Probiotics Supplementation Improves Intestinal Permeability, Obesity Index and Metabolic Biomarkers in Elderly Thai Subjects: A Randomized Controlled Trial

Intestinal integrity prevents the diffusion of allergens, toxins, and pathogens from the gastrointestinal lumen into the tissue and the circulatory system. Damage in intestinal integrity may cause mild to serious health issues, such as inflammation, gastrointestinal disorders, neurological diseases, and neurodegenerative disorders. Thus, maintaining a healthy intestinal barrier function is essential to sustain health. Probiotics are known for their ability to protect and restore intestinal permeability in vitro and in vivo. The multi-strain probiotics are more efficient than that of a single strain in terms of their protective efficacy. Therefore, the present study was planned and implemented to study the supplementation of probiotic mix (Lactobacillus paracasei HII01, Bifidobacterium breve, and Bifidobacterium longum) on intestinal permeability, lipid profile, obesity index and metabolic biomarkers in elderly Thai subjects. The results revealed that the supplementation of studied probiotics improved the intestinal barrier function (up to 48%), significantly increasing the high-density lipoprotein (HDL)-cholesterol. Moreover, the intervention improved obesity-related anthropometric biomarkers and short-chain fatty acid levels in human subjects. The current study strongly recommends further extended research to confirm the beneficial effect of probiotics, which may pave the way to formulate probiotic-based health supplements to adjuvant the treatment of several metabolic diseases.


Introduction
The most important protective function of the intestinal epithelium is the "barrier function," which prevents the diffusion of allergens, toxins, and pathogens from the gastrointestinal lumen into the tissue and the circulatory system [1,2]. A specialized complex structure is present in the lateral epithelial membranes' apical region, which is considered to be a significant module of epithelial barrier function known as tight junctions [3].

Study Design and Participants
All the participants approved the study procedure and provided their consent before the study. The ethical committee of Mae Fah Luang University agreed to the study protocol (Code: REH-62151). The study was performed according to the Declaration of Helsinki and following the Good Clinical Practices.
The effect of a probiotics mixture on intestinal permeation and other biomarkers were studied in Thai subjects in a randomized, double-blind, placebo-controlled study model.
For the screening, subjects were asked to consume mannitol and lactulose dissolved in water. Within 6 h of mannitol and lactulose consumption, subjects were required to collect their urine [20], and their intestinal permeability was analyzed using a colorimetric commercial kit (EnzyChromTM BioAssay, San Jose, CA, USA).
Any subjects with a history of cardiovascular events, suffering from kidney diseases, gastrointestinal tract (GI) disorders, or gouty arthritis were excluded from the study. In addition, those who have undergone treatment with probiotics, antibiotic drugs (or both) or any other drugs that are used to treat GI tract-related discomforts in the previous 14 days were also excluded.
Random Allocation Software was used to randomize the subjects, and the researchers and participants were blinded to the group assignment. The participants were randomized to receive either a probiotics supplement or placebo for 12 weeks and asked to come to the study center for follow-up. The screening and enrollment details are shown in Figure 1.

Treatment
The subjects in the probiotic group were provided with aluminum foil sachets containing a mixture of probiotics (2.0 × 10 10 CFU of Lactobacillus paracasei HII01; 2.0 × 10 10 CFU of Bifidobacterium breve; 1.0 × 10 10 CFU of Bifidobacterium longum), which was received from Lactomason Co., Ltd., Jinju-si, South Korea, and the placebo group were provided with 10 g of corn starch in a similar package of probiotics. The instructions for the consumption of the supplement were detailed in our previous report [18].

Treatment
The subjects in the probiotic group were provided with aluminum foil sachets containing a mixture of probiotics (2.0 × 10 10 CFU of Lactobacillus paracasei HII01; 2.0 × 10 10 CFU of Bifidobacterium breve; 1.0 × 10 10 CFU of Bifidobacterium longum), which was received from Lactomason Co., Ltd., Jinju-si, South Korea, and the placebo group were provided with 10 g of corn starch in a similar package of probiotics. The instructions for the consumption of the supplement were detailed in our previous report [18].

