Introduction

Metabolic syndrome (MetS), as a set of metabolic abnormalities including insulin resistance, abdominal obesity, dyslipidemia, and hypertension, has a substantial prevalence in the world1. Type 2 diabetes and cardiovascular diseases are the two most significant complications of MetS, which can be caused by a variety of factors, including lifestyle and dietary habits2.

Studies have demonstrated that the components of the MetS, particularly insulin resistance, can exacerbate the pathophysiology of the MetS, which is characterized by inflammation and oxidative stress as major contributors3. Insulin resistance increases oxidative stress and decreases antioxidant defense by increasing glycosylation and inducing lipolysis, which leads to the release of fatty acids4. According to the previous studies, people with MetS have poor antioxidant defense in the form of reduced superoxide dismutase (SOD) activity and higher levels of protein and lipid oxidation5. The definition of oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and free radicals and their elimination by the endogenous antioxidant defense system6. Increased levels of oxidative stress in the body can lead to fat accumulation, obesity, and high blood pressure by impairing glucose uptake, disturbing beta cells, and decreasing insulin secretion7. By influencing angiotensin II signaling, nitric oxide (NO) signaling, and other cellular processes, ROS contributes to the development and progression of hypertension8. Besides, oxidative stress can cause cell dysfunction by damaging proteins and lipids and enhance the formation of nitro-tyrosine, which is an indicator of cardiovascular diseases9. Furthermore, malondialdehyde (MDA), which is a marker of lipid peroxidation under conditions of oxidative stress, increases in people with MetS10. Free radicals and ROS are eliminated by endogenous antioxidant enzymes such as SOD and glutathione peroxidase (GPx), as well as antioxidant compounds11. Total Antioxidant Capacity (TAC) refers to the combined effect of all the antioxidants present in plasma, which play a crucial role in the elimination of ROS12.

Studies have shown that an imbalance in the gut microbiome can play an important role in causing oxidative stress, inflammation, insulin resistance, and, as a result, the MetS13.

Modifying the gut microbiota, especially with nutritional approaches, is one of the strategies that has been shown to improve the complications of MetS14. Previous studies have demonstrated the relationship between probiotic consumption and improvements in metabolic parameters15. Probiotics are defined as "live microorganisms that confer health benefits on the host when administered in sufficient quantities16. Probiotics can reduce oxidative stress and inflammation due to their effects on increasing glutathione (GSH) levels and the activity of antioxidant enzymes, including GPx and SOD17. Furthermore, they can help decrease oxidative stress by minimizing the expression of inflammatory cytokines in adipocytes and eliminating ROS. It has been shown that probiotics exert their antioxidant effects by absorbing metal ions, producing various metabolites, including bioactive peptides that create antioxidant capacity, and eliminating oxidant compounds18.

Probiotic supplements containing Lactobacillus plantarum have been proven to reduce oxidative stress by blocking the signaling pathways and boosting the antioxidant defense system19. In addition, a limited number of studies indicated that Lactobacillus plantarum-containing products can exert a positive impact on the oxidative and metabolic modifications associated with chronic diseases20. No studies have been conducted to evaluate the effect of consuming a food product containing Lactobacillus plantarum on subjects with MetS. Moreover, while there has been extensive research on the potential health benefits of probiotics, research on the use of synbiotic in the context of MetS has been relatively limited. The inclusion of Lactobacillus plantarum, Lactobacillus pentosus, and Chloromyces marcosianus in the synbiotic yogurt constitutes a novel blend of microorganisms that has not been widely studied in the context of MetS. Analyzing the effects of these specific microorganisms on oxidative stress presents an opportunity to uncover potentially unique benefits. So, the current study aims to investigate the impact of the newly developed synbiotic yogurt containing Lactobacillus plantarum, Lactobacillus pentosus, and Chloromyces marcosianus on oxidative stress status and some other risk factors for cardiovascular diseases in adults with MetS in order to develop efficient approaches for the prevention and management of MetS complications.

