Clinical Nutrition xxx (2015) 1e7

Contents lists available at ScienceDirect

Clinical Nutrition
journal homepage: http://www.elsevier.com/locate/clnu

Randomized control trials

Effects of beta-carotene fortified synbiotic food on metabolic control

of patients with type 2 diabetes mellitus: A double-blind randomized
cross-over controlled clinical trial
Zatollah Asemi a, Sabihe-Alsadat Alizadeh b, Khorshidi Ahmad c, Mohammad Goli d,
Ahmad Esmaillzadeh e, f, *
a
Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan, Iran
b
Department of Research and Development of Sekkeh Gaz Company, Isfahan, Iran
c
Department of Microbiology, Kashan University of Medical Sciences, Kashan, Iran
d
Department of Food Science and Technology, Islamic Azad University, Khorasgan Branch, Khorasgan, Iran
e
Food Security Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
f
Department of Community Nutrition, School of Nutrition and Food Science, Isfahan University of Medical Sciences, Isfahan, Iran

a r t i c l e i n f o s u m m a r y

Article history: Background & aims: The aim of the present study was to determine the beneficial effects of beta-carotene
Received 6 February 2015 fortified synbiotic food intake on metabolic status in patients with type 2 diabetes mellitus (T2DM).
Accepted 6 July 2015 Methods: This randomized double-blinded placebo-controlled crossover clinical trial was conducted
among 51 patients with T2DM. Individuals were randomly assigned to take either a beta-carotene for-
Keywords: tified synbiotic (n ¼ 51) or control food (n ¼ 51) for 6 weeks. The beta-carotene fortified synbiotic was
Synbiotic
containing Lactobacillus sporogenes (1  107 CFU), 0.1 g inulin and 0.05 g beta-carotene. Control food (the
Metabolic profiles
same substance without probiotic, inulin and beta-carotene) was packed in identical 9-g packages. Pa-
Inflammation
Oxidative stress
tients were requested to use the beta-carotene fortified synbiotic and control foods three times a day.
Type 2 diabetes Results: Beta-carotene fortified synbiotic food consumption resulted in a significant decrease in insulin
(1.00 ± 7.90 vs. þ3.68 ± 6.91 mIU/mL, P ¼ 0.002), HOMA-IR (0.73 ± 3.96 vs. þ1.82 ± vbnm4.09,
P ¼ 0.002), HOMA-B (0.52 ± 19.75 vs. þ8.71 ± 17.15, P ¼ 0.01), triglycerides (2.86 ± 49.53
vs. þ20.14 ± 50.10 mg/dL, P ¼ 0.02), VLDL-cholesterol levels (0.57 ± 9.90 vs. þ4.03 ± 10.02 mg/dL,
P ¼ 0.02) and total-/HDL-cholesterol ratio (0.01 ± 1.08 vs. þ0.64 ± 0.81, P ¼ 0.001) compared to the
control food. In addition, beta-carotene fortified synbiotic food consumption led to elevated plasma nitric
oxide (NO) (þ6.83 ± 16.14 vs. -3.76 ± 16.47 mmol/L, P ¼ 0.001) and glutathione (GSH) (þ36.58 ± 296.71
vs. -92.04 ± 243.05 mmol/L, P ¼ 0.01).
Conclusions: Beta-carotene fortified synbiotic food intake in patients with T2DM for 6 weeks had
favorable effects on insulin, HOMA-IR, HOMA-B, triglycerides, VLDL-cholesterol, total-/HDL-cholesterol
ratio, NO and GSH levels.
© 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

1. Introduction The prevalence of T2DM is 8.3% among adult population in the

Type 2 diabetes mellitus (T2DM), which accounts for 90e95% of
cases with diabetes [1], is a chronic disorder characterized by
impaired metabolism of glucose and lipids due to impaired insulin
secretion (beta cell dysfunction) or action (insulin resistance) [2].
* Corresponding author. Food Security Research Center, Isfahan University of
Medical Sciences, Isfahan, Iran. Tel.: þ98 311 7922720; fax: þ98 311 6681378.
E-mail address: Esmaillzadeh@hlth.mui.ac.ir (A. Esmaillzadeh).
world [3]. Main complications of T2DM are micro-as well as macro-
vascular pathologies that influence more than 17.5 million deaths
worldwide [4,5]. It has been reported that glycemic variability and
oxidative stress are the underlying causes of both macro- and
microvascular complications of T2DM [6]. The main strategy for
management of T2DM is the integrated control of hyperglycaemia,
dyslipidaemia and blood pressure [7,8]. Recently, some clinical
trials have reported that administration of synbiotic foods might
have beneficial effects on metabolic parameters, biomarkers of
inflammation and oxidative stress in these patients [9,10]. Our

