Clinical application of probiotics in type 2 diabetes mellitus: A randomized, double-blind, placebo-controlled study

Accepted Manuscript Clinical Application of Probiotics in Type 2 Diabetes Mellitus: a Randomized, DoubleBlind, Placebo-Controlled Study Livia Bordalo Tonucci, PhD, Professor, Karina Maria Olbrich dos Santos, PhD, Leandro Licursi de Oliveira, PhD, Sonia Machado Rocha Ribeiro, PhD, Hercia Stampini Duarte Martino, PhD PII:

S0261-5614(15)00331-3

DOI:

10.1016/j.clnu.2015.11.011

Reference:

YCLNU 2692

To appear in:

Clinical Nutrition

Received Date: 12 May 2015 Revised Date:

8 November 2015

Accepted Date: 12 November 2015

Please cite this article as: Tonucci LB, dos Santos KMO, Oliveira LLd, Ribeiro SMR, Martino HSD, Clinical Application of Probiotics in Type 2 Diabetes Mellitus: a Randomized, Double-Blind, PlaceboControlled Study, Clinical Nutrition (2016), doi: 10.1016/j.clnu.2015.11.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Clinical Application of Probiotics in Type 2 Diabetes Mellitus: a Randomized, Double-Blind, Placebo-Controlled Study Authors: Livia Bordalo Tonuccia, PhD

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Karina Maria Olbrich dos Santosb, PhD Leandro Licursi de Oliveirac, PhD Sonia Machado Rocha Ribeirod, PhD

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Hercia Stampini Duarte Martinod, PhD

Professor at Department of Nutrition, INTA College.

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Brazilian Agricultural Research Corporation (Embrapa). Address: Fazenda Três

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Lagoas, Estrada Sobral-Groaíras, Km 4. Postal Code 145. Zip Code 62011-970. c

Professor at Department of Biology, Federal University of Viçosa. Address: Av. PH

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Rolfs, s/n, Campus UVF, CCB II – Viçosa, MG, Brazil. Zip Code: 36.570-000. Professor at Department of Nutrition and Health, Federal University of Viçosa.

36.570-000.

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Address: Av. PH Rolfs, s/n, Campus UVF, CCB II – Viçosa, MG, Brazil. Zip Code:

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Corresponding author: Livia Bordalo Tonucci. Address: R. Antônio Rodrigues Magalhães, 359 - Dom Expedito Lopes, Sobral - CE. Zip Code: 62050-100. Postal code, 10. E-mail: [email protected]. Phones: 55 (88) 98809-9885/ Fax number: (31) 3899-3176. Short running head: Clinical Application of Probiotics in Diabetes Sources of support: EMBRAPA and FAPEMIG, Brazil Clinical trial registry: This trial is registered with ensaiosclinicos.gov.br/rg/RBR219644.

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ACCEPTED MANUSCRIPT ABSTRACT

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Background & Aims: Type 2 diabetes has been associated with dysbiosis and one of the

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possible routes to restore a healthy gut microbiota is by the regular ingestion of probiotics.

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We aimed to investigate the effects of probiotics on glycemic control, lipid profile,

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inflammation, oxidative stress and short chain fatty acids in T2D.

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Methods: In a double-blind, randomized, placebo-controlled trial, 50 volunteers consumed

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daily 120 g/d of fermented milk for 6 wk. Participants were assigned into two groups:

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probiotic group, consuming fermented milk containing Lactobacillus acidophilus La-5 and

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Bifidobacterium animalis subsp lactis BB-12 (109 colony-forming units/d, each) and control

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group, consuming conventional fermented milk. Anthropometric measurements, body

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composition, fasting blood and faecal samples were taken at baseline and after 6 wk.

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Results: 45 subjects out of 50 (90%) completed follow-up. After 6 wk, there was a

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significant decrease in fructosamine levels (- 9.91mmol/L; p = 0.04) and hemoglobin A1c

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tended to be lower (- 0.67%; p = 0.06) in probiotic group.

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significantly reduced in probiotic and control groups (- 1.5 and -1.3 pg/mL, -2.1 and -2.8

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ng/mL, respectively), while IL-10 was significantly reduced (- 0.65 pg/mL; p < 0.001) only

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in the control group. Fecal acetic acid was increased in both groups (0.58 and 0.59% in

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probiotic and control groups, respectively; p < 0.01). There was a significant difference

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between groups concerning mean changes of HbA1c (+ 0.31 for control group vs - 0.65 for

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probiotic group; p = 0.02), total cholesterol (+ 0.55 for control group vs - 0.15 for probiotic

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group; p = 0.04) and LDL-cholesterol (+ 0.36 for control group vs - 0.20 for probiotic group

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p = 0.03).

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Conclusions: Probiotic consumption improved the glycemic control in T2D subjects,

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however, the intake of fermented milk seems to be involved with others metabolic changes,

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such as decrease in inflammatory cytokines (TNF-α and resistin) and increase in the acetic

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acid.

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1. Introduction Diabetes mellitus is on the rise all over the world affecting more than 382 million

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people. Type 2 diabetes (T2D) accounts for 85% to 95% of all diabetes and is a complex

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chronic illness requiring multifactorial risk reduction strategies [1].

