Lotus Seed Resistant Starch Regulates Gut Microbiota and Increases

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Lotus Seed Resistant Starch Regulates Gut Microbiota and Increases SCFAs Production and Mineral Absorption in Mice Hongliang Zeng, Cancan Huang, Shan Lin, Mingjing Zheng, Chuanjie Chen, Baodong Zheng, and Yi Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02860 • Publication Date (Web): 27 Sep 2017 Downloaded from http://pubs.acs.org on September 29, 2017

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Journal of Agricultural and Food Chemistry

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Lotus Seed Resistant Starch Regulates Gut Microbiota and

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Increases SCFAs Production and Mineral Absorption in

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Mice

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Hongliang Zeng,†,‡ Cancan Huang,† Shan Lin,† Mingjing Zheng,† Chuanjie Chen,†

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Baodong Zheng,*†,‡ and Yi Zhang*†,‡

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†

College of Food Science, Fujian Agriculture and Forestry University, Fuzhou,

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Fujian, P. R. China 350002

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‡

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Special Starch, Fujian Agriculture and Forestry University, Fuzhou 350002, China

Fujian Provincial Key Laboratory of Quality Science and Processing Technology in

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ACS Paragon Plus Environment


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ABSTRACT:

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Lotus seed resistant starch, known as resistant starch type 3 (LRS3), was orally

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administered to mice to investigate its effects on the gut microbiota, short-chain fatty

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acids (SCFAs) production, and mineral absorption. The results showed that mice fed

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LRS3 displayed a lower level of gut bacterial diversity than other groups. The

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numbers of starch-utilizing and butyrate-producing bacteria, such as Lactobacillus

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and Bifidobacterium, and Lachnospiraceae, Ruminococcaceae, and Clostridium,

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respectively, in mice increased after the administration of medium and high doses of

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LRS3, while those of Rikenellaceae and Porphyromonadaceae decreased.

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Furthermore, SCFAs and lactic acid in mice feces were affected by LRS3, and

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lactate was fermented to butyrate by gut microbiota. LRS3 enhanced the intestinal

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absorption of calcium, magnesium, and iron, and this was dependent on the type and

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concentration of SCFAs, especially butyrate. Thus, LRS3 promoted the production

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of SCFAs and mineral absorption by regulating gut microbiota in mice.

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KEYWORDS: Lotus seed resistant starch, Gut microbiota, Short-chain fatty acids,

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Mineral absorption

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INTRODUCTION

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Type 3 resistant starch, also known as retrograded or crystalline starch, cannot be

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digested and absorbed in the small intestine of healthy humans but can be fermented

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or partially fermented by the gut microbiota in the large intestine, producing

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short-chain fatty acids (SCFAs), lactic acid and small amounts of gases.

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content of SCFAs increases in the large intestine and feces of humans eating diets

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having a high content of resistant starch, and the gut microbiota of the host changes

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correspondingly. 2 Currently, the identified human intestinal bacteria can be divided

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into 10 categories, such as Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria,

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Cyanobacteria, Verrucomicrobia ， Spirochaeates, Actinobacteria, VadinBE97 and

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Methanobrevibacter smithii. The bacteria belonged to Firmicutes, Bacteroidetes

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account for more than 87% of the total gut microbiota. 3 The types and quantities of

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intestinal flora are determined by microbial molecular ecology techniques, including

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fingerprinting technique, sequencing technology, nucleic acid hybridization and

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real-time quantitative PCR. SCFAs are the main energy sources of the colonic

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mucosa, especially the bottom of the colonic mucosa. 4 SCFAs can reduce the pH of

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the intestinal environment, thus affecting the intestinal flora’s structure, promoting

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the proliferation of beneficial bacteria and inhibiting the proliferation of pathogens,

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which promotes human health.

