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Probiotic yogurt improves antioxidant status in type 2 diabetic patients
Nutrition 28 (2012) 539–543
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Applied nutritional investigation
Probiotic yogurt improves antioxidant status in type 2 diabetic patients Hanie S. Ejtahed M.Sc. a, Javad Mohtadi-Nia Ph.D. a, Aziz Homayouni-Rad Ph.D. a, *, Mitra Niafar M.D., Ph.D. b, Mohammad Asghari-Jafarabadi Ph.D. a, Vahid Mofid M.Sc. c a
Faculty of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz, Iran Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran c Iran Dairy Industries Co., Tehran, Iran b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 March 2011 Accepted 19 August 2011
Objective: Oxidative stress plays a major role in the pathogenesis and progression of diabetes. Among various functional foods with an antioxidant effect, probiotic foods have been reported to repress oxidative stress. The objective of this clinical trial was to assess the effects of probiotic and conventional yogurt on blood glucose and antioxidant status in type 2 diabetic patients. Methods: Sixty-four patients with type 2 diabetes mellitus, 30 to 60 y old, were assigned to two groups in this randomized, double-blind, controlled clinical trial. The patients in the intervention group consumed 300 g/d of probiotic yogurt containing Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 and those in the control group consumed 300 g/d of conventional yogurt for 6 wk. Fasting blood samples, 24-h dietary recalls, and anthropometric measurements were collected at the baseline and at the end of the trial. Results: Probiotic yogurt significantly decreased fasting blood glucose (P < 0.01) and hemoglobin A1c (P < 0.05) and increased erythrocyte superoxide dismutase and glutathione peroxidase activities and total antioxidant status (P < 0.05) compared with the control group. In addition, the serum malondialdehyde concentration significantly decreased compared with the baseline value in both groups (P < 0.05). No significant changes from baseline were shown in insulin concentration and erythrocyte catalase activity within either group (P > 0.05). Conclusion: The consumption of probiotic yogurt improved fasting blood glucose and antioxidant status in type 2 diabetic patients. These results suggest that probiotic yogurt is a promising agent for diabetes management. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Probiotic yogurt Oxidative stress Type 2 diabetes Antioxidant enzyme activity Randomized clinical trial
Introduction Type 2 diabetes mellitus (T2DM) has rapidly increased in the world during the past few decades. Experimental and clinical evidence has suggested that oxidative stress plays a major role in the pathogenesis and progression of diabetes and its complications [1–3]. Diabetes is usually accompanied by an increased production of free radicals and impaired antioxidant defenses [2,4]. These conditions can lead to cellular organelle damage, the dysfunction of enzymes, an impairment of the binding of paraoxonase-1 to high-density lipoprotein and protection against lipid peroxidation, and the development of
The present study was supported by grant 5/4/3229 from the Vice-Chancellor for Research of Tabriz University of Medical Sciences, Iran. * Corresponding author. Tel.: þ98-411-335-7581; fax: þ98-411-334-0634. E-mail address: [email protected] (A. Homayouni-Rad). 0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2011.08.013
insulin resistance and may explain the presence of inflammation in T2DM [1,2,4–6]. Probiotics are live micro-organisms that, when administered in adequate amounts, confer health benefits on the host [7–9]. The consumption of probiotics have been shown to provide measurable health benefits, including the prevention and/or management of diarrhea, constipation, urinary tract infections, lactose intolerance, allergies, hepatic disease, inflammatory bowel disease, and diabetes mellitus. Certain species of bifidobacteria and lactobacilli used as probiotics can help balance intestinal microflora [10–13]. Studies have shown that special strains of lactic acid bacteria have antioxidant properties [14,15]. The antioxidative mechanisms of probiotics could be assigned to reactive oxygen species scavenging, metal ion chelation, enzyme inhibition, and the reduction activity and inhibition of ascorbate autoxidation [14]. In healthy persons, the consumption of goat milk fermented with
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Lactobacillus fermentum ME-3 has been shown to increase total antioxidative status (TAS) and decrease markers of oxidative stress [16,17]. The antioxidative properties of other probiotic strains have also been reported in healthy persons [18,19]. Studies using animal models of diabetes have also shown that Lactobacillus acidophilus and Lactobacillus casei attenuate oxidative stress and have antidiabetic effects [20,21]. Alterations in gut microbiota composition have recently been documented in patients with T2DM, providing a target for probiotic intervention [22]. Modification of gut microflora by probiotics may be seen as a novel means of regulating glucose metabolism and improving oxidative stress in T2DM. Thus, in this controlled trial, we tested the hypothesis that the consumption of probiotic yogurt containing L. acidophilus La5 and Bifidobacterium lactis Bb12 would improve blood glucose and antioxidant status in patients with T2DM.
until the assay. Blood samples were analyzed at the Drug Applied Research Center (Tabriz University of Medical Sciences, Tabriz, Iran). Fasting blood glucose was measured using the standard enzymatic method with a Parsazmun kit (Karaj, Iran). Glycated hemoglobin (HbA1c) was measured in the whole blood by cation exchange chromatography with a NycoCard HbA1c kit (Oslo, Norway). Insulin concentration was determined by a chemiluminescent immunoassay using a Liaison analyzer (DiaSorin, Salluggia, Italy). Erythrocyte superoxide dismutase (SOD) activity was measured spectrophotometrically using a Ransod kit (Randox Laboratories, Crumlin, UK) [24]. Erythrocyte glutathione peroxidase (GPx) activity was measured using the spectrophotometric technique and a Ransel kit (Randox Laboratories) according to the method described by Paglia and Valentine [25]. Erythrocyte CAT activity was measured by the method of Aebi [26]. Serum total antioxidant capacity was determined using a Randox TAS kit (Randox Laboratories) [27]. Serum malondialdehyde (MDA) concentration was determined using the thiobarbituric acid method described by Bilici et al. [28]. The present study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human persons were approved by the ethics committee at Tabriz University of Medical Sciences (no. 897). Written informed consent was obtained from all patients (the trial has been registered in the Iranian Registry of Clinical Trials, available at: http://www.irct.ir, identifier: IRCT 138903223533N1).
