INTRODUCTION

Obesity is a pathological condition characterized by excessive body mass accumulation, particularly in the abdominal region resulting from an imbalance between energy intake and expenditure. Importantly, obesity is a risk factor for many fatal diseases, especially diabetes, cardiovascular diseases, non-alcoholic fatty liver disease, and several cancer types. Obesity is associated with abnormal blood lipid levels (total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglyceride (TG)) which are the major risk factors for cardiovascular diseases (¹).

A recent meta-analysis observed a linear association between serum cholesterol levels and cardiovascular disease (CVD) mortality. One of the effective population-based strategies of cardiovascular disease prevention is associated with decreased cholesterol levels by improving the nutritional status of the population (²). Although dietary recommendations and exercise are the primary treatment for hypercholesterolemic patients, these methods can only modestly improve high blood cholesterol levels among patients (³). Owing to the known side effects of statin-like drugs that reduce serum cholesterol levels, there is a growing interest in non-drug therapies to improve the blood cholesterol profile. Probiotics, safe for human consumption and available as functional foods and nutritional supplements, have been shown to reduce serum cholesterol levels, offering a potential non-drug therapy for improving human blood cholesterol levels (⁴,⁵).

At present, the most widely accepted scientific definition of probiotic determined by WHO (World Health Organization) and FAO (Food and Agriculture Organization of the United Nations) is “live microorganisms that support health conditions in the host when given in appropriate amounts” (⁶). Several genera are used as probiotics, including Lactobacillus, Bifidobacterium, Bacillus, Pediococcus, and several yeasts (⁷). Probiotics are available in various forms, including food and dietary supplements in capsules, tablets, liquids, and powders. Many probiotic supplements contain 1-10 billion CFU per dose, in addition both specific strains and mixtures of specific strains in effective doses are important to make the connection between clinical benefits. Besides these live, active microorganisms can be found in fermented dairy products such as kefir, yoghurt, and cheese (⁵).

Different types of probiotics have different functions so that human health benefits have mainly been demonstrated for specific probiotic strains (⁷,⁸). The health benefits of fermented functional foods are expressed either directly through the interactions of ingested live microorganisms with the host (probiotic effect) or indirectly by ingesting microbial metabolites synthesized during fermentation (biogenic effect) (⁹,¹⁰). Among them, dairy products (particularly fermented milk and yogurt) are by far the most efficient and widely used (¹¹,¹²). The content and activity of a bioactive compound in dairy-fermented foodstuffs result from the type of food matrix, the individual bacterial strain properties, the processing conditions, and storage time. In this regard, it should be noted that the high bioactive biosynthetic rates observed in culture media might not always be extrapolated to dairy products. Therefore, factors such as optimal temperature for microbial growth and viability, food composition or bioactive stability, and shelf-life in the final foodstuff are paramount to reaching the final product's maximum concentration and activity of probiotics (⁹).

Probiotics have been shown to modulate intestinal flora, prevent the colonization of harmful bacteria in the intestine, strengthen the immune system, reduce and prevent symptoms related to diarrhea and constipation, and have beneficial effects on cancer and inflammatory bowel diseases. Thus, consuming probiotics is useful for maintaining health against pathogenic bacteria in the gut microbiota, and maintaining the normal balance of gut microbiota helps to improve digestive health as well as the immune system. Accordingly, the main evidence-based positive effects of probiotics have been shown to include antimicrobial and antimutagenic activities, also anticarcinogenic properties. The advantages of probiotics are associated with the increase in bioavailability of macro and micronutrients, mitigation of nutritional intolerances, beneficial effects on intestinal diseases and Crohn's disease, alleviation of allergic incidences and decrease of LDL and total cholesterol levels (⁷,⁸).

Supplementation with probiotics significantly reduced TC, LDL-C, and TGs and increased HDL-C in hypercholesteremic patients. Also, probiotic supplementation improved the anthropometric measurements (¹¹,¹³). Meta-analysis of randomized clinical trials revealed that probiotic supplementation could be useful in the primary prevention of hypercholesterolemia and may lead to reductions in risk factors for cardiovascular disease (¹⁴,¹⁵). Similarly, a systematic review study suggests that probiotic supplementation should be indicated as adjunctive treatment for dyslipidemias (¹¹).

