Anti-diabetic effects of Lactobacillus. casei fermented-yogurt through

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Bioactive Constituents, Metabolites, and Functions

Anti-diabetic effects of Lactobacillus. casei fermented-yogurt through re-shaping gut microbiota structure in type 2 diabetic rats ling qu, junli ren, lei huang, bo pang, xu liu, xiaodong liu, baolong li, and Yu-Juan Shan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04874 • Publication Date (Web): 06 Nov 2018 Downloaded from http://pubs.acs.org on November 6, 2018

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Journal of Agricultural and Food Chemistry

Anti-diabetic effects of Lactobacillus. casei fermented-yogurt through re-shaping gut microbiota structure in type 2 diabetic rats Ling Qu1, Junli Ren1, Lei Huang1, Bo Pang1, Xu Liu2, Xiaodong Liu1, Baolong Li2*, Yujuan Shan1* 1

Department of Food Science and engineering, Harbin Institute of Technology, No 92

West Dazhi Street, Harbin 150001, P. R. China 2 Center

of Drugs Safety and Evaluation, Heilongjiang University of Chinese

Medicine, Harbin, China *Correspondence author: [email protected] (Y.-J. Shan), [email protected] (B.-L. Li) [email protected] (Ling Qu), [email protected] (Junli Ren), [email protected] (Lei Huang), [email protected] (Bo Pang), [email protected] (Xu Liu), [email protected] (Xiaodong Liu)

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ACS Paragon Plus Environment


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Abstract

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The viable bacterial strains in conventional yogurt are intolerant of bile and acid,

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consequently cannot survive conditions and their beneficial bioactivities are thus lost.

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We have previously shown that Lactobacillus. casei Q14 (Lac-Q14), a probiotic has

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the potential to alleviate diabetes in rats. Herein, we used Lac-Q14 as the starter

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culture to ferment yogurt and explore the mechanisms of the bioactivity in diabetic

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rats. Results showed that Lac-Q14 yogurt improved blood glucose and insulin levels,

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and also lowered gene expression of critical enzymes involved in liver

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gluconeogenesis. Pyrosequencing showed an obvious change in the composition of

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intestinal microbiota in Lac-Q14 yogurt- treated rats. The abundance of 21 genera

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differed significantly between the Lac-Q14 yogurt group and diabetes group. Quite a

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few of short-chain fatty acids (SCFAs)-producing bacteria were selectively enriched,

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along with increased concentrations of SCFA and downstream Glucagon-like

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peptide-1 (GLP-1), Peptide YY (PYY) secretion.

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Keywords: gut microbiota, probiotic yogurt, short chain fatty acids, type 2 diabetes

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1. Introduction

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Type 2 diabetes mellitus (T2DM) is a multifactorial, chronic disease presenting a

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significant risk to public health. More and more evidence support the hypothesis that

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gut microbiota is crucial in the pathophysiology of T2DM. It is striking that gut

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microbiota (composition, function, richness, diversity) appears different between

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diabetic patients and a healthy population

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analysis of 345 Chinese individuals showed that the gut microbiota of T2DM patients

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was disordered in a moderate degree，with the universal butyrate-producing bacteria

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decreased, while the various opportunistic pathogens increased. Functionally

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speaking ， metabolic pathway related with oxidative stress resistance and sulphate

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reduction had an enrichment (1). Thus, gut microbiota composition has been identified

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as an important, potentially modifiable factor that can contribute to development of

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type 2 diabetes. Probiotics are named as ‘live micro-organisms that provide benefits

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to the host when administrating in sufficient quantities

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of the intestinal microbiota by probiotic has been proved both in clinical and animal

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trials (3).

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(1).

A metagenome-wide association

(2).