Materials and methods Subjects Sixty-four patients with T2DM 30 to 60 y old with a body mass index (BMI) lower than 35 kg/m2 were recruited for this study from the endocrinology clinic of Sina Hospital in Tabriz, Iran. Recruitment was done by telephone and advertisements. All patients had been diagnosed with T2DM for at least 1 y. Exclusion criteria were smoking; the presence of kidney, liver, or inflammatory intestinal disease, thyroid disorders, immunodeficiency diseases, or lactose intolerance; required insulin injections; use of nutritional supplements within the previous 3 wk of testing; use of cholesterol-lowering medication, estrogen, progesterone, or diuretics; pregnancy or breast-feeding; and consuming probiotic yogurt or any other probiotic products within the previous 2 mo of testing. The sample size was determined based on the primary information obtained from the study by Chamari et al. [19] for catalase (CAT). For an a value equal to 0.05 and a power of 80%, the sample size was computed as 21.788 (z22) per group [23]. This number was increased to 32 per group to accommodate the anticipated dropout rate. Study design and measurements The present study was a double-blinded, randomized controlled clinical trial in which subjects were randomly assigned to the probiotic (intervention) or conventional (control) yogurt group using a block randomization procedure with matched subjects in each block based on sex and age. The allocation of the intervention or control group was concealed from the researchers and the probiotic and conventional yogurt containers had an identical appearance. The yogurt containers had no labeled information about the type of yogurt inside. Therefore, neither the subjects nor the investigators were aware of the treatment assignments in this double-blinded study. Each group consisted of 32 patients. One week before the beginning of the trial, all patients refrained from eating yogurt or any other fermented foods. Over 6 wk, the probiotic and conventional groups consumed 300 g/d of probiotic and conventional yogurt, respectively. All patients were asked, throughout the 6-wk trial, to maintain their usual dietary habits and lifestyle and to avoid consuming any yogurt other than that provided to them by the researchers and any other fermented foods. The patients were instructed to keep the yogurt under refrigeration and to avoid any changes in medication, if possible. Arrangements were made so that the patients would receive a 1-wk supply of their probiotic or conventional yogurts every week. Compliance with the yogurt consumption guidelines was monitored by telephone interviews once a week. Information on food consumption, anthropometric measurements, and fasting blood samples were collected at the beginning and at the end of the trial. Nutrient intakes during 3 d were estimated using a 24-h dietary recall at the beginning and at the end of the study. Three-day averages of macro- and micronutrient intakes were analyzed by Nutritionist 4 software (First Databank, Hearst Corp, San Bruno, CA, USA). Anthropometric measurements were recorded by trained personnel. Body weights were measured using a scale (Seca, Hamburg, Germany) with 0.1-kg accuracy without shoes and with minimum clothing. Heights were measured using a stadiometer (Seca) with 0.1-cm accuracy without shoes. BMI was calculated by dividing body weight (kilograms) by height (meters) squared. A blood sample was drawn for each patient from the antecubital vein in the arm after a 12-h overnight fast. The serum samples were separated from whole blood by centrifugation at 3500 rpm for 10 min (Avanti J-25, Beckman, Brea, CA, USA). The serum and whole blood samples were frozen immediately at 70 C
Intervention The probiotic and conventional yogurts contained Lactobacillus bulgaricus and Streptococcus thermophilus. The probiotic yogurt was also enriched with B. lactis Bb12 and L. acidophilus La5 (Chr. Hansen, Hoersholm, Denmark) as Direct Vat Set cultures. The yogurts were produced weekly and distributed to the participants. Probiotic yogurts were sampled 1 d after manufacture (time of distribution) and microbiologically analyzed every week. Samples were refrigerated at 4 , with subsequent analyzing on day 7 of storage. MRS-bile agar medium was used for the differential enumeration of mixed probiotic bacteria in presence of yogurt bacteria [29]. All the samples were incubated at 37 C for 72 h under aerobic and anaerobic conditions. All experiments were performed in triplicate. Counts of L. acidophilus were achieved at the aerobic condition and viable counts of B. lactis were selectively achieved using the subtractive enumeration method [29]. Microbiological analyses of the probiotic yogurts showed that the average colony counts of L. acidophilus La5 and B. lactis Bb12 on day 1 were 7.23 106 and 6.04 106 cfu/g, respectively. Probiotic yogurts contained 1.85 106 cfu/g of L. acidophilus La5 and 1.79 106 cfu/g of B. lactis Bb12 on day 7. Both probiotic bacteria showed a steady survival rate during a 7-d storage time. The fat content was 2.5% and was comparable in both yogurt types. The probiotic and conventional yogurt containers were identical and the yogurts had a similar taste and appearance. The yogurts were specially prepared for this study by Iran Dairy Industries Co. (Tehran, Iran). Statistical analyses The experimental data were analyzed by SPSS 11.5 (SPSS, Inc., Chicago, IL, USA) and the results were expressed as mean standard deviation. The normality of the distribution of variables was tested by the Kolmogorov-Smirnov test. For the duration of diabetes, monounsaturated fatty acid, vitamin A, E, and C intakes, fasting blood glucose, and insulin that did not follow normal distributions, analyses were performed after log transformation. The background characteristics and nutrient intakes of patients in the two groups were compared using independentsamples t tests and chi-square tests. The use of diabetes medication in the two groups was compared using the Mann-Whitney U test. Differences between the two groups after the intervention were determined by analysis of covariance, adjusting for baseline measurements and covariates. In this study, duration of diabetes and polyunsaturated fatty acid intake were used as possible covariates. The changes in anthropometric measurements, nutrient intakes, fasting blood glucose, HbA1c, insulin, and oxidative stress markers of the patients between the beginning and the end of the trial were compared by paired-samples t test [30]. Results with P < 0.05 were considered statistically significant.
Results In this study, four patients were excluded from the statistical analysis because they needed to change their medication during the trial or they did not consume the yogurt according to the plan. Thus, data for 60 patients (23 male and 37 female) were analyzed (n ¼ 30 for each group). The patients demonstrated good compliance with the yogurt consumption and no adverse effects or symptoms were reported. The baseline characteristics of the patients in the two groups are listed in Table 1. The
H. S. Ejtahed et al. / Nutrition 28 (2012) 539–543 Table 1 Baseline characteristics of study participants
Age (y)* Men/womeny Weight (kg)* BMI (kg/m2)* Duration of diabetes (y)* Metformin/dz Glibenclamide/dz
Table 2 Dietary intakes of subjects throughout the study
Conventional yogurt (n ¼ 30)
Probiotic yogurt (n ¼ 30)
Variables
51.00 12/18 75.42 29.14 4.08 2 1
50.87 11/19 76.18 28.95 5.82 2 2
Energy (kcal) Baseline After intervention Carbohydrate (g) Baseline After intervention Protein (g) Baseline After intervention Total fat (g) Baseline After intervention Saturated fat (g) Baseline After intervention Monounsaturated fat (g) Baseline After intervention Polyunsaturated fat (g) Baseline After intervention Dietary fiber (g) Baseline After intervention Vitamin A (mg) Baseline After intervention Vitamin E (mg) Baseline After intervention Vitamin C (mg) Baseline After intervention Copper (mg) Baseline After intervention Zinc (mg) Baseline After intervention Calcium (mg) Baseline After intervention Phosphorus (mg) Baseline After intervention
7.32 11.28 4.30 4.28 1.25 1
541
7.68 10.94 3.65 4.95x 1.25 2
BMI, body mass index * Mean SD. y Frequency. z Median and interquartile range. x Significant difference between groups at baseline (P < 0.05, independentsamples t test).