Probiotics have been suggested to lowering plasma cholesterol levels through various mechanisms (⁵,¹⁶). Possible mechanisms than can be attributed to the hypocholesterolemic effect of probiotics in vitro and in vivo studies include; assimilation of dietary cholesterol into the cell surface, binding of cholesterol to the cellular surface, inhibition of de-novo synthesis of cholesterol, disruption of cholesterol micelles, and deconjugation of bile salt and bile salt hydrolase (BSH) activity (¹⁷-¹⁹).

The genera most often administered to groups treated with probiotics were Lactobacillus and Bifidobacterium. Less frequently than these other genera used were Saccharomyces, Streptococcus, and Enterococcus (¹¹). Probiotic strains, particularly Lactobacillus, play a significant role in lowering cholesterol levels (⁴). In a randomized, double-blind placebo-controlled study results showed that both single (Lactobacillus rhamnosus) and combined strains (Lactobacillus acidophilus and Bifidobacterium animalis) of probiotics, which are used regularly for 8 weeks, could be effective in hypercholesterolemic patients to reducing serum lipids (²⁰). No significant differences were observed for any probiotics for serum lipids in a 18-week, randomised, double-blind crossover study with healthy obese adults (²¹). Available evidence indicates that probiotics supplements can significantly reduce serum TC. Furthermore, higher baseline TC, longer intervention time, and probiotics in capsules form might contribute to a better curative effect. The present study was conducted to evaluate the effects of combined strains probiotic supplementation on weight loss and lipid profiles in obese hypercholesterolemic patients. This randomized, double-blind, placebo-controlled pilot trial were designed to evaluate the effects of probiotics on the lipid profile with Enterococcus faecium, Lactobacillus plantarum, Streptococcus thermophiles, Bifidobacterium lactis, Lactobacillus acidophilus, Bifidobacterium longum strains.

MATERIAL AND METHODS

RANDOMIZATION

Each participant was randomly assigned into intervention or placebo group, according to 1:1 equal proportion rule. Randomization assignment was performed using computer-generated random numbers. The randomized allocation sequence, enrolling and allocating participants to interventions were conducted by an independent investigator not involved in the assessment of the participants or in the data collection and analysis. All participants were blinded to treatment allocation (Fig. 1).

Figure 1. Flow diagram of the study. 

RESULTS

Daily dietary intakes of the participants are presented in table I. There were no significant differences in daily total energy and intakes between the probiotics and placebo groups at the beginning of the intervention (p > 0.05). There was no difference in the average energy amount recommended from the dietitian between the groups during intervention (p > 0.05). Also, comparisons showed no significant differences in age, marital status, physical activity level and education status between the two groups at baseline (p > 0.05) (data not shown).

Table I. Age, daily dietary intakes and recommended energy of the participants at the beginning of the intervention 

Values are expressed as mean ± (SD). *Mann-Whitney test was used to calculate the p value. RE: recommended daily energy intake from dietitian during intervention.

Table II shows the effect of probiotic supplementation on lipid profiles of the participants after 8 weeks of intervention. There were no significant differences in lipid profiles between the probiotics and placebo groups at the beginning of the intervention (p > 0.05). After 8 weeks of probiotics or placebo supplementation, changes in total cholesterol and LDL-C were found to be significant within both groups, notedly in women. No significant change in other parameters (HDL-C, and TG) were observed within groups. Overall, a non-significant decrease in serum TC (-36.50 ± 19.27 vs -25.91 ± 19.25, mg/dl, p = 0.193), LDL-C concentrations (-31.75 ± 18.11 vs -31.91 ± 31.00 mg/dl, p = 0.908), TG (-31.83 ± 67.37 vs -28.25 ± 59.09, p = 0.932) were detected following the supplementation with probiotic, compared to the placebo. Thus, taking probiotics resulted in a non-significant decrease in lipid profiles in comparison with the placebo (Table II).

Table II. shows the effect of probiotic supplementation on lipid profiles of the participants after 8 weeks of intervention. There were no significant differences in lipid profiles between the probiotics and placebo groups at the beginning of the intervention (p > 0.05). After 8 weeks of probiotics or placebo supplementation, changes in total cholesterol and LDL-C were found to be significant within both groups, notedly in women. No significant change in other parameters (HDL-C, and TG) were observed within groups. Overall, a non-significant decrease in serum TC (-36.50 ± 19.27 vs -25.91 ± 19.25, mg/dl, p = 0.193), LDL-C concentrations (-31.75 ± 18.11 vs -31.91 ± 31.00 mg/dl, p = 0.908), TG (-31.83 ± 67.37 vs -28.25 ± 59.09, p = 0.932) were detected following the supplementation with probiotic, compared to the placebo. Thus, taking probiotics resulted in a non-significant decrease in lipid profiles in comparison with the placebo (Table II). 