Clinical Data
The subjects' personal history was assessed, and their demographic characteristics were detailed. The body mass index (BMI) and weight of the subjects were measured using an electronic scale (Picooc ® , Model S1 Pro, Beijing, China) [18].

Clinical Data
The subjects' personal history was assessed, and their demographic characteristics were detailed. The body mass index (BMI) and weight of the subjects were measured using an electronic scale (Picooc ® , Model S1 Pro, Beijing, China) [18].

Laboratory Data
The samples (blood, urine, and feces) were collected at the screening point, baseline, follow-up, and the end of the study (Figure 2).

Treatment
The subjects in the probiotic group were provided with aluminum foil sachets containing a mixture of probiotics (2.0 × 10 10 CFU of Lactobacillus paracasei HII01; 2.0 × 10 10 CFU of Bifidobacterium breve; 1.0 × 10 10 CFU of Bifidobacterium longum), which was received from Lactomason Co., Ltd., Jinju-si, South Korea, and the placebo group were provided with 10 g of corn starch in a similar package of probiotics. The instructions for the consumption of the supplement were detailed in our previous report [18].

Clinical Data
The subjects' personal history was assessed, and their demographic characteristics were detailed. The body mass index (BMI) and weight of the subjects were measured using an electronic scale (Picooc ® , Model S1 Pro, Beijing, China) [18].

Laboratory Data
The samples (blood, urine, and feces) were collected at the screening point, baseline, follow-up, and the end of the study (Figure 2).  The biochemical parameters such as blood urea nitrogen (BUN), creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), HDLcholesterol (HDL-C), LDL-cholesterol (LDL-C), triglycerides (TG), and fasting blood sugar (FBS) levels were measured from blood samples using the automated machine at AMS Clinical Service Center, Chiang Mai University, Chiang Mai, Thailand.
Other biomarkers in the blood, such as Immunoglobulin A (IgA) and lipopolysaccharide (LPS), were measured using ELISA commercial kit (MyBioSource ® , San Diego, CA, USA for LPS, Elabscience ® , Houston, TX, USA for IgA).
Urine samples were collected from subjects to determine intestinal permeability. The samples were analyzed as detailed in our previous study [18] using a colorimetric commercial kit (EnzyChrom™, BioAssay, Hayward, CA, USA). Other biomarkers in the urine, such as quinolinic acid (QA) and 5-hydroxyindoleacetic acid (5-HIAA), were determined using ELISA commercial kit (Fivephoton Biochemicals™, San Diego, CA, USA for QA, and Immusmol, Bordeaux, France for 5-HIAA).

Statistical Analyses
The data were evaluated using the paired t-test of means using STATA version 15.1 (StataCorp, College Station, TX, USA) for windows licensed to the Faculty of Pharmacy, Chiang Mai University.
A descriptive analysis of the collected parameters was expressed as an absolute number and percentage. The continuous variables were expressed as mean ± standard deviation (SD) or standard error of the mean (SEM), depending on their statistical distribution. The group's data were also evaluated using Gaussian regression and Risk difference regression. The minimum level of statistical significance was set at p < 0.05.

The Study Participants
A total of 60 subjects were screened, and 48 subjects were selected for randomization. According to the study design, all enrolled subjects (men: 10, women: 38) completed the trial. The primary demographic data of the study subjects are detailed in Table 1. p-value at 95% confidence interval. The proportions were analyzed using an exact probability test, and the continuous demographic data were analyzed using a t-test.
The subjects in placebo and probiotic groups did not significantly differe at the beginning of the study. The distribution of male and female subjects did not show any significant differences (p > 0.05).