duration of diabetes was significantly different between the probiotic and conventional groups (P ¼ 0.039). However, other baseline characteristics of the patients (age, sex, weight, BMI, and medications) did not differ between the two groups (P > 0.05). There were no statistically significant differences in weight and BMI values between or within groups at the end of the study. The intake of polyunsaturated fatty acid was significantly different between the probiotic and conventional groups at the beginning of the study (P ¼ 0.033). No significant differences in energy and other nutrient intakes were observed between the two groups at baseline (P > 0.05). During the study, intakes of protein, zinc, calcium, and phosphorus significantly increased in both groups (P < 0.01). No significant changes from baseline were observed in the other nutrient intakes. At the end of the study, there were no statistically significant differences between the two groups for dietary intakes (P > 0.05; Table 2). There were no statistically significant differences in blood glucose, HbA1c, and insulin concentration between the two groups at the beginning of the study (P > 0.05). Fasting blood glucose and HbA1c were significantly decreased in the probiotic group compared with the control group (P ¼ 0.009 and P ¼ 0.019, respectively). Insulin concentration was not significantly different between groups at the end of the trial (P ¼ 0.955; Table 3). The intervention group showed 8.68% decrease in fasting blood glucose concentration from the baseline value (P ¼ 0.001). Although the differences were not statistically significant, HbA1c and insulin also decreased in the intervention group during the study (P ¼ 0.230 and P ¼ 0.654, respectively). In contrast, HbA1c increased significantly from the baseline in the control group (P ¼ 0.003; Table 3). Erythrocyte SOD activity was significantly different between the probiotic and conventional groups at baseline (P ¼ 0.004). There were no statistically significant differences between the two groups in the other variables at baseline (Table 3). Results of the analysis of covariance showed that there were statistically significant differences between the two groups in erythrocyte SOD and GPx activities and TAS at the end of the study when adjusted for duration of diabetes, polyunsaturated fatty acid intake, and baseline values (P ¼ 0.007, P ¼ 0.002, and P ¼ 0.014, respectively). No significant differences were detected in the CAT activity and MDA concentration between the two groups at the end of the trial (P > 0.05; Table 3). As presented in Table 3, erythrocyte SOD and GPx activities and TAS were significantly increased compared with the baseline values in the intervention group (P < 0.001, P < 0.001, and P ¼ 0.001, respectively). No significant changes from baseline were observed in erythrocyte CAT activity for either group
Conventional yogurt (n ¼ 30)
Probiotic yogurt (n ¼ 30)
1774.99 482.92 1809.67 434.30
1775.20 449.51 1776.67 392.28
232.69 76.15 241.86 68.08
242.88 70.44 239.64 59.54
70.08 20.22 80.82 21.34y
68.12 20.00 77.84 17.99y
68.01 18.27 65.93 16.53
65.49 19.23 61.10 18.63
20.62 7.56 19.21 5.45
20.86 7.19 18.78 5.96
22.76 7.07 21.73 6.28
23.61 10.24 21.88 9.52
18.13 6.42 16.06 5.77
15.03 4.33* 15.01 5.19
14.19 4.44 14.86 6.28
16.05 6.18 15.47 5.54
871.08 663.23 898.22 710.39
1152.05 814.67 1111.54 811.93
12.72 8.38 11.45 7.94
10.27 5.50 10.32 6.12
138.41 61.67 126.96 69.17
152.87 64.93 144.50 54.52
1.59 0.53 1.59 0.55
1.67 0.59 1.57 0.43
9.99 2.64 12.23 3.40y
10.13 3.35 11.43 3.09y
824.86 275.70 1215.11 213.56y
869.76 229.19 1264.69 227.63y
1185.39 319.75 1477.26 324.93y
1173.57 357.33 1462.60 337.15y
Data are presented as mean SD * Significant difference between groups at baseline (P < 0.05, independentsamples t test). y Significant difference within group throughout the study (P < 0.01, pairedsamples t test).
(P > 0.05; Table 3). The results of paired-samples t test showed that MDA concentration decreases were significant for the control and intervention groups (P ¼ 0.005 and P ¼ 0.013, respectively; Table 3).
Discussion In T2DM, free radicals are generated excessively. These free radicals cause lipid peroxidation and MDA generation [2]. Moreover, activities of SOD, GPx, and CAT, which scavenge reactive oxygen species, decrease in patients with T2DM [31]. It is important to increase the CAT and GPx activity, in addition to SOD activity, to remove reactive oxygen species. The improvement in oxidative stress status can contribute to diabetes management [2]. The present study showed that probiotic yogurt consumption significantly decreased fasting blood glucose and HbA1c and
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Table 3 Effects of 6 wk of probiotic and conventional yogurt consumption on blood glucose, hemoglobin A1c, insulin, and oxidative stress markers Variables Glucose (mmol/L) Baseline After intervention HbA1c (%) Baseline After intervention Insulin (mU/mL) Baseline After intervention SOD (U/g Hb) Baseline After intervention GPx (U/g Hb) Baseline After intervention CAT (K/g Hb) Baseline After intervention TAS (mmol/L) Baseline After intervention MDA (nmol/L) Baseline After intervention
Conventional yogurt (n ¼ 30)
Probiotic yogurt (n ¼ 30)
7.35 1.28 7.53 1.32
8.06 2.49 7.36 2.41y,z
6.87 0.81 7.17 0.66z
7.29 1.21 7.17 1.24y
6.31 3.72 6.50 3.57
7.47 4.89 6.97 4.49
1161.59 246.01 1136.54 256.26
975.80 238.34* 1113.69 177.77y,z
30.28 4.40 30.12 4.23
29.03 4.29 29.81 4.58yz
141.40 25.78 151.15 36.98
148.81 34.56 146.57 34.05
0.93 0.20 0.92 0.16
0.90 0.18 0.96 0.18y,z
2.81 0.65 2.55 0.60z
2.79 0.62 2.53 0.65z
CAT, catalase; GPx, glutathione peroxidase; Hb, hemoglobin; MDA, malondialdehyde; SOD, superoxide dismutase; TAS, total antioxidant status Data are presented as mean SD * Significant difference between groups at baseline (P < 0.01, independentsamples t test). y Significant difference between groups after intervention (P < 0.05, analysis of covariance, adjusted for duration of diabetes, polyunsaturated fatty acid intake, and baseline values). z Significant difference within group throughout the study (P < 0.01, pairedsamples t test).
increased erythrocyte SOD and GPx activities and TAS compared with conventional yogurt consumption. Furthermore, the MDA concentration significantly decreased in both groups. However, insulin concentration and erythrocyte CAT activity remained unchanged in the intervention group. In the present study, there were no statistically significant changes in weight, BMI, and energy within either group during the study. However, intakes of protein, zinc, calcium, and phosphorus significantly increased in both groups throughout the study. These increases most probably were caused by the yogurt consumption. There were no statistically significant differences between the two groups at the end of the study for these nutrients. Therefore, the observed results in the probiotic group could not have been caused by the changes in weight and dietary intakes. Chin [32] reported that T2DM could arise from imbalances of microflora in the gastrointestinal tract. Larsen et al. [22] documented that the intestinal microbiota of patients with T2DM was relatively enriched with gram-negative bacteria, belonging to the phyla Bacteroidetes and Proteobacteria. The proportion of Firmicutes to Bacteroidetes was significantly decreased in the patients with T2DM compared with the non-diabetic patients [22]. Therefore, we conducted the first randomized controlled trial investigating the effects of probiotic yogurt on blood glucose and antioxidant status in patients with T2DM. The findings support previously reported observational data on the antioxidant property of probiotic yogurt containing L. acidophilus and B. lactis in young healthy women [19]. Naruszewicz et al. [18] investigated the antioxidative effects of L. plantarum in smokers
