Effects of Î±-Galactooligosaccharides from Chickpeas on High-Fat-Diet

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Effects of a-Galactooligosaccharides from Chickpeas on High Fat Diet Induced Metabolic Syndrome in Mice Zhuqing Dai, Wanyong Lyu, Minhao Xie, Qingxia Yuan Yuan, Hong Ye, Bing Hu, Li Zhou, and Xiaoxiong Zeng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00489 • Publication Date (Web): 31 Mar 2017 Downloaded from http://pubs.acs.org on April 1, 2017

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Journal of Agricultural and Food Chemistry

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Effects of -Galactooligosaccharides from Chickpeas on High Fat Diet Induced

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Metabolic Syndrome in Mice

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Running title: Effects of Chickpea -GOS on Metabolic Syndrome in Mice

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Zhuqing Dai,† Wanyong Lyu,‡ Minhao Xie,† Qingxia Yuan,† Hong Ye,† Bing Hu,† Li

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Zhou,† Xiaoxiong Zeng†,*

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†

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210095, People’s Republic of China

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‡

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Beijing 100050, People’s Republic of China

College of Food Science and Technology, Nanjing Agricultural University, Nanjing

Nutrition and Food Branch of China Association of Gerontology and Geriatrics,



*To whom correspondence should be addressed. Tel & Fax: +86 25 84396791; E-mail: [email protected] (X. Zeng). 1

ACS Paragon Plus Environment


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ABSTRACT

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The gut microbiota has the ability to modulate host energy homeostasis, which may

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regulate metabolic disorders. Functional oligosaccharide may positively regulate the

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intestinal microbiota. Therefore, effects of -galactooligosaccharides (-GOS) from

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chickpea on high-fat diet (HFD) induced metabolic syndrome and gut bacterial

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dysbiosis were investigated. After 6-week intervention, HFD led to significant

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increases in levels of blood glucose, total cholesterol, triglyceride, glycated serum

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protein, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol

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of mice compared with normal chow fed. Meanwhile, all the -GOS treated groups

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significantly decreased above parameters compared with HFD group. HFD could

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significantly decrease the content of all bacteria especially Bacteroides (9.82 ± 0.09

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vs 10.3 ± 0.10, p < 0.05) and Lactobacilli (6.67 ± 0.18 vs 7.30 ± 0.24, p < 0.05), and

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the decrease in production of short-chain fatty acids was also observed. Treatment

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with -GOS significantly increased the number of Bifidobacteria (6.07 ± 0.23 of low

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dose treatment vs 5.65 ± 0.20 of HFD group) and Lactobacilli (7.22 ± 0.16 of low

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dose treatment). It also significantly promoted the secretion of propionic and butyric

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acids. These results indicate that -GOS from chickpeas may affect the metabolic

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disorders and gut bacterial ecosystem in a positive way.

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KEYWORDS: Metabolic syndrome; -Galactooligosaccharide; Chickpea; Gut

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microbiota; Short-chain fatty acid

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INTRODUCTION

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Metabolic syndrome is series of diseases including central obesity, hyperglycemia,

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insulin resistance, hyperlipidemia and hypertension.1 Humans are facing a devastating

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epidemic of metabolic syndrome because of the growing unhealthy diet. It has been

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reported that high-fat diet (HFD) induced obesity can cause oxidative damage,

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inflammatory response and metabolic endotoxemia, leading to various chronic

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metabolic abnormalities such as type 2 diabetes, carcinogenesis and cardiovascular

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disease.2,3 The mechanism of metabolic syndrome is a complex issue. Genetic,

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environmental and dietary factors are all considered to be the main elements in the

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development of metabolic syndrome. However, accumulating evidence suggests that

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the shift of gut microflora induced by HFD is a key factor in the development of

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obesity, insulin resistance and other indicators of metabolic syndrome.4-6 Reduction of

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beneficial bacteria and increase of pathogenic bacteria are consistently related to the

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development of obesity, systemic inflammation and metabolic comorbidity in both

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humans and rodents.7-9

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Galactooligosaccharides (GOS), formed by one to ten galactosyl moieties linked

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to a terminal glucose, or by exclusively galactosyl units (galactobiose, galactotriose,

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etc.), are considered to be one of the most common prebiotics.10,11 GOS plays a

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significant role in human health; it is considered to be utilized by gut microbiota and