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T2D is often associated with systemic inflammation [2] and increased oxidative stress

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[3]. Both molecular and metabolic mechanisms are links to β-cell dysfunction and/or insulin

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resistance [4]. However, the intake of probiotics has been demonstrated to reduce

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inflammation and oxidative stress markers, and to improve glycemic and insulin metabolism

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[5-6].

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In addition, the gut microbiota also seems to be important to the pathophysiology of

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T2D [7]. Findings from two studies that used faecal samples suggested that compositional

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and functional changes in the gut microbiota might be directly linked to the development of

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T2D [8-9]. Various others mechanisms have been proposed to explain the influence of the

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microbiota on insulin resistance and T2D, such as metabolic endotoxemia [10], modifications

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in the secretion of the incretins [11], and short-chain fatty acids (SCFA) production [12].

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Multiple cytokines are involved in the development of T2D, and are fundamental signals in

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the intestinal immune system, able to subvert the physiological status of inflammation in the

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gut [13].

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Alterations in the gut microbiota as a result of probiotics and prebiotics intake have

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been reported. Experimental studies have reported beneficial effects by strains of

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Lactobacillus on glycemic control, stress oxidative and/or inflammation in animal model of

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type 2 diabetes [5, 14]. However, the effects of probiotics on diabetes endpoints remain

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unknown.

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The objective of this study was to investigate the efficacy of the intake of fermented

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goat milk containing Lactobacillus acidophilus La-5 and Bifidobacterium animalis BB-12 on

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glycemic control, lipid profile, inflammation, oxidative stress and faecal SCFA in T2D

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subjects.

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2. Methods

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2.1 Subjects

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This randomised, double-blind, parallel-group, placebo-controlled trial was carried

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out in Ceará, Brazil, from June 2013 to February 2014. The study was performed on 50

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volunteers with T2D according to ADA criteria [15], age 35–60 y, body mass index (BMI)

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lower than 35 kg/m2 and type 2 diabetes diagnosed for at least one year, recruited from two

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endocrinology clinics. The appropriate sample size was calculated based on primary outcome

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of serum interleukin (IL)-6 levels obtained from Mazloom et al. [16], considering a alpha

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level of 0.05 and a power of 80%. The number of subjects required was 22.5 per group, but

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this number was increased to 25 per group to accommodate the withdrawals. The research

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team did recruitment within the clinics after indication of participants by endocrinologist.

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We excluded participants with clinical evidence of chronic illness or gastrointestinal

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disorders; presence of renal, hepatic, haematological or immunodeficiency diseases; history

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of cancer or cardiovascular diseases; current treatment with insulin, DPP-4 inhibitor or GLP-

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1 analogue; smoking; any intake of probiotics, supplements, antiobesity and anti-

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inflammatory drugs, and antibiotics in the three months preceding recruitment, and

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pregnancy or breast-feeding.

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The study protocol was approved by the Federal University of Viçosa Ethics

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Committee. All patients provided written informed consent. This trial is registered with

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ensaiosclinicos.gov.br/rg/RBR-219644.

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2.2 Randomisation and blinding

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Participants were randomly assigned (1:1) by computer (Research Randomizer,

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version 4.0) to either probiotic or control group in block sizes of four. The randomization was

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stratified by gender. Volunteers, investigators, and trial staff were masked to treatment

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allocation. However, investigators analysing study data were not masked. Blinding was done

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by a technical assistant, right after beverages production.

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2.3 Study protocol

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T2D subjects were randomly assigned to consume either 120 g/d of probiotic

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fermented goat milk containing 109 UFCs of Lactobacillus acidophilus La-5 and 109 UFCs of

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Bifidobacterium animalis subsp. lactis BB-12 (probiotic group) or 120 g/d of conventional

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fermented goat milk contained Streptococcus thermophilus TA-40 (control group) for 6 wk,

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during the breakfast. The fermented milk (FM) was delivered every 2 wk and volunteers were

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instructed to keep it under refrigeration (4 ºC).

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The probiotic and conventional FM were developed in parthership with Embrapa Goat

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and Sheep (Ceará, Brazil). Pasteurised milk was cooled to 43 ± 2oC for the addition of the

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starter culture (S. thermophilus TA-40; Danisco, Sassenage, France), and the probiotic

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cultures (L. acidophilus La-5 and B. animalis subsp. lactis BB-12; Chr. Hansen, Hoersholm,

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Denmark). The beverages were flavored with grape juice obtained from Embrapa Grape and

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Wine (Rio Grande do Sul, Brazil). Ingredients, chemical composition and antioxidants status

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of the probiotic FM are shown in Table supplementary 1 (see supplementary data). Both

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products had the same taste, color, and smell.

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Fermented milk was sampled immediately after manufacture and delivered on the

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following day. Microbiological analysis was conducted every week during the trial. S.

