Cholesterol Assimilation by Lactobacillus Probiotic Bacteria: An In Vitro Investigation

Excess cholesterol is associated with cardiovascular diseases (CVD), an important cause of mortality worldwide. Current CVD therapeutic measures, lifestyle and dietary interventions, and pharmaceutical agents for regulating cholesterol levels are inadequate. Probiotic bacteria have demonstrated potential to lower cholesterol levels by different mechanisms, including bile salt hydrolase activity, production of compounds that inhibit enzymes such as 3-hydroxy-3-methylglutaryl coenzyme A, and cholesterol assimilation. This work investigates 11 Lactobacillus strains for cholesterol assimilation. Probiotic strains for investigation were selected from the literature: Lactobacillus reuteri NCIMB 11951, L. reuteri NCIMB 701359, L. reuteri NCIMB 702655, L. reuteri NCIMB 701089, L. reuteri NCIMB 702656, Lactobacillus fermentum NCIMB 5221, L. fermentum NCIMB 8829, L. fermentum NCIMB 2797, Lactobacillus rhamnosus ATCC 53103 GG, Lactobacillus acidophilus ATCC 314, and Lactobacillus plantarum ATCC 14917. Cholesterol assimilation was investigated in culture media and under simulated intestinal conditions. The best cholesterol assimilator was L. plantarum ATCC 14917 (15.18 ± 0.55 mg/1010 cfu) in MRS broth. L. reuteri NCIMB 701089 assimilated over 67% (2254.70 ± 63.33 mg/1010 cfu) of cholesterol, the most of all the strains, under intestinal conditions. This work demonstrates that probiotic bacteria can assimilate cholesterol under intestinal conditions, with L. reuteri NCIMB 701089 showing great potential as a CVD therapeutic.


Introduction
Early studies by Anitschkow demonstrated that cholesterol administration results in symptoms of atherosclerosis [1], contributing to the lipid hypothesis, formulated by Duff and McMillan, which proposed an association between cholesterol and cardiovascular diseases (CVD) [2]. CVD are the leading cause of global mortality and morbidity and kill an estimated 16.7 million people worldwide [3]. Coronary artery disease (CAD), the most common CVD, is the leading cause of death and accounts for 7.25 million deaths globally [4]. The first line of treatment for CAD, dietary and lifestyle interventions, has proven inadequate. Pharmacological agents are being administered to target elevated low-density lipoprotein (LDL) levels [5,6], including 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), fibric acids, high-density lipoprotein stimulators (nicotinic acids), cholesterol absorption inhibitors (ezetimibe), and bile acid sequestrants. These pharmaceutics, however, have important limitations, with only 38% of dyslipidemia and 18% of CAD patients attaining the National Cholesterol Education Program goals [7]. Statins, the fundamental therapy for reducing LDL levels [8], fail to allow the majority of patients to meet their lipid goals [7,9,10]. There is a dire need for additional therapeutic modalities to lower cholesterol levels.
There has been increasing interest in probiotics, "microorganisms which when administered in adequate amounts confer a health benefit on the host, " research for the development of biotherapeutics [11,12]. In recent years, attention has been given to the ability of probiotic cells to reduce lipids and cholesterol levels [13], with several proposed mechanisms of action. One mechanism, bile salt hydrolase activity, is described in a recent review [14]. In addition, bacteria have been reported to assimilate cholesterol [15,16], thereby lowering luminal cholesterol levels available for absorption. Moreover, Lactobacillus bacteria can produce ferulic acid (FA) [17,18], which can inhibit hepatic HMG-CoA reductase and promote the excretion of acidic sterol [19]. With the demonstrated cholesterol-lowering properties of probiotic bacteria, further research is required to investigate the mechanism(s) by which the bacteria decrease cholesterol levels and to select bacteria capable of exerting cholesterollowering effects.
The goal of the presented work is to investigate Lactobacillus strains for their potential to assimilate cholesterol in both bacterial media and under simulated gastrointestinal conditions. This work provides grounds for future investigations to select a probiotic bacterial strain as a cholesterol-lowering therapeutic.

