Accepted Manuscript

Clinical Application of Probiotics in Type 2 Diabetes Mellitus: a Randomized, Double-

Blind, 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, Placebo-
Controlled Study, Clinical Nutrition (2016), doi: 10.1016/j.clnu.2015.11.011.

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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

Karina Maria Olbrich dos Santosb, PhD

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Leandro Licursi de Oliveirac, PhD

Sonia Machado Rocha Ribeirod, PhD

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Hercia Stampini Duarte Martinod, PhD

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a
Professor at Department of Nutrition, INTA College.
b

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

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

36.570-000.
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Corresponding author: Livia Bordalo Tonucci. Address: R. Antônio Rodrigues

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Magalhães, 359 - Dom Expedito Lopes, Sobral - CE. Zip Code: 62050-100. Postal
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code, 10. E-mail: livia_bordalo@hotmail.com. 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/RBR-
219644.
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1 ABSTRACT

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

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

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

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

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

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

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

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

10 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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12 Results: 45 subjects out of 50 (90%) completed follow-up. After 6 wk, there was a
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13 significant decrease in fructosamine levels (- 9.91mmol/L; p = 0.04) and hemoglobin A1c

14 tended to be lower (- 0.67%; p = 0.06) in probiotic group. TNF-α and resistin were
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15 significantly reduced in probiotic and control groups (- 1.5 and -1.3 pg/mL, -2.1 and -2.8
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16 ng/mL, respectively), while IL-10 was significantly reduced (- 0.65 pg/mL; p < 0.001) only

17 in the control group. Fecal acetic acid was increased in both groups (0.58 and 0.59% in
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18 probiotic and control groups, respectively; p < 0.01). There was a significant difference
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19 between groups concerning mean changes of HbA1c (+ 0.31 for control group vs - 0.65 for
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20 probiotic group; p = 0.02), total cholesterol (+ 0.55 for control group vs - 0.15 for probiotic

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

22 p = 0.03).

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

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

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

27 1. Introduction

28 Diabetes mellitus is on the rise all over the world affecting more than 382 million

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

30 chronic illness requiring multifactorial risk reduction strategies [1].

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

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32 [3]. Both molecular and metabolic mechanisms are links to β-cell dysfunction and/or insulin

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

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

35 [5-6].

36
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In addition, the gut microbiota also seems to be important to the pathophysiology of
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37 T2D [7]. Findings from two studies that used faecal samples suggested that compositional
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38 and functional changes in the gut microbiota might be directly linked to the development of

39 T2D [8-9]. Various others mechanisms have been proposed to explain the influence of the
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40 microbiota on insulin resistance and T2D, such as metabolic endotoxemia [10], modifications
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41 in the secretion of the incretins [11], and short-chain fatty acids (SCFA) production [12].

42 Multiple cytokines are involved in the development of T2D, and are fundamental signals in
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43 the intestinal immune system, able to subvert the physiological status of inflammation in the
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44 gut [13].
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45 Alterations in the gut microbiota as a result of probiotics and prebiotics intake have

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

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

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

49 unknown.

50 The objective of this study was to investigate the efficacy of the intake of fermented
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51 goat milk containing Lactobacillus acidophilus La-5 and Bifidobacterium animalis BB-12 on

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

53 subjects.

54

55 2. Methods

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56 2.1 Subjects

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

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

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

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

61
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endocrinology clinics. The appropriate sample size was calculated based on primary outcome
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62 of serum interleukin (IL)-6 levels obtained from Mazloom et al. [16], considering a alpha
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63 level of 0.05 and a power of 80%. The number of subjects required was 22.5 per group, but

64 this number was increased to 25 per group to accommodate the withdrawals. The research
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65 team did recruitment within the clinics after indication of participants by endocrinologist.
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66 We excluded participants with clinical evidence of chronic illness or gastrointestinal

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

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68 of cancer or cardiovascular diseases; current treatment with insulin, DPP-4 inhibitor or GLP-
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69 1 analogue; smoking; any intake of probiotics, supplements, antiobesity and anti-

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

71 pregnancy or breast-feeding.