and found that an L. plantarum intake for 6 wk decreased plasma F2-isoprostanes concentrations. In a study by Songisepp et al. [16], a significant improvement of TAS was seen with the daily consumption of fermented goat milk containing L. fermentum ME-3 in healthy persons after 3 wk. Kullisaar et al. [17] also reported that goat milk fermented by L. fermentum ME-3 increased total antioxidative activity and decreased lipid peroxidation markers in healthy persons. Previous evidence regarding the antidiabetic properties of probiotics has been limited to animal studies. Harisa et al. [21] showed that treatment with L. acidophilus alone or in combination with acarbose significantly decreased fasting blood sugar, HbA1c, and MDA concentration in diabetic rats. In an animal study, Yadav et al. [20] reported that probiotic dahi, a fermented milk product containing L. acidophilus and L. casei, had an antioxidative effect on the liver and pancreas tissues of highfructose–induced diabetic rats and delayed the onset of glucose intolerance, hyperglycemia, and hyperinsulinemia. In another study, Yadav et al. [33] found that probiotic dahi suppressed streptozotocin-induced oxidative damage in the pancreatic tissues of diabetic rats by inhibiting the lipid peroxidation and preserving the activity of SOD, GPx, and CAT. These effects of probiotic dahi may slow the decrease of insulin and increase of blood glucose [33]. Al-Salami et al. [34] reported that probiotic treatment had no effect on blood glucose concentration in healthy rats, but decreased blood glucose in diabetic rats because of an increased gliclazide (sulfonylurea) bioavailability. In another study, Matsuzaki et al. [35] showed that L. casei inhibited the production of proinflammatory cytokines and decreased plasma glucose concentration in non–insulin-dependent diabetic mice. The present study, indeed, has reported perhaps the first evidence of improved glucose metabolism in diabetic patients and the results are in accordance with the findings of the animal studies described. However, no statistically significant changes were observed in HbA1c, insulin concentration, and erythrocyte CAT activity in the probiotic yogurt group during the study, which may be explained by the short duration of the study. The precise mechanisms involved in the antidiabetic effects of probiotics remain largely unknown. These effects may be partly related to a probiotics-mediated decrease in oxidative stress. Moreover, the immune-modulatory and anti-inflammatory effects of probiotics and the modification of intestinal microflora could be other probable underlying mechanisms. The inhibition of ascorbate autoxidation, metal ion chelation, and reduction activity and scavenging of superoxide anion radicals, hydrogen peroxide, and free radicals are likely to underline the antioxidative effect of probiotic yogurt [14,36,37]. The relatively small but significant decrease in lipid peroxidation indicated by the decreased plasma MDA was not associated with changes in antioxidant status markers for the control group. The MDA concentration decrease in both yogurt groups could be due to the antioxidative effect of bioactive peptides released during yogurt fermentation by proteolytic lactic acid bacteria [38,39]. Fermented milks have been reported as dietary sources of natural antioxidants because of the presence of antioxidant peptides. Most identified bioactive peptides were derived from as-casein and have been shown to exhibit free radical scavenging and inhibit enzymatic and non-enzymatic lipid peroxidation [40]. The antioxidant peptides derived from whey protein are likely the result of the presence of cysteine-rich proteins that aid in the synthesis of glutathione, a potent intracellular antioxidant [41]. Although the yogurt for both trial groups can exert antioxidative effects by bioactive peptides, the results showed that
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probiotic yogurt was more effective in increasing antioxidative activity than conventional yogurt. The limitations of this study included its short duration and the absence of a control group that consumed no yogurt. Moreover, many exclusion criteria in this study could limit the generalizability of the results. Therefore, further investigations with longer duration and a no-yogurt control group, and a larger trial with some form of stratification are needed to confirm the positive effect of probiotic yogurt on the management of diabetes. Further studies on the effects of probiotics on intestinal microflora composition and intestinal transit time in diabetic patients would be useful. Conclusion This trial showed that consuming 300 g/d of probiotic yogurt containing L. acidophilus La5 and B. lactis Bb12 improved the antioxidant status and fasting blood glucose in patients with T2DM. These findings suggest that probiotic yogurt is a functional food that can exert antidiabetic and antioxidant properties. Acknowledgments The authors thank the Iran Dairy Industries Company for supplying the probiotic and conventional yogurts and supporting this study. References [1] Ceriello A, Motz E. Is oxidative stress the pathogenic mechanism underlying insulin resistance, diabetes, and cardiovascular disease? The common soil hypothesis revisited. Arterioscler Thromb Vasc Biol 2004;24:816–23. [2] Maritim AC, Sanders RA, Watkins JB. Diabetes, oxidative stress, and antioxidants: a review. J Biochem Mol Toxic 2003;17:24–38. [3] Stephens JW, Khanolkar MP, Bain SC. The biological relevance and measurement of plasma markers of oxidative stress in diabetes and cardiovascular disease. Atherosclerosis 2009;202:321–9. [4] Lipinski B. Pathophysiology of oxidative stress in diabetes mellitus. J Diabetes Complications 2001;15:203–10. [5] Fuhrman B, Volkova N, Aviram M. Pomegranate juice polyphenols increase recombinant paraoxonase-1 binding to high-density lipoprotein: studies in vitro and in diabetic patients. Nutrition 2010;26:359–66. [6] Zozulinska D, Wierusz-Wysocka B. Type 2 diabetes mellitus as inflammatory disease. Diabetes Res Clin Pract 2006;74:12–6. [7] Guarner F, Perdigon G, Corthier G, Salminen S, Koletzko B, Morelli L. Should yoghurt cultures be considered probiotic? Br J Nutr 2005;93:783–6. [8] Homayouni A. Letter to the editor. Food Chem 2009;114:1073. [9] Homayouni A, Azizi A, Ehsani MR, Yarmand MS, Razavi SH. Effect of microencapsulation and resistant starch on the probiotic survival and sensory properties of synbiotic ice cream. Food Chem 2008;111:50–5. [10] Higashikawa F, Noda M, Awaya T, Nomura K, Oku H, Sugiyama M. Improvement of constipation and liver function by plant-derived lactic acid bacteria: a double-blind, randomized trial. Nutrition 2010;26:367–74. [11] Goldin BR, Gorbach SL. Clinical indications for probiotics: an overview. Clin Infect Dis 2008;46:96–100. [12] Kaur IP, Kuhad A, Garg A, Chopra K. Probiotics: delineation of prophylactic and therapeutic benefits. J Med Food 2009;12:219–35. [13] Hoerr RA, Bostwick EF. Bioactive proteins and probiotic bacteria: modulators of nutritional health. Nutrition 2000;16:711–3. [14] Lin MY, Yen CL. Antioxidative ability of lactic acid bacteria. J Agric Food Chem 1999;47:1460–6. [15] Uskova MA, Kravchenko LV. Antioxidant properties of lactic acid bacteriaprobiotic and yogurt strains. Vopr Pitan 2009;78:18–23.
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[16] Songisepp E, Kals J, Kullisaar T, Mandar R, Hutt P, Zilmer M, et al. Evaluation of the functional efficacy of an antioxidative probiotic in healthy volunteers. Nutr J 2005;4:22–32. [17] Kullisaar T, Songisepp E, Mikelsaar M, Zilmer K, Vihalemm T, Zilmer M. Antioxidative probiotic fermented goats’ milk decreases oxidative stressmediated atherogenicity in human persons. Br J Nutr 2003;90:449–56. [18] Naruszewicz M, Johansson ML, Zapolska-Downar D, Bukowska H. Effect of Lactobacillus plantarum 299v on cardiovascular disease risk factors in smokers. Am J Clin Nutr 2002;76:1249–55. [19] Chamari M, Djazayery A, Jalali M, Sadrzadeh Yeganeh H, Hosseini S, et al. The effect of daily consumption of probiotic and conventional yoghurt on some oxidative stress factors in plasma of young healthy women. ARYA Atheroscler J 2008;4:175–9. [20] Yadav H, Jain S, Sinha PR. Antidiabetic effect of probiotic dahi containing Lactobacillus acidophilus and Lactobacillus casei in high fructose fed rats. Nutrition 2007;23:62–8. [21] Harisa GI, Taha EI, Khalil AF, Salem MM. Oral administration of Lactobacillus acidophilus restores nitric oxide level in diabetic rats. Aust J Basic Appl Sci 2009;3:2963–9. [22] Larsen N, Vogensen FK, Berg FWJ, Nielsen DS, Andreasen AS, Pedersen BK, et al. Gut microbiota in human adults with type 2 diabetes differs from non-diabetic adults. PLoS ONE 2010;5:e9085. [23] Pocock SJ. Clinical trials: a practical approach. New York: John Wiley & Sons; 1983. [24] Winterbourn CC, Hawkins RE, Brian M, Carrel RW. Red cell superoxide dismutase activity. J Lab Clin Med 1975;85:337–50. [25] Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158–69. [26] Aebi H. Catalase in vitro. Methods Enzymol 1984;105:121–6. [27] Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A. A novel method for measuring antioxidant capacity and its application for monitoring the antioxidant status in premature neonates. Clin Sci 1993;84:407–12. [28] Bilici M, Efe H, Arif Koroglu M, Avni Uydu H, Bekaroglu M, Deger O. Antioxidative enzyme activities and lipid peroxidation in major depression: alterations by antidepressant treatments. J Affect Disord 2001;64:43–51. [29] Mortazavian AM, Ehsani MR, Sohrabvandi S, Reinheimer JA. MRS-bile agar: its suitability for the enumeration of mixed probiotic cultures in cultured dairy products. Milchwissenschaft 2007;62:270–2. [30] Zar JH. Biostatistical analysis. New York: Pearson