Changes in the Study Parameters between the Group
Significant changes were observed in some of the study parameters in the probiotics group compared to the placebo after 12 weeks of the study. In detail, the body mass index (p ≤ 0.001), body fat (p = 0.016), muscle content (p = 0.022), waist (p = 0.001) and hip (p = 0.001) circumferences, creatinine (p = 0.001), AST (p = 0.024), HDL-C (p = 0.001), FBS (p = 0.001), IgA (p ≤ 0.001), LPS (p = 0.001), hsCRP (p = 0.029), lactulose-mannitol ratio (p ≤ 0.001), lactulose (p = 0.025), QA (p ≤ 0.008), and butyric acid (p = 0.014) content showed significant improvement in the probiotics group compared to the placebo. The results indicated that the probiotics intervention improved the study parameters in experimental subjects (Table 3). The Gaussian regression analysis revealed that the body fat, visceral fat, muscle content, body age, arm, waist and hip circumferences, creatinine, ALT, HDL-C, FBS, IgA, LPS, lactulose-mannitol ratio, QA, QA/5-HIAA ratio, and butyric acid content were significantly altered in probiotics group after 12 weeks of treatment ( Table 4). The risk difference analysis revealed that intestinal permeability was improved up to 48% in the probiotics supplemented group (Table 5).

