Hypercholesterolemia, characterized by excessive cholesterol in the blood, is a major risk factor for cardiovascular disease. Reducing serum cholesterol levels by 1% can decrease the likelihood of cardiovascular disease by as much as 3% (Manson et al., 1992).

Statins are representative drugs widely prescribed to treat hypercholesterolemia. They control cholesterol levels by inhibiting cholesterol synthesis in the liver. However, statin-related symptoms (SAS), such as muscle symptoms, diabetes, cognitive problems, depression, liver toxicity, and hemorrhagic stroke, have been reported (Thompson et al., 2016). Therefore, research is actively being conducted to lower cholesterol levels without side effects, and in particular, lactic acid bacteria are attracting attention as a potential cholesterol-lowering agent.

Lactic acid bacteria are Gram-positive bacteria that produce lactic acid, with representative examples including Lactobacillus spp., Bifidobacterium spp., and Streptococcus spp. Lactic acid bacteria are employed as probiotics to promote human health, such as lowering cholesterol (Anandharaj et al., 2014; Fuentes et al., 2016). The cholesterol-lowering mechanism of lactic acid bacteria has been elucidated, but further research is required because the mechanism is diverse and strain-specific. Representative mechanisms include bile acid deconjugation and cholesterol adsorption by lactic acid bacteria, both of which lower cholesterol by facilitating the excretion of bile acid and cholesterol (Reis et al., 2017).

Probiotics are live bacteria beneficial to humans when consumed appropriately; however, they require rigorous safety evaluation because they can cause side effects such as antibiotic resistance, deleterious metabolic activities, and bacteremia (Piqué et al., 2019). Additionally, factors such as temperature, acidity, and storage period affect the survival rate of probiotics (Fossi et al., 2017; Hossain et al., 2020). Heat-killed strains are a suitable alternative to address these problems. They are advantageous regarding safety and stability and exhibit effects similar to probiotics even in an inactivated state (Lin et al., 2007; Kang et al., 2020). Therefore, in this study, we selected lactic acid bacteria with excellent cholesterol-lowering ability. Heat-killed strains were administered to rats induced with hypercholesterolemia to assess the effect of lactic acid bacteria on cholesterol levels and to elucidate the cholesterol-lowering mechanism.

Lactic acid bacteria

Lactic acid bacteria used in this study were strains developed by MNHBio. Co., Ltd. (Korea). Strains were isolated from fermented food, and the process was conducted according to the internal regulations of MNHBio. Co., Ltd. The strains were cultured in MRS broth (Difco) at 37℃ for 24 h. After culturing, 20% glycerol stocks were prepared and stored at -80°C before use.

Measurement of cholesterol-lowering ability

The lactic acid bacteria were cultured in MRS broth (Difco) at 37℃ for 24 h. 1% of the culture (approximately 5.0 × 10⁸ CFU/ml) was inoculated into MRS broth supplemented with 0.2% (w/v) sodium thioglycolic acid (Aladdin Scientific), 0.3% (w/v) sodium taurocholic acid (Sigma) and cholesterol (100 μg/ml, Sigma) (Lim, 2011; Kim et al., 2020). Cholesterol mediums without bacteria or with Escherichia coli were used as control.

Cholesterol levels in the supernatant obtained by centrifugation at 3,000 rpm for 15 min at 4°C were measured by modifying the o-phthalaldehyde method (Rudel and Morris, 1973). 1 ml of the supernatant, 2 ml of 33% (w/v) potassium hydroxide, and 3 ml of absolute ethanol were mixed thoroughly and then incubated at 60°C for 15 min. After cooling the mixture, 5 ml of hexane and 1 ml of distilled water were added and vigorously mixed. The phase-separated hexane layer was transferred to another glass tube and evaporated under nitrogen. 10 min after adding 4 ml of o-phthalaldehyde reagent (0.5 mg/ml, Sigma) to the glass tube, 2 ml of concentrated sulfuric acid was slowly added. After 10 min, absorbance was measured at 550 nm. Cholesterol of various concentrations in the MRS broth was measured, and then a standard curve was drawn to calculate the cholesterol concentration in the supernatant.

Identification of cholesterol-lowering bacteria

To identify strains with excellent cholesterol-lowering abilities, 16S rRNA sequence analysis was conducted by Macrogen (Korea). The homology of the 16S rRNA sequences was analyzed using NCBI BLAST. A phylogenetic tree was constructed using the Neighbor-joining model with 1,000 bootstraps in the MEGA 11 program.

