E-Mail karger@karger.com

Original Paper

Ann Nutr Metab 2014;65:34–41 DOI: 10.1159/000365153

Effects of Daily Consumption of Synbiotic Bread on Insulin Metabolism and Serum High-Sensitivity C-Reactive Protein among Diabetic Patients: A Double-Blind, Randomized, Controlled Clinical Trial

Maryam Tajadadi-Ebrahimi   a Fereashteh Bahmani   c Hossein Shakeri   c Haleh Hadaegh   b Mohammad Hijijafari   c Fatemeh Abedi   c Zatollah Asemi   c  

a   Science Department, Science Faculty, Islamic Azad University, Tehran Central Branch, and b   Department of Research and Development, Sahar Bread Company, Tehran , and c   Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan , Iran

tance scores (–1.5 ± 2.7 vs. –0.2 ± 1.6 and 0.4 ± 3.5, respec-tively, p = 0.03), and homeostatic model assessment-β-cell function (–7.2 ± 16.3 vs. –0.7 ± 10.8 and 0.7 ± 8.2, respective-ly, p = 0.04) compared to the probiotic and control breads. We did not find any significant effect of synbiotic bread con-sumption on fasting plasma glucose, the quantitative insulin sensitivity check index, or serum hs-CRP levels compared to other breads. Conclusion: Consumption of the synbiotic bread among diabetic patients had beneficial effects on in-sulin metabolism. © 2014 S. Karger AG, Basel

Introduction

Obesity, increased adiposity in white adipose tissues, and impaired thermogenesis in brown adipose tissues [1, 2] among patients with type 2 diabetes mellitus (T2DM) result in impaired insulin function and inflammation. In-sulin resistance in patients with T2DM leads to a reduced insulin-mediated glucose uptake and metabolism, partic-

Key Words

Synbiotic · Probiotic · Insulin metabolism · Diabetes

Abstract

Background: This study was conducted to evaluate the ef-fects of daily consumption of synbiotic bread on the meta-bolic status of patients with type 2 diabetes mellitus. Meth-

ods: This randomized, double-blind, controlled clinical trial was performed in 81 diabetic patients. The subjects were randomly assigned to consumption of synbiotic (n  = 27), probiotic (n = 27), or control bread (n = 27) for 8 weeks 3 times a day in a 40-gram package. The synbiotic bread con-tained Lactobacillus sporogenes (1 × 10 8 CFU) and 0.07 g inu-lin per 1 g. The probiotic bread contained L. sporogenes (1 × 10 8 CFU per 1 g). Fasting blood samples were taken at base-line and after an 8-week intervention for quantification of related factors. Results: Consumption of the synbiotic bread resulted in a significant reduction in serum insulin levels (–3.2 ± 5.4 vs. –0.3 ± 3.4 and 0.6 ± 4.7 μIU/ml, respectively, p = 0.007), homeostatic model assessment for insulin resis-

Received: February 27, 2014 Accepted after revision: June 9, 2014 Published online: September 4, 2014

Zatollah Asemi, PhD Department of Biochemistry and Nutrition Kashan University of Medical Sciences, Bolvare Ghotbe RavandiKashan PO Box , Kashan 87198-441 (Iran) E-Mail asemi_r   @   yahoo.com

© 2014 S. Karger AG, Basel0250–6807/14/0651–0034$39.50/0

www.karger.com/anm

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ularly in skeletal muscle [3–5] , as well as dysregulated fat-ty acid metabolism and increased blood lipid profiles [6, 7] . Impaired insulin metabolism and increased inflam-matory factors could result in increased cardiovascular morbidity and mortality [8, 9] , damaged fibrinolysis [10] , plaque development [11] , and mental health issues (in particular an increased risk of depression) [8] .

Various strategies have been suggested for the man-agement of the glycemic status and inflammatory mark-ers in patients with T2DM, including diet therapy (es-pecially with a low glycemic index and glycemic load) [12] , lifestyle modifications [13] , weight loss in those who are obese [14] , vitamin B 1 supplementation [15] , and the use of blood lipid-lowering agents [16] . Recent-ly, a few studies have also reported that consumption of synbiotics and probiotics can improve insulin sensitiv-ity and decrease inflammatory factors [17, 18] . How-ever, such beneficial effects have mainly been seen in animal models or nondiabetic patients. In addition, data on the effects of synbiotics and probiotics on met-abolic status are conflicting. Administration of a synbi-otic food containing Lactobacillus sporogenes (27 × 10 7 CFU) and 1.08 g inulin to diabetic patients for 6 weeks compared to the control food resulted in decreased se-rum insulin levels [19] . However, no significant effects on fasting plasma glucose (FPG), serum insulin levels, and homeostatic model assessment for insulin resis-tance (HOMA-IR) were seen following the consump-tion of probiotic supplements among patients with T2DM after 8 weeks [20] .