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as increasing the intestinal absorption of minerals, lowering blood lipid effects and

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preventing colorectal cancer, mainly through the SCFAs. 6-8

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5

1

The

Resistant starch regulates the intestinal tract, such

Lotus seeds are the mature seeds from the genus Nelumbo, especially Nelumbo

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nucifera, which is an important commercial crop in China. 9 It contains a high level

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of amylose (~ 40%, w/w), which may contribute to the formation of lotus seed

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resistant starch type 3 (LRS3).

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Bifidobacterium.

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factors, including the surface microstructure, double-helix structure and biological

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mechanisms. However, these studies focused on the prebiotic effect of LRS3 in vitro.

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The study of gut microbiota in vivo is more complicated than in vitro.

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there is limited information on the effects of LRS3 on the in vivo gut microbiota.

12

10,11

LRS3 can stimulate the growth of

The prebiotic effect of LRS3 results from a combination of

13,14

Thus,

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The objective of this study was to investigate the effect of LRS3 on the gut

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microbiota in mice and the relationships among the gut bacterial community, SCFAs

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production and mineral absorption. Amplicons of the V3 variable regions of bacterial

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16S rDNA were analyzed using denaturing gradient gel electrophoresis (DGGE),

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cloning and sequencing. The contents of SCFAs and minerals in mice feces were

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determined

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spectrophotometry, respectively.

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MATERIALS AND METHODS

using

gas

chromatography

(GC)

and

atomic

absorption

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Materials. Formic, acetic, propionic, butyric, isobutyric, and lactic acids were

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purchased from Aladdin Reagent Co. (Shanghai, China). The standards of calcium

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(Ca), magnesium (Mg), and iron (Fe) were obtained from Zhongyijia Technology

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Development Co. (Tianjin, China). The Mo Bio DNA Extraction Kit was purchased

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from Mo Bio Laboratories Co. (Carlsbad, CA, USA) and the Poly-Gel DNA

88

Extraction Kit was purchased from Omega Bio-Tek Inc. (Norcross, GA, USA). The

4


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40% acrylamide/bis was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA,

90

USA). The other reagents used were of analytical grade and obtained from

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Sinopharm Group Chemical Reagent Co. (Shanghai, China). High amylose maize

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starch (HAMS) was purchased from Beststarch Creatmaterial Co., Ltd (Fujian,

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China). LRS3 was prepared and purified according to our previous methods. 11

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Animals and experimental design. BALB/c male mice, with weights of 18.0 ±

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1.0 g (Certification number: 2007000552260), were provided by the Experimental

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Animal Center, Fujian University of Traditional Chinese Medicine, Fujian Province,

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China. The animals were housed in stainless steel cages in a room with a controlled

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temperature (23 ± 1°C), relative humidity (60%–65%), and 12 h light/dark cycle

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(light on at 8:00 a.m. and off at 8:00 p.m.). All of the animal studies used in this

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study were performed in compliance with the Guidelines for the Care and Use of

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Laboratory Animals published by the U.S. National Institutes of Health (NIH

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Publication 85-23, 1996), and all procedures were approved by the Animal Care

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Review Committee (Approval NO. 2017019), Fujian University of Traditional

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Chinese Medicine, China. The basal diet was purchased from Jiangsu Xietong

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Organism Co. Ltd. (Nanjing, China). The compositions of 5 diets are shown in Table

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1 and its nutrition components are in accordance with GB 14924.3-2010 standards

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(China). The energy values were calculated using Mingwei Diet Pagoda

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Management System (Version 5.0, Fuzhou, China).