MD: mean difference; HDL: high-density lipoprotein; LDL: low-density lipoprotein; TG: triglyceride; TC: total cholesterol. Values are expressed as mean ± (SD). *Wilcoxon test. **Mann-Whitney test.

As shown in table III, BMI, weight, FM, and FM % decreased significantly by the end of study in both probiotics and placebo groups, although no statistically significant difference was observed between the groups at baseline and after intervention (p > 0.05). Within-group analyses indicated that anthropometric indices significantly decreased in response to adherence to low-calorie diet recommended by dietitian in both groups.

Table III. Effect of probiotic supplementation on anthropometric indices after 8 weeks of intervention 

MD: mean difference; BMI: body mass index; BW: boyd weight; FM: fat-mass; FFM: fat-free mass. Values are expressed as mean ± (SD). *Wilcoxon test. **Mann-Whitney test.

DISCUSSION

In the present study receiving 1.5 × 10² CFU/g combined strains probiotic capsules for 8 weeks along with individualized dietary intervention in hypercholesterolemic obese patients showed significant decreased in total cholesterol, LDL-C and TG concentrations. However, no intergroup statistical differences were determined in the reductions of total cholesterol, LDL-C, TG concentrations between the probiotic and placebo groups.

Daily dietary intakes of the participants are presented in table I. There were no significant differences in daily total energy and intakes between the probiotics and placebo groups at the beginning of the intervention (p > 0.05). There was no difference in the average energy amount recommended from the dietitian between the groups during intervention (p > 0.05). In this study, when the anthropometric values of male individuals in the probiotics and control groups were examined before and after the study, no significant statistical difference was found between the values at the beginning and end of the study within the groups (p < 0.05). However, when the pre and post-study values of the female participants in this study were examined, the values were found to be 79.16 ± 5.00 kg and 76.41 ± 7.00 kg, respectively, in the group receiving probiotics 86.73 ± 15.08 kg and 83.06 ± 15.85 kg, respectively, in the group receiving placebo. In addition, a significant decrease was observed between the pre and post-study values within the groups in some of the anthropometric measurements (p < 0.05) (Table III). This was attributed to the fact that there was no significant difference because the number of male individuals was lower than the number of female individuals, while the significant decrease in body weight in women revealed the effectiveness of the low-fat, low-cholesterol weight loss diet given by the dietitian. Regularly monitoring the groups depending on the nutritional treatment by the dietitian led to weight loss in both the probiotics and control groups. The most crucial step in the treatment of obesity is medical nutrition therapy, and the treatment aims to reduce obesity, and all kinds of complications related to obesity (²²). Meckling et al. examined 31 overweight and obese individuals to evaluate the effectiveness of a low-fat diet; the mean weight loss at the end of 10 weeks was 6.8 kg, and the mean decrease in BMI was 2.2 kg/m² (²³). Hu et al. examined the effect of two different diet programs containing low carbohydrate (< 40 g/day) and low fat (< 30 % kcal/day and 7 % saturated fat) for 12 months. The mean weight loss in the low-carbohydrate diet group was 5.3 kg, whereas it was 1.5 kg in the low-fat diet group (²⁴). In this study, weight loss in both the study and control groups due to the nutritional treatment created by the dietitian led to improved lipid levels. One of the reasons there was no significant difference in lipid levels between the group receiving probiotics and the group receiving placebo in both men and women was attributed to the improvement in lipid levels caused by the weight loss in the placebo group due to the diet recommended by the dietitian.

In this study, the mean weight loss in male participants was 2.46 kg in the probiotics group and 5.46 kg in the control group. The mean decrease in BMI was 0.90 kg/m² in the probiotics group and 1.56 kg/m² in the control group. Statistically, there was no significant difference between this weight loss and the decrease in BMI values (p > 0.05). Similarly, the mean weight loss in female participants was 2.75 kg in the probiotics group and 3.66 kg in the control group. The mean decrease in BMI was 1.12 kg/m² in the probiotics group and 1.17 kg/m² in the control group. Statistically, there was no significant difference between this weight loss and the decrease in BMI values (p > 0.05) (Table III). No statistically significant group differences were found in the anthropometric measurements taken at the beginning and end of the study, which was attributed to factors such as the fact that the study was conducted on obese individuals, the study period was limited (8 weeks), and the small sample size.