Press; 1998. [31] Rahbani-nobar ME, Rahimi-pour A, Rahbani-nobar M, Adi-beig F, Mirhashemi SM. Total antioxidant capacity, superoxide dismutase and glutathione peroxidase in diabetic patients. Med J IAS 1999;12:109–14. [32] Chin J. Prospects for beneficial health outcomes from intestinal microflora. Asia Pac J Clin Nutr 2005;14:64–5. [33] Yadav H, Jain S, Sinha PR. Oral administration of dahi containing probiotic Lactobacillus acidophilus and Lactobacillus casei delayed the progression of streptozotocin-induced diabetes in rats. J Dairy Res 2008;75:189–95. [34] Al-Salami H, Butt G, Fawcett JP, Tucker IG, Golocorbin-Kon S, Mikov M. Probiotic treatment reduces blood glucose levels and increases systemic absorption of gliclazide in diabetic rats. Eur J Drug Metab Pharmacokinet 2008;33:101–6. [35] Matsuzaki T, Yamazaki R, Hashimoto S, Yokokura T. Antidiabetic effects of an oral administration of Lactobacillus casei in a non-insulin–dependent diabetes mellitus (NIDDM) model using KK-Ay mice. Endocr J 1997;44: 357–65. [36] Wang YC, Yu RC, Chou CC. Antioxidative activities of soymilk fermented with lactic acid bacteria and bifidobacteria. Food Microbiol 2006;23: 128–35. [37] Lin MY, Chang FJ. Antioxidative effect of intestinal bacteria Bifidobacterium longum ATCC 15708 and Lactobacillus acidophilus ATCC 4356. Dig Dis Sci 2000;45:1617–22. [38] Farvin KHS, Baron CP, Nielsen NS, Jacobsen C. Antioxidant activity of yoghurt peptides: part 1din vitro assays and evaluation in u-3 enriched milk. Food Chem 2010;123:1081–9. [39] Farvin KHS, Baron CP, Nielsen NS, Otte J, Jacobsen C. Antioxidant activity of yoghurt peptides: part 2dCharacterisation of peptide fractions. Food Chem 2010;123:1090–7. [40] Korhonen H, Pihlanto A. Bioactive peptides: Production and functionality. Int Dairy J 2006;16:945–60. [41] Hayes M, Stanton C, Fitzgerald GF, Ross RP. Putting microbes to work: Dairy fermentation, cell factories and bioactive peptides. Part II: Bioactive peptide functions. Biotechnol J 2007;2:435–49.
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Applied nutritional investigation
Probiotic yogurt improves antioxidant status in type 2 diabetic patients Hanie S. Ejtahed M.Sc. a, Javad Mohtadi-Nia Ph.D. a, Aziz Homayouni-Rad Ph.D. a, *, Mitra Niafar M.D., Ph.D. b, Mohammad Asghari-Jafarabadi Ph.D. a, Vahid Mofid M.Sc. c a
Faculty of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz, Iran Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran c Iran Dairy Industries Co., Tehran, Iran b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 March 2011 Accepted 19 August 2011
Objective: Oxidative stress plays a major role in the pathogenesis and progression of diabetes. Among various functional foods with an antioxidant effect, probiotic foods have been reported to repress oxidative stress. The objective of this clinical trial was to assess the effects of probiotic and conventional yogurt on blood glucose and antioxidant status in type 2 diabetic patients. Methods: Sixty-four patients with type 2 diabetes mellitus, 30 to 60 y old, were assigned to two groups in this randomized, double-blind, controlled clinical trial. The patients in the intervention group consumed 300 g/d of probiotic yogurt containing Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 and those in the control group consumed 300 g/d of conventional yogurt for 6 wk. Fasting blood samples, 24-h dietary recalls, and anthropometric measurements were collected at the baseline and at the end of the trial. Results: Probiotic yogurt significantly decreased fasting blood glucose (P < 0.01) and hemoglobin A1c (P < 0.05) and increased erythrocyte superoxide dismutase and glutathione peroxidase activities and total antioxidant status (P < 0.05) compared with the control group. In addition, the serum malondialdehyde concentration significantly decreased compared with the baseline value in both groups (P < 0.05). No significant changes from baseline were shown in insulin concentration and erythrocyte catalase activity within either group (P > 0.05). Conclusion: The consumption of probiotic yogurt improved fasting blood glucose and antioxidant status in type 2 diabetic patients. These results suggest that probiotic yogurt is a promising agent for diabetes management. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Probiotic yogurt Oxidative stress Type 2 diabetes Antioxidant enzyme activity Randomized clinical trial
Introduction Type 2 diabetes mellitus (T2DM) has rapidly increased in the world during the past few decades. Experimental and clinical evidence has suggested that oxidative stress plays a major role in the pathogenesis and progression of diabetes and its complications [1–3]. Diabetes is usually accompanied by an increased production of free radicals and impaired antioxidant defenses [2,4]. These conditions can lead to cellular organelle damage, the dysfunction of enzymes, an impairment of the binding of paraoxonase-1 to high-density lipoprotein and protection against lipid peroxidation, and the development of
The present study was supported by grant 5/4/3229 from the Vice-Chancellor for Research of Tabriz University of Medical Sciences, Iran. * Corresponding author. Tel.: þ98-411-335-7581; fax: þ98-411-334-0634. E-mail address: [email protected] (A. Homayouni-Rad). 0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2011.08.013
insulin resistance and may explain the presence of inflammation in T2DM [1,2,4–6]. Probiotics are live micro-organisms that, when administered in adequate amounts, confer health benefits on the host [7–9]. The consumption of probiotics have been shown to provide measurable health benefits, including the prevention and/or management of diarrhea, constipation, urinary tract infections, lactose intolerance, allergies, hepatic disease, inflammatory bowel disease, and diabetes mellitus. Certain species of bifidobacteria and lactobacilli used as probiotics can help balance intestinal microflora [10–13]. Studies have shown that special strains of lactic acid bacteria have antioxidant properties [14,15]. The antioxidative mechanisms of probiotics could be assigned to reactive oxygen species scavenging, metal ion chelation, enzyme inhibition, and the reduction activity and inhibition of ascorbate autoxidation [14]. In healthy persons, the consumption of goat milk fermented with
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Lactobacillus fermentum ME-3 has been shown to increase total antioxidative status (TAS) and decrease markers of oxidative stress [16,17]. The antioxidative properties of other probiotic strains have also been reported in healthy persons [18,19]. Studies using animal models of diabetes have also shown that Lactobacillus acidophilus and Lactobacillus casei attenuate oxidative stress and have antidiabetic effects [20,21]. Alterations in gut microbiota composition have recently been documented in patients with T2DM, providing a target for probiotic intervention [22]. Modification of gut microflora by probiotics may be seen as a novel means of regulating glucose metabolism and improving oxidative stress in T2DM. Thus, in this controlled trial, we tested the hypothesis that the consumption of probiotic yogurt containing L. acidophilus La5 and Bifidobacterium lactis Bb12 would improve blood glucose and antioxidant status in patients with T2DM.
until the assay. Blood samples were analyzed at the Drug Applied Research Center (Tabriz University of Medical Sciences, Tabriz, Iran). Fasting blood glucose was measured using the standard enzymatic method with a Parsazmun kit (Karaj, Iran). Glycated hemoglobin (HbA1c) was measured in the whole blood by cation exchange chromatography with a NycoCard HbA1c kit (Oslo, Norway). Insulin concentration was determined by a chemiluminescent immunoassay using a Liaison analyzer (DiaSorin, Salluggia, Italy). Erythrocyte superoxide dismutase (SOD) activity was measured spectrophotometrically using a Ransod kit (Randox Laboratories, Crumlin, UK) [24]. Erythrocyte glutathione peroxidase (GPx) activity was measured using the spectrophotometric technique and a Ransel kit (Randox Laboratories) according to the method described by Paglia and Valentine [25]. Erythrocyte CAT activity was measured by the method of Aebi [26]. Serum total antioxidant capacity was determined using a Randox TAS kit (Randox Laboratories) [27]. Serum malondialdehyde (MDA) concentration was determined using the thiobarbituric acid method described by Bilici et al. [28]. The present study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human persons were approved by the ethics committee at Tabriz University of Medical Sciences (no. 897). Written informed consent was obtained from all patients (the trial has been registered in the Iranian Registry of Clinical Trials, available at: http://www.irct.ir, identifier: IRCT 138903223533N1).