Measurement of cholesterol in cell lysate

Cholesterol concentration in cell lysate was measured to confirm whether the removal of cholesterol by the strain occurred through cell wall adsorption. The strains were inoculated at 5.0 × 10⁸ CFU/ml into the cholesterol medium and incubated at 37°C for 24 h. The culture was centrifuged at 3,000 rpm for 15 min at 4°C. The cell pellet was washed twice with phosphate-buffered saline (PBS, pH 7.4). 500 μl of lysozyme (1 mg/ml, Sigma) was added to the cell pellet and incubated at 37°C for 4 h. The cell suspension was homogenized at an amplitude of 40%, 8 pulse cycles (15 sec on, 60 sec off) using an ultrasonic dismembrator (VCX-130; Invitrogen). Cell lysate was obtained as supernatant by centrifugation at 3,000 rpm for 15 min at 4°C. Cholesterol in the culture supernatant and cell lysate was measured as previously described.

Cholesterol-lowering ability of heat-killed bacteria

To compare the cholesterol-lowering ability of growing and heat-killed bacteria, heat-killed bacteria were prepared by autoclaving MRS cultures at 121°C for 15 min. Heat-killed bacteria were inoculated at 5.0 × 10⁸ CFU/ml, either alone or mixed, into the cholesterol medium and incubated at 37°C for 24 h. Cholesterol levels in the supernatant, obtained by centrifugation at 3,000 rpm for 15 min at 4°C, were measured as previously described.

Preparation of samples (MB2002)

The strains were cultured in MRS broth at 37℃ for 24 h. After autoclaving at 121℃ for 15 min, the cultures were centrifuged at 3,000 rpm for 15 min at 4℃. The heat-killed cells were diluted with supernatant to 1 × 10⁸, 1 × 10⁹, and 1 × 10¹⁰ CFU/ml and used as samples (MB-2002-L, -M, -H).

Animal model

Five-week-old male Sprague-Dawley rats (SD rats, Orient Bio Inc.) were acclimatized for one week and then were divided into six groups (n = 8 per group except for the normal diet group). Normal diet group (ND, n = 6) was fed a normal diet (D10001; JABIO), while the other groups were fed a high-cholesterol diet (HCD, D12336; JABIO) (Table 1). Rosuvastatin (Shandong Natural Micron Pharm Tech) was prepared at 10 mg/kg and orally administered 0.5 ml to the rosuvastatin group (HCD + R). Three experimental groups (HCD + L, HCD + M, and HCD + H) were orally administered 0.5 ml of the MB-2002-L, -M, or -H. The same amount of drinking water was orally administered to the control groups (ND and HCD). Rats were housed in an environment with a temperature of 22 ± 2℃, humidity of 55 ± 5%, and a 12 h light/dark cycle (08:00–20:00), and consumed water and food ad libitum. Administration was performed daily, and body weight and food intake were monitored twice weekly. The procedures for this study were approved by the Seoul National University Hospital Institutional Animal Care and Use Committee (SNUH-IACUC, BA-2210-353-003-03) and were conducted following regulations.

Table 1

Composition of the rat diets

Ingredient (g/kg)	NDa	HCDb
Casein	200.0	75.0
Soy protein	-	130.0
DL-Methionine	3.0	2.0
Corn starch	150.0	275.0
Maltodextrin 10	-	150.0
Sucrose	509.0	39.0
Cellulose, BW200	50.0	90.0
Soybean oil	50.0	50.0
Cocoa butter	-	75.0
Coconut oil, 76	-	35.0
Mineral mix, S10001	35.0	35.0
Calcium carbonate	-	5.5
Sodium chloride	-	8.0
Potassium citrate	-	10.0
Vitamin mix, 10001C	1.0	1.0
Choline bitartrate	2.0	2.0
Cholesterol, USP	-	12.5
Sodium cholic acid	-	5.0

^a ND, Normal diet

^b HCD, High-cholesterol diet

Measurement of lipids in plasma

Lipid profiles in plasma were analyzed using triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) kits (Asan Pharm) according to the manufacturer’s instructions. Low-density lipoprotein cholesterol (LDL-C) was calculated using Friedewald’s formula based on the TG, TC, and HDL-C concentrations (Friedewald et al., 1972).