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modify the composition of intestinal microbes.12 For example, GOS has been reported

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to promote the proliferation of beneficial bacteria (Bifidobacteria and Lactobacilli)

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and suppress the growth of pathogenic and putrefactive bacteria.13-15 In addition to

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being prebiotics, GOS exhibits other functions such as preventing constipation, 3



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reducing the level of blood total cholesterol (TC), improving mineral absorption and

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controlling some acute or chronic diseases.16-19

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Chickpea (Cicer arietinum L.), one of the most important pulse crops, is grown

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and consumed in a wide area, especially in the Asian and African countries. It

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accounts for a substantial proportion of human dietary nitrogen intake. Moreover, the

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nutritional values and health benefits of chickpeas also attract more and more

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attentions nowadays.20-22 It is not only a good source of protein, but also rich-in

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carbohydrates and fibers. The carbohydrates of chickpea consist of mono-, di-, oligo-

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and polysaccharides, and the amount of -GOS (based on dry mass) is around 10.4 to

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17.0% for different species of chickpeas.23,24 Ciceritol and stachyose are the most

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abundant -GOS in chickpeas, taking up 36-43% and 25% of the content of total

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soluble sugars in chickpea seeds, respectively. This makes chickpeas become a great

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source of -GOS. Recently, low-digestible carbohydrates have been widely

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demonstrated to be effective prebiotic factor. They are highly selective for the growth

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of Bifidobacterium and Lactobacilli, which stimulates the secretion of short-chain

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fatty acids (SCFAs) and promote alleviation of metabolic and inflammatory

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disorders.25-27 However, direct evidence of -GOS from chickpeas in preventing

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metabolic syndrome is still limited. The purpose of this study, therefore, was to

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evaluate the effects of -GOS from chickpea on HFD-induced metabolic syndrome.

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MATERIALS AND METHODS

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Chemicals and Diets. Chickpea seeds were obtained from Xinjiang Agricultural

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University (Urumchi, Xinjiang Uygur Autonomous Region of China). Normal chow 4


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(NC, 10% kcal from fat and total 3.85 kcal/g) and HFD (45% kcal from fat and total

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5.21 kcal/g) were purchased from Jiangsu Xietong Organism Co., Ltd. (Nanjing,

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China). Rat/mouse insulin ELISA kit, glycosylated serum protein assay kit, TC assay

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kit, triglyceride (TG) assay kit, high-density lipoprotein cholesterol (HDL-C) assay kit,

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low-density lipoprotein cholesterol (LDL-C) assay kit and uric acid assay kit were

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purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Other

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chemicals and reagents were obtained from Sigma-Aldrich Chemical Co., Ltd. (St.

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Louis, MO, USA) unless otherwise stated.

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Preparation of-GOS from Chickpea. -GOS from chickpea was extracted and

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partially purified according to previous method with some modifications.28 The

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powder of the dried chickpeas was soaked with 100% petroleum ether for 24 h at

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ambient temperature. The defatted residue was filtrated and extracted with 50% (v/v)

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aqueous ethanol twice by stirring at 50 ℃ for 2 h. The extracts were combined and

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centrifuged at 4000 rpm for 15 min, and the supernatant was concentrated by rotary

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evaporation (Heidolph Instruments, Schwabach of Germany). The concentrated

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solution was stirred with 30 g activated Charcoal-Celite (1:1, w/w) for 30 min. The

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mixture was then washed with 8% (v/v) aqueous ethanol (to remove mono- and

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disaccharides) and 50% aqueous ethanol, the latter elute was concentrated and

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freeze-dried to yield -GOS as a fine white powder. Agilent 1100 series HPLC system

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(Agilent Technologies, Santa Clara, CA, USA) equipped with a refraction index

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detector (RID) was used to analyze the composition of -GOS. The analysis was done

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with a Sugar-D column (4.6 × 250 mm, Nacalai Tesque Inc., Kyoto, Japan) eluted

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with mobile phase of acetonitrile-water (75:25, v/v) at a flow rate of 1.0 mL/min, and

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the sugars were identified by comparing the retention times with those of standard

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ones.

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Animal Trial. Fifty six-week old male CD-1® (ICR) IGS mice (body weight 26.2 ±

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3.4 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd.