Materials and methods Subjects Sixty-four patients with T2DM 30 to 60 y old with a body mass index (BMI) lower than 35 kg/m2 were recruited for this study from the endocrinology clinic of Sina Hospital in Tabriz, Iran. Recruitment was done by telephone and advertisements. All patients had been diagnosed with T2DM for at least 1 y. Exclusion criteria were smoking; the presence of kidney, liver, or inflammatory intestinal disease, thyroid disorders, immunodeficiency diseases, or lactose intolerance; required insulin injections; use of nutritional supplements within the previous 3 wk of testing; use of cholesterol-lowering medication, estrogen, progesterone, or diuretics; pregnancy or breast-feeding; and consuming probiotic yogurt or any other probiotic products within the previous 2 mo of testing. The sample size was determined based on the primary information obtained from the study by Chamari et al. [19] for catalase (CAT). For an a value equal to 0.05 and a power of 80%, the sample size was computed as 21.788 (z22) per group [23]. This number was increased to 32 per group to accommodate the anticipated dropout rate. Study design and measurements The present study was a double-blinded, randomized controlled clinical trial in which subjects were randomly assigned to the probiotic (intervention) or conventional (control) yogurt group using a block randomization procedure with matched subjects in each block based on sex and age. The allocation of the intervention or control group was concealed from the researchers and the probiotic and conventional yogurt containers had an identical appearance. The yogurt containers had no labeled information about the type of yogurt inside. Therefore, neither the subjects nor the investigators were aware of the treatment assignments in this double-blinded study. Each group consisted of 32 patients. One week before the beginning of the trial, all patients refrained from eating yogurt or any other fermented foods. Over 6 wk, the probiotic and conventional groups consumed 300 g/d of probiotic and conventional yogurt, respectively. All patients were asked, throughout the 6-wk trial, to maintain their usual dietary habits and lifestyle and to avoid consuming any yogurt other than that provided to them by the researchers and any other fermented foods. The patients were instructed to keep the yogurt under refrigeration and to avoid any changes in medication, if possible. Arrangements were made so that the patients would receive a 1-wk supply of their probiotic or conventional yogurts every week. Compliance with the yogurt consumption guidelines was monitored by telephone interviews once a week. Information on food consumption, anthropometric measurements, and fasting blood samples were collected at the beginning and at the end of the trial. Nutrient intakes during 3 d were estimated using a 24-h dietary recall at the beginning and at the end of the study. Three-day averages of macro- and micronutrient intakes were analyzed by Nutritionist 4 software (First Databank, Hearst Corp, San Bruno, CA, USA). Anthropometric measurements were recorded by trained personnel. Body weights were measured using a scale (Seca, Hamburg, Germany) with 0.1-kg accuracy without shoes and with minimum clothing. Heights were measured using a stadiometer (Seca) with 0.1-cm accuracy without shoes. BMI was calculated by dividing body weight (kilograms) by height (meters) squared. A blood sample was drawn for each patient from the antecubital vein in the arm after a 12-h overnight fast. The serum samples were separated from whole blood by centrifugation at 3500 rpm for 10 min (Avanti J-25, Beckman, Brea, CA, USA). The serum and whole blood samples were frozen immediately at 70 C
Intervention The probiotic and conventional yogurts contained Lactobacillus bulgaricus and Streptococcus thermophilus. The probiotic yogurt was also enriched with B. lactis Bb12 and L. acidophilus La5 (Chr. Hansen, Hoersholm, Denmark) as Direct Vat Set cultures. The yogurts were produced weekly and distributed to the participants. Probiotic yogurts were sampled 1 d after manufacture (time of distribution) and microbiologically analyzed every week. Samples were refrigerated at 4 , with subsequent analyzing on day 7 of storage. MRS-bile agar medium was used for the differential enumeration of mixed probiotic bacteria in presence of yogurt bacteria [29]. All the samples were incubated at 37 C for 72 h under aerobic and anaerobic conditions. All experiments were performed in triplicate. Counts of L. acidophilus were achieved at the aerobic condition and viable counts of B. lactis were selectively achieved using the subtractive enumeration method [29]. Microbiological analyses of the probiotic yogurts showed that the average colony counts of L. acidophilus La5 and B. lactis Bb12 on day 1 were 7.23 106 and 6.04 106 cfu/g, respectively. Probiotic yogurts contained 1.85 106 cfu/g of L. acidophilus La5 and 1.79 106 cfu/g of B. lactis Bb12 on day 7. Both probiotic bacteria showed a steady survival rate during a 7-d storage time. The fat content was 2.5% and was comparable in both yogurt types. The probiotic and conventional yogurt containers were identical and the yogurts had a similar taste and appearance. The yogurts were specially prepared for this study by Iran Dairy Industries Co. (Tehran, Iran). Statistical analyses The experimental data were analyzed by SPSS 11.5 (SPSS, Inc., Chicago, IL, USA) and the results were expressed as mean standard deviation. The normality of the distribution of variables was tested by the Kolmogorov-Smirnov test. For the duration of diabetes, monounsaturated fatty acid, vitamin A, E, and C intakes, fasting blood glucose, and insulin that did not follow normal distributions, analyses were performed after log transformation. The background characteristics and nutrient intakes of patients in the two groups were compared using independentsamples t tests and chi-square tests. The use of diabetes medication in the two groups was compared using the Mann-Whitney U test. Differences between the two groups after the intervention were determined by analysis of covariance, adjusting for baseline measurements and covariates. In this study, duration of diabetes and polyunsaturated fatty acid intake were used as possible covariates. The changes in anthropometric measurements, nutrient intakes, fasting blood glucose, HbA1c, insulin, and oxidative stress markers of the patients between the beginning and the end of the trial were compared by paired-samples t test [30]. Results with P < 0.05 were considered statistically significant.