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thermophilus enumeration was performed on M17 agar, containing lactose (Vetec, Duque de

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Caxias, Brazil, 5 g/L) and incubated aerobically at 37 ºC for 48 h. B. lactis and L. acidophilus

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were determined in modified De Man Rogosa Sharpe (MRS) agar (Oxoid, Basingstoke, UK),

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followed by incubation at 37 ºC for 72 h [17]. Microbiological analyses of the probiotic FM showed that the average colony counts

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of probiotic bacteria on days 1, 7 and 14 were 7.72 x 107, 5.82 x 107, and 1.62 x 106 CFUs/g

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of L. acidophilus La-5 and 4.45 x 108, 1.84 x 107, and 1.56 x 107CFUs/g of B. lactis BB-12,

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respectively. Both probiotic bacteria showed an appropiate survival rate during 14 days

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storage time (see Supplemental Table 2). Additionaly, sensory evaluation was carried out in

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FM samples during the trial through acceptability tests, using the hybrid hedonic scale (1 =

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disliked extremely, 5 = neither liked nor disliked, 09 = liked extremely) [18]. The overall

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acceptability of the probiotic and conventional FM was 8.17 and 8.20, respectively (see

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Supplemental Table 3).

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All anthropometric and biochemical measures were conducted in a fasting state taken

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at baseline and after 6 wk intervention. A single experienced examiner performed all

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anthropometric measurements. Body weight and body composition were assessed by bio-

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impedance (Tanita TBF-300, Tanita Corporation, Tokyo, Japan). Waist circumference

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(measured midway between lowest rib and iliac crest) was measured using a non-stretchable

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measuring tape (Sanny, São Paulo, Brazil).

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Dietary assessment was done using food records, which were conducted by the

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nutritionist in the first and the last week of intervention. Dietary intake was analysed using

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Avanutri Revolution software (Rio de Janeiro, Brazil). Regular level of physical activity was

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defined as three times a week for at least 30 min.

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Subjects were instructed to follow their usual diets, level of physical activity, and

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lifestyle, avoiding any changes in medication, and unusual or excessive food and alcohol

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drink consumption throughout the intervention period. We contacted the volunteers once a

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week to ask for adverse health events and to assess the adherence to treatment.

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2.4 Biochemical measurements

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For the biochemical measurements, blood samples were obtained after a 12 h

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overnight fast. Before the test day, the subjects were instructed not to consume alcohol and to

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refrain from heavy physical activity during 72 and 24 h, respectively. Samples were

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centrifuged at 1,000 g for 10 min at 4 °C, aliquoted and analysed or immediately stored at –

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80 °C for cytokines and oxidative stress analyses.

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Blood samples were analyzed for fasting plasma glucose (FPG) and lipid profile by

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enzymatic colorimetric method (Bioclin, Minas Gerais, Brazil, and Beckman Coulter,

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California, USA). Insulin was measure through electrochemiluminescence (Modular

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AnalyticsDxI800, Beckman Coulter, California, USA). Fructosamine was assayed by

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colorimetric method (BioSystems, Barcelona, Spain), and HbA1c with ion exchange high-

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performance liquid chromatography (Bio-rad kit, California, USA). The homeostasis model

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assessment index (HOMA-IR) was used as an indicator of insulin resistance and calculated as

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follows: HOMA-IR = fasting plasma insulin (µU/mL) x fasting plasma glucose (mmol/

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L)/22.5. Insulin resistance was diagnosed using a cut off value of 2.71 [19].

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For determination of oxidative stress markers, plasma samples were collected in

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vacutainers containing sodium citrate. Colorimetric assay (Sigma-Aldrich antioxidant assay

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kit, Missouri, USA; interassay coefficients of variation 3%) was used to measure plasma total

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antioxidant status (TAS). Plasma total F2–isoprostane was measured by ELISA kit (Caymans

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8-isoprostane EIA kit, Michigan, USA; interassay coefficients of variation 9.5% at 80 pg/mL

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and 6.4% at 32 pg/mL). Briefly, plasma samples (400 µL) were hydrolysed with (10N)

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NaOH (100 µL) at 45 °C for 2 h to measure both free and esterified isoprostane. After

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incubation, 100 µL of (10M) HCl was added and the samples were centrifuged at 1,500g for

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10 min to remove precipitated proteins.

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Cytokine concentrations were determined using a multiplexed bead immunoassay.

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This is a bead-based suspension array using the LuminexxMAP technology in which

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fluorescent-coded beads, known as microspheres, have cytokine capture antibodies on the

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bead surface to bind the proteins. The measures of 5 cytokines (IL-6, IL-10, TNF-α,

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adiponectin, and resistin) were measured using the human Cytokine/Adipokine magnetic

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bead kits (Millipore Coproration, Missouri, USA). Assays were performed in 96-well filter

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plates, as previously described [20]. The concentration of the samples was estimated from the

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standard curve using a fifth-order polynomial equation (Software xPonent/Analyst versão

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4.2). Intra-assay coefficient of variation was 2.0, 1.6, 2.6, 4.0, and 3.0 % for IL-6, IL-10,

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TNF-α, adiponectin, and resistin, respectively.

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2.5 Faecal analysis

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Feces samples (5-10g) were collected at enrollment and at the sixth week. Samples

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(blinded) were immediately placed at -80 °C and stored until analyzed. The extraction of

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SCFA (acetate, butyrate, and propionate) was based on the method of Smiricky-Tjardes et

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al.[21] and measured by GC (model GC-17A, Shimadzuw, Maryland, USA). N2 was used as

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the carrier gas and the flux in the column was 1.0 mL/min. The temperatures of the injector

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and detector were set at 220 and 250°C, respectively. Initial column temperature was 100°C

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sustained for 5 min, rising at 108°C/min until it reached 185°C. Next, the samples were

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injected (1 mL) through a Hamiltonw syringe (10 mL) in split system 5. The results were

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represented as per 100 mg of faeces (% w/w).