http://dx.doi.org/10.1016/j.clnu.2015.07.009
0261-5614/© 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Please cite this article in press as: Asemi Z, et al., Effects of beta-carotene fortified synbiotic food on metabolic control of patients with type 2
diabetes mellitus: A double-blind randomized cross-over controlled clinical trial, Clinical Nutrition (2015), http://dx.doi.org/10.1016/
j.clnu.2015.07.009
2 Z. Asemi et al. / Clinical Nutrition xxx (2015) 1e7
previous study also showed the favorable effects of a synbiotic food
on metabolic status of patients with T2DM [9], without any major
significant impact on biomarkers of oxidative stress. Due to the
antioxidant and anti-inflammatory effects [11], beta-carotene
might reinforce effects of the synbiotic food on metabolic profiles
in patients with diabetes. Taking antioxidant supplements con-
taining vitamin E, vitamin C, and beta-carotene for 8 weeks led to
decreased HOMA-IR among overweight young adults [12].
Furthermore, another study [13] reported that consumption of
beta-carotene plus D-galactose compared with D-galactose alone
resulted in increased glutathione (GSH), superoxide dismutase
(SOD) levels and glutathione peroxidase (GSH-Px) activities in rats
at weaning. However, supplementation with fruit and vegetable
juice concentrate (FVJC) for 6 months resulted in increased beta-
carotene levels but did not affect lipid profiles in overweight boys
[14].
The beneficial effects of synbiotics on metabolic profiles may be
attributed to decreased expression of the enzymes involved in fatty
acid synthesis [15]. It has also been suggested that synbiotics might
modulate gut microbiota-short chain fatty acid (SCFA)-hormone
axis [16]. Increased glutamate cysteine ligase (GCL) activity and
enhanced mRNA expression of both of the GCL subunits following
synbiotic and probiotic intake [17] and blocking the activities of
caspase-3 and attenuating lipid peroxidation after intake beta-
carotene [13] might also lead to anti-inflammatory and reduced
oxidative stress. As oxidative stress is a well known factor under-
lying diabetes related complications of diabetes, we hypothesized
that the administration of a beta-carotene fortified synbiotic food
might further influence the metabolic status, biomarkers of
inflammation and oxidative stress in patients with diabetes. To the
best of our knowledge, we are aware of no study indicating the
effects of beta-carotene fortified synbiotic food on metabolic status,
biomarkers of inflammation and oxidative stress in patients with
T2DM. The aim of the present study was, therefore, to assess the
favorable effects of a beta-carotene fortified synbiotic food on
metabolic status, biomarkers of inflammation and oxidative stress
in patients with T2DM.
2. Subjects and methods
2.1. Participants
The present study was a double-blinded controlled crossover
clinical trial that was carried out in Kashan, Iran, during January
2013 to May 2013. Patients aged 35e70 years and with a diagnosis
of T2DM were selected from a public clinic of Kashan University of
Medical Sciences, Kashan, Iran. Individuals with a fasting blood
sugar (FBS) of 126 mg/dL or 2-h postprandial glucose concen-
trations of 200 mg/dL were defined as having diabetes according
to the guidelines of the American Diabetes Association [18]. In this
study, we excluded subjects who were pregnant, using insulin or
vitamin supplements, and those with liver, inflammatory diseases,
coronary heart disease and allergies. Sample size was determined
according to serum insulin levels as a key variable in a previous
study in subjects with T2DM [9]. Considering the mean difference
of 2.2, the standard deviation of 4.0 and the type 2 error of 0.20, we
needed 46 patients per group. However we recruited 51 patients
per group to account for the possible dropouts. A total of 102 pa-
tients with T2DM were randomly allocated to receive either beta-
carotene fortified synbiotic (n ¼ 51) or control food (n ¼ 51) for 6
weeks. The current study was conducted according to the guide-
lines laid down in the Declaration of Helsinki. The ethical com-
mittee of Kashan University of Medical Sciences approved the study
and informed written consent was obtained from all participants.
Please cite this article in press as: Asemi Z, et al., Effects of beta-carotene fortified synbiotic food on metabolic control of patients with type 2
diabetes mellitus: A double-blind randomized cross-over controlled clinical trial, Clinical Nutrition (2015), http://dx.doi.org/10.1016/
j.clnu.2015.07.009
2.2. Study design
To obtain detailed information about the dietary intakes of
study participants, all patients entered into a 2-wk run-in period;
during which all subjects had to refrain from taking any other
synbiotic and probiotic foods. During the run-in period, partici-
pants were asked to record their dietary intakes for three non-
consecutive days. All patients were matched based on age, sex,
BMI, type and dosage of oral hypoglycemic agents (OHAs) they
were taking. At the end of run-in period, patients were randomly
allocated to the initial arm of the study to receive either a beta-
carotene fortified synbiotic or control food for 6 weeks. After this
time period, subjects were entered into a 3-week washout period.
Then the participants were crossed over to the alternate group for
an additional 6 weeks. Subjects were requested not to change their
routine physical activity or usual diets and not to use any synbiotic,
