Safety and Effects of Lactobacillus paracasei TISTR 2593 Supplementation on Improving Cholesterol Metabolism and Atherosclerosis-Related Parameters in Subjects with Hypercholesterolemia: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial

Probiotics have the potential as a multi-target approach to modulate hypercholesterolemia associated with premature atherosclerosis. Various strains of Lactobacillus paracasei have been reported to affect hypercholesterolemia positively. This study aimed to investigate the effects of L. paracasei TISTR 2593 on lipid profile, cholesterol metabolism, and atherosclerosis according to the registration of Thai Clinical Trial Registry as identification number TCTR 20220917002. A total of 50 participants with hypercholesterolemia were randomly and equally assigned to consume L. paracasei TISTR 2593 or a placebo in maltodextrin capsules daily. Biomarkers of lipid profiles, oxidative stress state, inflammatory state, and other biological indicators were examined on days 0, 45, and 90. The results showed that subjects taking the L. paracasei TISTR 2593 could significantly reduce the level of serum low-density lipoprotein-cholesterol (p < 0.05), malondialdehyde (p < 0.001), and tumor necrosis factor-α (p < 0.01). Moreover, L. paracasei TISTR 2593 increased the level of serum apolipoprotein E (p < 0.01) and adiponectin (p < 0.001) significantly. No changes in serum total cholesterol, high-density lipoprotein-cholesterol, triglyceride, total bile acids, and monocyte chemoattractant protein-1 were observed during L. paracasei TISTR 2593 supplementation. Therefore, L. paracasei TISTR 2593 could be an adjuvant probiotic supplement to ameliorate hypercholesterolemia and prevent or delay the development of atherosclerosis.


Introduction
Hypercholesterolemia is a major risk factor leading to cardiovascular diseases (CVDs), a major cause of death worldwide. High levels of total cholesterol, low-density lipoproteincholesterol (LDL-C), and triglyceride result in a build-up of fatty deposits inside the arteries leading to atherosclerosis [1]. According to the American College of Cardiology (ACC) This study was conducted following the rules of the Declaration of Helsinki. The protocol was approved by the Ethical Committee of the Human Experimentation Committee, Research Institute for Health Science (RIHES), Chiang Mai University, Thailand (Project No. 11/64), and the Thai Clinical Trials Registry (TCTR) is TCTR20220917002 (https://www.thaiclinicaltrials.org/show/TCTR20220917002, accessed on 6 September 2022). All subjects provided informed consent before participation in the study.
Subjects were randomly distributed into two groups: placebo or L. paracasei TISTR 2593. The inclusion criteria were male and female (non-pregnant), aged 30-65 years, mild and moderate hypercholesterolemia (defined by having serum LDL-C between 100 and 159 mg/dL), body mass index (BMI) between 19 and 30 kg/m 2 , no vegan, no vegetarian, and without cholesterol-and triglyceride-lowering drug consumption. In addition, subjects were excluded in the case of cardiovascular disease events and secondary dyslipidemia. Subjects were advised about lifestyle recommendations, but made no change in their exer-cise, eating habits, taking specific diet or other supplements during the whole study period. They were contacted twice weekly to ask about any adverse effects during the study.
In addition, subjects in this study underwent a general characteristics examination, including age, sex, BMI, systolic blood pressure, diastolic blood pressure, and heart rate, and were interviewed using a questionnaire to obtain information on their dietary status.

Study Design and Treatment
We conducted a single-center, prospective, randomized, double-blind, placebo-controlled, parallel-group trial. Subjects were kept blind regarding treatments. Blinded investigators performed data interpretation and analysis.
Subjects were randomly assigned using block randomization into two groups as follows: (1) L. paracasei TISTR 2593 in maltodextrin capsule obtained from the Expert Center of Innovative Health Food, Thailand Institute of Scientific and Technological Research, Thailand, at a daily dose of 1.05 × 10 10 CFU/g (350 mg per capsule); and (2) a placebo product which was a maltodextrin capsule. In addition, subjects consumed the assigned substance once daily before breakfast. Blood was collected to determine a lipid profile consisting of serum levels of total cholesterol (TC), triglyceride (TG), LDL-C, high-density lipoprotein-cholesterol (HDL-C) together with fasting blood glucose (FBG), blood safety parameters, and other parameters at a period prior to the intervention, then at 45-day and 90-day intervention periods. The study flowchart and enrollment are shown in Figure 1.
Subjects were randomly distributed into two groups: placebo or L. paracasei TISTR 2593. The inclusion criteria were male and female (non-pregnant), aged 30-65 years, mild and moderate hypercholesterolemia (defined by having serum LDL-C between 100 and 159 mg/dL), body mass index (BMI) between 19 and 30 kg/m 2 , no vegan, no vegetarian, and without cholesterol-and triglyceride-lowering drug consumption. In addition, subjects were excluded in the case of cardiovascular disease events and secondary dyslipidemia. Subjects were advised about lifestyle recommendations, but made no change in their exercise, eating habits, taking specific diet or other supplements during the whole study period. They were contacted twice weekly to ask about any adverse effects during the study.
In addition, subjects in this study underwent a general characteristics examination, including age, sex, BMI, systolic blood pressure, diastolic blood pressure, and heart rate, and were interviewed using a questionnaire to obtain information on their dietary status.