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(Beijing, China). All the experiments were approved by the Medical Research

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Committee on Animal Care and Use, Disease Control Centre of Jiangsu Province

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(SCXK2013−0005), China. The mice were maintained in cages (5 mice per cage) in a

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controlled environment (temperature 24-25 ℃, humidity 50-55%, 12 h light-dark

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cycles) and free to access food and water. They were randomly divided into five

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groups (10 mice for each group). One group was fed NC as blank control, one group

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was fed HFD as model control, and the other three groups were fed with HFD, as well

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as 0.083 g/d/kg (Low dose treatment, HFD + LDT), 0.42 g/d/kg (Medium dose

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treatment, HFD + MDT) and 0.83 g/d/kg (High dose treatment, HFD + HDT) -GOS

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in drinking water, respectively. During the experiment, body weight of mice, food

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consumption and water consumption were recorded weekly. Fresh feces were

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collected every two weeks and stored at -80 ℃. After 6 weeks of treatment, mice

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were fasted overnight (from 9:00 pm to 9:00 am), and the cage bedding was changed

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at 9:00 pm to avoid coprophagy. After chloral hydrate (200 mg/kg body weight)

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anesthesia, blood was collected by eyeball enucleating. Liver, spleen, kidney, and

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colon were quickly removed and stored at -80 ℃ until further analysis. Total cecum

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and colon contents were also collected, immediately frozen and stored at -80 ℃ until

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further analysis.

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Biochemical Analysis. Blood glucose levels were determined before sacrifice by

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using Ascensia contour blood glucose meter (Bayer Healthcare LLC, Mishawaka, IN,

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USA). Serum insulin level was measured using a rat/mouse insulin ELISA kit and

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insulin resistance index was calculated according to the homeostasis assessment

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model (HOMA-IR).29 Glycated serum protein level was measured by using a

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glycosylated serum protein assay kit. Serum TC and TG levels were measured by a

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TC assay kit and a TG assay kit, respectively. Serum HDL-C and LDL-C levels were

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measured using a HDL cholesterol assay kit and a LDL cholesterol assay kit,

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respectively. Uric acid levels were measured using a uric acid assay kit.

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Histology Analysis. The liver and colon tissues were cut into pieces (0.5-cm3),

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washed with saline and placed in a labeled cassette. The tissues were then fixed in

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12% formaldehyde solution for 24 h, and the residual fixative was washed away with

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distilled water. These tissues were then processed by using 30, 50, 70, 80, 90, 95 and

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100% ethanol for dehydration, respectively. After embedded in paraffin (BMJ-III

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embedding machine, Changzhou Electronic Instrument Factory, Jiangsu, China), the

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tissues were cut into 5-m thick sections by RM2235 microtome (Leica, Heidelberg,

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Germany). Hematoxylin and eosin (H&E) staining method was applied to stain the

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tissues, and two H&E-stained sections per liver were used for observation of steatosis

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with a microscope at 40× magnification. The histology was evaluated based on four

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parameters: macrovesicular steatosis, microvesicular steatosis,

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hypertrophy and inflammation.

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hepatocellular


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Analysis of SCFAs. Luminal samples were weighted and suspended in sufficient

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volume (600 L) of acidified water (pH 1-2) by vortex. After kept still at room

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temperature for 10 min and filtrated with 0.2 m nylon filter (Millipore Millex-GN),

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the extract was centrifuged at 6000 rpm for 20 min. The contents of SCFAs were then

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analyzed by using an Agilent 1100 series HPLC system with a diode array detector

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(DAD) set at 210 nm. Chromatographic separation of acetic, propionic and butyric

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acids was achieved at 30 ℃ using a Beckman Ultrasphere C18 column (4.6 × 250

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mm, 5 m particle size). The mobile phase was composed of KH2PO4 solution (phase

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A, 20 mM and pH 2.5) and methanol (phase B) with a gradient elution as follows:

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0-16 min, 5% B; 16-30 min, B from 5 to 30%; 30-40 min, 30% B. The flow rate was

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set at 0.8 mL/min, and injection volume was 20 L. Three biological replicates were

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processed in each group analyzed.