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For one week, all animals were allowed free access to distilled water and were fed

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the basal diet. Then, the mice were randomly divided into 5 groups of 14 mice each:

5



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1, normal control (NC) group in which mice were given the same volume of distilled

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water and the basal diet was provided ad libitum; 2, low dose of LRS3 (LD) group in

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which mice were given the same volume of distilled water and fed a modified basal

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diet with 5% LRS3; 3, medium dose of LRS3 (MD) group in which mice were given

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the same volume of distilled water and fed a modified basal diet with 10% LRS3; 4,

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high dose of LRS3 (HD) group in which mice were given the same volume of

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distilled water and fed a modified basal diet with 15% LRS3; and 5, HAMS group in

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which mice were given the same volume of distilled water and fed a modified basal

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diet with 10% high amylose maize starch. The food intakes and weights of mice

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were determined every day. The mice feces were collected on a 24-hour period

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during15 d and weighed, freeze-dried (LG-1.0 vacuum freeze drier, Xinyang,

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Shenyang, China), and stored at −80°C until further analysis.

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Extraction of DNA and PCR amplification. For each sample, 1.5 g of mice

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feces was thawed in ice water, suspended in 30 mL of 0.1 mol/L phosphate-buffered

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saline (pH 7.0) and stirred well. The supernatant was obtained by centrifuging three

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times at 200 ×g for 5 min. Then, the supernatant was centrifuged at 10,000 ×g for 5

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min at room temperature, and the bacterial pellets were collected. The pellets were

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resuspended in 3 mL of TE buffer (pH 8.0) and then the DNA was extracted using a

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PowerFecal® DNA Isolation Kit (Mo Bio Laboratories, Inc.) according to the

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manufacturer’s instructions. The DNA samples extracted from mice feces were

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amplified using a forward primer with a GC-clamp – GC-338F, (20 µM), sequence

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(5′−3′),CGCCCGGGGCGCGCCCCGGGGCGGGGCGGGGGCGCGGGGGGCCT

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ACGGGAGGCAGCAG – and a reverse primer – 518R, (20 µM), sequence (5′−3′),

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ATTACCGCGGCTGCTGG – to conserved sequences flanking the variable V3

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region of the bacterial 16S rDNA in a PTC220 PCR amplification system (Bio-Rad

136

Laboratories, Inc.). Each reaction mixture was prepared with 100 ng of template

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DNA, 0.5 µL of 20 µM GC-338F, 0.5 µL of 20 µM 518R, 5 µL of PCR buffer, 4 µL

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of 2.5 mM dNTPs, 0.5 µL of 5 U/µL rTaq, and 50 µL of ddH2O. The amplification

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program was composed of an initial denaturing step at 94°C for 5 min, followed by

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30 cycles of denaturing at 94°C for 30 s, annealing at 55°C for 30 s, and extension at

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72°C for 30 s. A final extension step was performed at 72°C for 10 min. The sizes

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and amounts of the amplicons were examined on a 2% agarose gel stained with

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ethidium bromide.

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Generation of microbiota profiles using DGGE. The DGGE was generated

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using the DCode Universal Mutation Detection System (Bio-Rad Laboratories, Inc.)

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as the reported method

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were

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(acrylamide/bisacrylamide = 37.5:1, Bio-Rad Laboratories, Inc.) containing a

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35−55% linear denaturing gradient in 50× TAE buffer (40 mmol/L Tris-acetate, 1

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mmol/L Na-EDTA, pH 8.0), which contained 0 and 100% denaturant (7 mol/L urea

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and 40% (v/v) formamide; Amersham Pharmacia Biotech, Amersham, UK). The

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electrophoresis was performed under a constant voltage of 150 V at 60°C for 16 h.

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After electrophoresis, the gel was silver-stained as previously described.

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obtained DGGE patterns were subsequently normalized and analyzed with Bio-Rad

applied

onto

15

with slight modifications. Briefly, 10 µL of PCR products 1

mm

thick

8%

(w/v)

7


polyacrylamide

gels

16

The


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Quantity One 4.3.0 software (Bio-Rad Laboratories, Inc.). The numbers and

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densities of electrophoresis bands in each sample were quantitatively analyzed to

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investigate the diversity and to perform a principal component analysis (PCA) of

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fecal flora. Clustering was accomplished using the Pearson correlation and the

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unweighted pair group mean average (UPGMA) method. The indices values of the

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DGGE profiles were compared using the following equations:

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S

(1)

H ' = - ∑ Pi ln Pi i= 1

H ' H 'm a x

=

H ' ln S

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EH =

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where H' is the Shannon–Weiner index, Pi is the ratio of the absorbance of the ith

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band to the absorbance sum of all the bands, EH is the evenness index, S is the value

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of richness, which is the number of the bands of each lane in the DGGE profile.