One meta-analysis has highlighted the effects of consuming fermented foods and positive impacts on weight maintenance, while separate studies have demonstrated that consumption of fermented yogurts and dairy foods can attenuate the likelihood of developing CVD and type 2 diabetes mellitus (²⁶). Milk hydrolysis generates peptides with satiety or anti-obesity effects. Consumption of fermented milk enriched with probiotics positively affects body weight reduction and serum lipids. In a study of 14 healthy individuals, it was observed that consumption of probiotic yoghurt (Lactobacillus acidophilus and Bifidobacterium lacti) caused a significant decrease in serum total cholesterol levels compared with normal yoghurt (p < 0.05) (¹²). In a study conducted by Larsen et al., 70 obese participants (20 men and 50 women) aged between 18 and 55 years were randomly assigned to five groups over an 8-week period. One group consumed the experimental yoghurt Gaio^®, while the second and third groups were given two newly developed yoghurts fermented with different bacterial cultures. The yoghurt provided to one of these groups was fermented with two strains of S. thermophilus and two strains of L. acidophilus, while the yoghurt given to the other group was fermented with two strains of S. thermophilus and one strain of L. rhamnosus. The fourth group received a placebo yoghurt, and the final group was given two daily placebo tablets. When all five treatment groups were compared after adjusting for small changes in body weight, a significant reduction in LDL cholesterol levels (8.4 %, p < 0.05) was observed after 8 weeks in the group consuming only Gaio^® product (²⁷). However, in fermented milk or yogurt consumption, it is more difficult to achieve standardization due to factors such as optimum temperature for microbial growth and viability, food composition, or bioactive stability (⁹). In this trial, we considered these standardization difficulties as a limitation and preferred to use probiotic strains. Standardization of used products affects quality in research. However, it is thought that planning and conducting this study with a fermented milk product may support similar positive results in obese individuals. We also preferred to use supplement forms to highlight the effects of probiotics on blood lipids in obese individuals. In addition to this, dietary supplements can differ in quality, purity, and consistency, leading to inconsistencies in dosing. To avoid this, all products are supplied from the same company in this research.

In this study, when the pre- and post-study values of male and female individuals in the probiotics group were compared, it was observed that total cholesterol and LDL cholesterol levels decreased significantly at the end of the study (p < 0.05) (Table II). In addition to this, when HDL levels and triglyceride levels of male and female individuals in the probiotics and control groups were compared before and after the study, no significant statistical difference was found (p > 0.05) (Table II). While no significant change was observed in HDL levels in 9 of the 11 meta-analyzed studies (p = 0.59), no significant change was observed in triglyceride levels in 8 (p = 0.89) (¹⁵). In the study conducted by Fuentes et al., no statistically significant differences were found in total cholesterol, triglycerides, LDL, and HDL cholesterol levels between individuals with cholesterol levels > 250 mg/dL who received either a placebo or L. plantarum at baseline and after 6 weeks. However, a significant decrease in LDL cholesterol and total cholesterol levels was observed after 12 weeks (⁴). In a meta-analysis of 11 randomized clinical trials that conducted to evaluate the effects of probiotics on blood lipids, a significant decrease in total cholesterol and LDL values was found (p = 0.001) (¹⁴). One of the reasons for these inconsistent results was that the study period was limited to 8 weeks, and another reason was that there was an insufficient number of people during randomization. Again, the same meta-analyse study demonstrated the duration of probiotic supplementation was positively associated with the LDL-C lowering effects of probiotics, also high-dose probiotics more effectively reduced LDL-C levels than low-dose probiotics (¹⁴). Based on these studies, it was thought that a significant decrease in blood lipid levels could be seen when the study period was increased above 8 weeks. Also, the effectiveness of each probiotic strain depended on the dosage.