Bacterial Growth Media and Chemicals.
De Man-Rogosa-Sharpe (MRS) broth was purchased from Fisher Scientific and prepared according to the manufacturer's instructions. Cholesterol-polyethylene glycol (PEG) 600 was purchased from Sigma-Aldrich (Oakville, ON, Canada). Water was purified with an EASYpure Reverse Osmosis System and a NANOpure Diamond Life Science (UV/UF) ultrapure water system from Barnstead Scientific Instrumentation (Dubuque, IA, USA). All other chemicals were of analytical or HPLC grade and purchased from commercial sources. Prior to any assay, a MRS-agar plate was streaked from the frozen stock to ensure purity and incubated at 37 ∘ C with 5% CO 2 for 24 h. One colony from the agar plate was used to inoculate 10 mL MRS broth which was then incubated at 37 ∘ C for 24 h, prior to any experimental assay. Bacterial cell viabilities were determined using standard colony counting methods. Briefly, 10-fold serial dilutions were prepared using 0.85% (w/v) NaCl. Diluted bacterial samples were streaked on MRS-agar plates which were then incubated at 37 ∘ C and 5% CO 2 for 48 h. Colonies were counted from each plate and the colony forming units (cfu) were recorded. All viability tests were performed in triplicate to ensure accuracy and reproducibility.

Determining Probiotic Cholesterol Assimilation in MRS.
The probiotic Lactobacillus strains were investigated for their capability to assimilate cholesterol in MRS broth. Cholesterol-PEG 600 was added to MRS broth at a final concentration of 100 g/mL. A 1% (v/v) inoculum of each overnight probiotic culture was added to MRS-cholesterol-PEG 600 and incubated at 37 ∘ C for 24 h. Following incubation, viability was measured by standard colony counting methods. For cholesterol analysis, the probiotic suspensions were centrifuged at 4000 rpm for 10 min at 4 ∘ C using a Napco 2028R centrifuge (Fisher Scientific, Ottawa, ON, Canada) and the supernatants containing nonassimilated cholesterol were collected. Cholesterol concentrations in the different suspensions were determined using a protocol modified from Rudel and Morris [30]. Briefly, 500 L of 33% (w/v) KOH and 1 mL absolute ethanol were added to 500 L of the samples. The solutions were then vortexed for 1 min and incubated at 37 ∘ C for 15 min followed by cooling to room temperature. For phase separation, 1 mL of deionized water and 1.5 mL of hexanes were added to the solutions and vortexed for 1 min. The phases were then allowed to separate at room temperature. Subsequently, 500 L of the hexane layer was transferred into a glass tube and the solvent was evaporated under a flow of nitrogen gas. Once dried, 1 mL of 50 mg/dL o-phthalaldehyde reagent prepared in acetic acid was added and the samples were mixed. Following mixing, 250 L of concentrated H 2 SO 4 was added to each tube and the solutions were vortexed for 1 min, followed by incubation for 20 min at room temperature. The resulting absorbance was read at 570 nm using a UV spectrophotometer Victor3 V 1420 Multilabel Counter (Perkin Elmer, Boston, MA, USA). A standard curve of absorbance versus cholesterol concentrations was generated using the cholesterol concentrations: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, and 500 g/mL cholesterol in MRS ( 2 = 0.9875).
The cholesterol assimilated by probiotic Lactobacillus strains was determined as follows: cholesterol assimilated ( g/mL) Cholesterol assimilated by each Lactobacillus strain was also calculated in terms of percent cholesterol assimilation: Cholesterol assimilated by each Lactobacillus strain was calculated considering a dose of 10 10 cells: cholesterol assimilated (mg/mL) probiotic cell viability (cfu/mL) × 10 10 . ( Samples and standards were tested in triplicate to ensure accuracy and reproducibility.

Determining Probiotic Cholesterol Assimilation under
Simulated Intestinal Conditions. The Lactobacillus strains were investigated for their capability to assimilate cholesterol under simulated intestinal conditions. Simulated intestinal fluid was prepared according to US Pharmacopeia, with modifications [31]. Briefly, simulated intestinal fluid consisted of 0.85% (w/v) NaCl, 6.8 g/L potassium phosphate monobasic, 1.5 g/L Oxgall, 3.5 g/L glucose, and 10 g/L pancreatin. The pH was adjusted to 6.8 by the addition of 2 M NaOH. Cholesterol-PEG 600 was added to the simulated intestinal fluid at a final concentration of 100 g/mL. A 1% (v/v) inoculum of each overnight probiotic culture was added to the simulated intestinal fluid. The tubes were then incubated at 37 ∘ C for 24 h on a rotary shaker set at 100 rpm. Following a 24-hour incubation, viability was determined by standard colony counting methods. For cholesterol analysis, the probiotic suspensions were centrifuged at 4000 rpm for 10 min at 4 ∘ C to collect the supernatant. Cholesterol assimilation was determined, as previously described. A standard curve of absorbance versus cholesterol concentrations in simulated intestinal fluid was generated using the concentrations: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, and 500 g/mL cholesterol ( 2 = 0.9875). Samples and standards were tested in triplicate to ensure accuracy and reproducibility.  Tukey's homogeneous subsets generated from pairwise comparisons are represented as a, b, c, d, and e, with "a" representing the most significant subset from control. significance was set at P <0.05 and P values less than 0.01 were considered highly significant.