probiotic and fermented products other than the one provided to
them by the investigators. Participants were not asked to provide
home blood monitoring (BM) checks to the investigators. In addi-
tion, as the duration of intervention was short (6 weeks), they were
requested not to change the dosage and type of OHAs they were
taking to avoid their possible effect on the study findings. Beta-
carotene fortified synbiotic or control foods were provided to par-
ticipants every 3 weeks. Compliance with the consumption of foods
was monitored once a week through phone interviews. To increase
the compliance, all individuals were receiving short messages on
their cell phones to receive beta-carotene fortified synbiotic or
control foods each day. Subjects' nutritional status was evaluated
by a three-day food records at 3 time points (baseline, week 3 and 6
of intervention) during the course of the study. Then, data on food
records were analyzed using Nutritionist IV software (First Data-
bank, San Bruno, CA, USA) modified for Iranian foods.
2.3. Beta-carotene fortified synbiotic and control foods
A beta-carotene fortified synbiotic food contained a probiotic
bacteria of Lactobacillus sporogenes (1  107 CFU), 0.1 g inulin (HPX)
as prebiotic, 0.05 g beta-carotene with 0.38 g isomalt, 0.36 g sor-
bitol and 0.05 g stevia per 1 g as sweetener. Control food contained
the same substance without L. sporogenes, inulin and beta-carotene.
Individuals were requested to take the synbiotic and control foods
in 9-g packages three times a day.
2.4. Assessment of variables
Height and weight were measured at the beginning of the study
and the end-of-trial in a standing position by using a digital scale
(Seca, Hamburg, Germany). BMI was calculated for each patient by
dividing weight in kg to the height in m2. All the measurements
were obtained by the same person (a nutritionist). Patients were
visited every week by the same physician (an endocrinologist).
After a 12 h fast, 10 mL blood samples were obtain from patients to
determine lipid concentrations, insulin, hs-CRP, liver enzymatic,
calcium, iron and magnesium concentrations. Serum lipid con-
centrations were also determined on the day of blood collection.
Then, the samples were stored at 70  C before analysis at the
KUMS reference laboratory. Commercial kits were used to measure
fasting plasma glucose (FPG), serum levels of cholesterol, tri-
glycerides, VLDL-, LDL-, HDL-cholesterol, alanine aminotransferase
(ALT), aspartate aminotransferase (AST), alkalin phosphatase (ALP),
calcium, iron and magnesium (Pars Azmun, Tehran, Iran). All inter-
and intra-assay CVs for FPG, lipid profiles, liver enzymes, calcium,
iron and magnesium measurements were less than 5%. Serum
levels of insulin were quantified by using ELISA kit (Monobind,
California, USA). HOMA-IR and b-cell function (HOMA-B) and
 Z. Asemi et al. / Clinical Nutrition xxx (2015) 1e7
QUICKI were calculated based on suggested formulas. Circulating
levels of serum high sensitivity C-reactive protein (hs-CRP) was
quantified using ELISA kit (LDN, Nordhorn, Germany). The plasma
nitric oxide (NO) concentrations were measured by Griess method,
total antioxidant capacity (TAC) by the use of FRAP method devel-
oped by Benzie and Strain and total glutathione (GSH) using the
method of Beutler et al. We measured malondialdehyde (MDA)
concentrations using the thiobarbituric acid reactive substance
(TBARs) spectrophotometric test. Systolic blood pressure (SBP) and
diastolic blood pressure (DBP) were measured via a sphygmoma-
nometer (ALPK2, Zhejiang, China).
2.5. Statistical analysis
To examine normal distribution of variables, we applied Kol-
mogroveSmirnov test. All analyses were conducted based on
intention-to-treat principle. Missing values were treated based on
Last-Observation-Carried-Forward (LOCF) method. Independent
samples Student's t test was used to detect differences in general
characteristics and dietary intakes between the two groups. To
determine the effects of beta-carotene fortified synbiotic food
intake on markers of insulin metabolism, lipid concentrations,
biomarkers of inflammation and oxidative stress; we used one-way
repeated measures analysis of variance. In this analysis, the treat-
ment (beta-carotene fortified synbiotic vs. control food) was
regarded as between-subject factor and time with two time-points
(the beginning study and end-of-trial) was considered as within-
subject factor. To evaluate whether baseline values of biomarkers
and baseline BMI can affect the findings, we adjusted the analyses
for these measures. These analyses were done using analysis of
covariance. P < 0.05 was considered as statistically significant. All
statistical analyses were done using the Statistical Package for So-
cial Science version 17 (SPSS Inc., Chicago, Illinois, USA).
3. Results
Of the 51 patients (16 males and 35 females) enrolled in the
study, 48 patients (16 males and 32 females) completed the entire
crossover study. Among individuals in beta-carotene fortified syn-
biotic food group, 1 patient [withdrawn (n ¼ 1)] dropped out. The
exclusions in the control group were 2 subjects [withdrawn
(n ¼ 2)]. Finally, 99 participants [beta-carotene fortified synbiotic
group (n ¼ 50) and control group (n ¼ 49)] completed the trial
(Fig. 1). The number of dropouts in the intervention group was 2
people and that in the non-intervention group was 1 individual.