Study Design and Treatment
We conducted a single-center, prospective, randomized, double-blind, placebo-controlled, parallel-group trial. Subjects were kept blind regarding treatments. Blinded investigators performed data interpretation and analysis.
Subjects were randomly assigned using block randomization into two groups as follows: (1) L. paracasei TISTR 2593 in maltodextrin capsule obtained from the Expert Center of Innovative Health Food, Thailand Institute of Scientific and Technological Research, Thailand, at a daily dose of 1.05 × 10 10 CFU/g (350 mg per capsule); and (2) a placebo product which was a maltodextrin capsule. In addition, subjects consumed the assigned substance once daily before breakfast. Blood was collected to determine a lipid profile consisting of serum levels of total cholesterol (TC), triglyceride (TG), LDL-C, high-density lipoprotein-cholesterol (HDL-C) together with fasting blood glucose (FBG), blood safety parameters, and other parameters at a period prior to the intervention, then at 45-day and 90-day intervention periods. The study flowchart and enrollment are shown in Figure 1.

Blood Sampling and Biochemical Measurements
Blood samples were collected in the morning after 12 h fasting at each visit and kept in a test tube to analyze for blood lipid profiles (TG, TC, LDL-C, and HDL-C), serum biochemistry (FBG, blood urea nitrogen (BUN), creatinine, and alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP), blood electrolytes (sodium, potassium, and chloride), and complete blood count examinations as the following parameters: white blood cell count (WBC), hemoglobin concentration, hematocrit, platelet count (PLT), neutrophil, lymphocyte, monocyte, eosinophil, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). The tests were done in the Laboratory Unit of Chiang Mai Medical Lab, Chiang Mai, Thailand. Additionally, other blood samples were allowed to clot and were spun at 1450 RCF or g force for 10 min at 4 • C. Then, the serum was transferred into dry well-labeled specimen plastic tubes and stored at −80 • C until analysis. Serum analysis was performed on a spectrophotometry (SPECTROstarNano, BMG LABTECH, Biotechnology division, Scientific Promotion Ltd., Ortenberg, Germany) analyzer to study a change of oxidative stress status, an inflammation marker, lipid metabolism enzymes, and MCP-1 parameter.
The oxidative stress and antioxidant markers were used to evaluate the oxidative stress status. The lipid peroxidation level, excess production of reactive oxygen species (ROS), was investigated using the thiobarbituric acid reactive substances (TBARS) method according to Bhutia et al. [24], and glutathione (GSH) level, which is an antioxidant capacity to mitigate oxidative stress, was determined according to the previous study [25].

Statistical Analysis
The sample size of fifty subjects was calculated using the superiority design method (calculating sample size when the outcome measure is a continuous variable) [26] based on the mean difference of LDL-C reductions with a significance level of 0.05 [27]. Twenty-five subjects were enrolled in each group. A total of fifty patients (twenty-five subjects in each group) was calculated to achieve a statistical power of 80%, giving a 10% dropout rate).
Statistical analysis was performed using SPSS software version 22 (SPSS Inc., Chicago, IL, USA). Data were ensured using the normal distribution of variables by the Kolmogorov-Smirnov test, and variables that were non-normally distributed were log-transformed before statistical analysis.
The descriptive analysis was expressed as an absolute number and percentage. Continuous variables were expressed using means and standard deviations (SDs) and associated two-tailed 95% confidence intervals (CIs). We applied the means substitution method to handle missing values. The sensitivity analysis in which the missing values were not imputed was performed. Outcomes were compared to the baseline during a study period, and a paired t-test was used to identify a significant difference. The differences between the group's outcomes were analyzed using repeated measure ANOVA followed by an LSD post hoc test. The statistical significance was considered when the p-value was less than 0.05.