Discussion
The study subjects completed the experimental procedures successfully and showed significant clinical improvements in intestinal permeability, lipid profile, and short-chain fatty acids.
A recent study revealed that the use of Lactobacillus species could improve the intestinal microbiota and reduce gut permeability [23]. According to Ohland and MacNaughton [24], probiotics improved the intestinal barrier function by increasing the production of mucus, secretory IgA and antimicrobial peptides, and increased tight junction integrity of epithelial cells and competitive adherence for pathogens.
Zhang et al. [27] demonstrated that the surface-layer associated proteins (SLAP) of L. paracasei ssp. paracasei M5-L and L. casei Q8-L protect the bacteria-mediated epithelial barrier disruption by suppressing the occludin production and inhibiting the delocalization of zonula occludens-1. Similarly, the use of L. paracasei ssp. paracasei L. casei W8 ® improved the intestinal barrier function and reduced the inflammation in high-fat diet-fed rats [28].
Laval et al. [29] showed that L. rhamnosus CNCM I-3690 enhanced intestinal integrity by increasing the level of occludin and E-cadherin.
Al-Sadi et al. [30] screened some probiotic species such as Bifidobacterium bifidum, B. breve, B. longum, Escherichia coli strain Nissle, and probiotic species or strains of Lactobacillus acidophilus, L. brevis, L. casei, L. helveticus, L. johnsonii, L. plantarum, and L. rhamnosus to identify the effective probiotic species or strain that prevents intestinal inflammation by increasing the tight junction. Among the screened probiotic bacterial species and strains, L. acidophilus LA1 strain showed an effective increase in Caco-2 trans-epithelial resistance and reduced paracellular permeability indicating the improvement of Caco-2 tight junction barrier function. Oral supplementation of LA1 showed TLR-2 dependent improvement of tight junction barrier and protection against intestinal inflammation, thereby preventing dextran sodium sulfate (DSS)-induced colitis in the mouse model [30].
In the present study results, the level of the lactulose-mannitol ratio and lactulose were reduced significantly in the probiotic supplemented group compared to baseline values and the placebo (Tables 2 and 3). Moreover, the intestinal permeability of the probioticsupplemented subjects was improved up to 48% ( Table 5). The increased intestinal barrier function was observed in the serum level LPS; a significant level of reduction was observed in LPS concentration after probiotic intervention (Tables 3 and 4). Accordingly, the studied probiotic mixture might have the ability to improve intestinal barrier function.
The reduction in the fecal concentrations of short-chain fatty acids (SCFAs) is associated with diseases and aging [31,32]. Cai et al. (2016) reported that the centenarians have a high concentration of SCFAs, associated with the high dietary fiber intake [33].
The probiotic strains of Lactobacillus and Bifidobacterium species were propionic, lactic, and butyric acid producers [34,35]. The consumption of L. plantarum P-8 significantly reduced the opportunistic pathogens and increased Bifidobacterium level. Moreover, the levels of propionate and acetate were increased [36]. Moens et al. reported that the growth of probiotic bacteria might increase the lactate concertation, which facilitates lactate-consuming microbial growth, subsequently increasing SCFAs production, particularly butyrate [37]. The further microbial analysis is required to confirm the association between changes in SCFAs levels and probiotic interventions. In the present study, probiotics intervention significantly increased propionic and butyric acid levels, whereas changes in lactic and acetic acids levels were non-significant (Table 2). Gaussian regression analysis revealed that butyric acid level increased notably (p = 0.008) after the probiotic intervention ( Table 4).
In the present study, BMI, body fat, muscle, and waist and hip circumferences were improved in the probiotic group compared to placebo ( Table 3). The body and visceral fat, muscle, body age, and arm, waist and hip circumferences were significantly improved after the 12-week course of probiotic intervention, as per the Gaussian regression analysis ( Table 4). The level of HDL-C was increased significantly in the probiotic-treated group, while a noted level of reduction was observed in the placebo (Table 3). No significant changes were observed in TC, TG, LDL-C, and hsCRP values after the study period in the probiotic-treated group (Table 4). These results indicated that the studied multispecies probiotic mix improved the lipid profile and obesity-related biomarkers in studied human subjects.
The gut microbiota and its secreted compounds may affect the tryptophan metabolism and gut inflammation, which affects the kynurenine and QA levels [39]. L. paracasei may influence the central 5-hydroxytryptamine (5-HT) system and brain-derived neurotrophic factor (BDNF) expression through butyrate. B. breve and B. longum affect the glutaminergic system and neural activities in the brain through humoral and neural routes, respectively [40].
The supplementation of L. paracasei HII01 increased the levels of short-chain fatty acids in obese, hypercholesterolemic, and diabetic human subjects [17,37]. 5-HIAA and QA/5-HIAA ratio was not significantly affected by the supplementation of L. paracasei HII01, B. breve, B. longum, inulin, and fructooligosaccharide. [18]. The present study results indicated that the supplementation of the studied probiotic mixture decreased the QA level (Tables 3 and 4). Thus, the intervention might influence the tryptophan metabolism and expression of BDNF.
The probiotic intervention improved liver aminotransferases in patients with nonalcoholic fatty liver disease [41], and alcohol-induced liver disease [42]. In this study, ALT level was reduced, and AST level was not significantly changed ( Table 4).
The studies on the influence of probiotics on renal function are very limited and the reported studies showed that probiotic supplementation improved renal function through increased intestinal barrier function [43,44]. In the present study also BUN values were not changed significantly, whereas creatinine levels were reduced significantly in the probiotic treated group (Table 4).
Altogether, the results of the current study provided the basic information about the influence of studied (L. paracasei HII01, B. breve, and B. longum) probiotic mixture on intestinal permeability, lipid profile, body fat, liver and kidney function, neurotransmitter levels, and short-chain fatty acids in elderly Thai subjects.

Conclusions
The current study has limitations, such as its limited sample size, questionnaire regarding eating habits, exercise, work activity, and overcoming the disease, lack of extended follow-up, and microbiota analysis. Nevertheless, the present study represents the effects of the intervention of a probiotic mixture composed of Lactobacillus and Bifidobacterium on kidney and liver function, lipid profile, intestinal integrity, microbial metabolites, bacterial endotoxin (LPS) level, and biomarkers of gut-brain communication pathways in elderly Thai subjects.
The results revealed that the supplementation of studied probiotics improved the intestinal barrier function, the lipid profile and obesity-related biomarkers in human subjects. Further studies are strongly recommended to confirm the beneficial effect of probiotics, which may pave the way to formulate probiotic-based health supplements to adjuvant the treatment of several metabolic diseases.

Data Availability Statement:
The data presented in the manuscript is available on request from the corresponding author.