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2.6 Statistical Analysis

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Statistical analysis was done using SPSS Statistics version 20 (IBM, NY, USA). All

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data were checked for normal distribution using Shapiro-Wilk test and Skewness/Kurtosis.

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Data were reported as mean (SD), and median and interquartile interval (P25 and P75%),

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since some variables were not normally distributed (HbA1c, HOMA-IR, IL-6, and acetic

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acid). Comparison between probiotic and control groups at baseline was tested using

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unpaired Student’s t test or Mann-Whitney test. Paired Student’s t test or Wilcoxon matched-

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pairs signed-rank test were used to analyze differences between baseline and endpoint values.

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Pearson or Spearman’s correlation tests were performed to measure the degree of correlation

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between cytokine concentrations and glycemic control. A p value < 0.05 was considered

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statistically significant.

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3. Results

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A total of 45 (90 %) subjects aged 35 to 60 y (mean 51.40 ± 6.80) concluded this

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study. Five patients were excluded according to reasons shown in Figure 1. Abdominal

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discomfort was the only reported adverse effect. This subject (n = 1) belonged to the control

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group and was withdrawn from the study.

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Baseline characteristics are shown in Table 1. The main drugs used by the volunteers

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were metformin (94% in the probiotic group and 95.5% in the control group) and

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glibenclamide (44% in the probiotic group and 41% in the control group). At the baseline, no

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relevant differences could be detected in the general characteristics between control and

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probiotics group, except for HbA1c (p = 0.04), which was higher in the probiotic group.

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There were no statistically significant differences in anthropometric and body composition

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values between or within groups at the end of the study (data not shown). Similarly, at the

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beginning of the study, no significant differences were found between the two groups in

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terms of dietary intakes. Comparing the dietary intakes throughout the study separately in

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each group, we observed no significant differences within group (see Supplemental Table 4).

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3.1 Impact of the intervention on glicemic control

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Biochemical markers after probiotic treatment are show in Table 2. At 6-week follow-

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up, the consumption of probiotic fermented milk significantly decreased fructosamine levels

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(-9.91 mmol/L; p = 0.04) and HbA1c levels tended to be reduced (-0.67%; p = 0.06), while in

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the control group no significant effect was detected on glycemic control (p > 0.05). When the

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median changes in HbA1c were compared between groups, there was a significant difference

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(+ 0.31 for control group vs - 0.65 for probiotic group; p = 0.02). The fasting plasma glucose

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(FPG), insulin concentrations, as well as insulin resistance, evaluated by the HOMA index,

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did not change significantly throughout the follow-up period in both groups (p > 0.05 for all).

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3.2 Effect of probiotics on lipid profile Within group comparisons of lipid profile revealed that consumption of probiotic

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fermented milk prevented a rise in total cholesterol (TC) and LDL-C, while in the control

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group we observed a significant increase in the TC and LDL-c (11.35 and 16.10%; P =0.01

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and P =0.04, respectively). Furthermore, when the mean changes were compared between

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the two groups, there was a statistically significant difference in the TC (- 0.58mmol/L, 95%

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CI: – 1.13 to – 0.03; p = 0.04) and LDL-C (-0.20 mmol/L, 95% CI: -1.02 to -0.03; p = 0.03).

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At the end of trial, no significant effect on HDL-C, VLDL, and triglycerides were found in

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both groups (p > 0.05 for all). The TC:HDL-C ratio was significantly increased by 8.94%

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(0.28 mmol/L) in the control group during the study (p =0.03), while no statistically

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significant changes (p = 0.75) was reported in probiotic group (Table 2).

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3.3 Effect of probiotics on markers of oxidative stress

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At baseline, there was no difference between groups in TAS (0.01mM; 95% CI: –

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0.06 to 0.07; p = 0.86) and F2-isoprostane levels (11.66 pg/dL; 95% CI: – 8.77 to 32.11; p

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=0.25). No significant difference was detected in plasma TAS concentrations from baseline

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to post intervention in probiotic or control groups (p =0.40 and P =0.23, respectively) (Table

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2). Similarly, plasma F2-isoprostane concentrations did not change in the probiotic (p = 0.76)

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or control (p = 0.94) group over time.

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3.4 Effect of probiotics on cytokines levels

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Anti-inflammatory markers, such as IL-10 and adiponectin, decreased after

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intervention in control group (- 0.64 pg/mL [95% CI: -0.47 to -0.81; p = 0.001] for IL-10, -

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0.76 µg/mL [95% CI: -0.75 to -1.59; p = 0.07] for adiponectin), while no significant change

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was observed in the probiotic group (p = 0.38 and p = 0.14, respectively). At the end of trial,

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the two groups exhibited reduction in TNF- α (- 1.4 pg/mL [95% CI: - 0.01 to 2.79; p =0.04]

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for probiotic, -1.36 pg/mL [95% CI: -0.24 to -2.5; P =0.02] for control group) and resistin (-

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2.06 ng/mL [95% CI: -0.66 to -3.44; p = 0.006] for probiotic, - 2.80ng/mL [95% CI: -1.45 to

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- 4.16; p =0.001] for control group). No significant differences in IL-6 levels were observed

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in any groups post intervention. Reductions in resistin were positively correlated with

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reductions in HbA1c (r = 0.320; p = 0.03). No correlation was found between other cytokines

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and markers of glycemic control (data not shown). In Fig. 1 it is possible to compare the

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cytokines concentration between the two groups .