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Quantitative PCR (qPCR) Analysis. In the present study, qPCR was used to

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evaluate the effects of -GOS on microbial composition after 6-week treatment in

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comparison with control. Briefly, total DNA was extracted from fresh fecal sample

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(200 mg) by using a TIANamp Stool DNA kit (Tiangen Biotechnology Co., Ltd.,

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Beijing, China) according to the manufacturer’s instructions, and the resulting DNA

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samples were stored at -80 ℃ . The concentration of DNA was assessed

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spectrophotometrically by using a NanoDrop 2000 spectrophotometer (NanoDrop

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Technologies, Wilmington, DE, USA). Different microbial groups including total

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bacteria, Bacteroides, Lactobacilli, Bifidobacteria, Eubacterium rectale/Clostridium

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coccoides, and Clostridium leptum were distinguished and quantified by qPCR

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(Applied Biosystems® 7500 Real-Time PCR System (ABI Co., Ltd., USA)). The 16S

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rRNA gene targeted group-specific primers used in this study are listed in Table 1.

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The reaction mixture (20 L) comprised 10 L of SYBR Premix Ex (Takara

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Biotechnology Co., Ltd., Dalian, China), 0.4 L of ROX Reference Ⅱ, 0.4 L of each

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of the specific primers (10 M; Sangon Biotechnology Co., Ltd., Shanghai, China),

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6.8 L of sterile distilled water, and 2 L of DNA template. The PCR reaction was

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initiated by a 5 min activation at 95 ℃, followed by 40 cycles at 95 ℃ for 10 s, and

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60 ℃ for 35 s to anneal the primer and elongate the product. The sample DNA copy

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number was calculated by absolute quantification. For standard curves, a series of 10

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times gradient dilutions of the standard products were used and at least six nonzero

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standard concentrations per assay were applied. The concentration of each bacterium

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was expressed as log10 copy number. Each reaction was carried out in triplicate.

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Statistical Analysis. Statistical analysis was performed using SPSS software version

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15.0. The data were presented as mean ± SD and analyzed using one-way ANOVA

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followed by Tukey’s post hoc test to compare multiple groups and Student’s t-test to

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determine the differences in 2 groups. The level of statistical significance was set at p

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< 0.05.

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RESULTS

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Effect of -GOS on Body Weight. As shown in Figure 1A, the soluble sugars

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extracted from chickpea were effectively separated by HPLC. According to the

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retention times of standard sugars, the main soluble sugars in extract were fructose,

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sucrose, raffinose, ciceritol and stachyose, which is consistence with the previous 9



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reports.30-32 After purification, most of the fructose and sucrose were removed, and the

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resulting -GOS was mainly composed of 15.0% raffinose, 25.1% ciceritol and

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51.5% stachyose.

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Throughout the animal experiment, there were no significant differences in food

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and water consumption among the five groups (data not shown). After 6-week

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treatment, mice fed with HFD had higher body weight compared with those fed with

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NC (Figure 2). The growth of body weight of NC, HFD, HFD + LDT, HFD + MDT,

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HFD + HDT was 30.0, 36.5, 24.8, 34.6 and 32.2%, respectively. Mice fed with

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-GOS showed lower increase in body weight compared with HFD group, however,

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there were no significant differences on the body weight among these four groups (p >

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0.05).

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Effects of -GOS on Blood Glucose Related Index. As shown in Figure 3A and

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3B, HFD fed mice showed significant increases in blood glucose level and glycated

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serum protein level (p < 0.05) after 6-week dietary intervention. Compared with HFD

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group, the three -GOS treatments reduced blood glucose level by 38.1, 28.2 and

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34.0%, respectively. Among them, HFD + LDT and HFD + HDT showed significant

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differences (p < 0.05). Reduced glycated serum protein levels were also observed for

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-GOS treatments, however, there was no significant difference compared with HFD

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group. The insulin level showed no significant change for mice fed with HFD (Figure

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3C), while HOMA-IR significantly increased for HFD group (Figure 3D). All the

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-GOS treatments reduced HOMA-IR compared with HFD group, but there were no

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statistical differences when analyzed by Tukey’s post hoc test. For the three

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treatments of -GOS, HFD + LDT showed better effects on reducing blood glucose

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level and glycated serum protein level, while HFD + HDT showed better effect on

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reducing HOMA-IR.

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Effects of -GOS on Serum Lipid. The serum concentrations of TC, TG, HDL-C

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and LDL-C were measured at the end of the animal experiment and the results are

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shown in Table 2. All the four serum lipid related indexes for HFD fed mice were

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significantly higher than those for NC fed mice (p < 0.05). -GOS treatments

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significantly decreased the elevation in serum TC, TG, HDL-C and LDL-C (p < 0.05).