Measurement of lipids in the liver and feces

One g of the liver was suspended in 1 ml 0.9% sodium chloride (saline) solution and homogenized at an amplitude of 40%, 8 pulse cycles (15 sec on, 60 sec pause) using an ultrasonic dismembrator (VCX-130; Invitrogen). 4 ml of saline solution and 5 ml of chloroform-methanol (2:1) were added to the suspension (Folch et al., 1957). The solution was vigorously vortexed for 1 min and left at room temperature for 20 min to extract lipids. This was centrifuged at 2,000 rpm for 10 min, and the solvent phase was collected using 1 ml sterile syringes (Kraus et al., 2015). The solvent was evaporated using a rotary evaporator (Buchi, R-114) and then dissolved in 1 ml of methanol. Using this solution as a sample, TG and TC were analyzed using the kits.

Feces obtained for 2 days before the autopsy were ground with a pestle and suspended in 5 ml saline solution and 5 ml of chloroform-methanol (2:1). The subsequent processes (extraction, evaporation, and dissolving) were the same as for the liver lipid extraction. The TC and bile acid (BA) were analyzed using the kits (BA kit; Crystal Chem) according to the manufacturer’s instructions.

Western blot analysis

Expression levels of ATP-binding cassette subfamily G member 5/8 (ABCG5/8) were determined by western blot analysis. Proteins were extracted from the liver and cecum using RIPA buffer (HanLab) with phosphatase and protease inhibitors (Roche) and ultrasonication under the same conditions as lipid extraction. Protein concentrations were standardized to 50 µg using a Pierce BCA assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The proteins were fractionated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were sequentially immunoblotted with 5% skim milk and primary antibodies (Abcam, Cell Signaling Technology), then incubated with secondary antibodies (Cell Signaling Technology). Signals were generated by treating with the ECL western blot substrate (Thermo Fisher Scientific, USA). Protein band images were obtained through the ChemiDoc MP imaging system (Bio-Rad), and band intensities were quantified using ImageJ software.

Statistical analysis

All experiments were conducted in triplicate, and the results are expressed as mean and standard deviation (mean ± SD). One-way ANOVA followed by Tukey’s multiple comparisons test was used to evaluate significant differences (p < 0.05). The statistical analysis was performed using Prism 10 (GraphPad Software).

Cholesterol-lowering bacteria

Eight strains developed by MNHBio. Co., Ltd. were screened for cholesterol-lowering ability. As a result of measuring the residual cholesterol concentration in the supernatant after culturing in MRS broth containing 0.1 mg/ml cholesterol, three strains showed cholesterol-lowering ability (Fig. 1). UBC-15 had the lowest cholesterol concentration of 0.032 ± 0.015 mg/ml, followed by UBC-61 (0.058 ± 0.009 mg/ml) and UBC-U90 (0.088 ± 0.01 mg/ml). The cholesterol concentration in the control groups was not different from the initial cholesterol concentration, confirming that environmental factors did not affect cholesterol levels. The cholesterol reduction rates of UBC-15 (69.1%) and UBC-61 (44.6%) were higher than those of previously reported strains (Dambekodi and Gilliland, 1998; Lee et al., 2009; Tomaro-Duchesneau et al., 2014; Elaby et al., 2018). Therefore, we selected UBC-15 and UBC-61 for their excellent cholesterol-lowering ability. The strains were identified by 16S rRNA sequence analysis and phylogenetic tree. UBC-15 and UBC-61 were highly similar (> 99.0%) with Bifidobacterium longum and Limosilactobacillus reuteri, respectively (Supplementary data Figs. S1 and S2). These strains have been deposited in the Korea Collection for Type Cultures (KCTC) and assigned the following numbers: B. longum UBC-15 (KCTC15677BP) and L. reuteri UBC-61 (KCTC15676BP).

Fig. 1. Cholesterol-lowering ability of lactic acid bacteria. Lactic acid bacteria were inoculated into MRS broth containing 100 μg/ml cholesterol and cultured at 37℃ for 24 h. After incubation, the cholesterol concentration in the supernatant was measured. Control was not inoculated with the bacteria; negative control was inoculated with E. coli. Results are presented as means ± SD, n = 3. *p < 0.05, ***p < 0.001 compared to the control.

