1. Introduction
Hyperlipidemia is a major risk factor for the development of serious cardiovascular diseases such as myocardial infarction, stroke, and atherosclerosis, especially when accompanied by chronic diseases such as hypertension and diabetes [
1]. In particular, high blood triacylglyceride (TG) levels due to intake of a high-fat diet can contribute to the progression of arteriosclerosis [
2,
3]. Accordingly, there has been substantial research focused on identifying or developing functional food ingredients to control or prevent hypertriglyceridemia by managing TG levels.
Moreover, accumulating evidence points to the important role of intestinal microflora as an environmental factor affecting obesity and related diseases, including hyperlipidemia. An increase in harmful intestinal bacteria activates inflammatory pathways that affect lipid metabolism, leading to the development of obesity-related diseases [
4]. With recognition of specific patterns of intestinal microbial metabolism associated with obesity, there has been increased interest in the development of probiotics that can reduce TG levels and lipids in the body. For example, consumption of shakes containing
Lactobacillus acidophilus,
Bifidobacterium bifidum, and fructo-oligosaccharide resulted in a significant increase in HDL-cholesterol and a reduction in glycemia in elderly people with type 2 diabetes mellitus [
5]. In addition, consumption of yogurt containing
Lb. acidophilus and
Bifidobacterium lactis caused a significant decrease in cholesterol in patients with hypercholesterolemia [
6], and consumption of milk fermented with
Lb. acidophilus 145 and
Bifidobacterium longum BB536 reduced LDL-cholesterol levels [
7]. Furthermore, research on various probiotics on improving blood lipids has been preceded [
8]. These beneficial effects were directly attributed to the improved intestinal microbial environment from ingestion of probiotics.
Among the available probiotics,
Lactobacillus plantarum Q180 (LPQ180) is a safe plant-derived lactic acid bacterium that has been isolated from the feces of adults, which has demonstrated the ability to reduce blood TG levels both in vitro and in vivo animal experiments [
9,
10]. LPQ180 was confirmed to inhibit adipocyte differentiation of 3T3-L1 cells in vitro due to the reduction of C/EBPα and PPARγ levels [
11]. In vivo experiments confirmed that obese mice fed a high-fat diet and supplemented with the LPQ180 strain exhibited reduced fat cell uptake and size [
12]. However, there have been no human intervention trials about LPQ180 conducted to date.
Based on the hypothesis that LPQ180 has a positive effect on lipid metabolism by improving intestinal microbiota composition and related metabolites, we conducted a double-blind, randomized, controlled trial to determine the effects of LPQ180 consumption postprandial lipid responses after oral lipid tolerance test (OLTT) using TNO fat challenge formula [
13] in men and women over 20 years of age with a healthy and slightly higher fasting TG levels (<200 mg/dL). In addition, the effect on fecal environments was evaluated. These findings can provide a scientific basis for the development of LPQ180 as a functional ingredient to maintain healthy postprandial lipid metabolism.
2. Materials and Methods
2.1. Subjects
Adults over the age of 20 who had blood TG levels less than 200 mg/dL were recruited to participate in this trial through posters and online advertising. All participants provided written consent and the protocol received approval from the Internal Review Board (IRB) of Ewha Woman’s University Mokdong Hospital (IRB No. EUMC 2016-06-040). This study was also registered on the International Clinical Trials Registry Platform of the WHO (ICTRP, No. KC T0002085). The exclusion criteria were as follows: Recommended Food Score (RFS) > 36 to exclude participants having healthy dietary habit [
14,
15]; body mass index (BMI) below 23 kg/m
2 and over 35 kg/m
2; regular intensity exercise (≥10 h/week); chronic smokers (≥20 cigarettes/day); alcoholism and those who consume 420 g alcohol per week; diagnosis of hyperlipidemia, diabetes, hypertension, liver disease, bowel disease, kidney disease, cardiovascular disease, cerebrovascular disease, pancreatitis, cancer, thyroid disease, dementia, Parkinson’s disease, depression, anorexia/bulimia, or multiple sclerosis; taking medication or herbal medicine at least two months before the first visit; taking any probiotics or prebiotics or sustained intake of dietary supplements that affect body fat lipid metabolism and bowel health within the previous month before the first visit; participation in another clinical research trial within three months before the first visit; and pregnant or lactating women
2.2. Study Design
The study was designed as a randomized, double-blind, placebo-controlled, and parallel trial. The selected subjects ingested LPQ180 or placebo capsules daily for 12 weeks after a two-week run-in period. Seventy subjects who met the selection criteria were randomly assigned to the placebo or LPQ180 group by block randomization method (
Figure 1).