Characteristics of the Subjects
A total of 50 hypercholesterolemic subjects were assessed for eligibility, and 4 subjects were excluded due to the presence of secondary dyslipidemia (n = 3) and relocation (n = 1).
Subjects were randomly allocated into the L. paracasei TISTR 2593 group (n = 23) and the placebo group (n = 23). Four subjects dropped out: two from the placebo group and two from the L. paracasei TISTR 2593 group. Finally, 42 subjects remained and were included in the analysis (Figure 1).
Subjects in the L. paracasei TISTR 2593 group were 48.5 ± 5.3 years old and 65.2% female whereas subjects in the placebo group were 46.0 ± 5.1 years old and 82.6% female. All subjects reported no serious adverse effects or clinical symptoms throughout the study. The within-group and between-group analysis showed that general characteristics of BMI, systolic blood pressure, diastolic blood pressure, heart rate, and calorie intake had no statistically significant difference at baseline and the end of the study. The general characteristics data of all subjects at baseline and the end of the study were presented in Table 1.

Effect of L. paracasei TISTR 2593 Supplementation on Blood Safety Parameters
Blood samples were tested for FBG, BUN, creatinine, ALT, AST, ALP, blood electrolytes, and hematology representing safety parameters when repetitively consuming L. paracasei TISTR 2593 supplementation for 90 days.
A comparison of within-group and between-group was analyzed, and results are shown in Table 2. FBG, BUN, creatinine, ALT, AST, ALP, blood electrolytes, and completed blood count showed no significant difference when comparing within-group and betweengroup throughout the intervention.

Effect of L. paracasei TISTR 2593 Supplementation on Blood Lipid Profiles
To investigate the effect of L. paracasei TISTR 2593 on blood lipid profiles, TC, TG, LDL-C, and HDL-C were tested in hypercholesteremic subjects. The results are shown in Table 3. Consumption of a 1.05 mg/day x 10 10 of L. paracasei TISTR 2593 for 45 and 90 days showed significant (p < 0.05 and p < 0.05, respectively) reduction of LDL-C level together with the mean difference from baseline at −17.75 mg/dL (95% CI: −23.84 to 18.94) and −17.52 mg/dL (95% CI: −41.59 to 1.19), respectively. In addition, a significant reduction was found in the serum level of LDL-C compared to the placebo group (p < 0.01). However, TC, TG, and HDL-C showed no significant differences when compared within and between groups.

Effect of L. paracasei TISTR 2593 Supplement on Oxidative Stress and Inflammation
Hypercholesterolemia alters vascular endothelial cell membranes, enhancing oxidative stress and inflammation mediators implicated in the pathogenesis of atherosclerosis [28,29]. The oxidative stress status including lipid peroxidation product (MDA) levels, antioxidant activity (GSH), and inflammatory parameters including IL-10, IL-6, TNF-α, and MCP-1 were also investigated to observe the possible beneficial effects of L. paracasei TISTR 2593.
Additionally, a significant difference in the reduction in MDA (p < 0.001) and TNF-α (p < 0.05) levels was also found when compared to the placebo group. However, GSH, IL-10, IL-6, and MCP-1 levels did not change when compared within and between groups. The results are shown in Table 4.   ized, placebo-controlled crossover study found that Bifidobacterium animalis subsp. lactis B94 or Bacillus subtilis R0179 supplementation for six weeks increased plasma deconjugated bile acids in subjects with a BMI ≥30 kg/m 2 , while there was no effect of Lactobacillus plantarum HA-119 on plasma bile acids [51]. It seems that some strains of probiotics can modulate plasma bile acid profiles, leading to the clinical benefits mentioned earlier. To understand the effect of probiotics on bile acid metabolism, plasma concentrations of deconjugated bile salts are required for further investigation. Even though there were no differences in baseline characteristics between groups, some limitations were still found in the study. Our study did not analyze changes in the gut microbiota community in feces, which is an essential indicator to confirm the colonization of the probiotic L. paracasei TISTR 2593. In addition, the participants' diet, lifestyle, mood, and physical activity could affect or modulate the underlying mechanisms because the study was conducted in a free-living setting; however, we asked them to maintain their diet and exercise patterns during the study period and ensured by measuring calorie intake at the start and the end of the study which showed no significant changes.

Conclusions
In summary, the current study findings demonstrated that supplementation with L. paracasei TISTR 2593 capsules for 90 days has a lowering effect on LDL-C, anti-oxidative stress, and anti-inflammation in Thai adults with high cholesterol levels. Thus, L. paracasei TISTR 2593 could be an adjuvant probiotic supplement to help manage LDL-C levels and potentially delay the development of atherosclerosis. However, the underlying mechanism of how L. paracasei TISTR 2593 exerts the reduction in blood lipids and prevents the development of atherosclerosis, such as the connection with the gut microbiota community, should be performed in further studies which may support probiotic-based food supplementations for managing hypercholesterolemia associated with cardiovascular diseases.