Reduction of cholesterol by cell wall adsorption

One of the well-known cholesterol-lowering mechanisms through which lactic acid bacteria reduce cholesterol is adsorbing it to the cell wall (Dambekodi and Gilliland, 1998; Lee et al., 2015). Cholesterol concentration in cell lysates was measured to investigate this mechanism in B. longum UBC 15 and L. reuteri UBC-61. The cholesterol concentrations in cell lysates were 0.046 ± 0.008 mg/ml for B. longum UBC-15 and 0.04 ± 0.016 mg/ml for L. reuteri UBC-61 (Fig. 2). Most of the cholesterol removed by the strains was recovered in the cell lysates, resulting in total cholesterol concentrations that were not significantly different from the control. The results suggest that B. longum UBC-15 and L. reuteri UBC-61 lower cholesterol concentration by adsorbing cholesterol to the cell wall. Previous studies have reported that lactic acid bacteria and cholesterol adsorption are associated with the production of exopolysaccharides (Jurášková et al., 2022). Exopolysaccharide production correlates with cholesterol concentrations, affecting its lowering ability (Tok and Aslim, 2010). Therefore, it is inferred that B. longum UBC-15 and L. reuteri UBC-61 produced more exopolysaccharides than the other six strains, resulting in their high cholesterol-lowering ability.

Fig. 2. Cholesterol concentration in the cell lysate and supernatant of selected strains. B. longum UBC-15 and L. reuteri UBC-61 were inoculated into MRS broth containing 100 μg/ml cholesterol and cultured at 37℃ for 24 h. After incubation, the cell pellet and supernatant were obtained by centrifugation. The cell pellet was then disrupted using ultrasonication to extract cell lysate. Cholesterol concentrations in the cell lysate and supernatant were measured. Control was not inoculated with the bacteria. Results are presented as means ± SD, n = 3.

Cholesterol-lowering ability of heat-killed B. longum UBC-15 and L. reuteri UBC-61

Several studies reported that strains maintained their cholesterol-lowering ability even after heat-killing (Kim et al., 2009; Ting et al., 2015). Comparative experiments with growing cells were conducted to evaluate the cholesterol-lowering ability of heat-killed B. longum UBC-15 and L. reuteri UBC-61. The cholesterol-lowering ability of heat-killed B. longum UBC-15 and L. reuteri UBC-61 was lower than that of growing cells, but no significant difference was observed (Fig. 3). This finding is consistent with previous reports indicating that heat-killed cells have a lower cholesterol-lowering ability than growing cells (Choi, 2014; Tjandrawinata et al., 2022). In contrast, Kim et al. (2020) have reported that the cholesterol-lowering ability is the same for both growing and heat-killed cells. This difference appears to depend on the method of heat-killing. The autoclaving method used for heat-killing appears to have affected the cell surface, thereby reducing the cholesterol-lowering ability compared to growing cells. In other words, it can be inferred that cholesterol reduction occurred through cell adsorption in heat-killed cells, similar to growing cells. However, in this study, considering the insignificant difference between growing and heat-killed cells, as well as the safety and stability issues associated with growing cells, heat-killed cells are considered to be more practical.

Fig. 3. Cholesterol-lowering ability of heat-killed bacteria. B. longum UBC-15 and L. reuteri UBC-61 were autoclaved at 121℃ for 15 min. The growing (□) and heat-killed (■) cells of the two strains were inoculated in MRS broth containing 100 μg/ml cholesterol and cultured at 37℃ for 24 h. After incubation, the cholesterol concentration in the supernatant was measured. Control was not inoculated with the bacteria. Results are presented as means ± SD, n = 3. **p < 0.01, ***p < 0.001 compared with the control; ns: no significant difference between growing and heat-killed cells.

Recent studies have reported the potential benefits of using mixed strains (Chapman et al., 2011). Fukushima and Nakano (1996) reported that a mixture of bacteria was more effective in reducing hypercholesterolemia than a single strain. Accordingly, the difference in cholesterol-lowering ability between heat-killed strains treated alone and mixed was compared. As shown in Fig. 4, the mixture of heat-killed B. longum UBC-15 and L. reuteri UBC-61 reduced cholesterol concentration to 0.01± 0.006 mg/ml, which is lower than when treated individually. The residual cholesterol concentration of the mixed strains was approximately half that of the single strains, suggesting that the cholesterol reduction rate was enhanced by the additive effect of the two strains.