The beneficial effects of synbiotics and probiotics on insulin metabolism and inflammatory markers might be mediated by the production of short-chain fatty acids [21, 22] , modulation of the gut flora composition [22] , and a decreased expression of inflammation-relevant genes [23] . To our knowledge, there is no study indicating the effects of daily consumption of synbiotic and probiotic breads on insulin metabolism and serum high-sensitivity C-reactive protein (hs-CRP) in patients with T2DM. The aim of the current study is, therefore, to investigate the effects of daily consumption of synbiotic and probiotic breads on insulin metabolism and serum hs-CRP in pa-tients with T2DM.

Materials and Methods

Participants This randomized, double-blind, controlled clinical trial was

performed in Kashan, Iran, from October 2013 to December 2013. To estimate the required sample size, we used an appropri-

ate formula, where the type 1 (α) and type 2 errors (β) were con-sidered 0.05 and 0.20 (power = 80%), respectively. In addition, the HOMA-IR score was defined as the key variable, and based on earlier studies [24] the SD of this variable was 2.6. We consid-ered 2.0 as the meaningful difference in mean HOMA-IR scores between the 2 groups. Therefore, the required sample size was estimated to be 27 subjects in each group. The diagnosis of T2DM was made based on criteria of the American Diabetes Association [25] , i.e. patients fulfilling one of the following criteria were con-sidered to have T2DM: FPG ≥ 126 mg/dl, 2-hour plasma glucose ≥ 200 mg/dl, and HbA1C ≥ 6.5%. In the current study, we includ-ed individuals with T2DM and aged 35–70 years recruited from among patients attending the Golabchi Diabetes Clinic affiliated with the Kashan University of Medical Sciences (Kashan, Iran). We excluded pregnant women, those using insulin or vitamin supplements, and those with chronic kidney disease, liver or lung disease, and chronic or acute inflammatory disease, heart valve disease, short-bowel syndrome, and allergies. This study was conducted according to the guidelines of the Declaration of Hel-sinki. The ethics committee of the Kashan University of Medical Sciences approved this study, and written informed consent was obtained from all participants.

Study Procedure To obtain detailed information about the dietary intakes of

the study participants, all patients entered a 2-week run-in pe-riod during which they had to refrain from eating any other synbiotic and probiotic food. During the run-in period, par-ticipants were asked to record their dietary intakes for 3 non-consecutive days. At the end of the run-in period, subjects were randomly assigned to synbiotic, probiotic, or control bread for 8 weeks. Subjects were stratified based on age, sex, BMI, and the type and dosage of oral hypoglycemic medications they were taking, and they were then randomly assigned to the synbiotic, probiotic, or control group based on computer-generated ran-dom number lists. Randomization and allocation were con-cealed from the researcher and the participants until after the main analyses had been completed. A trained nutritionist at a diabetes clinic did the randomized allocation sequence, en-rolled the participants, and assigned the participants to the in-terventions. Participants were asked not to alter their routine physical activity or usual diet and not to consume any synbi-otic, probiotic, or fermented products other than the one pro-vided to them by the investigators. Individuals were advised to contact research staff immediately if they suspected a reaction to the synbiotic or probiotic bread. Synbiotic, probiotic, or con-trol bread was provided to the participants every 3 days. The breads were provided at the clinic. They were stored in a cool, dry place, and participants were told to store it the same way at home. Participants were instructed to consume one whole bread for each portion, as the size of the breads was designed for this purpose. The breads all had the same appearance, so it was not possible to distinguish one type from another. Compliance with the consumption of breads was monitored once a week through phone interviews. Compliance was also double-checked via the use of 3-day dietary records completed throughout the study. The dietary intakes of participants in the same period were assessed by means of 3-day dietary records (2 week days and 1 weekend day at the study baseline and at weeks 3, 6, and 8 of  intervention). The dietary records were kept according

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to  estimated values in household measurements. To obtain the  nutrient intakes of the participants based on the 3-day food  diaries in each phase, we used Nutritionist IV software (First Databank, San Bruno, Calif., USA) modified for Iranian foods.  This study is registered at http://www.irct.ir (No. IRCT201311215623N13).

Assessment of Variables Anthropometric measurements were obtained at baseline and

after 8 weeks of intervention in each arm. Body weight was mea-sured in an overnight fasted state, without shoes, and in minimal clothing using a digital scale (Seca, Hamburg, Germany) to the nearest 0.1 kg. Height was measured using a nonstretch tape mea-sure (Seca) to the nearest 0.1 cm. The BMI was calculated as weight in kilograms divided by height in meters squared.