(2)

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Identification of DGGE DNA bands. The bands of specific interest were excised

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from the DGGE gel with a sterile razor and placed in a sterile centrifuge tube. Then,

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50 µL of the sterile water was added, and the mixture was mashed and placed at 4°C

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for 12 h to obtain the bacterial DNA from the gel. The DNA sample was amplified

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using

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CCTACGGGAGGCAGCAG – and a reverse primer – 518R, (20 µM), sequence

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(5′−3′), ATTACCGCGGCTGCTGG – under the same conditions as described above.

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The PCR products were obtained from a 1% agarose gel and purified using a

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Poly-Gel DNA Extraction Kit (OMEGA Engineering, Inc., Norwalk, CT, USA). The

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cloning and sequencing analysis of DNA samples were carried out according to the

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methods of Zhou et al. 17 These sequences were submitted to the GenBank database,

a

forward

primer

–

338F,

(20

µM),

8


sequence

(5′−3′),

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and the homology was compared using the BLAST algorithm-based program. The

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closest matched sequences were obtained, and the neighbor-joining phylogenetic tree

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was constructed by MEGA 5.0 software. The reliability of the tree topology was

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gauged by performing a bootstrap analysis using 1,000 replicates.

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qPCR analysis of Lactobacillus and Bifidobacterium. The quantifications of

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Lactobacillus and Bifidobacterium were performed using an MJ OpticonR-2

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quantitative real-time PCR system (Bio-Rad Laboratories, Inc.). The construction

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and verification of the standard DNA were carried out according to a previous

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method.

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copy number and cycle threshold. Each reaction mixture was prepared with 10 µL of

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2× SYBR Premix Ex Taq (TaKaRa, Dalian, China), 0.5 µL of 10 µM forward primer,

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0.5 µL of 10 µM reverse prime, 1 µL of DNA, and 20 µL of ddH2O. The

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amplification program consisted of pre-incubation at 90°C for 3 min, 40 cycles at

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90°C for 10 s, 58–63°C for 10 s, and 72°C for 10 s, and finally one cycle for the

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melting curve analysis. The real-time PCR of Lactobacillus used a forward primer –

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F-Lacto, sequence (5′−3′), GAGGCAGCAGTAGGGAATCTTC – and a reverse

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primer – R-Lacto, sequence (5′−3′), GGCCAGTTACTACCTCTATCCTTCTTC.

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The real-time PCR of Bifidobacterium used a forward primer – F-Bifido, sequence

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(5′−3′), CGCGTC YGGTGTGAAAG – and a reverse primer – R-Bifido, sequence

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(5′−3′), CCCCA CATCCAGCATCCA. The number of each strain was expressed as

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a logarithm of the copy number of the 16S rDNA gene per dry fecal weight.

198

18

The standard curve was constructed from the relationship between the

Determination of SCFAs and lactic acid. SCFAs and lactic acid in mice feces

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19

199

were determined according to a previously reported method

with some

200

modifications. Briefly, the standard solutions of formic, acetic, propionic, butyric,

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isobutyric, and lactic acids were prepared at concentrations of 5, 10, 15, 20, and 25

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mmol/L, respectively. The quantitative analysis of SCFAs was performed by GC

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(Agilent7890, Palo Alto, CA, USA) on a HP-INNOWAX chromatographic column

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(30 m × 0.320 mm × 0.25 µm; Agilent) at the conditions of 100°C of initial

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temperature for 0.5 min, then heating to 200°C at 4°C/min. The flow rates of carrier

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gas, fuel gas and oxidant gas were 20 mL/min of N2, 30 mL/min of H2 and 300

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mL/min of air, respectively. The signal was detected at 240°C with a FID detector.