The LPQ180 group (n = 35) received two 400 mg capsules containing 4.0 × 109 colony-forming units of LPQ180 per day. The placebo group (n = 35) took two capsules containing the same amount of maltodextrin, which did not contain any probiotic strains. Both products were obtained from CKD Bio Corp., Ltd. (Seoul, Korea). The quality of all products such as harmful microbial levels and heavy metal levels were also checked by CKD Bio Corp.
2.3. Sample Collection
Blood collection was performed after fasting for 12 h, and postprandial blood collection was performed at 0, 6, and 12 weeks after ingestion of a 500-mL TNO fat challenge formula (palm oil 60 g, dextrose 83.5 g, protein powder 20 g, and 320 mL water) along with the study capsules. Blood was collected using EDTA Vacutainer blood collection tubes (Becton Dickinson, Franklin Lakes, NJ, USA) and stored in a 1.5-mL Eppendorf tube at −80 °C. Plasma and red blood cells were separated from the blood by centrifugation (1500×
g, 4 °C, 15 min). Chylomicrons were isolated by adding sodium chloride solution to the plasma [
16].
The fecal samples were collected at Weeks 0 and 12 using commercial kit for stool sampling (Medi4you, Seoul, Korea). Participants sent stool samples with ice pack and immediately stored at −80 °C without any treatment until analysis.
2.4. Blood Lipid Biomarker in Plasma
Biochemical parameters were analyzed in ISO 15180 certificated lab (Green Cross Laboratory, Seoul, Korea) or Ewha Woman’s University Mokdong Hospital. TG and chylomicron (CM) TG plasma levels were analyzed using an enzyme analyzer (BioTek Instruments Inc., Winooski, VT, USA), and the results were detected with a triglyceride colorimetric assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). Apolipoprotein (Apo)B-48 and ApoB-100 plasma levels were analyzed by LBIS Human Apo-48 ELISA Kit (Shibayagi Co., Ltd., Gunma, Japan) and Human Apolipoprotein B ELISA PRO kit (Mabtech AB, Stockholm, Sweden), respectively, and the absorbance was detected on a SpectraMax 190 and StatFax 2600 microplate reader (Molecular Devices, San Jose, CA, USA).
2.5. Intestinal Microbial Community Analysis
DNA extraction, library preparation, and next-generation sequencing (NGS) for microbiome analysis were all commissioned by Macrogen, Inc. (Seoul, Korea). In total, 134 fecal samples were collected and stored below −80 °C. To prepare the library, DNA was extracted from the fecal samples using a PowerSoil® DNA Isolation Kit (MO BIO Laboratories, Carslbad, CA, USA). Thereafter, PCR was performed to amplify the V3-V4 region of 16S rRNA genes according to the Miseq sequencing Library Prep guide (Illumina Inc., San Diego, CA, USA) with the 16S V3-V4 primer 16S Amplicon PCR Forward Primer 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and 16S Amplicon PCR Reverse Primer 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC.
A subsequent limited-cycle amplification step was then performed to add multiplexing indices and Illumina sequencing adapters. The final products were normalized and pooled, and the size of the libraries were verified using TapeStation DNA screentape D1000 (Agilent Technologies, Santa Clara, CA, USA). Sequencing was then performed on the HiSeq2500 platform (Illumina Inc.).
2.6. Intestinal Microbial Community Analysis
2.6.1. Fecal Microbial metaBolites: Biogenic Amines, SCFAs, Indoles and Phenols
Analysis of the biogenic amines was performed with reference to the method described by Saarinen [
17] using a Shiseido SI-2 HPLC system (Kyoto, Japan) equipped with a fluorescence detector. Separation was carried out on a Cadenza 5CD-C18 (5 µm, 250 mm × 3 mm) column equipped with a Cadenza 5CD-C18 Guard Cartridge (5 µm, 5 mm × 2 mm; Imtakt USA, Portland, OR, USA). Total biogenic amines were defined as the sum of agmatine, methylamine, ethylamine, pyrrolidine, dimethylamine, propylamine, tryptamine, butylamine, 2-phenyl ethylamine, putrescine, cadaverine, histamine, tryamine, spermidine, and sperimine (Sigma Aldrich Corporation. Ltd., St. Louis, MO, USA). Heptylamine was used as the internal standard (IS).
The short chain fatty acids (SCFAs) in the feces were determined according to the method described by Costabile et al. [
18]. Unbranched SCFAs, including acetic acid, propionic acid, butyric acid, and valeric acid, and branched SCFAs, including, iso-butyric acid and iso-valeric acid (Sigma Aldrich Corporation), were used as external standard solutions. The sum of the branched and unbranched SCFAs was defined as the total SCFA content. 2-Ethyl butyric acid was used as the internal standard. The SCFAs were analyzed using GC with a flame ionization detector (FID) on an Agilent 6890N system (Agilent Technologies) equipped with a DB-FATWAX UI column (0.25 µm, 30 m × 0.25 mm; Agilent Technologies).