Biochemical Assessment Fasting blood samples (10 ml) were taken at baseline and the

end of the study at the Kashan reference laboratory early in the morning after an overnight fast. FPG levels were quantified with commercially available kits (Pars Azmun, Tehran, Iran). Serum in-sulin levels were assayed using enzyme-linked immunoassay kits (DiaMetra, Milan, Italy). The intra- and interassay coefficents of variation for insulin were 2.7 and 5.5%, respectively. The HOMA-IR, homeostatic model assessment-β-cell function (HOMA-B), and quantitative insulin sensitivity check index (QUICKI) were calculated based on suggested formulas [26] . The serum hs-CRP concentration was assayed using ELISA kits (LDN, Nordhorn, Germany). The inter- and intra-assay coefficients of variations for the hs-CRP assays ranged from 4.9 to 7.1%. Measurements of FPG, insulin, and hs-CRP were done in a blinded fashion, in duplicate, in pairs (before/after the intervention) at the same time, in the same analytical run, and in random order to reduce systematic error and interassay variability.

Synbiotic, Probiotic, and Control Breads The synbiotic bread contained the viable and heat-resistant

probiotic L. sporogenes (1 × 10 8 CFU) and 0.07 g inulin as a prebi-otic per 1 g. The probiotic bread contained L. sporogenes (1 × 10 8 CFU per 1 g). The control bread (the same substance without pro-biotic bacteria and prebiotic inulin) was packed in identical pack-aging and coded by the producer to guarantee blinding. Patients were asked to consume the synbiotic, probiotic, or control bread 3 times a day in a 40-gram package for a total of 120 g/day. In the current study, we selected L. sporogenes instead of any other Lac-tobacillus species due to its highly beneficial characteristics, in-cluding its viability at high temperatures, in the acidity of the stom-ach, and in bile acids, as well as its growth under physiological conditions and its beneficial effects on the intestinal environment and stool frequency and characteristics [27] . During production, the content of the breads was measured routinely and the survival of the probiotic bacteria was also measured on days 0, 3, and 5, as some samples from each production process were held in the lab-oratory for these tests. The probiotic bacteria were alive until the end of the shelf life of the bread (day 5). We had produced probi-otic bread with these bacteria without any prebiotics before, and the bacteria were alive. L. sporogenes are spores and are encapsu-lated, which would result in their increased protection. The synbi-otic, probiotic, and control breads were provided by the Sahar Bread Company (Tehran, Iran). The Sahar Bread Company knew

the identity of each bread, as they sent the samples with codes printed on the packaging. All others were blinded to the identity of the samples. The L. sporogenes for this study was provided by the Tak Gen Zist Company (Tehran, Iran).

Statistical Analysis To ensure a normal distribution of the variables, the Kolmo-

grov-Smirnov test was applied. The analyses were done based on an intention-to-treat approach. Missing values were treated using the last-observation-carried-forward method, which ignores whether the participant’s condition was improving or deteriorat-ing at the time of dropout but instead freezes outcomes at the val-ue observed before dropout (i.e. the last observation). One-way analysis of variance (ANOVA) was used to detect differences in general characteristics and dietary intakes and to determine the effects of synbiotic, probiotic, and control breads on insulin me-tabolism and hs-CRP levels between the 3 groups. Changes across the 3 groups were compared using Bonferoni post hoc pairwise comparisons. To assess whether the magnitude of the change de-pended on the baseline values (FPG, insulin, HOMA-IR, HOMA-B, QUICKI, and hs-CRP), age, and the baseline BMI, we condi-tioned all analyses to the baseline values, age, and the baseline BMI to avoid potential biases. These adjustments were done using anal-ysis of covariance (ANCOVA). All statistical analyses were con-ducted using the Statistical Package for the Social Sciences version 17 (SPSS Inc., Chicago, Ill., USA).

Results

A total of 81 patients (15 males: 5 in each group, and 66 females: 22 in each group) with T2DM were recruit-ed into this study and randomly assigned to synbiotic (n = 27), probiotic (n = 27), or control bread (n = 27) for 8 weeks. One patient was excluded from the control group due to a need for insulin therapy. Among the in-dividuals in the probiotic group, 1 person was excluded due to a need for antibiotic treatment and 1 withdrew themselves from the study; in the symbiotic group, 1 person withdrew themselves from the study and 1 per-son was excluded due to a need for supplement therapy. Finally, 76 participants (synbiotic bread, n = 25; probi-otic bread, n = 25, and control bread, n = 26) completed the trial ( fig. 1 ). However, as the analysis was done based on an intention-to-treat approach, all 81 patients (n = 27 in each group) were included in the final analysis.