Methods

Study design and participants

This was a randomized, placebo-controlled clinical study conducted in Yasuj, Iran, from December 2022 to March 2023. According to the study protocol, participants were recruited from clients of health institutions affiliated to Yasuj University of Medical Sciences. The criteria for inclusion in the study consisted of a confirmed diagnosis of the MetS, age raging between 30 and 50 years, and a body mass index (BMI) within the range of 25–35 kg/m2. MetS diagnosis was confirmed in accordance with the ATP III criteria when a minimum of three of the specified criteria were satisfied: waist circumference (WC) > 102 cm in men and > 88 cm in women, triglyceride (TG) ≥ 150 mg/dl, high-density lipoprotein (HDL) ≤ 40 mg/dl in men and ≤ 50 mg/dl in women, blood pressure ≥ 130 to 85 mmHg, and FBG ≥ 100 mg/dl. Subjects were not included in the study who followed weight loss programs or had weight changes of more than 10% of initial weight during the last six months. Professional athletes or subjects with any changes in the intensity and duration of physical activity in the previous four weeks, smokers, alcoholic beverage consumers as well as pregnant, lactating, and postmenopausal women were also not included. Other exclusion criteria were as following: having allergy to dairy products and probiotics; routinely consumption of probiotic or synbiotic yogurts; patients with diagnosed diseases including cardiovascular, lung, nervous system, kidney, liver, thyroid, and other endocrine diseases, diabetes, and cancers; taking drugs that affect appetite, body weight, and lipid metabolism such as corticosteroids, oral contraceptives, antidepressants, antipsychotics, and anti-hyperlipidemics, antibiotics; and receiving probiotics and other dietary supplements in the last three months.

Before the primary intervention, all of the selected participants took part in a two-week "run-in" period. During this time, relevant questionnaires were utilized to collect data on sociodemographic factors, medical history, medication and supplement usage, dietary habits, and levels of physical activity. This study was accepted by the ethics committee of Tehran University of Medical Sciences (ethics number: IR.TUMS.MEDICINE.REC.1401.080).

The sample size was calculated based on the change in MDA levels in the intervention (2.3 ± 0.6) and control (3.2 ± 1.2) groups, as published in a study conducted by Karamali et al.21, with a confidence level of 95% and a power of 80%, which computed 18 participants per group. Considering an 18% dropout rate, 22 individuals were assigned to each group.

Randomization and intervention

Following the end of the run-in period and before the beginning of the intervention, the participants were subjected to random allocation into either the intervention or control groups. This randomization process was conducted using a block randomization procedure with block sizes of 2 and 4, resulting in 22 participants being allocated to each group. The process of randomization was conducted using Random Allocation Software (RAS), version 1.0, with stratification based on sex and BMI. The participants in both the intervention and control groups were instructed to consume 300 g per day of synbiotic yogurt or regular yogurt, respectively for 12 weeks. Participants were also asked to adhere to their regular dietary habits and lifestyle, avoiding consuming any additional probiotic or synbiotic products throughout the study. The participants' dietary intake and physical activity level were evaluated at baseline, at the end of the sixth week, and at the end of the trial. For the dietary intake, a 3-day food recall questionnaire and nutritionist IV software were used for analysis, and the International Physical Activity Questionnaire (IPAQ)22, which has been validated for the Iranian population, was used for measuring physical activity level. Participants were categorized based on their total weekly activity levels: individuals with activity levels less than 600 min were classified as mild activity, those with activity levels between 600 and 3000 min were regarded as displaying moderate activity, and individuals exceeding 3000 min were classified as having severe activity.

Origin and composition of the synbiotic yogurt

The synbiotic yogurt that was used in this study contains strains of Lactobacillus plantarum, Lactobacillus pentosus (2×108 CFU), Chloromyces marcosianos yeast, and 3% of various natural plants (celery, shallot, chicory, and mint)23. The microbiological analysis demonstrated a colony rate of 2×108 for both bacterial strains. Skimmed cow's milk was used in the production of yogurt. Initially, the skim milk experienced the processes of pasteurization and homogenization at 95 °C for 5–10 min, followed by further cooling to 42–43 °C. Subsequently, the starter was added to the mixture of milk and water at the rate of 3%. After that, the combination of milk and water completed an incubation process lasting 3–4 h at a temperature of 42–43 °C, resulting in the formation of yogurt. Following this, the yogurt underwent a cooling process at a temperature of 4 °C for 24 h in order to facilitate maturation. Herb powder was delivered in a spice container next to yogurts every two weeks. After the full preparation of the yogurts, they were measured in terms of the live microbial population, and their amount was equal to our desired amount.