The beneficial modulation

In recent years, probiotics have been increasingly applied in food processing as 3



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nutritional supplements(4). Yogurt is one of the most popular probiotic products(5). It

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contains digested lactose and viable bacterial strains, typically Streptococcus

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thermophilus and Lactobacillus bulgaricus, as well as several nutrients such as

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protein, calcium, phosphorus, potassium and vitamins B2 and B12

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initial starter of yogurt is not resistant to bile or acid and therefore not survive in the

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gastrointestinal tract

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enhance its nutritional and physiological value, and these products are called

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probiotic yogurt. Probiotic yogurt has nutritional and therapeutic benefits such as

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control of intestinal infections, improving lactose intolerance, reducing cholesterol

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levels and antitumor activity (5, 8)

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(7).

(6).

However, the

As a result, yogurt is supplemented with many probiotics to

Lactobacillus. casei Q14, a probiotic extracted from traditional Chinese fermented

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foods, presents beneficial effects on T2DM via modification of gut microbiota

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Herein, we have used Lactobacillus. casei Q14 to produce a functional yogurt to

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investigate its nutritional-physiological value. This study focused on the effects of

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Lactobacillus. casei Q14 fermented yogurt on T2DM, and explored the underlying

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mechanisms linked to changes in gut microbiota. The present study can be used as a

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basis for the bio-therapeutic potential of traditional fermented foods as a form of 4


(9, 10).

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treatment for diabetes.

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2. Materials and methods

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2.1 Ethics statement

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All procedures involving rats caring were conformed with the guidelines of the

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Institutional Animal Care Use Committee, Heilongjiang province, China. The

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protocols were approved by the Ethics Committee of Experimental Animals

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(Qualified number: SCXK-Hei-2012-016) at Heilongjiang University of Chinese

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Medicine.

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2.2 Reagents and materials

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Lactobacillus Q14 was separated from Yak yoghurt, a traditional Chinese

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fermented diary product in Qinghai Province of China. The strain was identified by

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16S rRNA gene sequencing and stored at -80℃.The animal feed was bought from Ke

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Ao Xie Li Feed Co., Ltd (Beijing, China). Streptozotocin (STZ) was from Sigma

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Chemical Co. (St. Louis, MO, USA). Metformin was obtained from Sino-American

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Shanghai Squibb Pharmaceuticals Ltd (Shanghai, China). Total cholesterol (TC),

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triacylglycerol (TG), low density lipoprotein cholesterol (LDL) kits were purchased

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from Jiancheng Bioengineering Institute (Nanjing, China). Glucagon-like peptide-1 5



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(GLP-1), Peptide YY (PYY), Ghrelin and Neuropeptide Y (NPY) kits were from

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Milliplex. Tissue RNA Extraction kit, TransScript One-Step gDNA Removel and

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cDNA Synthesis SuperMix kit, TransStart Top Green qPCR SuperMix kit were

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bought from TransGen Biotech (Beijing, China).

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2.3. Preparation of Lac-Q14 yogurt

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The probiotic yogurt and plain yogurt both contained Lactobacillus bulgaricus and

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Streptococcus thermophilus. In addition, Lactobacillus. casei Q14 was supplemented

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into the probiotic yogurt as a starter culture. Microbiological analysis of the probiotic

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yogurt indicated that the average colony counts of Lactobacillus. casei Q14 were 2.3

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×109 cfu/mL.

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2.4. Animals and experimental design

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Male Wistar rats, specific pathogen free (SPF), weighing 160-200 g were housed in

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Center of Drugs Safety and Evaluation, Heilongjiang University of Chinese Medicine.

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The environment of the animal house is followings: temperature 22 ± 2 ℃, humidity

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55 ± 5% with 12h:12 h light and dark cycle. Rats were freely accessd to food and

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water. The high-fat diet (HFD) contained 20% carbohydrate, 45% fat and 35% protein

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(a percentage of total kcal 100%, according to D12451; Research Diets Inc.). The 6


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normal feed contains 63.9% carbohydrate, 15.8% fat, and 20.3% protein ( AIN-93G

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diet, total energy 100%). The formulas of the diets are shown in Table S1. In the first

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6 weeks of the experiment, rats of normal control were fed on normal chow, and other

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groups were fed on HFD. At the 7th week, diabetic rats were intraperitoneally injected

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with STZ (35 mg / kg, dissolved in 0.05 M sodium citrate buffer, pH 4.5), and rats in

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normal control group (N) were injected with vehicle citrate buffer (0.5 mL / kg).