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The three treatments of -GOS even lowered TG and HDL-C levels to normal ones,

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which showed no significant difference with those for NC group. Serum TG

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concentration was significant lower (p < 0.05) following administration of HFD +

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LDT than that of HFD + MDT.

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Liver Steatosis. The histopathology of liver sections is shown in Figure 4. For

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HFD fed mice, increased lipid deposition in the livers was observed, the cytoplasm of

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the centrilobular hepatocytes showed microvesicular steatosis with numerous of small

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lipid droplets, as well as macrovesicular steatosis with large lipid droplets. In addition,

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the clusters of inflammatory cells were observed in the livers of HFD group. For HFD

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+ LDT and HFD + MDT groups, the livers showed slight fatty degeneration, which

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the average percentages of microvesicular steatosis and macrovesicular steatosis were

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less than 20%. Several mice from these groups showed a markedly attenuated degree

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of hepatic steatosis compared to mice from HFD group, with the fatty change

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primarily consisting of the widely scattered large lipid droplets in the central leaflet

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region and little evidence of microvesicular involvement. For HFD + HDT, the livers

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showed almost none fatty degeneration, with none appearance of steatosis nor

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inflammation. Thus, the treatment of-GOS significantly reduced the incidence of

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fatty liver.

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Effects of -GOS on SCFAs. The concentrations of acetic, lactic, propionic and

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butyric acids and total SCFAs amount were reduced after HFD feeding (Table 3).

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Supplementation of -GOS prevented the decrease in total SCFAs, and even

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promoted the secretion of SCFAs. When compared to NC and HFD groups, all the

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three treatments of -GOS significantly increased the concentrations of propionic and

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butyric acids (p < 0.05). High dose of -GOS also significantly increased the

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secretion of acetic and lactic acids, while HFD + LDT and HFD + MDT did not.

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When compared the three doses of treatments, the promoting effect of -GOS on

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secretion of SCFAs showed a dose-dependent relationship.

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Effects of -GOS on Gut Microbiota. After a period of 6-week treatment, HFD

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feeding significantly decreased gut total bacterial quantity (p < 0.05) and altered the

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composition of gut microbiota (Table 4). Briefly, HFD significantly decreased the

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amount of Bacteroides, Lactobacilli and C. leptum groups compared with NC group,

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while no significant differences were observed in the number of Bifidobacteria and E.

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rectale/C. coccoides groups between HFD group and NC group. Compared with HFD

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group, the -GOS diet significantly stimulated (p < 0.05) the growth of Bifidobacteria

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and Lactobacilli. The numbers of Bacteroides, E. rectale/C. coccoides and C. leptum

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groups did not show significant changes (p > 0.05) between HFD group and -GOS

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groups. High dose of -GOS treatment significantly increased the proliferation of

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total bacteria compared with HFD group, mainly due to the stimulation of

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Bifidobacteria, Lactobacilli and C. leptum groups.

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DISCUSSION

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HFD feeding resulted in weight gain, high levels of blood glucose and insulin

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resistance, TC and TG. It also led to hepatic steatosis and changes of selected fecal

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microbial abundances and SCFAs. These results are similar to some reports that

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diet-induced obesity is shown to be correlated with the alteration of gut microbiota.

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For example, HFD reduced the amount of Bifidobacteria and Lactobacilli in a study

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conducted by Singh et al. 33 Daniel et al. found that HFD caused changes of the

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diversity of dominant gut bacteria, decreased the amount of Ruminococcaceae and

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increased the amount of Rikenellaceae.34 After -GOS intervention, significant

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increases of Bifidobacteria and Lactobacilli were observed. It is believed that

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Bifidobacteria and Lactobacilli play a protective role against pathogens by producing

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antimicrobial agents and/or blocking of adhesion of pathogens. In this way, they were

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thought to protect gut integrity and to regulate the host metabolism. Besides,

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increased levels of SCFAs were observed in -GOS fed mice. SCFAs are produced

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when dietary fiber is fermented in the colon which supplies the host with an additional

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amount of energy. Two-third of the energy supply for normal colonic epithelia was

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from SCFAs, particularly butyric acid. The alteration of the microbial cross feeding

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patterns and promotion of microflora might also cause high levels of fecal SCFAs.35

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Because of its non-digestible characteristic in gastrointestinal, GOS has been shown 13



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to be an excellent dietary source for production of SCFAs and health-promoting

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bacteria such as Bifidobacteria and Lactobacilli.10 The present study also

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demonstrated the positive effects.