Laboratory analyses

Blood samples were collected following a 12-h period of fasting during the night, both at the beginning and the end of the study. The separation of serum from whole blood was performed via centrifugation at a speed of 3500 rpm for a duration of 10 min. The serum samples were immediately frozen and stored at a temperature of − 70 °C until the time of the assay. Blood samples were analyzed at the Negin Salamat Laboratory (Yasouj, Iran). The determination of serum MDA concentration was conducted using the spectrophotometric method, which is based on the reaction between MDA and thiobarbituric acid (TBA). The serum values of TAC, total oxidant status (TOS), SOD, and GPx were measured using commercial kits (Navand Salamat Co., Urmia, Iran) and the spectrophotometric method.

Statistical analysis

SPSS version 26 was used to analyze the experimental data, and the findings were presented as a mean ± SD. The analyses were carried out based on per protocol principle. The data distribution was analyzed using the Kolmogorov–Smirnov test. The independent sample t test and Chi-square were used to analyze the differences between the groups at baseline. Differences between groups post-intervention were determined using analysis of covariance (ANCOVA) adjusted for baseline values, age, and gender as covariates. Within-group differences were evaluated by using the paired t-test. P < 0.05 was considered statistically significant.

Adverse events

Each participant was requested to provide information on any health problems experienced over the course of the trial. Compliance with yogurt consumption was evaluated on a weekly basis through telephone interviews and through 24-h dietary recall interviews.

Results

Figure 1 shows the flowchart of the study. Dropouts occurred in the placebo group due to unwillingness (n = 2) and travel (n = 1). The patients demonstrated good compliance with yogurt consumption. There were no reported adverse effects or complications. The general characteristics of the study participants in both the intervention and control groups are listed in Table 1. There were no significant differences in terms of sex, age, educational level, physical activity level, energy, macronutrients, and fiber intake, body weight, BMI, waist circumference, and blood pressure at baseline (p > 0.05) between two groups.

Fig. 1
figure 1

Flowchart of the study. Including patient screening, enrollment, and randomization. Dropouts occurred in the placebo group due to unwillingness (n = 2) and travel (n = 1).

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Table 1 Baseline characteristics of the study participants.
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Table 2 shows the oxidative parameters of the study subjects throughout the study. After the 12 weeks intervention, TAC, SOD, and GPx levels significantly increased (p = 0.002, p = 0.02, p = 0.005; respectively) and serum TOS levels decreased (p = 0.01) in the intervention group compared to the baseline levels. However, MDA levels remained unchanged. In the control group, serum TAC, MDA, and GPx levels decreased significantly (p < 0.05) compared to the baseline values. Results of the ANCOVA showed a statistically significant increase in the level of GPx (p = 0.01) and significant decrease (p = 0.006) in TOS level in the intervention group compared to the control group. No significant differences were observed in the level of MDA, SOD, and TAC between two groups at the end of the study by using ANCOVA test adjusted for age, gender, and baseline values.

Table 2 Biochemical parameters of the study subjects at baseline and after the intervention.
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Discussion

The present randomized clinical trial aimed to investigate the effect of consuming a newly developed synbiotic yogurt on oxidative stress status in adults with MetS. Elevated systemic levels of oxidative stress biomarkers and lipid peroxidation, as well as decreased antioxidant levels, have been found in patients with MetS. Extensive evidence has also shown that oxidative stress may be one of the key mediators of CVD in subjects with MetS24,25.