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3.5 Effect of probiotics on faecal SCFA analysis

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Data from faecal analysis showed a significant increase in the acetic acid in both

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groups (0.58 % [95% CI: 0.97 to 1.96; p = 0.005] for probiotic, 0.59 % [95% CI: 0.98 to

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1.89; p = 0.006] for control group) at the end of trial. However, there were no significant

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differences in the butyric and propionic acids after intervention in control and probiotic

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groups, as shown in Fig. 2. Additionally, no significant difference was found between

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changes of the two groups in butyric, acetic and propionic acids (p > 0.05). Interestingly, a

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higher proportion of propionic acid and a lower proportion of butyric acid were recorded in

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both groups. At end of trial, the proportion of propionic: acetic: butyric acids, taking into

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account the mean values, was also similar: 10: 8: 1 in the control group and 14: 10: 1 in the

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probiotic group.

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4. Discussion

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This is the first clinical trial to assess the impact of probiotic use on fructosamine,

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faecal SCFA, IL-10, resistin, adiponectin, and F2-isoprostane in T2D patients. It is speculated

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that improvement in the inflammatory markers, stress oxidative status, and SCFA contributes

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to diabetes control [14, 22]. The present study showed that fermented milk consumption significantly decreased

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inflammatory cytokines (TNF-α and resistin) in both treatments (control vs probiotic), as well

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as caused a significant increase in the acetic acid, but only the probiotic group showed

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improvement in glycemic control, as demonstrated by fructosamine and HbA1c. The main

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differences between the results were: significant decrease in IL-10, a tendence of decreasing

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in adiponectin levels, and significant increase in TC and LDL-C, which were observed only

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in control group. These results indicate that these last factors hinder the improvement in

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glycemic control of subjects in the control group. Furthermore, total phenolic content and

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antioxidant activity were significantly higher in the probiotic fermented milk (see

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Supplemental Table 1).

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Most of the previous animal studies that evaluated the effect of probiotics on glycemic

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control in T2D, related that probiotics, especially Lactobacillus, can reduced FPG, HbA1c,

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and insulin, beyond inflammatory markers, such as IL-2, INF-γ, IL-6, and TNF-α [5, 23-24].

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The intervention time ranged from 8 to 14 wk. Increase in GLUT4 mRNA expression levels

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in adipose tissue has also been reported [5, 25].

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Concerning clinical trials, previous studies including diabetic subjects have shown

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controversial results, especially regarding glycemic control and oxidative stress. Ejtahed et

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al., [26] reported reductions in HbA1c (p < 0.05) after 6 wk of yogurt intake containing 1010

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CFUs of L. acidophilus La-5 and B. lactis BB-12. On the other hand, L. acidophilus NCFM

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capsule (1010 CFUs) intake during 4 wk, did not change the glycemic control, insulin, IL-1,

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IL-6, TNF- α, and protein C reactive (CRP) in T2D subjects [27]. Also, multispecies

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probiotic supplementation (L. acidophilus, L. casei, L. rhamnosus, L. bulgaricus, B. breve, B.

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longum, and Streptococcus thermophilus - 109 CFUs each) associated with fructo-

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oligosaccharide (100 mg) during 8 wk did not result in significant reductions in FPG and a

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significant increase in the levels of insulin, HOMA-IR, and LDL-C was found in both groups

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[28]. Subsequently, this same research group reported that daily consumption of Bacillus

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coagulans (107 CFUs) and 1.08g of inulin during 6 wk, did not result in changes in FPG and

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HOMA-IR (p > 0.05), although a significant decrease in hs-CRP and a significant increase in

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glutathione peroxidase (GPx) activity were found [29].

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The measurement of plasma F2-isoprostane is considered the best measure of plasma

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oxidative stress and TAS gives the total sum of both exogenous and endogenous antioxidants

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[30] showing the complete antioxidant status picture. It has been suggested that the effects of

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probiotic on glycemic control can be partly explained by the effects on oxidative stress [31].

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However, the present study did not show a significant impact of probiotics on F2-isoprostane

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or TAS plasma levels, as well as, earlier clinical trial that evaluated TAS after 6 wk intake of

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B. coagulans (107 CFUs) and 1g of inulin in 124 diabetic patients [29]. On the order hand,

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after consumption of probiotic yogurt containing L. acidophilus La-5 and B. lactis BB-12 for

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6 wk, there was an increase in erythrocyte superoxide dismutase (SOD) and GPx activities,

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and TAS (p < 0.05), although no significant changes were observed in HbA1c, insulin, and

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erythrocyte catalase activity [26].

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We also hypothesized that the improvement in glycemic control after probiotic

293

treatment could be mediated, in part, through immune-modulatory effects. To capture these

294

interrelations, were selected a set of key inflammatory and anti-inflammatory cytokines,

295

including adipokines produced primarily by adipocytes (adiponectin), macrophages (resistin

296

and IL-6), or both (TNF-α), all related to glycemic control and insulin resistance [32-33].