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

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

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

75 2.2 Randomisation and blinding

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

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

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

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

80 by a technical assistant, right after beverages production.

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81 2.3 Study protocol

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

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

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

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

86
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during the breakfast. The fermented milk (FM) was delivered every 2 wk and volunteers were
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87 instructed to keep it under refrigeration (4 ºC).
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88 The probiotic and conventional FM were developed in parthership with Embrapa Goat

89 and Sheep (Ceará, Brazil). Pasteurised milk was cooled to 43 ± 2oC for the addition of the
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90 starter culture (S. thermophilus TA-40; Danisco, Sassenage, France), and the probiotic
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91 cultures (L. acidophilus La-5 and B. animalis subsp. lactis BB-12; Chr. Hansen, Hoersholm,

92 Denmark). The beverages were flavored with grape juice obtained from Embrapa Grape and
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93 Wine (Rio Grande do Sul, Brazil). Ingredients, chemical composition and antioxidants status
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94 of the probiotic FM are shown in Table supplementary 1 (see supplementary data). Both
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95 products had the same taste, color, and smell.

96 Fermented milk was sampled immediately after manufacture and delivered on the

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

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

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

100 were determined in modified De Man Rogosa Sharpe (MRS) agar (Oxoid, Basingstoke, UK),
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101 followed by incubation at 37 ºC for 72 h [17].

102 Microbiological analyses of the probiotic FM showed that the average colony counts

103 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

104 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,

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

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

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

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

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

110 Supplemental Table 3).

111
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All anthropometric and biochemical measures were conducted in a fasting state taken
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112 at baseline and after 6 wk intervention. A single experienced examiner performed all
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113 anthropometric measurements. Body weight and body composition were assessed by bio-

114 impedance (Tanita TBF-300, Tanita Corporation, Tokyo, Japan). Waist circumference
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115 (measured midway between lowest rib and iliac crest) was measured using a non-stretchable
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116 measuring tape (Sanny, São Paulo, Brazil).

117 Dietary assessment was done using food records, which were conducted by the
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118 nutritionist in the first and the last week of intervention. Dietary intake was analysed using
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119 Avanutri Revolution software (Rio de Janeiro, Brazil). Regular level of physical activity was
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120 defined as three times a week for at least 30 min.

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

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

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

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

125 2.4 Biochemical measurements

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

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

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

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

130 80 °C for cytokines and oxidative stress analyses.

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

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

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

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

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

136
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performance liquid chromatography (Bio-rad kit, California, USA). The homeostasis model
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137 assessment index (HOMA-IR) was used as an indicator of insulin resistance and calculated as
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138 follows: HOMA-IR = fasting plasma insulin (µU/mL) x fasting plasma glucose (mmol/

139 L)/22.5. Insulin resistance was diagnosed using a cut off value of 2.71 [19].
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140 For determination of oxidative stress markers, plasma samples were collected in
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141 vacutainers containing sodium citrate. Colorimetric assay (Sigma-Aldrich antioxidant assay

142 kit, Missouri, USA; interassay coefficients of variation 3%) was used to measure plasma total
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143 antioxidant status (TAS). Plasma total F2–isoprostane was measured by ELISA kit (Caymans
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144 8-isoprostane EIA kit, Michigan, USA; interassay coefficients of variation 9.5% at 80 pg/mL
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145 and 6.4% at 32 pg/mL). Briefly, plasma samples (400 µL) were hydrolysed with (10N)

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

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

148 10 min to remove precipitated proteins.