Cholesterol Assimilation in MRS.
The capability of 11 probiotic Lactobacillus strains to assimilate cholesterol in MRS media was determined. The viability (expressed as cfu/mL) of the probiotic cells was investigated upon incubation with 100 g/mL water-soluble cholesterol-PEG 600 in MRS. All probiotic cells under investigation were viable after incubation with cholesterol in the growth media for 24 h at 37 ∘ C, as shown in Figure 1(a). The cell viability ranged Data is expressed as mean ± SEM, = 3. Tukey's homogeneous subsets generated from pairwise comparisons are represented as a, b, c, d, and e, with "a" representing the most significant subset from control. from 2.87 ± 0.176 × 10 7 cfu/mL for L. plantarum ATCC 14917 to 1.18 ± 0.0504 × 10 9 cfu/mL for L. reuteri NCIMB 701359. All the strains investigated were successful (P < 0.001) at assimilating cholesterol following 24 h of incubation in cholesterol-containing MRS, as seen in Figure 1(b) The amount of cholesterol assimilated by the probiotic Lactobacillus strains in terms of a dose of 10 10 cells was calculated. The results indicate that all of the strains significantly (P < 0.001) assimilated cholesterol, in terms of mg cholesterol assimilated per 10 10 cells in MRS, as shown in Table 2. The results obtained, when bacterial cell counts were taken into account, were different from those previously described. Indeed, when normalized for viability counts, one probiotic strain, L. plantarum ATCC 14917, assimilated the most cholesterol, with 15.18 ± 0.55 mg of cholesterol assimilated per 10 10 cells (P < 0.05, subset "a"). The Lactobacillus strains that assimilated the least cholesterol, in terms of assimilation by 10 10 cfu (subset "e"), were L. reuteri NCIMB 11951 (0.33 ± 0.09 mg/10 10 cfu), L. reuteri NCIMB 701359 (0.19 ± 0.02 mg/10 10 cfu), L. reuteri NCIMB 702655 (0.96 ± 0.07 mg/10 10 cfu), L. reuteri NCIMB 701089 (0.99 ± 0.12 mg/10 10 cfu), L. fermentum NCIMB 8829 (1.16 ± 0.05 mg/10 10 cfu), L. rhamnosus ATCC 53103 GG (0.52 ± 0.07 mg/10 10 cfu), and L. acidophilus ATCC 314 (1.79 ± 0.08 mg/10 10 cfu).

Cholesterol Assimilation under Simulated Intestinal Conditions.
The Lactobacillus strains were further investigated for their capability to assimilate cholesterol under simulated intestinal conditions. The viability of the probiotic Lactobacillus strains was investigated upon incubation with 100 g/mL water-soluble cholesterol-PEG 600 in simulated intestinal fluid. Following 24 h of incubation at 37 ∘ C, all the probiotic cells under investigation were found to be viable, as shown in Figure 2(a). Specifically, the bacterial cell viability ranged from 1 × 10 5 ± 0.01 × 10 5 cfu/mL for L. plantarum ATCC 14917 to 1.41 × 10 7 ± 0.046 × 10 7 cfu/mL for L. reuteri NCIMB 11951. In terms of cholesterol assimilation, the control, containing no probiotic, demonstrated no cholesterol assimilation, as expected. All the strains of Lactobacillus under investigation, except L. rhamnosus ATCC 53103 GG (−0.43 ± 0.61 g/mL), were successful (P < 0.001) at assimilating cholesterol (expressed as g/mL), as demonstrated in Figure 2(b). Three Lactobacillus strains (subset "a") were shown to be significantly the best (P < 0.05) at assimilating cholesterol under simulated intestinal conditions: L. reuteri NCIMB 11951 (35.36 ± 0.72 g/mL), L. reuteri NCIMB 701089 (37.58 ± 1.06 g/mL), and L. acidophilus ATCC 314 (41.20 ± 1.92 g/mL).
Similar to the studies in MRS broth, the amount of cholesterol assimilated by the probiotic Lactobacillus strains in terms of a dose of 10 10 cells following 24 h of incubation under simulated conditions was calculated, as shown in Table 3. Indeed, when bacterial cell counts of each strain are accounted for, the best strains are different from those previously described. However, the results indicate that all of the strains significantly (P < 0.05) assimilated cholesterol, in terms of mg cholesterol assimilated per 10 10 cells under simulated intestinal conditions, except for L. rhamnosus