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Then, 0.2 µL of the respective standard solutions was loaded on the GC system. The

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standard curves of SCFAs and lactic acid were obtained with the concentration as the

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abscissa (x) and the peak area as the ordinate (Y). The standard curve equations were

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follows: Y=89.67x+56.62 (Formic acid, R2=0.9961), Y=57.68x+13.40 (Acetic acid,

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R2=0.9912), Y=150.23x+11.59 (Propionic acid, R2=0.9994), Y=199.26x+7.58

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(Butyric acid, R2=0.9956), Y=282.52x+12.38 (Isobutyric acid, R2=0.9985), and

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Y=64.23x+48.41 (Lactic acid, R2=0.9897).

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Then, 0.5 g of the mice feces was added to a centrifuge tube containing 4 mL of

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distilled water and mixed thoroughly on a vortex-mixer for 2 min. After mechanical

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shaking at 4°C for 30 min and centrifugation at 10,000 ×g at 4°C for 20 min)

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(Biofuge Strato, Thermo Electron Corporation, Waltham, MA, USA), the

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supernatants were filtered through a 0.45-µm filter. The contents of SCFAs and lactic

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acid were measured as described above.

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Determination of the mineral absorption. Dried samples of 0.5 g of feces or

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diet (sampling at the late of experimental period) were individually added to 9 mL of

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HNO3/H2O2 (8:1) in a microwave digestion instrument (XT-9900, Tuoxin Analysis

224

Instrument Technology co., LTD, Shanghai, China), and then, in order, digested at 2

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Kg/cm−2 pressure with a microwave power of 400 W for 60 s, 5 Kg/cm−2 pressure

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with a microwave power of 800 W for 60 s, 10 Kg/cm−2 pressure with a microwave

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power of 800 W for 120 s, 15 Kg/cm−2 pressure with a microwave power of 800 W

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for 120 s, 20 Kg/cm−2 pressure with a microwave power of 800 W for 180 s, and 25

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Kg/cm−2 pressure with a microwave power of 800 W for 180 s until discoloration.

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The digestion solution was dry-ashed in a crucible. The final sample dilution was

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made in 0.075 mol/L HNO3 to 10 mL and filtered through a 0.45-µm filter. Mineral

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concentrations were determined by atomic absorption spectrophotometry (AA6300,

233

Shimadzu Co., Kyoto-fu, Japan) in an acetylene-air flame at the following

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wavelengths: 422.7 nm (Ca), 285.2 nm (Mg) and 248.3 nm (Fe). The mineral’s

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apparent absorption was calculated as follows:

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Apparent absorption (%) = (Total intake-Total excretion)/ Total intake ×100

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Statistical analysis. All of the experiments were conducted in triplicate. The

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results were expressed as mean ± standard deviations. Data were analyzed by

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one-way analysis of variance and Duncan’s post-hoc test at a 5% confidence level

240

using SPSS (Version 16.0, Chicago, IL, USA). The matrix of intensity and relative

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position was subjected to a PCA using SPSS software.

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RESULTS AND DISCUSSION

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(3)


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Effects of LRS3 on mouse general health, weight and feed efficiency.

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Throughout the experiments, all of the animals appeared healthy and lively, with

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smooth hair and no shedding. The fecal pellets were normal, and there were no

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treatment-related deaths and diseases. No mice experienced diarrhea or constipation.

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The growth of mice was normal during the 15-d experimental period. Different body

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weights of mice were observed among the NC, LD, MD, HD, and HAMS groups

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(Figure 1). At the beginning of the experiment, the body weights of the mice were

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18.0 ± 1.0 g. The body weights of mice in each group increased over time, and the

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gain rate of mice in the NC group was the greatest among these groups (p

Lotus Seed Resistant Starch Regulates Gut Microbiota and Increases

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