Fig. 4. Cholesterol-lowering ability of mixed strains. B. longum UBC-15 and L. reuteri UBC-61 were autoclaved at 121℃ for 15 min. The two heat-killed strains were inoculated individually or simultaneously in MRS broth containing 100 μg/ml cholesterol and cultured at 37℃ for 24 h. After incubation, the cholesterol concentration in the supernatant was measured. Control was not inoculated with the bacteria. Results are presented as means ± SD, n = 3. ***p < 0.001, ****p < 0.0001 compared with the control.

Consequently, we found that heat-killed B. longum UBC-15 and L. reuteri UBC-61 had excellent cholesterol-lowering ability, with higher ability when combined rather than treated individually. Based on these results, a mixture of the two heat-killed strains was developed as MB-2002 and used in subsequent animal studies.

Body weight, food intake, and liver weight

Body weight gain, food intake, and liver weight were measured in rats fed a high-cholesterol diet and MB-2002 for five weeks. These results are shown in Table 2. The groups fed a high-cholesterol diet exhibited increased body weight and liver weight compared to the normal diet (ND) group. There were no significant differences among the test groups. Similarly, Kim et al. (2018) showed that rats fed a high-cholesterol diet tended to gain weight, and Kim et al. (2020) reported an increase in liver weight with a high-cholesterol diet, finding no significant difference between the test groups. In contrast, food intake did not significantly differ among the groups. These results suggest that administering MB-2002 to rats fed a high-cholesterol diet had no significant effect on body weight gain, food intake, or liver weight.

Table 2

Effects of MB-2002 on the body weight gain, food intake, and liver weight of rats fed a high-cholesterol diet

	ND	HCD	HCD + R	HCD + L	HCD + M	HCD + H
Weight gain (g/day)	6.18 ± 1.22a	8.12 ± 0.94	7.97 ± 0.91	7.55 ± 1.38	8.07 ± 0.78	7.53 ± 1.38
Food intake (g/day)	23.68 ± 1.7	22.81 ± 1.62	23.14 ± 1.42	22.68 ± 1.47	22.97 ± 1.37	22.50 ± 1.24
Liver weight (g)	14.74 ± 1.90b	30.21 ± 1.50	28.30 ± 2.08	29.74 ± 2.45	29.41 ± 1.95	26.78 ± 3.36

Data was expressed as mean ± SD, n = 8 (except for ND; n = 6).

ND, Normal diet; HCD, high-cholesterol diet; R, Rosuvastatin; L, MB-2002-L 1 × 10⁸ dead cells/rat; M, MB-2002-M 1 × 10⁹ dead cells/rat; H, MB-2002-H 1 × 10¹⁰ dead cells/rat.

^a p < 0.05, ^b p < 0.0001 compared with the HCD.

Effect of MB-2002 on lipid profile in plasma, liver, and feces

The effects of a high-cholesterol diet and MB-2002 on lipid profiles in plasma, liver, and feces are shown in Table 3. Plasma total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels significantly increased in the HCD group. Hepatic TG and TC levels were significantly increased in the HCD group. It has been reported that a continuous high-cholesterol diet increases cholesterol and triglycerides in the blood and liver (Lee et al., 2008; Kim et al., 2018). This study showed similar results, however, plasma TG levels were decreased in the HCD group. Similarly, Wang et al. (2010) reported that serum TG levels were significantly lower than those in the control group, which was attributed to the unique metabolic state of the animals and the diet composition. MB-2002 (HCD + L, HCD + M, HCD + H) significantly decreased plasma TC and LDL-C levels in a dose-dependent manner. High-density lipoprotein cholesterol (HDL-C) levels were lowest in the HCD group. In the MB-2002 groups, HDL-C significantly increased dose-dependently, and the HCD + H group showed no significant difference from the ND group. Liver TG levels significantly decreased in the HCD + H group, and TC levels were significantly decreased in all the MB-2002 groups. Fecal TC and bile acid (BA) levels increased in the HCD group and further increased significantly in a dose-dependent manner with MB-2002 administration. The cholesterol-lowering mechanism of intestinal lactic acid bacteria is known to include excretion of cholesterol through cell wall adsorption, the conversion of cholesterol to coprostanol, and bile acid excretion by deconjugating bile acids (Jang et al., 2008; Reis et al., 2017). In in vitro experiments confirmed that MB-2002 reduced cholesterol concentration in the medium through cell wall adsorption. Since it was administered in an inactivated state, it appears that cholesterol excretion occurred through cell wall adsorption, leading to an increase in TC levels in feces. Similarly, based on reports that BA is directly adsorbed to exopolysaccharides, it appears that BA is excreted through cell wall adsorption, resulting in increased fecal BA levels (Pigeon et al., 2002).