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Fasting blood glucose was measured on the 3rd and 7th day after injection. Rats with

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more than (> )11.1 mmol / L of the blood glucose were defined as T2DM. T2DM rats

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were then randomized into 5 groups (n = 8 per group): Diabetic control group (D);

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Metformin treatment group (M); Lactobacillus Q14 treatment group (Q14); Plain

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yogurt treatment group (PY), Lactobacillus Q14 fermented yogurt treatment group

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Lac-(Q14Y). The experimental scheme was shown in Figure 1.

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2.5. Oral glucose tolerance test (OGTT)

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The rats were fasted for 12 hours and given by gavage with 40% glucose (2 g / kg)

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solution. Tail vein blood samples were collected at a series of time-points after the

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intake of glucose, respectively, and blood glucose level was measured by a blood

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glucose meter (Roche Diagnostics, Germany). The glucose curve was mapped and the 7



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area under it (AUC glucose) was calculated using GRAPH PAD（9）.

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2.6 Collection of samples

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Before the end of the experiment, rats were fasted for 16 hours, then anesthetized by

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intraperitoneal administration of 10% chloral hydrate. Blood samples were collected

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through abdominal aorta. Then serum was carefully removed from the plain tubes and

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stored at -80 ℃. After blood collection, livers were collected, cut into small pieces

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and immediately frozen in liquid nitrogen. Feces were then collected by five-point

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sampling method. All samples were stored at −80 °C for further measurement.

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2.7. Serological indicators measurement

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TC, TG, LDL, insulin, GLP-1, PYY, Ghrelin and NPY were quantified by ELISA kits

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according to the manufacturer’s protocols.

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2.8 Quantitative real-time PCR

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TRIzol reagent (Invitrogen, CA, USA) was applied to extract total RNA from

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approximately liver tissue. The RNA concentration was measured by a NanoVue

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spectrophotometer (GE Life Sciences). The cDNA was synthesized by oligo (dT) and

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Superscript III reverse transcriptase (Invitrogen). Quantitative real-time PCR

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(RT-qPCR) was performed with Step One Plus Real-Time PCR System (Life 8


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Technologies, CA, USA) and SYBR Green mix (Invitrogen). Primers for qPCR used

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in this study were shown in Table 2. The real-time PCR amplifications were

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performed as follows: forty-four cycles of 95 °C for 10 s, 60 °C for 15 seconds,

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60-95 °C with a heating rate of 0.1 °C/s. The equal volume of deionized water in

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negative controls was added instead of cDNA sample. The relative expression ratio

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(RQ) of mRNA was calculated as the method of 2−ΔΔCT（9）.

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2.9 SCFAs measurement by gas chromatography (GC)

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The stool sample (400mg) was mixed with distilled water (2mL), and centrifuged at

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18800g for 10 minutes. The original supernatant was centrifuged again for 15 min.

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The supernatant was filtrated by 0.45 μm membrane. Filtrate (1 mL) and formic acid

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(0.1 mL) were injected into a gas-phase flask for SCFAs measurement（9）.

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3.0 MetaVx™ Library Preparation and Illumina MiSeq Sequencing

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TIANamp Stool DNA Kit (Tiangen, Beijing, China) was used to extract bacterial

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DNA according to manufacturer’s instructions. Prokaryotic 16S rDNA V3 and V4

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hypervariable regions were selected for generating amplicons and followed by

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taxonomic analysis. The specific primers targeting 16S rDNA V3 and V4

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hypervariable regions of bacteria were designed by GENEWIZ. The sequence of the 9



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forward

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“CCTACGGRRBGCASCAGKVRVGAAT”

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“GGACTACNVGGGTWTCTAATCC” respectively. DNA libraries were validated

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by Agilent 2100 Bioanalyzer, and quantified by Qubit 2.0 Fluorometer. Image

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analysis and base calling were performed using the MiSeq Control Software (MCS)

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（10）.