However, as the analysis was done based on intention-to-treat
principle, all 51 patients (51 in each group) were included in the
final analysis. Mean age of patients was 52.9 ± 8.1 years.
No serious adverse reactions were reported following the con-
sumption of beta-carotene fortified synbiotic food in patients with
T2DM throughout the study. Comparing the anthropometric mea-
sures at baseline and also after intervention, we did not observe a
significant difference in weight and BMI between the two groups
(Table 1).
Based on the three-day dietary records throughout the study, no
statistically significant difference was seen between the two groups
in terms of dietary intakes of energy, carbohydrates, protein, fat,
saturated fatty acids (SFA), polyunsaturated fatty acids (PUFA),
monounsaturated fatty acids (MUFA), cholesterol, total dietary fiber
(TDF), magnesium, iron, calcium and beta-carotene (Table 2).
Beta-carotene fortified synbiotic food consumption resulted in a
significant decrease in serum insulin (1.00 ± 7.90
vs. þ3.68 ± 6.91 mIU/mL, P ¼ 0.002), HOMA-IR (0.73 ± 3.96
vs. þ1.82 ± 4.09, P ¼ 0.002), HOMA-B (0.52 ± 19.75
vs. þ8.71 ± 17.15, P ¼ 0.01), serum triglycerides (2.86 ± 49.53
Please cite this article in press as: Asemi Z, et al., Effects of beta-carotene fortified synbiotic food on metabolic control of patients with type 2
diabetes mellitus: A double-blind randomized cross-over controlled clinical trial, Clinical Nutrition (2015), http://dx.doi.org/10.1016/
j.clnu.2015.07.009
3
vs. þ20.14 ± 50.10 mg/dL, P ¼ 0.02), VLDL-cholesterol levels
(0.57 ± 9.90 vs. þ4.03 ± 10.02 mg/dL, P ¼ 0.02) and total-/HDL-
cholesterol ratio (0.01 ± 1.08 vs. þ0.64 ± 0.81, P ¼ 0.001)
compared to the control food (Table 3). In addition, consumption of
beta-carotene fortified synbiotic food led to elevated plasma NO
levels (þ6.83 ± 16.14 vs. 3.76 ± 16.47 mmol/L, P ¼ 0.001). Similarly,
elevated plasma GSH (þ36.58 ± 296.71 vs. 92.04 ± 243.05 mmol/L,
P ¼ 0.01) and serum magnesium levels (þ0.08 ± 0.37
vs. 0.26 ± 0.41 mg/dL, P < 0.001) were observed in the beta-
carotene fortified synbiotic-fed group compared with controls.
We did not observe any significant effect of beta-carotene fortified
synbiotic food consumption on FPG, QUICKI, other lipid profiles, hs-
CRP, MDA, liver enzymes, calcium, iron levels and blood pressure.
After adjustment for baseline levels, no significant changes in
our findings occurred, except for serum magnesium levels
(P ¼ 0.07) (Data not shown). Additional adjustments for baseline
BMI did not affect the findings.
4. Discussion
The current cross-over study demonstrated that administration
of beta-carotene fortified synbiotic food among patients with
T2DM for 6 weeks had beneficial effects on insulin metabolism,
serum triglycerides, VLDL-, total/HDL-cholesterol ratio, magne-
sium, plasma NO and GSH levels; however, it did not influence
other metabolic profiles compared with the control food.
Patients with T2DM are subject to impaired insulin metabolism,
dyslipidemia, increased inflammation and oxidative stress. To the
best of our knowledge, the present study is the first evaluating the
favorable effects of beta-carotene fortified synbiotic food in pa-
tients with T2DM. The current study showed that beta-carotene
fortified synbiotic food consumption for 6 weeks led to signifi-
cant improvements in insulin resistance indicators, serum tri-
glycerides, VLDL-cholesterol levels and total-/HDL-cholesterol
ratio, while FPG, QUICKI index and other serum lipid profiles
remained unchanged. In agreement with our study, Malaguarnera
et al. [19] observed a significant reduction in the HOMA-IR score
following the intake Bifidobacterium longum with fructo-
oligosaccharides (Fos) among patients with non alcoholic steato-
hepatitis, but no detectable changes in serum insulin concentra-
tions were reported. Our previous study among healthy pregnant
women revealed a decreased serum triglycerides and VLDL-
cholesterol levels after consumption of synbiotic food containing
L. sporogenes (1  107 CFU) and 0.04 g inulin as prebiotic per 1 g for
9 weeks, but we did not observe any effect on other lipid profiles
[20]. Possible mechanisms by which beta-carotene fortified syn-
biotic food intake might improve insulin resistance results from the
impact on gene expression and gut microbiota-SCFA-hormone axis
[16]. In addition, inhibited free radical generation and recovery of
cell redox status following beta-carotene supplementation might
result in preserved insulin receptor structure and action, and in
turn decrease insulin resistance [12]. Furthermore, the main
mechanism of decreased serum triglycerides and VLDL-cholesterol
levels has not been elucidated yet; however, possible mechanisms
have been suggested. One of these mechanisms is that SCFA pro-
duction especially propionate in the gut could inhibit the synthesis
of fatty acids in the liver, thereby decreasing the triglycerides and
VLDL-cholesterol levels secretion rate [21].
Findings from this study demonstrated that beta-carotene for-
tified synbiotic food consumption resulted in a significant rise in
NO levels, while serum hs-CRP concentrations remained un-
changed. In agreement with our study, an increase in NO produc-
tion in human umbilical vein endothelial cells was observed
following the administration of soy milk fermented with Lactoba-
cillus plantarum or Streptococcus thermophilus for 48 h [22]. In
4 Z. Asemi et al. / Clinical Nutrition xxx (2015) 1e7