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Administration of prebiotics is helpful in lowering hepatic steatosis, colon

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inflammation, level of blood glucose and insulin concentration, which has been

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demonstrated by animal experiments and human trials.1,36,37 In the present study,

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significantly lower concentrations of blood lipid, blood glucose and serum insulin

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were detected after the administration of -GOS. Liver histopathology also showed

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that treatment of -GOS inhibited the microvesicular steatosis and macrovesicular

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steatosis. The results are consistent with the proliferation of prebiotics.

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The exact mechanisms by which GOS exert its protective effect on metabolic

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syndrome are still not fully understood. However, some studies have demonstrated

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that the promotion of gut microbiota might play an important role. Oligosaccharides

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are metabolized by the colonic microbiota and fermented to produce SCFAs, mainly

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including acetic, propionic and butyric acids. SCFAs are absorbed in the colon, where

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butyric acid provides energy for colonic epithelial cells. Acetic and propionic acids

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are the substrates for gluconeogenesis and lipogenesis in liver and peripheral organs.

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Except as energy sources, SCFAs have the ability to regulate colonic gene expression

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by inhibiting histone deacetylase and modulating the signaling pathway through

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G-protein-coupled receptors (GPRs) such as GPR41 or GPR43.27,38 In another aspect,

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GOS may promote mucosal barrier function by directly stimulating the intestinal

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goblet cells.39 Other oligosaccharides like human milk oligosaccharides and bovine

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colostrum oligosaccharides have also been reported to have effects on gene expression

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of colonic epithelial cells.40 The third possible explanation for the findings might be

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that -GOS reduced oxidative stress, particularly in the liver, which is known to be

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present in HFD-induced metabolic syndrome. Likewise, a study indicated that western

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HFD induced marked whole-body oxidative stress and elevated body adiposity,

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without changing body weight.41 -GOS from soybean was reported to have effective

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effect in reducing the oxidative stress.42

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In conclusion, administration of-GOS from chickpeas to mice fed HFD led to an

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increased production of SCFAs and incrassation of beneficial Bifidobacteria and

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Lactobacilli. Furthermore, there were significant effects on some metabolic syndrome

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markers, namely blood glucose, insulin, TC, TG, HDL-C and LDL-C due to the

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administration of -GOS. The shift in gut microbiota might be responsible for the

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alleviation of metabolic syndrome. Therefore, dietary intervention using -GOS from

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chickpeas should be an advisable method to enhance the gastrointestinal systems and

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effective in alleviating some of the parameters of metabolic syndrome.

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Acknowledgements

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This work was supported by Grants-in-Aid for scientific research from the National

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Natural Science Foundation of China (31171750), a grant funded by Jiangsu Key

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Laboratory of Quality Control and Further Processing of Cereals & Oils, and a project

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funded by the Priority Academic Program Development of Jiangsu Higher Education

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Institutions (PAPD).

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Figure Captions

470

Figure 1. HPLC chromatograms of soluble sugars (A) and -GOS (B) from chickpea.

471

Peaks: 1, fructose; 2, sucrose; 3, raffinose; 4, ciceritol; 5, stachyose.

472

Figure 2. Changes of body weight (the body weight was recorded weekly) of controls

473

and -GOS fed mice for 6 weeks.

474

Figure 3. Effect of controls and -GOS on levels of serum blood glucose, glycated

475

protein, insulin and insulin resistance in mice fed for 6 weeks, a-d represent

476

significant differences between different groups (p < 0.05).

477

Figure 4. Representative images of liver microsections stained with hematoxylin and

478

eosin and observed with 40 × magnification. A, NC group; B, HFD group; C, HFD +

479

LDT group; D, HFD + MDT group; E, HFD + HDT group.