149 Cytokine concentrations were determined using a multiplexed bead immunoassay.

150 This is a bead-based suspension array using the LuminexxMAP technology in which
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151 fluorescent-coded beads, known as microspheres, have cytokine capture antibodies on the

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

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

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

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

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

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157 4.2). Intra-assay coefficient of variation was 2.0, 1.6, 2.6, 4.0, and 3.0 % for IL-6, IL-10,

158 TNF-α, adiponectin, and resistin, respectively.

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159 2.5 Faecal analysis

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

161
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(blinded) were immediately placed at -80 °C and stored until analyzed. The extraction of
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162 SCFA (acetate, butyrate, and propionate) was based on the method of Smiricky-Tjardes et
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163 al.[21] and measured by GC (model GC-17A, Shimadzuw, Maryland, USA). N2 was used as

164 the carrier gas and the flux in the column was 1.0 mL/min. The temperatures of the injector
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165 and detector were set at 220 and 250°C, respectively. Initial column temperature was 100°C
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166 sustained for 5 min, rising at 108°C/min until it reached 185°C. Next, the samples were

167 injected (1 mL) through a Hamiltonw syringe (10 mL) in split system 5. The results were
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168 represented as per 100 mg of faeces (% w/w).

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169 2.6 Statistical Analysis

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

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

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

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

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

175 unpaired Student’s t test or Mann-Whitney test. Paired Student’s t test or Wilcoxon matched-
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176 pairs signed-rank test were used to analyze differences between baseline and endpoint values.

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

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

179 statistically significant.

180 3. Results

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

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

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

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184 group and was withdrawn from the study.

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

186
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were metformin (94% in the probiotic group and 95.5% in the control group) and
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187 glibenclamide (44% in the probiotic group and 41% in the control group). At the baseline, no
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188 relevant differences could be detected in the general characteristics between control and

189 probiotics group, except for HbA1c (p = 0.04), which was higher in the probiotic group.
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190 There were no statistically significant differences in anthropometric and body composition
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191 values between or within groups at the end of the study (data not shown). Similarly, at the

192 beginning of the study, no significant differences were found between the two groups in
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193 terms of dietary intakes. Comparing the dietary intakes throughout the study separately in
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194 each group, we observed no significant differences within group (see Supplemental Table 4).
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195 3.1 Impact of the intervention on glicemic control

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

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

198 (-9.91 mmol/L; p = 0.04) and HbA1c levels tended to be reduced (-0.67%; p = 0.06), while in

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

200 median changes in HbA1c were compared between groups, there was a significant difference
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201 (+ 0.31 for control group vs - 0.65 for probiotic group; p = 0.02). The fasting plasma glucose

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

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

204 3.2 Effect of probiotics on lipid profile

205 Within group comparisons of lipid profile revealed that consumption of probiotic

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

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

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

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

210 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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212 both groups (p > 0.05 for all). The TC:HDL-C ratio was significantly increased by 8.94%
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213 (0.28 mmol/L) in the control group during the study (p =0.03), while no statistically

214 significant changes (p = 0.75) was reported in probiotic group (Table 2).
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215 3.3 Effect of probiotics on markers of oxidative stress

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

217 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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218 =0.25). No significant difference was detected in plasma TAS concentrations from baseline
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219 to post intervention in probiotic or control groups (p =0.40 and P =0.23, respectively) (Table
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220 2). Similarly, plasma F2-isoprostane concentrations did not change in the probiotic (p = 0.76)

221 or control (p = 0.94) group over time.

222 3.4 Effect of probiotics on cytokines levels

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

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

225 0.76 µg/mL [95% CI: -0.75 to -1.59; p = 0.07] for adiponectin), while no significant change
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226 was observed in the probiotic group (p = 0.38 and p = 0.14, respectively). At the end of trial,

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

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

229 2.06 ng/mL [95% CI: -0.66 to -3.44; p = 0.006] for probiotic, - 2.80ng/mL [95% CI: -1.45 to

230 - 4.16; p =0.001] for control group). No significant differences in IL-6 levels were observed

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

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

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

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234 cytokines concentration between the two groups .