Response to advertisement
n=80
Ineligible subjects by screening
for inclusion criteria
n=29
Eligible for participation
n=51

Run-in period
n=51

Synbiotic food Control food

n=25 n=26

Washout period
n=51

Withdrawals (n=2) Withdrawals (n=1)

Control Food Synbiotic food

n=23 n=25

Completed the trial and analyzed

n=48

Final analyzed with intention-to-treat

n=51

Fig. 1. Patient flow diagram.

Table 1
General characteristics of the study participants.a

Control food Beta-carotene fortified synbiotic food (n ¼ 51) Pb

(n ¼ 51)

Weight at study baseline (kg) 78.28 ± 13.42 77.59 ± 13.65 0.79

Weight at end-of-trial (kg) 78.19 ± 13.28 77.43 ± 13.49 0.77
Weight change 0.08 ± 1.58 0.15 ± 1.32 0.81
BMI at study baseline (kg/m2) 30.15 ± 5.07 29.88 ± 4.77 0.78
BMI at end-of-trial (kg/m2) 30.11 ± 5.01 29.84 ± 4.87 0.78
BMI change 0.03 ± 0.60 0.03 ± 0.47 0.99
a
All values are means ± standard deviation.
b
Obtained from independent t test.

contrast, some investigators did not observe such effects of syn-
biotic administration on NO levels. For example, Lactobacillus GG
for 4 weeks could not increase intestinal NO concentrations among
children with active inflammatory bowel disease [23]. The evidence
on the anti-inflammatory properties of synbiotics is inconclusive. In
line with our study, taking antioxidant supplements containing
10,000 IU beta-carotene, 200 IU alpha-tocopherol, 250 mg vitamin
C, 50 mg selenium and 15 mg zinc did not affect plasma CRP con-
centration in trained men [24]. However, in a study by Giamarellos-
Bourboulis et al. [25], synbiotics supplementation for 15 days was
accompanied by a reduction of CRP concentrations among subjects
who did or did not develop sepsis. Serum beta-carotene

Please cite this article in press as: Asemi Z, et al., Effects of beta-carotene fortified synbiotic food on metabolic control of patients with type 2
diabetes mellitus: A double-blind randomized cross-over controlled clinical trial, Clinical Nutrition (2015), http://dx.doi.org/10.1016/
j.clnu.2015.07.009
 Z. Asemi et al. / Clinical Nutrition xxx (2015) 1e7 5

Table 2
Dietary intakes of patients that received either beta-carotene fortified synbiotic or control foods throughout the study.a

Control food Beta-carotene fortified synbiotic food (n ¼ 51) Pb

(n ¼ 51)

Energy (kcal/d) 2194 ± 233 2147 ± 230 0.33

Carbohydrates (g/d) 299.2 ± 59.2 296.0 ± 49.8 0.77
Protein (g/d) 80.4 ± 13.0 80.3 ± 18.8 0.95
Fat (g/d) 78.4 ± 8.3 75.3 ± 15.6 0.20
SFAc (g/d) 23.8 ± 4.5 23.5 ± 5.9 0.73
PUFAd (g/d) 24.9 ± 4.6 25.3 ± 4.3 0.67
MUFAe (g/d) 20.4 ± 3.6 20.8 ± 6.5 0.73
Cholesterol (mg/d) 194.4 ± 85.7 184.2 ± 84.0 0.54
TDFf (g/d) 18.3 ± 5.1 19.2 ± 4.4 0.35
Magnesium (mg/d) 239.4 ± 56.8 252.2 ± 58.7 0.26
Iron (mg/d) 13.5 ± 3.5 12.8 ± 3.1 0.30
Calcium (mg/d) 1071.9 ± 157.1 1088.8 ± 190.4 0.62
Beta-carotene (mg/d) 1012.4 ± 513.1 1277.2 ± 695.3 0.59
a
All values are means ± standard deviations.
b
Obtained from independent t test.
c
SFA: Saturated fatty acid.
d
PUFA: Polyunsaturated fatty acid.
e
MUFA: Monounsaturated fatty acid.
f
TDF: Total dietary fiber.

Table 3
Metabolic profiles, inflammatory factors and biomarkers of oxidative stress at study baseline and after 6-wk intervention in patients that received either beta-carotene fortified
synbiotic or control foods.a