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Although other studies mention the relationship between inflammation and gut

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microbiota [10, 34], few experimental and clinical studies investigated this association in the

299

diabetes context [5, 27, 35]. Some experimental studies reported reductions in inflammatory

300

cytokines, and/or improvements in glucose and insulin metabolims after ingestion of

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Lactobacillus spp during 14 wk [5, 35]. Two randomized trials did not observe effects on

302

inflammatory cytokines after the intake during 4 or 6 wk of L. acidophilus NCFM (1010

303

CFUs) or L. acidophilus, L. bulgaricus, L. bifidum, and L. casei (CFUs and strain not

304

reported), respectively [16, 27]. In agreement with these previous studies, IL-6 did not

305

change after intervention, however, TNF- α and resistin levels decreased in both treatments in

306

our study. It has been shown that anti-inflammatory activities of goat milk oligosaccharides

307

reside in their ability to function either as prebiotics or as a receptor for microorganisms

308

(Daddaoua et al., 2006). Others studies shows the ability of bioactive peptides derived from

309

food fermentation in the immune system regulation, including inhibition of NF-κB, a pro-

310

inflammatory transcription factors [36-37].

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In regard to anti-inflammatory cytokines (IL-10 and adiponectin), no changes were

312

observed in the probiotic group, unlike the control group, which showed a significant

313

reduction at the end of trial. Thus, our results suggest that the reduction in these cytokines

314

reflected on lower glycemic control observed in the control group. Recently, Mohamadshahi

315

et al. [38], also reported that the consumption of probiotic yogurt enriched with L.

316

acidophilus La-5 and B. lactis BB-12 (106 CFUs) for 8 wk caused significant decrease in

317

HbA1c and TNF-α levels in the intervention group, but no changes were observed in IL-6 and

318

hs-CRP levels. In mice, L. rhamnosus GG orally administrated for 13 wk improves insulin

319

sensitivity by stimulating adiponectin secretion and consequent activation of AMPK [39],

320

highlighting the important role of adiponectin in glycemic control. Some contradictory results

321

may be explained by complex regulation of cytokines, and we point out that the production of

322

IL-6 and IL-10 is stimulated by TNF-α, which was reduced in the present study.

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323

Additionally, microbiota components account for the production of SCFA, which are

324

linked to anti-inflammatory mechanisms (inhibition of NF-κB) and also exerting a protective

325

function in favor of intestinal epithelium [40]. However, reports on the ability of lactic acid

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bacteria to modulate SCFA at the intestinal level are limited. In our literature review [41], we

327

didn’t find experimental or clinical trials that evaluated the effect of probiotics intake on the

328

SCFA in T2D context. Interestingly, growing evidence suggests a cross talk between SCFA

329

and improves in glycemic control, especially butyric and propionic acids via the G protein

330

coupled receptors FFA2 and FFA3 [42-43]. However, we found a significant increase in

331

acetic acid after intervention in both groups, which has little effect on incretins (GLP-1, PYY,

332

and GIP) secretion [42] compared to butyric and propionic acids. This increase in the acetic

333

acid in both treatments may be attributed to phenolic compounds present in FM [44].

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With regard to lipid profile, contradictory results have also been shown in the

335

literature. L. acidophilus La-5 and B. lactis BB-12 (106 CFUs) use contributed to the decrease

336

of TC (4.5%) and LDL-C (7.5%) levels (p < 0.05) after 6 wk in T2D subjects [45]. In a

337

different study, the same probiotics taken for 8 wk, caused reductions in LDL-C/ HDL-C (p

338

= 0.01) and increase HDL-C levels [46]. However, other studies using different species of

339

Lactobacillus or B. coagulans and inulin for 6 wk did not observe effects on the lipid profile

340

in T2D subjects [16, 29].

341

prevented a rise in TC and LDL-C compared to the control group, which can be attributed, in

342

part, by the higher total phenolic concentrations and antioxidant capacity in probiotic FM,

343

however it was not enough to cause significant reductions in these parameters in the probiotic

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group.

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Our study demonstrated that consumption of probiotic FM

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Finally, probiotics as functional foods offer great potential to improve health and/or

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help prevent certain diseases when taken as part of a balanced diet and healthy lifestyle. Our

347

results showed low intake of micronutrients and dietary fiber, and high intake of saturated

348

fatty acids (see Supplemental Table 4).

349

In conclusion, this trial suggests that probiotic consumption improves the glycemic

350

control in T2D subjects, however, the intake of fermented goat’s milk seems to be involved

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with changes in inflammatory cytokines (TNF-α and resistin) and in the acetic acid

352

concentrations. The major findings of this study are the following: (i) L. acidophilus and B.

353

lactis intake seem to be associated with a decrease of the fructosamine and HbA1c levels; (ii)

354

fermented goat’s milk consumption appears to be related to acetic acid content in faecal

355

samples; (iii) the role of probiotic in averting an increase of CT and LDL-C and a reduction

356

of anti-inflammatory cytokines. Because our study was designed to be a short-term trial, our

357

results need to be confirmed in long-term trials testing the hypothesis that probiotic

358

supplementation is effective in improving glycemic control, regulating SCFA levels and

359

decreasing oxidative stress in T2D subjects.

360

Conflict of interest

361

None.