The indoles and phenols were analyzed according to the methods described by Flickinger et al. [
19] and Park et al. [
20]. GC-FID was performed essentially as described for the SCFA analysis, with the exception of the use of a DB-17 column (0.25 µm, 30 m × 0.25 mm; Agilent Technologies). Indole, phenol,
p-cresol, and skatole were used as external standards in the range of 30–500 µmol/L, and 4-isopropylphenol (Sigma Aldrich Corporation) was used as the internal standard.
2.6.2. Fecal Excretion Fat-Related Metabolites: Neutral Sterols and Bile Acids
Neutral sterols and bile acids were analyzed as described by Anita Fechner et al. [
21] and Sylvia Keller [
22] using an Agilent GC 7890B-5977B MSD GC-MS system (Agilent Technologies) equipped with a DB-5MS column (0.25 µm, 30 m × 0.25 mm). Total sterols were defined as the sum of cholesterol, coprostanol (Steraloids Inc., Newport, RI, USA), coprostanone (Carbosynth Ltd., Newbury, UK), cholestanol, cholestanone, and cholestenone, and 5α-cholestane was used as the internal standard. All reagents except for coprostanol and coprostanone were purchased from Sigma Aldrich Corporation
Sterol extracts were saponified by the addition of NaOH for bile acid analysis. The primary external standards were cholic acid and chenodeoxycholic acid, and the secondary external standards were iso-lithocholic acid (Steraloids Inc.), lithocholic acid, iso-deoxycholic acid (Steraloids Inc.), and deoxycholic acid; hyodeoxycholic acid was used as the internal standard. All reagents were purchased from Sigma-Aldrich Corporation unless otherwise indicated.
2.7. Statistical Analysis
The sample size was estimated to be 35 subjects per group to provide an 80% power of demonstrating a significant difference in TG levels, based on similar previous studies and a drop-out rate of 20% [
23,
24,
25]. Cross-group comparisons of all randomized subjects were performed for intent of treatment (ITT) analysis. Correlation analysis was performed with a per-protocol (PP) analysis including only those who completed the study. Based on the normality assessment, the data were log transformed to obtain a normal distribution prior to analysis. Student’s
t-test was used to compare the characteristics of the subjects at baseline for continuous variables and to compare microbial communities between treatment groups, whereas categorical variables were analyzed by the chi-square test. In all functional assessments, a linear mixed-effect model was used with group, time, and group-by-time interactions (group×time) as fixed-effect variables for analyzing changes at baseline from Weeks 0 to 12, and as random-effects variables to analyze the difference between groups according to the intake period. The area under the curve (AUC) was calculated using the trapezoidal rule for postprandial lipid and related metabolic parameters at both 0 and 12 weeks. Pearson’s correlation coefficients were used to determine the correlations among variables. Statistical analysis was performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). When a
p-value was less than 0.05, the difference was considered to be statistically significant for all parameters.
4. Discussion
There has been increasing interest in studying the ability of probiotics to maintain healthy blood lipid levels. In a 12-week study of 128 subjects, ingestion of strains
L. curvatus HY7601 and
L. plantarum KY1032 reduced TG levels, and increased the Apo AV and LDL particle size [
26]. In another clinical study, low-fat yogurt made with
B. longum was consumed by 32 subjects for four weeks, resulting in a reduction in TC levels [
27]. In addition, 60 subjects that received an
L. plantarum strain for 12 weeks showed reduced LDL-cholesterol levels [
28]. These studies have motivated further research regarding the lipid improvement function of probiotics; however, elucidating the precise mechanism has been difficult. Based on promising results from in vitro studies and animal models, we sought to identify the effects of LPQ180 on the improvement of lipid levels in human subjects with relatively healthy or slightly higher TG levels and to establish the underlying mechanisms through analysis of intestinal microbial metabolites and microorganisms from fecal samples.
Previous clinical studies only focused on the effect of probiotics on plasma lipids, whereas the present study further highlights the effects of strain LPQ180 on the intestinal environment, as well as providing insight into mechanisms that further improve lipid levels under certain conditions. Intake of LPQ180 in the current study reduced LDL-cholesterol and ApoB-100 plasma levels, the major lipid markers in the blood on fasting, which concurred with results of previous studies [
28,
29,
30]. Although only LDL-cholesterol and ApoB-100 levels were decreased by LPQ180 supplementation for 12 weeks, postprandial blood levels of lipid markers such as TG, ApoB-48, and ApoB-100 were also significantly decreased in the LPQ180 group compared to those in the placebo group. Previous studies have shown that CM TG levels in arterial plasma increased during the postprandial period with a peak at 240–300 min [
31]. LPQ180 was screened using inhibitory effect for porcine pancreatic lipase activity [
9]. Therefore, our results indicate that LPQ180 may help maintain healthy postprandial lipid metabolism, as the AUC value of CM TG was significantly reduced compared with that of the placebo group 4–6 h after TNO fat formula ingestion.