Control food (n ¼ 51) Beta-carotene fortified synbiotic food (n ¼ 51) Pb

Wk0 Wk6 Change Wk0 Wk6 Change

c
FPG (mg/dL) 142.82 ± 45.05 146.49 ± 55.07 3.67 ± 35.14 137.43 ± 46.84 135.82 ± 38.63 1.61 ± 43.65 0.50
Insulin (mIU/mL) 8.96 ± 6.43 12.64 ± 9.76t 3.68 ± 6.91 11.00 ± 11.73 10.00 ± 8.85 1.00 ± 7.90 0.002
HOMA-IRd 3.08 ± 2.23 4.90 ± 5.10t 1.82 ± 4.09 4.20 ± 6.14 3.47 ± 3.64 0.73 ± 3.96 0.002
HOMA-Be 21.21 ± 19.80 29.92 ± 25.69t 8.71 ± 17.15 24.96 ± 25.22 24.44 ± 25.01 0.52 ± 19.75 0.01
QUICKIf 0.33 ± 0.04 0.32 ± 0.04 0.01 ± 0.03 0.34 ± 0.05 0.34 ± 0.06 0.001 ± 0.06 0.16
Total cholesterol (mg/dL) 168.50 ± 36.44 160.41 ± 29.42t 8.09 ± 23.56 169.08 ± 53.92 161.21 ± 42.69 7.87 ± 26.90t 0.96
Triglycerides (mg/dL) 144.84 ± 60.33 164.98 ± 81.77t 20.14 ± 50.10 153.53 ± 85.67 150.67 ± 81.02 2.86 ± 49.53 0.02
VLDL-cholesterolg (mg/dL) 28.96 ± 12.06 32.99 ± 16.35t 4.03 ± 10.02 30.70 ± 17.13 30.13 ± 16.20 0.57 ± 9.90 0.02
LDL-cholesterolh (mg/dL) 85.10 ± 31.13 84.57 ± 25.34 0.53 ± 22.25 88.07 ± 45.57 87.98 ± 34.71 0.09 ± 27.74 0.92
HDL-cholesteroli (mg/dL) 54.42 ± 13.17 42.84 ± 8.19t 11.58 ± 11.18 50.30 ± 12.90 43.10 ± 11.84 7.20 ± 16.73t 0.12
Total: HDL cholesterol ratio 3.23 ± 0.93 3.87 ± 1.03t 0.64 ± 0.81 3.60 ± 1.70 3.61 ± 0.90 0.01 ± 1.08 0.001
hs-CRPj (ng/mL) 5975.62 ± 4597.65 5763.60 ± 4733.88 212.02 ± 2943.09 5159.80 ± 4596.17 4885.10 ± 4752.86 274.70 ± 3560.67 0.92
NOk (mmol/L) 40.92 ± 11.34 37.16 ± 14.36 3.76 ± 16.47 41.44 ± 10.94 48.27 ± 17.55 6.83 ± 16.14t 0.001
TACl (mmol/L) 967.12 ± 201.44 957.09 ± 211.65 10.03 ± 170.15 995.53 ± 241.76 988.56 ± 200.78 6.97 ± 203.51 0.93
GSHm (mmol/L) 641.80 ± 193.01 549.76 ± 166.44t 92.04 ± 243.05 682.67 ± 313.51 719.25 ± 291.71 36.58 ± 296.71 0.01
MDAn (mmol/L) 3.88 ± 0.77 2.93 ± 0.85t 0.95 ± 0.88 4.06 ± 1.12 2.78 ± 0.76 1.28 ± 1.33t 0.16
ALTo (IU/L) 22.10 ± 8.73 22.77 ± 10.16 0.67 ± 6.21 21.75 ± 6.63 21.08 ± 9.58 0.67 ± 7.42 0.32
ASTp (IU/L) 18.69 ± 7.39 20.69 ± 9.49 2.00 ± 8.55 20.40 ± 10.82 21.92 ± 18.84 1.52 ± 11.93 0.81
ALPq (U/L) 203.98 ± 50.40 194.58 ± 57.41t 9.40 ± 21.17 205.62 ± 54.51 192.71 ± 50.94 12.91 ± 32.65t 0.52
Calcium (mg/dL) 9.18 ± 0.69 8.87 ± 0.46t 0.31 ± 0.49 9.00 ± 0.65 8.80 ± 0.43 0.20 ± 0.57t 0.26
Iron (mg/dL) 71.04 ± 41.29 70.71 ± 28.86 0.33 ± 34.5 68.95 ± 38.90 74.12 ± 30.41 5.17 ± 24.09 0.35
Magnesium (mg/dL) 1.91 ± 0.39 1.65 ± 0.40t 0.26 ± 0.41 1.58 ± 0.29 1.66 ± 0.26 0.08 ± 0.37 <0.001
SBPr (mmHg) 132.92 ± 22.93 134.15 ± 12.46 1.23 ± 19.32 132.98 ± 12.49 135.90 ± 13.24 2.92 ± 11.60 0.59
DBPs (mmHg) 85.41 ± 10.10 85.98 ± 8.70 0.57 ± 6.16 81.86 ± 13.44 84.25 ± 9.77 2.39 ± 15.80 0.44
a
All values are means ± standard deviations. Baseline values of magnesium were significantly different between the two groups.
b
Obtained from repeated measures ANOVA.
c
FPG: Fasting plasma glucose.
d
HOMA-IR: Homeostasis model of assessment-insulin resistance.
e
HOMA-B: Homeostatic model assessment-Beta cell function.
f
QUICKI: Quantitative insulin sensitivity check index.
g
VLDL-cholesterol: Very low density lipoprotein-cholesterol.
h
LDL-cholesterol: Low density lipoprotein-cholesterol.
i
HDL-cholesterol: High density lipoprotein-cholesterol.
j
Hs-CRP: High sensitivity C-reactive protein.
k
NO: Nitric oxide.
l
TAC: Total antioxidant capacity.
m
GSH: Total glutathione.
n
MDA: Malondialdehyde.
o
ALT: Alanine aminotransferase.
p
AST: Aspartate aminotransferase.
q
ALP: Alkalin phosphatase.
r
SBP: Systolic blood pressure.
s
DBP: Diastolic blood pressure.
t
Different from wk 0, P < 0.05.