362

Acknowledgments

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This study was supported by Brazilian Agricultural Research Corporation

364

(EMBRAPA) and Foundation for Research Support of the State of Minas Gerais

365

(FAPEMIG). We thank study volunteers for their participation; Mariana Rodrigues and

366

Etianne Lopes (INTA) for trial staff; Adriano Lima (EMBRAPA) for providing data

367

management support; Renata Toledo (Federal University of Viçosa) and José Ricardo

368

(EMBRAPA) for providing laboratory support. We thank Tabosa Santos and Isabel Cristina

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(EMBRAPA) for technical assistance and Marcelo Amadei for providing study recruitment

370

support. The authors also thank the Embrapa Goats and Sheep for generous support.

371

Authors contributions

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LBT, KMOS, and HSDM designed the trial. LBT and KMOS were responsible for the

373

analysis and production of fermented milk. LBT and HSDM conducted the reseach- were

374

responsible for study recruitment, screening, and delivery of interventions. LBT and HSDM

375

did the statistical analysis. LBT and LLO analysed and interpreted stress oxidative datas. All

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authors participated in data interpretation. LBT wrote the first draft of the report, and all other

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authors commented on the draft and approved the final version. Authorship order reflects

378

overall contribution to the work presented. None of the authors had a conflict of interest.

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Appendix A. Supplementary data

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[28] Asemi Z, Zare Z, Shakeri H, Sabihi S, Esmaillzadeh A. Effect of Multispecies Probiotic Supplements on Metabolic Profiles, hs-CRP, and Oxidative Stress in Patients with Type 2 Diabetes. Annals of Nutrition and Metabolism. 2013;63:1-9. [29] Asemi Z, Khorrami-Rad A, Alizadeh S-A, Shakeri H, Esmaillzadeh A. Effects of

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synbiotic food consumption on metabolic status of diabetic patients: A double-blind randomized cross-over controlled clinical trial. Clinical Nutrition. 2014;33:198-203.

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plasma markers of oxidative stress in diabetes and cardiovascular disease. Atherosclerosis. 2009;202:321-9.

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[31] Wang X, Bao W, Liu J, OuYang Y-Y, Wang D, Rong S, et al. Inflammatory Markers and Risk of Type 2 Diabetes: A systematic review and meta-analysis. Diabetes Care. 2013;36:166-75.

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Adiponectin, resistin and IL-6 plasma levels in subjects with diabetic foot and possible correlations with clinical variables and cardiovascular co-morbidity. Cardiovascular Diabetology. 2010;9:50.

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[36] Martínez-Augustin O, Rivero-Gutiérrez B, Mascaraque C, Sánchez de Medina F. Food Derived Bioactive Peptides and Intestinal Barrier Function. International Journal of Molecular Sciences. 2014;15:22857-73. [37] Marcone S, Haughton K, Simpson P, Belton O, Fitzgerald D. Milk-derived bioactive

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peptides inhibit human endothelial-monocyte interactions via PPAR-gamma dependent

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Effects of probiotic yogurt consumption on inflammatory biomarkers in patients with type 2 diabetes. Bioimpacts. 2014;4:83-8.

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[42] Lin HV, Frassetto A, Kowalik Jr EJ, Nawrocki AR, Lu MM, Kosinski JR, et al. Butyrate and Propionate Protect against Diet-Induced Obesity and Regulate Gut Hormones via Free Fatty Acid Receptor 3-Independent Mechanisms. PLoS ONE. 2012;7:e35240. [43] Kasubuchi M, Hasegawa S, Hiramatsu T, Ichimura A, Kimura I. Dietary Gut Microbial Metabolites, Short-chain Fatty Acids, and Host Metabolic Regulation. Nutrients. 2015;7:2839-49.

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[44] Parkar SG, Trower TM, Stevenson DE. Fecal microbial metabolism of polyphenols and its effects on human gut microbiota. Anaerobe. 2013;23:12-9. [45] Ejtahed HS, Mohtadi-Nia J, Homayouni-Rad A, Niafar M, Asghari-Jafarabadi M, Mofid V, et al. Effect of probiotic yogurt containing Lactobacillus acidophilus and Bifidobacterium

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lactis on lipid profile in individuals with type 2 diabetes mellitus. Journal of dairy science. 2011;94:3288-94.

[46] Mohamadshahi M, Veissi M, Haidari F, Javid AZ, Mohammadi F, Shirbeigi E. Effects

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controlled clinical trial. J Res Med Sci. 2014;19:531-6.

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of probiotic yogurt consumption on lipid profile in type 2 diabetic patients: A randomized

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ACCEPTED MANUSCRIPT Tables Table 1. Baseline characteristics of the type 2 diabetes participants

(n = 22)

(n = 23)

Age (y)

50.95 ± 7.20

51.83 ± 6.64

0.73

Sex (M/F)

14/8 (63/37)

12/11 (53/47)

0.45

Diabetes duration (y)

4.5 (2 – 15)

6.0 (2 -17)

0.51

Regular physical activity

12 (54.5)

0.31

Weight (kg)

77.15 ± 13.85

71.70 ± 12.43

0.16

BMI (kg/m2)

27.94 ± 4.15

27.49 ± 3.97

0.65

Total body fat (%)

32.95 ± 8.96

33.92 ± 8.24

0.70

HbA1c (%)

5.35 (4.86 – 6.15)