Results In this study, four patients were excluded from the statistical analysis because they needed to change their medication during the trial or they did not consume the yogurt according to the plan. Thus, data for 60 patients (23 male and 37 female) were analyzed (n ¼ 30 for each group). The patients demonstrated good compliance with the yogurt consumption and no adverse effects or symptoms were reported. The baseline characteristics of the patients in the two groups are listed in Table 1. The
H. S. Ejtahed et al. / Nutrition 28 (2012) 539–543 Table 1 Baseline characteristics of study participants
Age (y)* Men/womeny Weight (kg)* BMI (kg/m2)* Duration of diabetes (y)* Metformin/dz Glibenclamide/dz
Table 2 Dietary intakes of subjects throughout the study
Conventional yogurt (n ¼ 30)
Probiotic yogurt (n ¼ 30)
Variables
51.00 12/18 75.42 29.14 4.08 2 1
50.87 11/19 76.18 28.95 5.82 2 2
Energy (kcal) Baseline After intervention Carbohydrate (g) Baseline After intervention Protein (g) Baseline After intervention Total fat (g) Baseline After intervention Saturated fat (g) Baseline After intervention Monounsaturated fat (g) Baseline After intervention Polyunsaturated fat (g) Baseline After intervention Dietary fiber (g) Baseline After intervention Vitamin A (mg) Baseline After intervention Vitamin E (mg) Baseline After intervention Vitamin C (mg) Baseline After intervention Copper (mg) Baseline After intervention Zinc (mg) Baseline After intervention Calcium (mg) Baseline After intervention Phosphorus (mg) Baseline After intervention
7.32 11.28 4.30 4.28 1.25 1
541
7.68 10.94 3.65 4.95x 1.25 2
BMI, body mass index * Mean SD. y Frequency. z Median and interquartile range. x Significant difference between groups at baseline (P < 0.05, independentsamples t test).
duration of diabetes was significantly different between the probiotic and conventional groups (P ¼ 0.039). However, other baseline characteristics of the patients (age, sex, weight, BMI, and medications) did not differ between the two groups (P > 0.05). There were no statistically significant differences in weight and BMI values between or within groups at the end of the study. The intake of polyunsaturated fatty acid was significantly different between the probiotic and conventional groups at the beginning of the study (P ¼ 0.033). No significant differences in energy and other nutrient intakes were observed between the two groups at baseline (P > 0.05). During the study, intakes of protein, zinc, calcium, and phosphorus significantly increased in both groups (P < 0.01). No significant changes from baseline were observed in the other nutrient intakes. At the end of the study, there were no statistically significant differences between the two groups for dietary intakes (P > 0.05; Table 2). There were no statistically significant differences in blood glucose, HbA1c, and insulin concentration between the two groups at the beginning of the study (P > 0.05). Fasting blood glucose and HbA1c were significantly decreased in the probiotic group compared with the control group (P ¼ 0.009 and P ¼ 0.019, respectively). Insulin concentration was not significantly different between groups at the end of the trial (P ¼ 0.955; Table 3). The intervention group showed 8.68% decrease in fasting blood glucose concentration from the baseline value (P ¼ 0.001). Although the differences were not statistically significant, HbA1c and insulin also decreased in the intervention group during the study (P ¼ 0.230 and P ¼ 0.654, respectively). In contrast, HbA1c increased significantly from the baseline in the control group (P ¼ 0.003; Table 3). Erythrocyte SOD activity was significantly different between the probiotic and conventional groups at baseline (P ¼ 0.004). There were no statistically significant differences between the two groups in the other variables at baseline (Table 3). Results of the analysis of covariance showed that there were statistically significant differences between the two groups in erythrocyte SOD and GPx activities and TAS at the end of the study when adjusted for duration of diabetes, polyunsaturated fatty acid intake, and baseline values (P ¼ 0.007, P ¼ 0.002, and P ¼ 0.014, respectively). No significant differences were detected in the CAT activity and MDA concentration between the two groups at the end of the trial (P > 0.05; Table 3). As presented in Table 3, erythrocyte SOD and GPx activities and TAS were significantly increased compared with the baseline values in the intervention group (P < 0.001, P < 0.001, and P ¼ 0.001, respectively). No significant changes from baseline were observed in erythrocyte CAT activity for either group
Conventional yogurt (n ¼ 30)
Probiotic yogurt (n ¼ 30)
1774.99 482.92 1809.67 434.30
1775.20 449.51 1776.67 392.28
232.69 76.15 241.86 68.08
242.88 70.44 239.64 59.54
70.08 20.22 80.82 21.34y
68.12 20.00 77.84 17.99y
68.01 18.27 65.93 16.53
65.49 19.23 61.10 18.63
20.62 7.56 19.21 5.45
20.86 7.19 18.78 5.96
22.76 7.07 21.73 6.28
23.61 10.24 21.88 9.52
18.13 6.42 16.06 5.77
15.03 4.33* 15.01 5.19
14.19 4.44 14.86 6.28
16.05 6.18 15.47 5.54
871.08 663.23 898.22 710.39
1152.05 814.67 1111.54 811.93
12.72 8.38 11.45 7.94
10.27 5.50 10.32 6.12
138.41 61.67 126.96 69.17
152.87 64.93 144.50 54.52
1.59 0.53 1.59 0.55
1.67 0.59 1.57 0.43
9.99 2.64 12.23 3.40y
10.13 3.35 11.43 3.09y
824.86 275.70 1215.11 213.56y
869.76 229.19 1264.69 227.63y
1185.39 319.75 1477.26 324.93y
1173.57 357.33 1462.60 337.15y
Data are presented as mean SD * Significant difference between groups at baseline (P < 0.05, independentsamples t test). y Significant difference within group throughout the study (P < 0.01, pairedsamples t test).
(P > 0.05; Table 3). The results of paired-samples t test showed that MDA concentration decreases were significant for the control and intervention groups (P ¼ 0.005 and P ¼ 0.013, respectively; Table 3).
Discussion In T2DM, free radicals are generated excessively. These free radicals cause lipid peroxidation and MDA generation [2]. Moreover, activities of SOD, GPx, and CAT, which scavenge reactive oxygen species, decrease in patients with T2DM [31]. It is important to increase the CAT and GPx activity, in addition to SOD activity, to remove reactive oxygen species. The improvement in oxidative stress status can contribute to diabetes management [2]. The present study showed that probiotic yogurt consumption significantly decreased fasting blood glucose and HbA1c and
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Table 3 Effects of 6 wk of probiotic and conventional yogurt consumption on blood glucose, hemoglobin A1c, insulin, and oxidative stress markers Variables Glucose (mmol/L) Baseline After intervention HbA1c (%) Baseline After intervention Insulin (mU/mL) Baseline After intervention SOD (U/g Hb) Baseline After intervention GPx (U/g Hb) Baseline After intervention CAT (K/g Hb) Baseline After intervention TAS (mmol/L) Baseline After intervention MDA (nmol/L) Baseline After intervention
Conventional yogurt (n ¼ 30)
Probiotic yogurt (n ¼ 30)
7.35 1.28 7.53 1.32
8.06 2.49 7.36 2.41y,z
6.87 0.81 7.17 0.66z
7.29 1.21 7.17 1.24y
6.31 3.72 6.50 3.57
7.47 4.89 6.97 4.49
1161.59 246.01 1136.54 256.26
975.80 238.34* 1113.69 177.77y,z
30.28 4.40 30.12 4.23
29.03 4.29 29.81 4.58yz
141.40 25.78 151.15 36.98
148.81 34.56 146.57 34.05
0.93 0.20 0.92 0.16
0.90 0.18 0.96 0.18y,z
2.81 0.65 2.55 0.60z
2.79 0.62 2.53 0.65z
CAT, catalase; GPx, glutathione peroxidase; Hb, hemoglobin; MDA, malondialdehyde; SOD, superoxide dismutase; TAS, total antioxidant status Data are presented as mean SD * Significant difference between groups at baseline (P < 0.01, independentsamples t test). y Significant difference between groups after intervention (P < 0.05, analysis of covariance, adjusted for duration of diabetes, polyunsaturated fatty acid intake, and baseline values). z Significant difference within group throughout the study (P < 0.01, pairedsamples t test).
