turnbaugh, P. J. (2013). Diet rapidly and reproducibly alters the human gut microbiome. Nature, 505, 559. https://doi.org/10.1038/nature12820 https://www. nature.com/articles/nature12820#supplementary-information.
10
Journal of Functional Foods xxx (xxxx) xxxx
D. Hou, et al.
Zonja, B., ... Andres-Lacueva, C. (2016). Biomarkers of Morbid obesity and prediabetes by metabolomic profiling of human discordant phenotypes. Clinica Chimica Acta, 463, 53–61. https://doi.org/10.1016/j.cca.2016.10.005. Venn, B. J., Perry, T., Green, T. J., Skeaff, C. M., Aitken, W., Moore, N. J., ... Williams, S. (2010). The effect of increasing consumption of pulses and Wholegrains in Obese People: A randomized controlled trial. Journal of the American College of Nutrition, 29(4), 365–372. https://doi.org/10.1080/07315724.2010.10719853. Wang, J., Wang, P., Li, D., Hu, X., & Chen, F. (2019). Beneficial effects of ginger on prevention of obesity through modulation of gut microbiota in mice. European Journal of Nutrition. https://doi.org/10.1007/s00394-019-01938-1. Watanabe, H., Inaba, Y., Kimura, K., Asahara, S.-I., Kido, Y., Matsumoto, M., ... Inoue, H. (2016). Dietary Mung Bean Protein Reduces Hepatic Steatosis, Fibrosis, and Inflammation in Male Mice with Diet-Induced, Nonalcoholic Fatty Liver Disease. The Journal of Nutrition, 147(1), 52–60. https://doi.org/10.3945/jn.116.231662. Wu, S. J., Wang, J. S., Lin, C. C., & Chang, C. H. (2001). Evaluation of hepatoprotective activity of Legumes. Phytomedicine, 8(3), 213–219. https://doi.org/10.1078/09447113-00033. Xie, J., Du, M., Shen, M., Wu, T., & Lin, L. (2019). Physico-chemical properties, antioxidant activities and angiotensin-I converting enzyme inhibitory of protein hydrolysates from Mung bean (Vigna radiate). Food Chemistry, 270, 243–250. https://doi. org/10.1016/j.foodchem.2018.07.103. Xue, B., Xie, J., Huang, J., Long, C., Gao, L., Ou, S., . . . Peng, X. (2016). Plant Polyphenols Altering a Pathway of Energy Metabolism by Inhibiting Fecal Bacteriodetes and Firmicutes In Vitro. Food & Function, 7(3), 10.1039.C1035FO01438G. doi: https:// doi.org/10.1039/C5FO01438G. Yao, Y., Zhu, Y., & Ren, G. (2014). Mung bean protein increases plasma cholesterol by upregulation of hepatic HMG-CoA Reductase, and CYP7A1 in mRNA Levels. Journal of Food and Nutrition Research, 2(11), 770–775. https://doi.org/10.12691/jfnr-2-11-2. Yeap, S. K., Beh, B. K., Ho, W. Y., Mohd Yusof, H., Mohamad, N. E., Ali, N. M., ... Long, K. (2015). In vivo antioxidant and hypolipidemic effects of fermented mung bean on hypercholesterolemic mice. Evidence-based Complementary and Alternative Medicine: eCAM, 2015, 508029. https://doi.org/10.1155/2015/508029. Zhao, L., Zhang, Q., Ma, W., Tian, F., Shen, H., & Zhou, M. (2017). A combination of quercetin and resveratrol reduces obesity in high-fat diet-fed rats by modulation of gut microbiota. Food & Function, 8(12), 4644–4656. https://doi.org/10.1039/ c7fo01383c.
obesity: Insights from human studies and preclinical models. Neuroscience & Biobehavioral Reviews, 76, 154–162. https://doi.org/10.1016/j.neubiorev.2017.01. 026. Rebello, C. J., Greenway, F. L., & Finley, J. W. (2014). A review of the nutritional value of legumes and their effects on obesity and its related co-morbidities. Obesity Reviews, 15(5), 392–407. https://doi.org/10.1111/obr.12144. Rowland, I., Gibson, G., Heinken, A., Scott, K., Swann, J., Thiele, I., & Tuohy, K. (2018). Gut microbiota functions: Metabolism of nutrients and other food components. European Journal of Nutrition, 57(1), 1–24. https://doi.org/10.1007/s00394-0171445-8. Saad, M. J. A., Santos, A., & Prada, P. O. (2016). Linking Gut microbiota and inflammation to obesity and insulin resistance. Physiology, 31(4), 283–293. https://doi. org/10.1152/physiol.00041.2015. Schneeberger, M., Everard, A., Gómez-Valadés, A. G., Matamoros, S., Ramírez, S., Delzenne, N. M., ... Cani, P. D. (2015). Akkermansia muciniphila inversely correlates with the onset of inflammation, altered adipose tissue metabolism and metabolic disorders during obesity in mice. Scientific Reports, 5, 16643. https://doi.org/10. 1038/srep16643. Seganfredo, F. B., Blume, C. A., Moehlecke, M., Giongo, A., Casagrande, D. S., Spolidoro, J. V. N., ... Mottin, C. C. (2017). Weight-loss interventions and gut microbiota changes in overweight and obese patients: A systematic review. Obesity Reviews, 18(8), 832–851. https://doi.org/10.1111/obr.12541. Sharma, V., Smolin, J., Nayak, J., Ayala, J. E., Scott, D. A., Peterson, S. N., & Freeze, H. H. (2018). Mannose Alters Gut Microbiome, Prevents Diet-Induced Obesity, and Improves Host Metabolism. Cell Reports, 24(12), 3087–3098. https://doi.org/10. 1016/j.celrep.2018.08.064. Stephens, R. W., Arhire, L., & Covasa, M. (2018). Gut Microbiota: From microorganisms to metabolic organ influencing obesity. Obesity, 26(5), 801–809. https://doi.org/10. 1002/oby.22179. Swee Keong, Y., Hamidah, M. Y., Nurul Elyani, M., Boon Kee, B., Yong, H. W., Norlaily Mohd, A., . . . Kamariah, L. (2013). In Vivo Immunomodulation and Lipid Peroxidation Activities Contributed to Chemoprevention Effects of Fermented Mung Bean against Breast Cancer. Evidence-Based Complementray and Alternative Medicine,2013,(2013-4-22), 2013(2013), 708464. doi: http://dx.doi.org/10.1155/ 2013/708464. Tulipani, S., Palau-Rodriguez, M., Miñarro Alonso, A., Cardona, F., Marco-Ramell, A.,
11