Please cite this article in press as: Asemi Z, et al., Effects of beta-carotene fortified synbiotic food on metabolic control of patients with type 2
diabetes mellitus: A double-blind randomized cross-over controlled clinical trial, Clinical Nutrition (2015), http://dx.doi.org/10.1016/
j.clnu.2015.07.009
6 Z. Asemi et al. / Clinical Nutrition xxx (2015) 1e7
concentrations of below the median was linked with the increased
CRP levels in HIV-1-seropositive women [26]. The accurate mech-
anisms by which synbiotics might influence NO concentrations are
unknown. Decreased superoxide anion may result in elevated NO
levels. Nitric oxide might be inactivated through the reaction with
superoxide. This reaction would result in the production of per-
oxynitrite, or more stable hydrogen peroxide radicals [27]. Lack of
the effect of beta-carotene fortified synbiotic food intake on hs-CRP
concentrations might be mediated by the dosage of probiotics and
inulin used the patients under examination as well as by the
duration of intervention.
Our study revealed that beta-carotene fortified synbiotic food
intake could significantly increase plasma GSH concentrations, but
did not influence TAC and MDA levels. In consistent with our study,
a 9-week administration of L. sporogenes (1  10⁷ CFU) and 0.04 g
inulin (HPX)/g as the prebiotic among pregnant women resulted in
a significant rise in plasma GSH concentrations, but did not affect
TAC levels [20]. Consumption of the probiotic Lactobacillus sali-
varius (5  108 CFU) per day for 3 weeks in rats increased the
colonic GSH content [28]. The useful effects of synbiotics intake on
GSH levels might be attributed to the effect on SCFA generation
especially butyrate in the gut. Butyrate provides NADPH for syn-
thesis of GSH, apoptosis induction and up-regulation of oxidative
pentose pathway activity [29]. In addition, beta-carotene might
decrease oxidative stress by blocking the activities of caspase-3 and
attenuating lipid peroxidation [13]. Lack of the significant effect of
beta-carotene fortified synbiotic food intake on plasma TAC and
MDA concentrations in this study might be explained by trial
duration, the strains of probiotic bacteria used as well as the dosage
of bacterial strain and inulin.
Some of the main strengths of our study were assessment of
markers of insulin metabolism, its randomized design and the in-
clusion of washout period. In addition, differences in the compo-
sition of the two groups were minimized because the patient
population was well defined by the inclusion and exclusion criteria.
Our study has some limitations. Fecal bacteria loads were not
quantified before and after beta-carotene fortified synbiotic food
intake. Although, it would be helpful to know the average HbA1c in
both groups at recruitment/cross over and end of study, due to
budget limitations we did not assess average HbA1c.
In conclusion, this randomized, double-blind, cross-over trial
demonstrated that beta-carotene fortified synbiotic food intake for
6 weeks in type 2 patients had beneficial effects on insulin meta-
bolism, triglycerides, VLDL-cholesterol, total/HDL-cholesterol ratio,
NO, GSH, magnesium levels; however, it did not influence FPG,
QUICKI, other lipid concentrations, hs-CRP, TAC, MDA, liver en-
zymes, calcium, iron levels and blood pressure.
Funding
This study was supported by a grant (no. 91113) from Kashan
University of Medical Sciences.
Conflicts of interest
None of the authors had any personal or financial conflict of
interest.
Author contributions
ZA contributed in conception, design, statistical analysis and
drafting of the manuscript. S-S A, AKh and MG contributed in
conception, data collection and manuscript drafting. AE contrib-
uted in conception, design and statistical analysis. All authors read
and approved the final version of the paper.
Please cite this article in press as: Asemi Z, et al., Effects of beta-carotene fortified synbiotic food on metabolic control of patients with type 2
diabetes mellitus: A double-blind randomized cross-over controlled clinical trial, Clinical Nutrition (2015), http://dx.doi.org/10.1016/
j.clnu.2015.07.009
Acknowledgments
The present study was supported by a grant from the Vice-
chancellor for Research, KUMS, and Iran. The authors would like
to thank the staff of Naghavi diabetes Clinic (Kashan, Iran) for their
assistance in this project.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.clnu.2015.07.009.
References
[1] American Diabetes Association. Diagnosis and classification of diabetes mel-
litus. Diabetes Care 2014;37(Suppl 1):S81e90.
[2] DeFronzo RA. Insulin resistance, lipotoxicity, type 2 diabetes and atheroscle-
rosis: the missing links. The Claude Bernard Lecture 2009. Diabetologia
2010;53:1270e87.
[3] Guariguata L. Contribute data to the 6th edition of the IDF diabetes Atlas.
Diabetes Res Clin Pract 2013;100:280e1.
[4] Vaidya V, Gangan N, Sheehan J. Impact of cardiovascular complications among
patients with type 2 diabetes mellitus: a systematic review. Expert Rev
Pharmacoecon Outcomes Res 2015:1e11.
[5] Banerjee M, Vats P. Reactive metabolites and antioxidant gene polymorphisms
in type 2 diabetes mellitus. Indian J Hum Genet 2014;20:10e9.
[6] Johnson EL. Glycemic variability in type 2 diabetes mellitus: oxidative stress
and macrovascular complications. Adv Exp Med Biol 2012;771:139e54.
[7] Astrup AS, Tarnow L, Rossing P, Pietraszek L, Riis Hansen P, Parving HH.
Improved prognosis in type 1 diabetic patients with nephropathy: a pro-
spective follow-up study. Kidney Int 2005;68:1250e7.
[8] Fioretto P, Solini A. Antihypertensive treatment and multifactorial approach
for renal protection in diabetes. J Am Soc Nephrol 2005;16(Suppl 1):S18e21.
[9] Asemi Z, Khorrami-Rad A, Alizadeh SA, Shakeri H, Esmaillzadeh A. Effects of
synbiotic food consumption on metabolic status of diabetic patients: a double-
blind randomized cross-over controlled clinical trial. Clin Nutr 2014;33:
198e203.
[10] Shakeri H, Hadaegh H, Abedi F, Tajabadi-Ebrahimi M, Mazroii N, Ghandi Y,
et al. Consumption of synbiotic bread decreases triacylglycerol and VLDL
levels while increasing HDL levels in serum from patients with type-2 dia-
betes. Lipids 2014;49:695e701.
[11] Ciccone MM, Cortese F, Gesualdo M, Carbonara S, Zito A, Ricci G, et al. Dietary
intake of carotenoids and their antioxidant and anti-inflammatory effects in
cardiovascular care. Mediat Inflamm 2013;2013:782137. http://dx.doi.org/
10.1155/2013/782137 [Epub 31.12.2013].
[12] Vincent HK, Bourguignon CM, Weltman AL, Vincent KR, Barrett E, Innes KE,
et al. Effects of antioxidant supplementation on insulin sensitivity, endothelial
adhesion molecules, and oxidative stress in normal-weight and overweight
young adults. Metabolism 2009;58:254e62.
[13] Yu F, Hao S, Zhao Y, Yang H, Fan XL, Yang J. In utero and lactational beta-
carotene supplementation attenuates D-galactose-induced hearing loss in
newborn rats. Food Chem Toxicol 2011;49:1697e704.
[14] Canas JA, Damaso L, Altomare A, Killen K, Hossain J, Balagopal PB. Insulin
resistance and adiposity in relation to serum beta-carotene levels. J Pediatr
2012;161. 58e64 e1e2.
[15] Williams CM. Effects of inulin on lipid parameters in humans. J Nutr
1999;129:1471Se3S.
[16] Yadav H, Lee JH, Lloyd J, Walter P, Rane SG. Beneficial metabolic effects of a
probiotic via butyrate induced GLP-1 secretion. J Biol Chem 2013;288(35):
25088e97.
[17] Lutgendorff F, Trulsson LM, van Minnen LP, Rijkers GT, Timmerman HM,
Franzen LE, et al. Probiotics enhance pancreatic glutathione biosynthesis and
reduce oxidative stress in experimental acute pancreatitis. Am J Physiol
Gastrointest Liver Physiol 2008;295:G1111e21.
[18] American Diabetes Association. Diagnosis and classification of diabetes mel-
litus. Diabetes Care 2012;35(Suppl 1):S64e71.
[19] Malaguarnera M, Vacante M, Antic T, Giordano M, Chisari G, Acquaviva R, et al.
Bifidobacterium longum with fructo-oligosaccharides in patients with non
alcoholic steatohepatitis. Dig Dis Sci 2012;57:545e53.
[20] Taghizadeh M, Hashemi T, Shakeri H, Abedi F, Sabihi SS, Alizadeh SA, et al.
Synbiotic food consumption reduces levels of triacylglycerols and VLDL, but
not cholesterol, LDL, or HDL in plasma from pregnant women. Lipids
2014;49(2):155e61.
[21] Trautwein EA, Rieckhoff D, Erbersdobler HF. Dietary inulin lowers plasma
cholesterol and triacylglycerol and alters biliary bile acid profile in hamsters.
J Nutr 1998;128:1937e43.
[22] Cheng CP, Tsai SW, Chiu CP, Pan TM, Tsai TY. The effect of probiotic-fermented
soy milk on enhancing the NO-mediated vascular relaxation factors. J Sci Food
Agric 2013;93:1219e25.
 Z. Asemi et al. / Clinical Nutrition xxx (2015) 1e7 7