In this study a non-significant decrease in serum TC, TG, and LDL-C concentrations were detected following the supplementation with probiotic, compared to the placebo. Thus, taking probiotics resulted in a non-significant decrease in lipid profiles in comparison with the placebo (Table II). If we discuss this information by emphasizing important studies in the literature; in a study conducted by Jones et al. on hypercholesterolemic adults by giving yoghurt containing BSH-active Lactobacillus reuteri NCIMB 30242, no statistically significant difference was observed when total cholesterol, LDL, HDL and triglyceride levels were compared between the two groups receiving placebo yoghurt and yoghurt containing L. reuteri after 3 weeks (p > 0.05). When the differences of the same values were compared after 6 weeks, significant decreases were observed in total cholesterol (p = 0.031) and LDL cholesterol (p = 0.016) levels, whereas no statistically significant difference was observed in HDL (p = 0.808) and triglyceride (p = 0.230) levels (²⁸). In another study conducted by Jones et al. using a capsule containing Lactobacillus reuteri NCIMB 30242, when total cholesterol, LDL, HDL and triglyceride levels were compared after 9 weeks, significant decreases were observed in total cholesterol and LDL cholesterol levels (p < 0.001), while no statistically significant difference was observed in HDL and triglyceride levels (²⁹). While several clinical trials have shown that probiotics have cholesterol-lowering activity (²⁷,³⁰), some studies have shown the opposite result (³¹). These negative results may be related to poor bacterial strain selection, the study method or the clinical design of the trial, and these studies did not specify BSH activity as a characteristic of the strain administered. In contrast, a BSH-active strain (L. plantarum and L. reuteri) has been shown to significantly reduce total cholesterol and LDL cholesterol when administered as a synbiotic in humans (³²). Probiotics with BSH activity have often been found to have cholesterol-lowering effects in vivo studies (³³,³⁴). In a meta-analysis, it was determined that when yoghurt containing two strains of S. thermophilus and E. faecium was given, these strains showed a cholesterol-lowering effect (³⁵). These results suggest that the use of probiotics may improve lipid metabolism by decreasing total and LDL cholesterol concentrations. However, both the efficacy of probiotics for cholesterol lowering and safety should be investigated further in well-designed clinical trials. The findings of recent meta-analysis suggest that probiotics can enhance SCFA content in the body, improve oxidative stress and inflammation, and effectively alleviate hyperlipidemia. The primary lipid-lowering mechanisms of probiotics encompass: a) the production of BSH, which enhances cholesterol excretion; b) the promotion of cholesterol catabolism and the inhibition of cholesterol synthesis in the body via key intestinal metabolites, specifically SCFAs; and c) the modulation of immune regulation and the intestinal barrier through signaling molecules (³⁶). In a recent study results demonstrated that regular and strain-specific use of probiotics (probiotics containing Lactobacillus or Bifidobacterium) could be effective to reducing plasma lipid level in hypercholesterolemic patients (²⁰). In this study, a probiotic product supplement containing 1.5 × 10² CFU active probiotic microorganisms (Lactobacillus acidophilus, Enterococcus faecium, Lactobacillus plantarum, Streptococcus thermophillus, Bifidohacterium lactis, Bifidobacteriurn longum) was used. Also the other reason for inconsistent results may attributed to the different type of strains that have been used (¹¹). Finally, the confusing and interconnected reasons for this study's results between groups may be attributed to the different types of probiotic strains used, doses of probiotics, delivery matrix, study duration, and study population. However, not all probiotic interventions are effective against dyslipidemia. The results are controversial and depend on several factors such as probiotic strain, dose, duration of the treatment, lifestyle changes, etc. Additional studies are highly recommended on the cholesterol-lowering property of probiotics, which could help to reduce the risk of CVD and other dyslipidemia-associated health issues.

CONCLUSION

In this randomized controlled clinical trial, dietary supplementation of probiotics for 8 weeks had no prominent favorable effect on serum total cholesterol, LDL-C, TG levels, and body weight loss in obese subjects with hyperlipidemia. However, further well-designed large sample size randomized controlled trials, with longer supplementation durations are needed for a stronger assessment of the lipid-modulating properties of probiotics on obese subjects. Thus, more specific data regarding the duration and dosage of probiotic use in obese individuals may reveal significant beneficial effects on health.

LIMITATIONS

Some limitations might explain the contradictory findings of the present trial. Firstly, the reduced number of patients is one of the major limitations of this study, this is probably the reason why there are no differences in most of the parameters evaluated. Secondly, the short follow-up time is another limitation of the study. Moreover, doses of probiotics and type of strains that have been used may attributed to the inadequate results. Additionally, it was emphasized that the efficacy of probiotics would be strengthened by increasing the treatment period to ≥ 8 weeks. Also, during the regulation of nutritional intake, the individual declaration is taken into consideration so that, unbalanced and higher consumption amounts may also affect the results.