The study results showed that after 12 weeks of intervention, the synbiotic yogurt consumption demonstrated significant improvements in TAC, TOS, SOD, and GPx levels compared to their baseline values. In contrast, the control group exhibited a significant decrease in serum TAC and GPx levels from the baseline values. However, when the two groups compared at the end of the study, only the levels of GPx and TOS level showed significant improvement in the intervention group. These findings indicate an enhanced antioxidant defense and a reduction in oxidative stress by consuming the synbiotic yogurt. These results are similar to the findings of some previous studies. In a study conducted on breast cancer patients, improvement was seen in MDA and SOD, but not in GPx and TAC levels after 10 weeks of synbiotic supplementation26. In another study that was conducted on patients with Parkinson's disease, both TAC and MDA were significantly improved compared to the control group after 12 weeks of synbiotic supplementation27. However, in another study, daily consumption of a synbiotic food did not have any significant effects on TAC levels in pregnant women during 9 weeks28. A meta-analysis concluded that the effect of intervention with synbiotic on oxidative stress factors is significant only if the duration of the intervention is more than 12 weeks, and subgroup analysis for periods of 6 and 8 weeks indicates the weak effectiveness of these compounds29. In addition to the duration of the intervention, the number and type of the probiotics used in supplements or foods may also affect the study results.

The mechanism of action underlying the antioxidant effects of probiotics encompasses various pathways. Probiotics have been shown to modulate gut microbiota composition30,31,32, leading to the production of bioactive metabolites, such as short-chain fatty acids (SCFAs), which possess antioxidant properties33,34. Probiotics scavenge superoxide and hydroxyl radicals, modulate the production of short-chain fatty acids, generate bioactive peptides with antioxidant and radical scavenging activities, and may alter the lipid profile, thereby reducing the serum levels of malondialdehyde (MDA), a marker of lipid oxidation29,35. It has been shown that probiotics could elevate the redox capacity through various ways including chelate metal ions; generate metabolites with antioxidant activity, such as GSH and butyrate; and regulate enzymes that produce reactive oxygen species36. Inhibiting ferrous Fe accumulation leads to modulation of the redox state in the colonic fermentation system and as a result boosting antioxidative capacity37. SCFAs can scavenge ROS, reduce oxidative damage, and mitigate systemic inflammation which may ultimately improve oxidative stress status38. Synbiotic maintain the balance of intestinal microbial flora and empower the intestinal mucosal barrier, enhance immune tolerance of the intestinal cells, and interact with the inflammatory response39. The increase in the production of SOD and catalase as a result of the consumption of synbiotic prevents the accumulation of oxidant compounds such as superoxide and hydrogen peroxide40. Moreover, probiotics facilitate the absorption of polyphenols in the intestine, transforming inactive glycosylated polyphenols into their active form, thus rendering them absorbable41. Additionally, probiotics contribute to the production of certain B vitamins with antioxidant properties, while also exhibiting a competitive behavior that reduces pathogenic bacteria, consequently lowering endotoxin levels and inhibiting the secretion of pro-inflammatory cytokines such as TNF-α and IL-142,43. Furthermore, the reduction of superoxide anions and hydroperoxides such as MDA, attributed to probiotics and synbiotics, may be due to the increase in nitric oxide (NO) levels44. Previous studies suggest that NO reacts with peroxides and hydroxides, converting into stable and inactive species, thus preserving the availability of NO in endothelial tissue45. Additionally, lactobacilli exhibit specific biological properties, including the metabolization of prebiotics to regulate intestinal permeability and enhancement of the immune system through bacterial translocation and colonization in the host29,46,47.

The results of our study suggest that the synbiotic yogurt intervention effectively improved oxidative stress status in individuals with MetS. To the best of our knowledge, this was the first study evaluated the effects of synbiotic yoghurt on oxidative stress biomarkers in patients with MetS. However, the duration of the intervention was relatively short, and therefore, the long-term effects of synbiotic yogurt consumption on oxidative stress should be investigated in future studies.

Conclusions

In conclusion, the findings of this randomized clinical trial demonstrate that consuming the synbiotic yogurt containing Lactobacillus plantarum, Lactobacillus pentosus, and Chloromyces marcosianos yeast significantly improved oxidative stress status in adults with MetS. These results support the potential of this new developed synbiotic yogurt as a dietary intervention for individuals with MetS, offering a novel approach to ameliorating oxidative stress and potentially reducing the risk of associated complications. Further research is warranted to explore the long-term effects of synbiotic yogurt consumption and its impact on various metabolic parameters in individuals with MetS.