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2.10 Statistical Analysis

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All data were analysed with GraphPad software and represented as mean ± standard

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difference (SD). Statistical significance of difference was determined using one-way

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ANOVA followed by multiple comparisons with a Tukey’s test. P < 0.05 was

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considered as the statistical significance.

primers

and

reverse

primers

was and

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3. Results 3.1 Lac-Q14 yogurt improved the glucose tolerance and insulin level

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The rat T2DM model was successfully established as before. During the first six

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weeks, body weight has been continuously increasing (Fig. 2A). The injection of STZ

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(35mg/kg) (at the beginning of week 7) caused a significant weight loss in all HFD 10


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feeding groups from week 8 to week 9. After six-week-intervention, metformin, Q14

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and Q14 yogurt delayed the decrease of body weight, and the obvious significance

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was exhibited compared with D group.

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Oral glucose intolerance test (OGTT) is a publicly identified criterion for the

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diagnosis of insulin resistance in T2DM. At the end of week 7, glucose tolerance was

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impaired in the HFD-STZ groups (Fig. 2B) and the AUC values were more than 3

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times higher (Fig. 2b) than in the N group. After the 6-week treatment, the glucose

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intolerance and insulin levels were improved in metformin, Q14, and Q14 yogurt

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groups (Fig. 2C & Fig. 2D), although a little higher than that in control group. These

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results demonstrated that Q14 yogurt has the potential to improve glucose tolerance in

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T2DM.

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3.2 Effects of Lac-Q14 yogurt on blood lipid profile

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Diabetes is often accompanied by disorders of lipid metabolism. As show in

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Supplementary Fig. S1, serum lipid markers including total cholesterol (TC),

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triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in diabetic rats

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were significantly higher than normal rats. Q14 yogurt reduced serum TC, TG and

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LDL-C levels, but no statistical significance. 11



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3.3 Effects of Lac-Q14 yogurt on liver gluconeogenesis and de-novo lipogenesis

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Hepatic glucose 6-phosphate (G6P) and phosphoenolpyruvate carboxykinase

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(PEPCK) are critical enzymes in gluconeogenesis which response to the activation by

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glucagon and inactivation by insulin

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G6P and PEPCK in diabetic rats were significantly higher than normal rats, which

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caused the increased fasting blood glucose level. After Q14 yogurt intervention,

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expression levels of G6P and PEPCK decreased by 58.7, and 51.5%, respectively.

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Acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) are two rate-limiting

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enzymes in de-novo lipogenesis, and sterol regulatory element-binding protein-1

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(SREBP-1) as well as carbohydrate-responsive element binding protein (ChREBP)

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could transcriptionally activate the expression of them

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differences among the groups in mRNA levels of ACC, FAS and SREBP-1, excepting

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ChREBP, which decreased by 64% in the Q14Y group (Fig. 4).

(11).

We observed that the expression levels of

(12).

There were no significant

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3.4 Lac-Q14 yogurt reshaped gut microbiota structure

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A total of 6711082 usable pyrosequencing reads were obtained from 48 samples.

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After quality filtering, we identified 750 OTUs at the 97% similarity level. First, we

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discovered the distinctions and similarities of intestinal microbiota in six groups. As 12


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shown in Fig. S1, we found that there were 414 shared OTUs among the six groups. It

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is noteworthy that only 10 OTUs were found in N group, 4 OTUs in M group, and 1

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OTU in D, Q14, Q14Y group respectively.

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Fig. 5 showed that the intestinal microbial composition at different taxonomic

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levels in six groups. Our data showed that gut microbiota in rats was largely

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dominated by the phyla Bacteroidetes and Firmicutes. Microflora in diabetes rats was

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sharply shifted with a reduced proportion of the phyla Bacteroidetes (by 16.9%), and

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an increased proportion of the phyla Firmicutes by 16.5% (P

Anti-diabetic effects of Lactobacillus. casei fermented-yogurt through

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