Table 3

Effects of MB-2002 on plasma, liver, and fecal lipid profiles in rats fed a high-cholesterol diet

	ND	HCD	HCD + R	HCD + L	HCD + M	HCD + H
Plasma
TG (mg/dl)	57.20 ± 12.45c	42.28 ± 11.28	49.79 ± 14.01	41.93 ± 6.66	43.09 ± 8.83	46.44 ± 5.02
TC (mg/dl)	74.50 ± 18.63d	300.0 ± 75.22	286.3 ± 59.07	241.3 ± 20.04b	230.3 ± 59.06c	215.2 ± 32.76d
LDL-C (mg/dl)	19.48 ± 7.58d	275.5 ± 77.43	258.1 ± 65.64	209.2 ± 29.06b	198.0 ± 61.94d	172.4 ± 35.71d
HDL-C (mg/dl)	43.58 ± 19.94d	16.00 ± 8.80	18.21 ± 8.48	22.90 ± 9.87	23.65 ± 9.33	34.63 ± 8.56c
Liver
TG (mg/g)	3.98 ± 0.43d	64.59 ± 0.38	44.26 ± 7.59d	59.35 ± 9.40	55.54 ± 5.52	52.49 ± 2.19a
TC (mg/g)	1.78 ± 0.19d	30.16 ± 0.94	25.93 ± 2.43	25.03 ± 3.54a	24.93 ± 4.03a	24.59 ± 0.68a
Fecal
TC (mg/g)	2.32 ± 1.21d	17.6 ± 6.63	28.26 ± 4.87c	26.27 ± 1.19b	32.52 ± 0.62d	35.24 ± 2.65d
BA (µmol/g)	12.22 ± 2.45b	19.11 ± 3.34	22.66 ± 3.36	23.95 ± 1.63a	27.40 ± 1.79c	27.75 ± 1.56d

Data was expressed as mean ± SD, n = 8 (except for ND; n = 6).

ND, Normal diet; HCD, high-cholesterol diet; R, Rosuvastatin; L, MB-2002-L 1 × 10⁸ dead cells/rat; M, MB-2002-M 1 × 10⁹ dead cells/rat; H, MB-2002-H 1 × 10¹⁰ dead cells/rat.

TG, triglyceride; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; BA, bile acid.

^a p < 0.05, ^b p < 0.01, ^c p < 0.001, ^d p < 0.0001 compared with the HCD group.

In conclusion, the results of this study suggest that MB-2002 dose-dependently not only reduced TC and LDL-C levels but also increased HDL-C, showing the possibility of reducing the rate of cardiovascular events (Park and Koh, 2011).

Effect of MB-2002 on ABCG 5/8 expression level

We examined the effects of a high-cholesterol diet and MB-2002 on the expression of ABCG5 and ABCG8 (ABCG5/8) in the liver and intestine (cecum) (Fig. 5A). As shown in Fig. 5B and 5C, a, a high-cholesterol diet downregulated the expression of liver ABCG5/8. MB-2002 upregulated liver ABCG5/8 expression by up to approximately 58.2% and 133.1%, respectively, compared to the HCD group. Similarly, a high-cholesterol diet downregulated the expression of cecum ABCG5/8, whereas MB-2002 upregulated it, with ABCG5/8 increasing by approximately 154.9% and 270.8%, respectively, in the HCD + H compared to the HCD group (Fig. 5D and 5E).

Fig. 5. Effects of MB-2002 on ABCG5 and ABCG8 expression in the liver and cecum rats fed a high-cholesterol diet. For western blot analysis, proteins were extracted from the liver and cecum, and (A) the expression of ABCG5 and ABCG8 was examined. The expression levels of ABCG5 and ABCG8 were calculated as a ratio to β-actin expression and expressed relative to the ND group. (B), (C) relative expression levels of ABCG5 and ABCG8 isolated from the liver and (D), (E) relative expression levels of ABCG5 and ABCG8 isolated from the cecum are presented as means ± SD, n = 3. ND, Normal diet; HCD, high-cholesterol diet; R, Rosuvastatin; L, MB-2002-L 1 × 10⁸ dead cells/rat; M, MB-2002-M 1 × 10⁹ dead cells/rat; H, MB-2002-H 1 × 10¹⁰ dead cells/rat. *p < 0.05, **p < 0.01 compared with the HCD group.