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Table 1. Group-specific Primers Based on 16S rRNA Sequences Used for qPCR Target bacterial group

Sequence (5’-3’)

PCR

product

size (bp) all bacteria

F-Eub338 ACTCCTACGGGAGGCAGCAG

192

R-Eub518 ATTACCGCGGCTGCTGG Bacteroides

F-AllBac296 GAGAGGAAGGTCCCCCAC

108

R-AllBac412 CGCTACTTGGCTGGTTCAG Bifidobacteria

F-Bifido CGCGTCYGGTGTGAAAG

244

R-Bifido CCCCACATCCAGCATCCA Lactobacilli

F-Lacto GAGGCAGCAGTAGGGAATCTTC

126

R-Lacto GGCCAGTTACTACCTCTATCCTTCTTC Clostridium leptum group

F-sg-Clept GCACAAGCAGTGGAGT

242

R3-sg-Clept CTTCCTCCGTTTTGTCAA Clostridium coccoides

F-g-Ccoc AAATGACGGTACCTGACTAA R-g-Ccoc CTTTGAGTTTCATTCTTGCGAA

24


441

Page 25 of 32


Table 2. Effects of -GOS on Concentrations of Serum TC, TG, HDL-C and LDL-C in Mice Fed for 6 Weeks Treatments NC HFD HFD + LDT HFD + MDT HFD + HDT

TC mmol/L 3.53 ±0.62 a 6.41 ±1.31 d 5.04 ±0.99 b 5.87 ±0.74 c 5.14 ±0.47 bc

TG mmol/L 0.38 ±0.17 a 0.99 ±0.36 b 0.39 ±0.18 a 0.41 ±0.11 a 0.54 ±0.26 a

HDL-C mmol/L 0.69 ±0.38 a 1.62 ±0.29 b 0.68 ±0.22 a 0.73 ±0.29 a 0.94 ±0.47 a

LDL-C mmol/L 0.92 ±0.26 a 3.02 ±0.67 d 2.32 ±0.44 bc 2.53 ±0.65 c 1.99 ±0.60 b

Values are mean ± SD (n = 10), a-d represent significant differences between different groups (p < 0.05).

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Table 3. Effects of -GOS on Secretion of Total SCFAs in Mice Fed for 6 Weeks Treatments NC HFD HFD + LDT HFD + MDT HFD + HDT

acetic acid 100 ±12.5 ab 92.5 ±10.4 a 103 ±10.6 abc 116 ±14.8 bc 118 ±16.0 c

Production of SCFAs (mM) lactic acid propionic acid 63.9 ±13.8 a 57.1 ±11.1 a 55.2 ±8.82 a 51.1 ±14.0 a 60.5 ±10.4 a 94.5 ±7.22 b 61.1 ±5.90 a 88.5 ±10.7 b 79.2 ±8.68 b 94.3 ±10.2 b

butyric acid 47.5 ±8.54 a 37.9 ±6.47 a 63.6 ±6.98 b 66.6 ±10.9 b 70.0 ±7.41 b

total SCFAs 269 ±24.1 ab 237 ±24.7 a 302 ±42.7 bc 332 ±21.5 cd 362 ±36.9 d

Values are mean ± SD (n = 10), a-d represent significant differences between different groups (p < 0.05).

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Table 4. Effects of -GOS on Colonic Microbiota Composition in Mice Fed for 6 Weeks Bacterial group

NC 11.2 ±0.10 c 10.3 ±0.10 c 5.79 ±0.34 ab 7.30 ±0.24 b 9.76 ±0.20 a 9.39 ±0.32 c

HFD 10.6 ±0.08 a 9.82 ±0.09 ab 5.65 ±0.20 a 6.67 ±0.18 a 9.61 ±0.26 a 8.90 ±0.23 ab

Treatments HFD + LDT 10.6 ±0.16 a 9.76 ±0.14 a 6.07 ±0.23 bc 7.22 ±0.16 b 9.62 ±0.24 a 9.03 ±0.19 abc

HFD + MDT 10.7 ±0.14 a 9.92 ±0.18 ab 6.25 ±0.31 c 7.21 ±0.20 b 9.42 ±0.39 a 8.85 ±0.33 a

HFD + HDT 10.8 ±0.16 a 10.0 ±0.23 b 6.16 ±0.28 c 7.29 ±0.25 b 9.68 ±0.25 a 9.23 ±0.26 bc

all bacteria Bacteroides Bifidobacteria Lactobacilli Eubacterium rectal/Clostridium coccoides group Clostridium leptum group Data are mean ± SD (n = 10), expressed as log10 copy number/g of freeze-dried luminal sample, a-c represent significant differences between

different groups of certain bacterial group (p < 0.05).

27



Figure 1.

28


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Figure 2.

29



Figure 3.

30


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Figure 4.

31



Graphic Abstract

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Effects of Î±-Galactooligosaccharides from Chickpeas on High-Fat-Diet

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