235 3.5 Effect of probiotics on faecal SCFA analysis

236
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Data from faecal analysis showed a significant increase in the acetic acid in both
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237 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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238 1.89; p = 0.006] for control group) at the end of trial. However, there were no significant

239 differences in the butyric and propionic acids after intervention in control and probiotic
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240 groups, as shown in Fig. 2. Additionally, no significant difference was found between
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241 changes of the two groups in butyric, acetic and propionic acids (p > 0.05). Interestingly, a

242 higher proportion of propionic acid and a lower proportion of butyric acid were recorded in
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243 both groups. At end of trial, the proportion of propionic: acetic: butyric acids, taking into
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244 account the mean values, was also similar: 10: 8: 1 in the control group and 14: 10: 1 in the
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245 probiotic group.

246

247 4. Discussion

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

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

250 that improvement in the inflammatory markers, stress oxidative status, and SCFA contributes
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251 to diabetes control [14, 22].

252 The present study showed that fermented milk consumption significantly decreased

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

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

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

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

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

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

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

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

261 Supplemental Table 1).

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262 Most of the previous animal studies that evaluated the effect of probiotics on glycemic
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263 control in T2D, related that probiotics, especially Lactobacillus, can reduced FPG, HbA1c,

264 and insulin, beyond inflammatory markers, such as IL-2, INF-γ, IL-6, and TNF-α [5, 23-24].
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265 The intervention time ranged from 8 to 14 wk. Increase in GLUT4 mRNA expression levels
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266 in adipose tissue has also been reported [5, 25].

267 Concerning clinical trials, previous studies including diabetic subjects have shown
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268 controversial results, especially regarding glycemic control and oxidative stress. Ejtahed et

al., [26] reported reductions in HbA1c (p < 0.05) after 6 wk of yogurt intake containing 1010
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269
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270 CFUs of L. acidophilus La-5 and B. lactis BB-12. On the other hand, L. acidophilus NCFM

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

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

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

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

275 oligosaccharide (100 mg) during 8 wk did not result in significant reductions in FPG and a
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276 significant increase in the levels of insulin, HOMA-IR, and LDL-C was found in both groups

277 [28]. Subsequently, this same research group reported that daily consumption of Bacillus

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

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

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

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

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

283 [30] showing the complete antioxidant status picture. It has been suggested that the effects of

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

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

286
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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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287 B. coagulans (107 CFUs) and 1g of inulin in 124 diabetic patients [29]. On the order hand,
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288 after consumption of probiotic yogurt containing L. acidophilus La-5 and B. lactis BB-12 for

289 6 wk, there was an increase in erythrocyte superoxide dismutase (SOD) and GPx activities,
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290 and TAS (p < 0.05), although no significant changes were observed in HbA1c, insulin, and
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291 erythrocyte catalase activity [26].

292 We also hypothesized that the improvement in glycemic control after probiotic
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293 treatment could be mediated, in part, through immune-modulatory effects. To capture these
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294 interrelations, were selected a set of key inflammatory and anti-inflammatory cytokines,
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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].

297 Although other studies mention the relationship between inflammation and gut

298 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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301 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

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

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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

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

310 inflammatory transcription factors [36-37].

311
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In regard to anti-inflammatory cytokines (IL-10 and adiponectin), no changes were
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312 observed in the probiotic group, unlike the control group, which showed a significant
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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
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315 et al. [38], also reported that the consumption of probiotic yogurt enriched with L.
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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
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318 hs-CRP levels. In mice, L. rhamnosus GG orally administrated for 13 wk improves insulin
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319 sensitivity by stimulating adiponectin secretion and consequent activation of AMPK [39],
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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.

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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326 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

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

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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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334 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
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of TC (4.5%) and LDL-C (7.5%) levels (p < 0.05) after 6 wk in T2D subjects [45]. In a
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337 different study, the same probiotics taken for 8 wk, caused reductions in LDL-C/ HDL-C (p
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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
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340 in T2D subjects [16, 29]. Our study demonstrated that consumption of probiotic FM
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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,
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343 however it was not enough to cause significant reductions in these parameters in the probiotic
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344 group.
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345 Finally, probiotics as functional foods offer great potential to improve health and/or

346 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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351 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

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

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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

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359 decreasing oxidative stress in T2D subjects.