The heterodimer ABCG5/8 is a sterol channel found in the liver and intestines. Liver ABCG5/8 promotes biliary excretion of sterols and intestinal ABCG5/8 limits sterol absorption (Yu et al., 2014; Wang et al., 2015).

It has been reported that an atherogenic diet suppresses the expression of ABCG5/8 in the liver but not the intestines (Côté et al., 2013). Another study reported that a cholesterol-free and high-fat diet suppresses the expression of ABCG5/8 in the intestines (De Vogel-van Den Bosch et al., 2008). In this study, it is inferred that the expression of ABCG5/8 decreased in both the liver and intestines in the HCD group due to the high cholesterol and fat content of the diet. This reduction appears to have increased plasma TC and LDL-C levels in the HCD group. MB-2002 reversed the HCD-induced inhibition of ABCG5/8 expression. Some studies have reported that overexpression of ABCG5/8 increases biliary cholesterol excretion and inhibits cholesterol adsorption, leading to cholesterol excretion (Yu et al., 2002; Wu et al., 2004; Yoon, 2010). However, when only hepatic ABCG5/8 was overexpressed, biliary cholesterol excretion increased, but cholesterol adsorption in the intestine was not inhibited, and therefore plasma TC levels did not decrease (Wu et al., 2004). This suggests that ABCG5/8 in the liver and intestines function independently and that intestinal ABCG5/8 is relatively important in controlling cholesterol concentration. Furthermore, the results of this study were consistent with reports indicating that HDL-C levels increase when ABCG-5/8 expression is upregulated (De Souza et al., 2012; Yu et al., 2014). Therefore, MB-2002 dose-dependently upregulated the expression of ABCG5/8 in both the liver and cecum, thereby increasing cholesterol concentration in the intestinal lumen. The increased cholesterol was adsorbed by MB-2002 and then excreted, resulting in decreased plasma TC and LDL-C levels but increased plasma HDL-C.

본 연구에서 엠앤에이치바이오㈜에서 개발한 8종의 균주의 콜레스테롤 저하 능력을 조사하였다. Bifidobacterium longum UBC-15 (KCTC15677BP)와 Limosilactobacillus reuteri UBC-61 (KCTC15676BP)이 각각 69.1%와 44.6%의 콜레스테롤 감소율을 보여, 콜레스테롤 저하 능력이 우수한 균주로써 두 균주를 선발하였다. B. longum UBC-15와 L. reuteri UBC-61은 세포 흡착을 통해 콜레스테롤을 제거했으며, 두 균주 모두 열에 의한 사멸 후에도 콜레스테롤 저하 능력을 유지했다. B. longum UBC-15 및 L. reuteri UBC-61 혼합 처리 시 개별 처리보다 약 2배 정도 콜레스테롤을 감소시켰다. 이러한 균주 혼합물(MB-2002)을 고콜레스테롤혈증을 유도한 쥐에 투여하여 고콜레스테롤혈증 개선 효과를 확인하였다. 대조군(HCD)과 비교했을 때, MB-2002 투여군(HCD + L, HCD + M, HCD + H)의 혈중 총 콜레스테롤(TC)과 저밀도 지단백 콜레스테롤(LDL-C)이 용량 의존적으로 유의하게 감소한 반면, 고밀도 지단백 콜레스테롤(HDL-C)은 HCD + H군에서 유의미하게 증가하였다. 간의 중성지방(TG)과 총 콜레스테롤(TC)은 감소하였고, 분변의 콜레스테롤과 담즙산이 용량 의존적으로 유의하게 증가하였다. 또한, MB-2002이 간과 장의 ABCG5/8의 발현을 상향조절하여 콜레스테롤 배설을 유도하는 것으로 나타났다. 이러한 결과는 MB-2002가 콜레스테롤 배설을 촉진하여 고콜레스테롤혈증을 개선시키는, 더 나아가 심혈관 질환에 대한 위험을 줄일 수 있는 기능성 원료로서의 잠재력을 보여주었다.

Dae-Jung Kang is the founder of MNHBio Co., Ltd. and Min Ji Cho is an employee of MNHBio Co., Ltd. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.