There is growing recognition of the importance and role of gut microorganisms in metabolic disorders such as obesity, metabolic syndrome, and diabetes [
32,
33,
34]. In particular, a greater abundance of beneficial intestinal microflora is considered to be effective in improving intestinal immunity and overall intestinal health [
35,
36]. Although we did not find a significant effect of LPQ180 on gut microflora diversity, analysis of the microbial community at the genus level showed that abundance of
Eggerthella spp. tended to decrease in the LPQ180 group after 12 weeks. According to Leo Lahti [
37], Actinobacteria, the phylum to which
Eggerthella belongs, plays a role in endogenous lipid metabolism and is positively correlated with plasma cholesterol. In addition, Fu et al. [
38] analyzed the correlation between intestinal microflora and lipid level by age, sex, and host genetics, finding a significant positive correlation with
Eggerthella spp. abundance. In the above study, the
Eggerthella spp. has been shown to increase blood TG levels and reduce HDL-cholesterol. Therefore, the present results suggest that intake of LPQ180 may help improve blood lipids by reducing levels of
Eggerthella spp.
Polysaccharides that cannot be digested by humans are decomposed by beneficial intestinal bacteria such as probiotics, resulting in the production of SCFAs as byproducts [
39,
40]. The accumulation of SCFAs reduces the pH of the intestinal environment, which consequently inhibits the growth of harmful bacteria in the colon and results in a suitable environment for beneficial intestinal microorganisms [
41]. Conversely, harmful bacteria such as
Escherichia coli,
Clostridium sp.,
Bacteroides fragilis,
Enterococcus faecalis, and
Proteus sp. produce decay products such as ammonia, amines, indoles, and secondary bile acids [
42,
43,
44,
45]. Previous studies have demonstrated that high levels of branched SCFAs in hypercholesterolemic rats were correlated with a high prevalence of
Odoribacter (Bacteroidetes) producing iso-valeric acid and with
Ruminococcus (Firmicutes) requiring branched SCFAs [
46]. Metabolite levels in the above in vivo study were associated with changes in intestinal microflora. The Shannon index is a kind of species diversity index. It is interpreted that the higher is the value, the higher is the diversity of microorganisms. In addition, in the case of NMDS analysis of the microbial community by sample, the more distinctly separated are the groups, the more it can be interpreted that the change of microflora occurred significantly. Unfortunately, the current study did not show a difference in microbiota diversity based on the count of OTUs. This lack of difference may be related to large individual variations because of the small number of subjects. We also did not find a significant effect of LPQ180 intake on changes in SCFAs. It seems that LPQ180 as probiotics have a limit to increase gut beneficial bacteria. However, the significant reduction of indole and phenols confirmed that intake of LPQ180 affected gut environments, positively.
Correlation analysis further confirmed the link between the intestinal microbial community and changes in the lipid profile induced by LPQ180. Reductions of TG, TC, ApoB, and LDL-cholesterol lipid markers were more notable in subjects with high baseline levels of gut bacteria
R. bromii and
K. alysoides, which produce SCFAs by decomposing indigestible components [
47,
48]. Subjects with a higher baseline level of
B. intestinihominis, which has immunomodulatory effects by increasing inflammatory control cytokines such as IFNγ that have antiviral and antitumor properties [
49], exhibited reduced levels of TG and biogenic amines. Subjects with higher baseline levels of
F. plautii, which is known to degrade flavonoids in the intestine [
50], also exhibited increased HDL-cholesterol levels after LPQ180 intake for 12 weeks. With respect to intestinal microbial metabolites, a higher baseline level of oral and intestinal commensal bacteria, including anti-inflammatory species
Streptococcus salivarius [
51], resulted in an increase in SCFAs and a decrease in primary bile acids in the LPQ180 group. In addition, subjects with a higher baseline level of
R. mucilaginosa, which causes infection through induction of inflammatory marker cyclooxygenase-2 in hosts with compromised immunity [
52], exhibited increased SCFAs and decreased primary bile acids. Moreover, correlation analysis demonstrated that LPQ180 significantly help to maintain postprandial lipid responses in subjects with high levels of metabolites such as biogenic amines and indole and phenols. No significant correlations of any of these biomarkers were detected in the placebo group, and higher indole and phenol levels were actually associated with increased changes in TG, VLDL, CM TG, and ApoB-48, demonstrating the opposite effects observed in the LPQ180 group.