6.07 (5.39 – 7.0)

0.04*

7.97 ± 2.31

0.41

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FPG (mmol/L)

7.38 ± 2.39

09 (39.1)

2.15 (1.71 – 3.26) 2.63 (1.57 – 3.51)

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HOMA-IR

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Probiotic

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p1 value

Control

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Characteristics

0.55

997.74 ± 460.50

1123.91 ± 578.76

0.36

Glibenclamide (mg)

5.02 ± 12.60

9.78 ± 23.84

0.83

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Metformin (mg)

Data are mean ± SD, n (%) or median (P25-P75). 1Significant difference between groups at baseline (p < 0.05 from Student’s t test or Mann–Whitney test). M = masculine; F = female; BMI = body mass index; HbA1c = haemoglobin A1c; FPG = fasting plasma glucose.

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24 Table 2: Metabolic parameters of type 2 diabetes subjects at baseline and endpoint by fermented milk treatment Probiotic (n = 23)

Measure Week 0

Week 6

Mean/median change

p1

Week 0

7.38 (2.37)

7.54 (2.50)

0.16

0.65

7.89 (2.28)

Fru, mmol/L

295.5 (62.23)

296.90 (59.64)

1.36

0.85

302.82 (51.31)

0.11

0.82

-1.65

0.95

5.35 (4.9 to 6.1) 6.7) 6.65 (5.2 to

Insulin, µU/mL

8.3 (5.0 to 10.1)

2.15 (1.71 to

2.3 (1.55 to

3.26)

3.27)

TC, mmol/L

4.85 (1.32)

5.30 (1.19)

LDL-C, mmol/L

2.24 (1.24)

2.60 (1.11)

1.51 (0.26)

3.20 (1.06)

mmol/L

Effect (95% CI)

p2

n change 0.52

0.14

6.35 (- 0.64 to 1.34)

0.48

292.90 (53.25)

-9.91

0.04

-12.06 (-28.51 to 4.40)

0.15

..

6.07 (5.4 to 7.0)

5.40 (5.1to 7.3)

-0.67

0.06

7.5 (5.2 to 11.9)

6.8 (5.2 to 11.4)

-0.70

0.73

2.63 (1.57 to

2.65 (1.71 to 0.02

0.41

0.02

..

3.52)

4.21)

0.72

.. 0.77

0.55

0.01

4.66 (1.38)

4.51 (1.11)

- 0.15

0.52

-0.58 (-1.13 to -0.003)

0.04

0.36

0.04

2.31 (1.17)

2.11 (0.82)

- 0.20

0.31

-0.52 (-1.02 to -0.03)

0.03

1.53 (0.34)

0.02

0.59

1.56 (0.29)

1.53 (0.33)

- 0.03

0.50

3.48 (0.97)

0.28

0.03

2.98 (0.64)

2.94 (0.72)

- 0.05

0.70

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HDL-C,

p1

8.41 (2.41)

0.73

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0.15

HOMA-IR

TC:HDL-C

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11.4)

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5.66 (4.9 to HbA1c, %

Mean/media

Week 6

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FPG, mmol/L

Intervention effect

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Control (n = 22)

-0.006 (-0.21 to 0.08) 0.38

-0.17 (-0.53 to 0.19)

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25 1.48 (0.93)

1.69 (0.83)

0.21

0.08

1.48 (0.64)

1.37 (0.58)

- 0.11

0.30

-0.22 (-0.56 to 0.12)

0.19

TAG, mmol/L

1.83 (0.72)

1.99 (0.91)

0.16

0.73

1.60 (0.63)

1.68 (0.63)

0.08

0.28

-0.008 (-0.44 to 0.27)

0.62

TAS, (mM)

0.31 (0.12)

0.33 (0.08)

0.02

0.23

0.31 (0.09)

0.33 (0.11)

0.02

0.39

0.01 (-0.07 to 0.08)

0.87

62.99 (33.67)

63.42 (32.35)

- 0.43

0.94

74.66 (30.20)

72.49 (34.70)

- 2.17

0.76

-2.59 (-21.41 to 16.23)

0.78

F2-iso, (pg/mL)

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LDL-C:HDL-C

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Data are means (SD) or median (P25-P75). 1Difference between baseline and endpoint. p value obtained from paired t test/Wilcoxon matched-pairs signed-rank test for the within-group comparisons. 2Obtained from unpaired Student’s t test or Mann–Whitney test, as statistical differencece between changes (control vs probiotic). FPG = fasting

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blood glucose; Fru = fructosamine; TC = total cholesterol; TAG = triglyceride; TAS = total antixodant status; F2-iso = F2-isoprostane.

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Figures

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Fig 1. Trial profile.

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*

†

*

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†

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Fig 2. Effects of the fermented milks intake on cytokine levels Data are shown as mean (SD). *p < 0.05, †p = 0.001 from paired t test or for Wilcoxon matched-pairs signed-rank test, as statistical within group differences (baseline vs endpoint). IL = interleukin; TNF- α = tumor necrosisfactor alpha.

28

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Fig 3. Effects of the fermented milks intake on faecal short-chain fatty acid concentrations

Data are shown as mean (SD). †p ≤ 0.01 from Wilcoxon matched-pairs signed-rank test, as

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statistical within group differences (baseline vs endpoint).