No side effects were reported following the consump-tion of synbiotic and probiotic breads in patients with T2DM throughout the study. Comparing the anthropo-metric measures at baseline and after the intervention, we did not find a significant difference in weight or BMI be-tween the 3 groups ( table 1 ).

Based on the 3-day dietary records throughout the study, no statistically significant difference was seen be-

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tween the 3 groups in terms of dietary intakes of energy, carbohydrates, protein, fat, saturated fatty acids, polyun-saturated fatty acids, monounsaturated fatty acids, cho-lesterol, total dietary fiber, insoluble fiber, calcium, mag-nesium, or vitamin C (data not shown).

Consumption of the synbiotic bread resulted in a sig-nificant reduction in serum insulin levels (–3.2 ± 5.4 vs. –0.3 ± 3.4 and 0.6 ± 4.7 μIU/ml, respectively, p = 0.007), HOMA-IR scores (–1.5 ± 2.7 vs. –0.2 ± 1.6 and 0.4 ± 3.5, respectively, p  = 0.03), and HOMA-B (–7.2 ± 16.3 vs. –0.7 ± 10.8 and 0.7 ± 8.2, respectively, p = 0.04) compared to the probiotic and control breads ( table 2 ). No signifi-

cant effect of synbiotic bread consumption on FPG, QUICKI, or serum hs-CRP levels was seen compared to the probiotic and control breads. In addition, in a pair-wise comparison between the synbiotic and probiotic groups, no significant differences were seen, except for serum insulin levels (p = 0.02) and HOMA-IR (p = 0.02).

When we adjusted the analysis for baseline values, the above mentioned findings remained significant, except for serum insulin levels (p  = 0.08) and HOMA-B (p  = 0.17) ( table 3 ). Adjustment for age and baseline BMI did not produce any significant changes in our findings, ex-cept for HOMA-B (p = 0.06).

Table 1. General characteristics of the study participants

Control breada

(n = 27)Probiotic breada

(n = 27)Synbiotic breada

(n = 27)pb

Age, years 53.4±7.5 52.0±7.2 51.3±10.4 0.65Height, cm 158.8±7.7 158.9±9.1 162.1±10.1 0.32Weight at study baseline, kg 76.8±12.1 75.1±14.3 80.6±15.2 0.34Weight at the end of the trial, kg 76.8±11.9 75.0±14.4 80.1±15.3 0.32BMI at the study baseline, kg/m2 30.5±4.1 29.8±5.7 30.8±5.9 0.79BMI at the end of the trial, kg/m2 30.4±4.1 29.8±5.9 30.8±5.9 0.78Metformin use, n/day 2.0±0.8 1.9±1.2 2.0±1.1 0.87Glibenclamide use, n/day 2.1±0.9 2.0±1.2 2.1±1.1 0.98

Data are means ± SD. a Five males and 22 females in each group. b Obtained from an ANOVA test.

Allocated to the synbioticbread (n = 27)

Allocated to the probioticbread (n = 27)

Allocated to the controlbread (n = 27)

Allo

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Lost to follow-up (n = 2) Withdrawal (n = 1) Supplement use (n = 1)

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Excluded (n = 19) Not living in Kashan (n = 6) Taking excluded insulin therapy

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Analyzed (n = 27)Analyzed (n = 27)Analyzed (n = 27)

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Fig. 1. Patient flow diagram.

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Discussion

Our study indicated that consumption of the synbi-otic bread for 8 weeks among diabetic patients had ben-eficial effects on serum insulin levels, HOMA-IR scores, and HOMA-B but did not affect FPG, QUICKI, or serum hs-CRP levels compared to the probiotic and control breads. To the best of our knowledge, this is the first study to examine the effects of synbiotic and probiotic breads on the insulin metabolism and inflammatory factors of diabetic patients.

