This 12-week randomized, double-blind, placebo-controlled clinical trial was conducted at Diabetes and Endocrine diseases Hospital in Sulaymaniyah, Iraq. The number of participants was 68 adult males and females, who were diagnosed as T2D for at least two years. The protocol was approved by both Ethics Committee of Tehran University of Medical Sciences (NO. IR.TUMS.MEDCINE.REC.1400.702) and Research Ethics Committee in Sulaymaniyah general directorate of health. In accordance to the contents of the 1975 Declaration of Helsinki as revised in 1983 and was registered at the Iranian Registry of Clinical Trials (IRCTID: IRCT20210529051435N1). Sample size was defined based on primary information in previous studies [22]. Glycemic and lipid profile variables were used to define the sample size, individually and finally total cholesterol (TC) level was found to be as the key variable which resulted in a maximum determined sample size [22, 23]. The findings from previous trials suggested plasma Lp-PLA2 mass to be positively associated with TC [24]. For an expected change of 0.25 mmol/L (9.65 mg/dL) between intervention and control groups and by considering α = 0.05 and a power of 80%, the sample size was computed to be 29.9 (≈ 30) per group which was increased to 34 to accommodate the expected dropout rate.

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Eligible participants include those with established T2D for at least 2 years prior to the commencement of the study, aged 50–65 years, BMI between 25 and 34.9 kg / m2.

Exclusion criteria were, type 1 diabetes or non-diabetic patients, individuals who had irregular diet and unstable body weight (body-weight change > 5% within 3 months before screening), changing and/or addition in their routine medications within 3 months prior and during the intervention, history of cardiovascular disease, hypertension, heart attack, angina pectoris, cerebrovascular disease, stroke, Thyroid disease, and other chronic diseases and transmitted diseases in the past year. Current smokers, menopause, individuals on non-steroidal anti-inflammatory drugs and multivitamin or use of any nutritional supplements within the previous 3 weeks prior to study initiation, as well as the presence of liver, kidney, inflammatory or immunodeficiency diseases; thyroid disorders, use of any type of estrogen, progesterone, or diuretics; pregnancy or breast-feeding, and consumption of any type of probiotic product and/or antibiotic in the previous 2 months of testing. Participants who agreed to participate in the study were asked to sign a permission form after the lead researcher described the study protocol.

68 individuals were randomly assigned to two groups using a stratified block randomization approach and the principal researchers and all participants were blinded to the contents of the capsules throughout the study procedure and the final analysis. Participants were asked to receive either multispecies probiotic supplements (n = 34, i.e., 12 males and 22 females) or the placebo (n = 34, i.e., 8 males and 26 females). Supplements were either placebo (Placebo; containing starch and inactive ingredient excipients) or probiotics with dosages of Bacillus Coagulans, Lactobacillus plantarum, Lactobacillus acidophilus and Bifdobacterium bifdum, 3 billions/capsules (3 × 109 colony forming units (cfu) per capsule). Oral capsules of Biodiab containing the mentioned strains which was produced by Tak-Gene Zist pharmaceutical manufacturer were taken by participants. Both probiotic and placebo capsules were produced and coded by the same manufacturer company (A or B). The packaging, odor, and appearance of placebo containing capsules were just similar to the supplement containing capsules. Each capsule was swallowed orally with a full glass of water. Participants from each group took one Biodiab capsules daily. A full pack of capsule was given to each participant monthly, then at any visits (biweekly) we counted the capsules to be sure about participant compliance, and if at the end a participant did not take ≥ 80% of the supplement we would exclude it from our results. A general information sheet was completed for each patient.

Body weight was measured by a calibrated scale with 0.1 kg accuracy (Seca, Hamburg, Germany) with barefoot and minimal clothes. Height was measured by a wall-mounted tape, barefoot and at a straight standing posture with 0.5-centimeter accuracy. BMI was calculated by dividing weight in kilograms by height in meters squared. Waist circumference (directly on the skin) was measured at the umbilical level after normal expiration with the subject in an upright standing posture using a plastic measuring tape with measurements to the nearest 0.1 cm. Anthropometric parameters was assessed at screening, baseline and 12th week. Body composition and fluid status was assessed by bioimpedance analysis (BIA) using the Inbody 770 (InBody co, Seoul, Korea). Bioimpedance measurements performed at a spectrum of 50 frequencies between 5 and 1000 kHz enable to differentiate between intra- and extracellular fluid, as low electronic currents cannot pass cell membranes and flow through extracellular water only. We recorded the full report of BIA while we analyzed fat mass (FM) and fat free mass (FFM).

To assess participant’s food intake, we collected 24 h food recalls at the baseline and end of the study. A trained nutritionist asked the patients to recall their intake by using a specific set of questions to gain as much detailed information as possible. To take the best result, for better estimating the portion size of foods and drinks, we used the Household Measures and Food Model booklet. Dietary intake was analyzed in terms of energy and macro - micronutrients (with a focus on micronutrients with fermented food) intake by Nutritionist software version IV.

To evaluate the physical activity levels, the short form of International Physical Activity Questionnaires (IPAQ) was used at the baseline and the end of the study. The short form of IPAQ includes seven questions that explore walking and moderate-intensity and vigorous-intensity activity during the past seven days. Frequency (days per week) and duration (time per day) have been collected separately for each specific type of activity. To analyze the activities, metabolic equivalents per minute (MET-minute) score was used. The MET-minute score was computed by multiplying the MET score by the minutes performed and used categorical score. The patients with at least 3000 met/minute score was obtained from the combination of all the activities were considered category 3 (high) and with at least 600 met/minute score were considered category 2 (moderate), and patients not included in category 2 or 3 were considered category 1 (low) [25].

Venous blood samples (10 ml) were drawn by venipuncture after 8 to 12 h fasting at baseline and the end of the study. The blood samples were centrifuged for 10 min at 3000 rpm using (Hettich centrifuge “Germany”), and serum will aliquot into separate micro tubes and were stored in -80 °C until biochemical analyses.

Serum levels of glucose, insulin, total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL), triglyceride (TG) and blood level of glycated hemoglobin (HbA1c) were measured at once and at the same place using an auto analyzer (Cobass 6000 Roche “Germany”). Insulin resistance was defined by calculating the HOMA-IR using Matthews et al.’s equation [26]: fasting glucose concentration (mg/dl) × fasting insulin level (mU/l)/405.

Lp-PLA2 serum level was measured by using an ELISA kit (diaDexus, Inc. South San Francisco, CA, USA).

The obtained data was analyzed with SPSS, version 25 (SPSS Inc. Chicago, IL, USA). An Intention-To-Treat (ITT) approach was used for data analysis. In this approach, all the enrolled participants who take ≥ one capsule were included. Results were expressed as mean ± standard deviation (SD) and percentage for quantitative and qualitative variables, respectively. At the baseline, quantitative variables were analyzed by independent sample t-test. Chi square test was used to compare qualitative variables. The repeated measure analysis of variance (ANOVA) was used to report the results after correcting for sex, total energy intake and body fat mass. A P value equal and less than 0.05 was considered statistically significant.