360 Conflict of interest

361 None.
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362 Acknowledgments
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363 This study was supported by Brazilian Agricultural Research Corporation

364 (EMBRAPA) and Foundation for Research Support of the State of Minas Gerais
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365 (FAPEMIG). We thank study volunteers for their participation; Mariana Rodrigues and
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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
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368 (EMBRAPA) for providing laboratory support. We thank Tabosa Santos and Isabel Cristina
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369 (EMBRAPA) for technical assistance and Marcelo Amadei for providing study recruitment
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370 support. The authors also thank the Embrapa Goats and Sheep for generous support.

371 Authors contributions

372 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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376 authors participated in data interpretation. LBT wrote the first draft of the report, and all other

377 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.

379 Appendix A. Supplementary data

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Tables

Table 1. Baseline characteristics of the type 2 diabetes participants

Characteristics Control Probiotic p1 value

(n = 22) (n = 23)

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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

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Diabetes duration (y) 4.5 (2 – 15) 6.0 (2 -17) 0.51

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Regular physical activity 12 (54.5) 09 (39.1) 0.31

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Weight (kg) 77.15 ± 13.85 71.70 ± 12.43 0.16
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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

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HbA1c (%) 5.35 (4.86 – 6.15) 6.07 (5.39 – 7.0) 0.04*

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FPG (mmol/L) 7.38 ± 2.39 7.97 ± 2.31 0.41

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HOMA-IR 2.15 (1.71 – 3.26) 2.63 (1.57 – 3.51) 0.55

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Metformin (mg) 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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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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Table 2: Metabolic parameters of type 2 diabetes subjects at baseline and endpoint by fermented milk treatment

Control (n = 22) Probiotic (n = 23) Intervention effect

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Measure Mean/media
Week 0 Week 6 Mean/median change p1 Week 0 Week 6 p1 Effect (95% CI) p2
n change

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FPG, mmol/L 7.38 (2.37) 7.54 (2.50) 0.16 0.65 7.89 (2.28) 8.41 (2.41) 0.52 0.14 6.35 (- 0.64 to 1.34) 0.48

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Fru, mmol/L 295.5 (62.23) 296.90 (59.64) 1.36 0.85 302.82 (51.31) 292.90 (53.25) -9.91 0.04 -12.06 (-28.51 to 4.40) 0.15

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5.66 (4.9 to ..
HbA1c, % 5.35 (4.9 to 6.1) 0.11 0.82 6.07 (5.4 to 7.0) 5.40 (5.1to 7.3) -0.67 0.06 0.02

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6.7)

6.65 (5.2 to ..

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Insulin, µU/mL 8.3 (5.0 to 10.1) -1.65 0.95 7.5 (5.2 to 11.9) 6.8 (5.2 to 11.4) -0.70 0.73 0.72
11.4)

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2.15 (1.71 to 2.3 (1.55 to 2.63 (1.57 to 2.65 (1.71 to ..

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HOMA-IR 0.15 0.73 0.02 0.41 0.77
3.26) 3.27) 3.52) 4.21)
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TC, mmol/L 4.85 (1.32) 5.30 (1.19) 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

LDL-C, mmol/L 2.24 (1.24) 2.60 (1.11) 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
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HDL-C, -0.006 (-0.21 to 0.08)

1.51 (0.26) 1.53 (0.34) 0.02 0.59 1.56 (0.29) 1.53 (0.33) - 0.03 0.50 0.38
mmol/L

TC:HDL-C 3.20 (1.06) 3.48 (0.97) 0.28 0.03 2.98 (0.64) 2.94 (0.72) - 0.05 0.70 -0.17 (-0.53 to 0.19) 0.35
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LDL-C:HDL-C 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

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F2-iso, (pg/mL) 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

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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

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within-group comparisons. 2Obtained from unpaired Student’s t test or Mann–Whitney test, as statistical differencece between changes (control vs probiotic). FPG = fasting

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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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.

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

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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).
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