T2DM can result in metabolic complications includ-ing insulin resistance, dyslipidemia, and increased in-flammatory markers [28] . We demonstrated that con-sumption of the synbiotic bread significantly decreased serum insulin levels, HOMA-IR scores, and HOMA-B but did not affect FPG, QUICKI, or serum hs-CRP lev-els compared to the probiotic and control breads. Few studies have reported beneficial effects of synbiotics and probiotics on insulin metabolism. In line with our study, Raso et al. [18] observed improvement in many aspects of insulin resistance, such as the fasting re-sponse, hormonal homeostasis, and glycemic control, following the consumption a synbiotic composed of L. paracasei plus arabinogalactan and fructo-oligosac-charides in a rat model of high-fat feeding. Further-more, a significant decrease in serum insulin levels in our previous study in diabetic patients was seen after administration of a synbiotic food containing L. sporo-genes (27 × 10 7 CFU) and 1.08 g inulin for 6 weeks com-pared to the control food [19] . The same findings were reported among type 2 diabetic patients who received L. acidophilus for 4 weeks [29] , among patients with T2DM who received multispecies probiotic supple-ments for 8 weeks [20] , and among pregnant women who received 2 strains of L. acidophilus and Bifidobac-terium animalis with a total of minimum 1 × 10 7 CFU for 9 weeks [30] . However, some researchers did not find such beneficial effects. For instance, no significant dif-ference in FPG, serum insulin levels, or HOMA-IR scores was seen following the consumption of probiotic supplements among patients with T2DM after 8 weeks compared to the placebo [20] . In addition, supplementa-tion with the probiotic strain L. casei Shirota did not show any significant effects on insulin sensitivity or β-cell function [31] . Several mechanisms can explain the beneficial effects of synbiotic bread on insulin me-tabolism. The beneficial effects of synbiotic bread on insulin sensitivity might be attributed to their impact on gene expression [32] and modulation of the gut mi-

crobiota-short-chain fatty acid-hormone axis, and es-pecially with regard to increased promotion of the hor-mone glucagon-like peptide-1 from intestinal L cells [22] . In addition, changes in the levels of gut hormones like peptide YY [33] and activation of the lipopolysac-charide Toll-like receptor-2 [34] by synbiotics may ex-plain its effects on insulin metabolism.

The present study showed that the intake of synbiotic bread did not affect serum hs-CRP levels. Divergent evi-dence is available on the anti-inflammatory properties of synbiotics and probiotics. In agreement with our study, consumption of the synbiotic containing 2 Lactobacillus strains, 1 Bifidobacterium strain, 1 Propionibacterium strain, and galacto-oligosaccharides (32 g/l) did not influ-ence serum CRP levels among men with a low serum en-terolactone concentration after cross-over at 6 weeks [35] . Furthermore, consumption of 1,500-mg probiotic capsules containing L. acidophilus, L. bulgaricus, L. bifi-dum, and L. casei twice daily did not show any significant changes in serum hs-CRP levels among diabetic patients after 6 weeks compared to the placebo [24] . Similar re-sults were seen among patients with Crohn’s disease who received Saccharomyces boulardii (1 g/day) for 52 weeks [36] and patients with rheumatoid arthritis who received L. rhamnosus GG for 12 months [37] . However, our pre-vious study showed that multispecies probiotic supple-mentation resulted in decreased serum hs-CRP levels among diabetic patients for 8 weeks [20] . In addition, the same findings were reported with consumption of a syn-biotic food containing L. casei, B. breve, and galacto-oli-gosaccharides among patients undergoing hepatobiliary resection [38] and with the use of a synbiotic food in pa-tients with severe multiple injuries [39] . The absent effect of synbiotic bread consumption on serum hs-CRP levels in the current study may be the result of different study designs, the dosages of probiotic and inulin used, the pa-tients studied, and the duration of supplementation.

Several limitations must be considered when inter-preting our findings. First of all, due to budget limita-tions, we were unable to assess the beneficial effects of the synbiotic and probiotic breads on signaling pathways and other inflammatory markers including IL-1, IL-6, and tumor necrosis factor-α. Secondly, we did not to ad-minister the synbiotic and probiotic breads for more than 8 weeks. Long-term interventions might lead to greater changes.

In conclusion, consumption of the synbiotic bread for 8 weeks among patients with T2DM had beneficial effects on insulin metabolism but did not affect FPG, QUICKI, or serum hs-CRP levels.

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Tajadadi-Ebrahimi/Bahmani/Shakeri/Hadaegh/Hijijafari/Abedi/Asemi

Ann Nutr Metab 2014;65:34–41DOI: 10.1159/000365153

40

Acknowledgments

The present study was supported by a grant (No. 92107) from the Vice Chancellor for Research, Kashan University of Medical Sciences. The authors would like to thank the staff of the Gholab-chi Clinic (Kashan, Iran) for their assistance with this project. We are grateful to the Research and Development Department of the Sahar Bread Company, which provided the synbiotic and probiotic products for the present study. Furthermore, we are

grateful to the Research and Development Department of the Tak Gen Zist Company, which provided the L. sporogenes for this study.

Disclosure Statement

None of the authors has any personal or financial conflict of interests.

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