Discussion
The risk of developing CAD, the leading cause of death, is directly associated with elevated cholesterol levels. With the increasing prevalence of CAD and the lack of a successful therapeutic, there is an important need for a novel therapeutic approach. Recent work on the gut microbiome has led to investigations of probiotic formulations for health disorders, including metabolic syndrome, inflammatory bowel disease, and allergies [11,21,32,33]. Probiotic bacteria are advantageous as they are naturally found in foods such as yoghurt, are inexpensive, and are generally regarded as safe (GRAS). Of interest are the recent results demonstrating that probiotic bacteria have significant cholesterol-lowering properties [14,21]. The hypocholesterolemic effects of probiotic bacteria have been linked to intrinsic bile salt hydrolase activity [14], cholesterol assimilation and incorporation in cellular membranes [15,16], and the production of compounds, such as FA [17,18], that can inhibit the activity of enzymes, including HMG-CoA reductase [19]. Cholesterol assimilation by probiotic bacteria in the gastrointestinal tract would allow for the reduction of cholesterol absorption by enterocytes and excretion of the cholesterol from the host, as depicted in Figure 3. This would, in turn, lead to a decreased risk of developing CAD. The goal of the presented work was to investigate probiotic strains for their ability to assimilate cholesterol from bacterial culture media, as well as under simulated intestinal conditions.
Previous groups have demonstrated that certain probiotic bacterial strains can assimilate cholesterol [34,35]. Screening for cholesterol-lowering properties, in vitro, has become an important criterion in the selection of bacterial strains for in vivo probiotic investigations. We investigated Lactobacillus strains, selected from previous studies, for their ability to assimilate cholesterol. Initially, MRS bacterial culture media was supplemented with cholesterol and the bacterial strains were added for 24 h of incubation. All the bacterial strains were shown to successfully assimilate cholesterol but with high variability across the species and strains.   We also investigated how much cholesterol would be assimilated based on 10 10 bacterial cells, representative of a typical probiotic dose. When cell counts of each strain were accounted for, under simulated intestinal conditions, L. reuteri NCIMB 701089 was the best (P < 0.05) assimilator with 2254.70 ± 63.33 mg of cholesterol assimilated per 10 10 cells. Hypercholesterolemia is defined as having a serum cholesterol level over 240 mg/dL [38]. With this number in mind, we hypothesize that the administration of the probiotic strains, especially L. reuteri NCIMB 701089, could lower cholesterol levels significantly, although animal studies are required to evaluate its efficacy. One concern is the fact that recent work, by Madani et al., questions the use of in vitro cholesterol reducing activity assays as predictors of in vivo cholesterol-lowering activity [39]. With this in mind, it is clear that there is a need for additional work into strains, such as L. reuteri NCIMB 701089, prior to its use as a cholesterol-lowering therapeutic. Future work may focus on investigations into other cholesterol-lowering properties, including screening for bile salt hydrolase activity. In terms of cholesterol assimilation, the specific mechanism by which the cholesterol is removed from the supernatant should be determined. Ideally, a probiotic that would influence multiple targets, using bile salt hydrolase activity, reducing HMG-CoA reductase activity, and assimilating cholesterol, would be developed. In addition, a probiotic formulation could be developed as a combination therapy with pharmaceutics such as statins.

Conclusion
These results provide an initial screening of probiotic strains for their efficacy as cholesterol-lowering therapeutics via cholesterol assimilation. The capability of probiotic Lactobacillus strains to remove cholesterol from media, especially under simulated intestinal conditions, demonstrates their potential use as cholesterol-lowering agents. Moreover, the data suggests that L. reuteri NCIMB 701089 should be further characterized for its capability to lower cholesterol, using both in vitro and in vivo investigations. This work is an initial step for the development of a successful cholesterol-lowering probiotic therapeutic.