[23] Bruzzese E, Raia V, Gaudiello G, Polito G, Buccigrossi V, Formicola V, et al.
Intestinal inflammation is a frequent feature of cystic fibrosis and is reduced
by probiotic administration. Aliment Pharmacol Ther 2004;20:813e9.
[24] Hagobian TA, Jacobs KA, Subudhi AW, Fattor JA, Rock PB, Muza SR, et al.
Cytokine responses at high altitude: effects of exercise and antioxidants at
4300 m. Med Sci Sports Exerc 2006;38:276e85.
[25] Giamarellos-Bourboulis EJ, Bengmark S, Kanellakopoulou K, Kotzampassi K.
Pro- and synbiotics to control inflammation and infection in patients with
multiple injuries. J Trauma 2009;67:815e21.
[26] Baeten JM, McClelland RS, Wener MH, Bankson DD, Lavreys L, Mandaliya K,
et al. Relationship between markers of HIV-1 disease progression and serum
beta-carotene concentrations in Kenyan women. Int J STD AIDS 2007;18:
202e6.
[27] Komers R, Anderson S. Paradoxes of nitric oxide in the diabetic kidney. Am J
Physiol Ren Physiol 2003;284:F1121e37.
[28] Peran L, Camuesco D, Comalada M, Nieto A, Concha A, Diaz-Ropero MP, et al.
Preventative effects of a probiotic, Lactobacillus salivarius ssp. salivarius, in
the TNBS model of rat colitis. World J Gastroenterol 2005;11:5185e92.
[29] Matthews GM, Howarth GS, Butler RN. Short-chain fatty acid modulation of
apoptosis in the Kato III human gastric carcinoma cell line. Cancer Biol Ther
2007;6:1051e7.

Please cite this article in press as: Asemi Z, et al., Effects of beta-carotene fortified synbiotic food on metabolic control of patients with type 2
diabetes mellitus: A double-blind randomized cross-over controlled clinical trial, Clinical Nutrition (2015), http://dx.doi.org/10.1016/
j.clnu.2015.07.009