increased erythrocyte SOD and GPx activities and TAS compared with conventional yogurt consumption. Furthermore, the MDA concentration significantly decreased in both groups. However, insulin concentration and erythrocyte CAT activity remained unchanged in the intervention group. In the present study, there were no statistically significant changes in weight, BMI, and energy within either group during the study. However, intakes of protein, zinc, calcium, and phosphorus significantly increased in both groups throughout the study. These increases most probably were caused by the yogurt consumption. There were no statistically significant differences between the two groups at the end of the study for these nutrients. Therefore, the observed results in the probiotic group could not have been caused by the changes in weight and dietary intakes. Chin [32] reported that T2DM could arise from imbalances of microflora in the gastrointestinal tract. Larsen et al. [22] documented that the intestinal microbiota of patients with T2DM was relatively enriched with gram-negative bacteria, belonging to the phyla Bacteroidetes and Proteobacteria. The proportion of Firmicutes to Bacteroidetes was significantly decreased in the patients with T2DM compared with the non-diabetic patients [22]. Therefore, we conducted the first randomized controlled trial investigating the effects of probiotic yogurt on blood glucose and antioxidant status in patients with T2DM. The findings support previously reported observational data on the antioxidant property of probiotic yogurt containing L. acidophilus and B. lactis in young healthy women [19]. Naruszewicz et al. [18] investigated the antioxidative effects of L. plantarum in smokers
and found that an L. plantarum intake for 6 wk decreased plasma F2-isoprostanes concentrations. In a study by Songisepp et al. [16], a significant improvement of TAS was seen with the daily consumption of fermented goat milk containing L. fermentum ME-3 in healthy persons after 3 wk. Kullisaar et al. [17] also reported that goat milk fermented by L. fermentum ME-3 increased total antioxidative activity and decreased lipid peroxidation markers in healthy persons. Previous evidence regarding the antidiabetic properties of probiotics has been limited to animal studies. Harisa et al. [21] showed that treatment with L. acidophilus alone or in combination with acarbose significantly decreased fasting blood sugar, HbA1c, and MDA concentration in diabetic rats. In an animal study, Yadav et al. [20] reported that probiotic dahi, a fermented milk product containing L. acidophilus and L. casei, had an antioxidative effect on the liver and pancreas tissues of highfructose–induced diabetic rats and delayed the onset of glucose intolerance, hyperglycemia, and hyperinsulinemia. In another study, Yadav et al. [33] found that probiotic dahi suppressed streptozotocin-induced oxidative damage in the pancreatic tissues of diabetic rats by inhibiting the lipid peroxidation and preserving the activity of SOD, GPx, and CAT. These effects of probiotic dahi may slow the decrease of insulin and increase of blood glucose [33]. Al-Salami et al. [34] reported that probiotic treatment had no effect on blood glucose concentration in healthy rats, but decreased blood glucose in diabetic rats because of an increased gliclazide (sulfonylurea) bioavailability. In another study, Matsuzaki et al. [35] showed that L. casei inhibited the production of proinflammatory cytokines and decreased plasma glucose concentration in non–insulin-dependent diabetic mice. The present study, indeed, has reported perhaps the first evidence of improved glucose metabolism in diabetic patients and the results are in accordance with the findings of the animal studies described. However, no statistically significant changes were observed in HbA1c, insulin concentration, and erythrocyte CAT activity in the probiotic yogurt group during the study, which may be explained by the short duration of the study. The precise mechanisms involved in the antidiabetic effects of probiotics remain largely unknown. These effects may be partly related to a probiotics-mediated decrease in oxidative stress. Moreover, the immune-modulatory and anti-inflammatory effects of probiotics and the modification of intestinal microflora could be other probable underlying mechanisms. The inhibition of ascorbate autoxidation, metal ion chelation, and reduction activity and scavenging of superoxide anion radicals, hydrogen peroxide, and free radicals are likely to underline the antioxidative effect of probiotic yogurt [14,36,37]. The relatively small but significant decrease in lipid peroxidation indicated by the decreased plasma MDA was not associated with changes in antioxidant status markers for the control group. The MDA concentration decrease in both yogurt groups could be due to the antioxidative effect of bioactive peptides released during yogurt fermentation by proteolytic lactic acid bacteria [38,39]. Fermented milks have been reported as dietary sources of natural antioxidants because of the presence of antioxidant peptides. Most identified bioactive peptides were derived from as-casein and have been shown to exhibit free radical scavenging and inhibit enzymatic and non-enzymatic lipid peroxidation [40]. The antioxidant peptides derived from whey protein are likely the result of the presence of cysteine-rich proteins that aid in the synthesis of glutathione, a potent intracellular antioxidant [41]. Although the yogurt for both trial groups can exert antioxidative effects by bioactive peptides, the results showed that
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probiotic yogurt was more effective in increasing antioxidative activity than conventional yogurt. The limitations of this study included its short duration and the absence of a control group that consumed no yogurt. Moreover, many exclusion criteria in this study could limit the generalizability of the results. Therefore, further investigations with longer duration and a no-yogurt control group, and a larger trial with some form of stratification are needed to confirm the positive effect of probiotic yogurt on the management of diabetes. Further studies on the effects of probiotics on intestinal microflora composition and intestinal transit time in diabetic patients would be useful. Conclusion This trial showed that consuming 300 g/d of probiotic yogurt containing L. acidophilus La5 and B. lactis Bb12 improved the antioxidant status and fasting blood glucose in patients with T2DM. These findings suggest that probiotic yogurt is a functional food that can exert antidiabetic and antioxidant properties. Acknowledgments The authors thank the Iran Dairy Industries Company for supplying the probiotic and conventional yogurts and supporting this study. References [1] Ceriello A, Motz E. Is oxidative stress the pathogenic mechanism underlying insulin resistance, diabetes, and cardiovascular disease? The common soil hypothesis revisited. Arterioscler Thromb Vasc Biol 2004;24:816–23. [2] Maritim AC, Sanders RA, Watkins JB. Diabetes, oxidative stress, and antioxidants: a review. J Biochem Mol Toxic 2003;17:24–38. [3] Stephens JW, Khanolkar MP, Bain SC. The biological relevance and measurement of plasma markers of oxidative stress in diabetes and cardiovascular disease. Atherosclerosis 2009;202:321–9. [4] Lipinski B. Pathophysiology of oxidative stress in diabetes mellitus. J Diabetes Complications 2001;15:203–10. [5] Fuhrman B, Volkova N, Aviram M. Pomegranate juice polyphenols increase recombinant paraoxonase-1 binding to high-density lipoprotein: studies in vitro and in diabetic patients. Nutrition 2010;26:359–66. [6] Zozulinska D, Wierusz-Wysocka B. Type 2 diabetes mellitus as inflammatory disease. Diabetes Res Clin Pract 2006;74:12–6. [7] Guarner F, Perdigon G, Corthier G, Salminen S, Koletzko B, Morelli L. Should yoghurt cultures be considered probiotic? Br J Nutr 2005;93:783–6. [8] Homayouni A. Letter to the editor. Food Chem 2009;114:1073. [9] Homayouni A, Azizi A, Ehsani MR, Yarmand MS, Razavi SH. Effect of microencapsulation and resistant starch on the probiotic survival and sensory properties of synbiotic ice cream. Food Chem 2008;111:50–5. [10] Higashikawa F, Noda M, Awaya T, Nomura K, Oku H, Sugiyama M. Improvement of constipation and liver function by plant-derived lactic acid bacteria: a double-blind, randomized trial. Nutrition 2010;26:367–74. [11] Goldin BR, Gorbach SL. Clinical indications for probiotics: an overview. Clin Infect Dis 2008;46:96–100. [12] Kaur IP, Kuhad A, Garg A, Chopra K. Probiotics: delineation of prophylactic and therapeutic benefits. J Med Food 2009;12:219–35. [13] Hoerr RA, Bostwick EF. Bioactive proteins and probiotic bacteria: modulators of nutritional health. Nutrition 2000;16:711–3. [14] Lin MY, Yen CL. Antioxidative ability of lactic acid bacteria. J Agric Food Chem 1999;47:1460–6. [15] Uskova MA, Kravchenko LV. Antioxidant properties of lactic acid bacteriaprobiotic and yogurt strains. Vopr Pitan 2009;78:18–23.
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