Probiotic Potential of Lactobacillus Strains on the Adipogenesis of 3T3-L1 Cells

Obesity is the most common health problem in developed countries and is considered a significant risk factor for many major human diseases. This study aimed to evaluate the inhibitory effect of lactic acid bacteria isolated from the human vagina on adipocyte differentiation in 3T3-L1 preadipocytes. We screened 26 strains of lactic acid bacteria to test their effects against obesity. Among the tested strains, Lactobacillus gasseri MG2855 exhibited a lipase inhibitory activity of 11.84% and inhibited relative lipid content of 3T3-L1 cells (96.21%) at a concentration of 1,000 μg/mL. The survival rate of L. gasseri MG2855 in 0.3% bile salt was 88.47%, and the survival rate after incubation for 3 h in pH 3.0 was 78.64%. L. gasseri MG2855 showed higher sensitivity to erythromycin, chloramphenicol, tetracyclin, and cephalothin in 16 antibiotic sensitivity tests. These results demonstrate that L. gasseri MG2855 can be employed as a potential probiotic with anti-obesity effects. Probiotic Potential of Lactobacillus Strains on the Adipogenesis of 3T3-L1 Cells


Introduction
Lactic acid bacteria (LAB) possess special physiological activities and are generally regarded as safe bacteria (GRAS). LAB have been widely used in the production of a number of fermented foods, particularly in dairy and vegetable products with functional and probiotic properties [1]. Recently, as the consumer demand for natural and chemical preservatives has increased, a novel and alternative method for the preservation of human and animal foods is required. The term "probiotic" is described as a feed supplement with beneficial viable microbial culture that yields positive effects in host animals by significantly improving intestinal microbial balance and enhancing nutrient absorption [2]. Furthermore, probiotics exhibit potent antagonist effect on pathogens in the gastrointestinal tract [3]. LAB produces essential antimicrobial metabolites that exterminate other pathogenic bacteria. There are numerous microorganisms that can classify as probiotics, including those that belong to the Lactobacillus and Bifidobacterium genera. Lactobacilli are considered enteric organisms and are used in the industrial processing of fermented dairy, meat, vegetable, and cereal products. According to the World Health Organization, for an organism to be classified as a probiotic, it has to comply with the principles related to their safety and biological properties. In terms of safety, the probiotic should not exhibit any pathogenicity, should not be able to transfer antibiotic-resistance genes, and should sustain genetic stability.
In spite of the current public awareness of obesity, the cases continue to increase. Obesity is the most common health problem in developed countries and is considered a significant risk factor for many major human diseases such as heart disease, cancer, arthritis, and diabetes [4]. Obesity develops because of an increase in the number of fat cells and their lipid content as a result of adipocyte differentiation (adipogenesis). The primary role of adipocytes involves the synthesis and storage of triglycerides during periods of caloric excess. As a result of adipogenesis, the size or number of the fat cells increases and lipid drops accumulate inside the cell [4]. Thus, preadipocyte cell lines are useful models for investigating the adipogenic process. An enhanced understanding of the process of adipogenesis would help in preventing the initiation and progression of adipogenesis, which leads to obesity and obesity-related diseases in humans. For a microorganism to be qualified for use as a probiotic, it should exhibit beneficial effects, including the modulation of immune responses [5] and anticarcinogenic and anti-oxidative activities [6]. Moreover, certain LAB has been found to be effective in regulating the number of fat cells in adipose tissue in overweight adults [7] and in a diet-induced obese animal model [8].
The aim of this work was (a) to screen LAB strains with potentially significant anti-obesity activity and (b) to study the characteristics related to the probiotic potential of these microorganisms in vitro.

LAB sample and treatment
After culturing, the isolated strains were harvested in a refrigerated centrifuge (1,100 × g for 3 min at 4°C) and washed three times with distilled water to remove the MRS broth. The washed isolated strains were freeze-dried and resuspended in phosphate buffered saline (PBS; pH 7.2) at a concentration of 10 mg/mL and homogenized for 50 sec followed by a 1 min rest period (repeated 3 times) using a sonicator (VCX 400, Sonics and Materials Inc., CT, USA). The suspension was centrifuged at 1,100 × g for 15 min at 4°C. The 3T3-L1 cells were treated with 1,000 µg/mL concentrations of the supernatant.

Lipase activity
The method of Lee et al. [9] for lipase activity determination was modified in this study. Pancreatic lipase activity was measured using porcine pancreatic lipase (Sigma, USA). For the enzymatic reaction, 0.1 mg/mL of the sample solution dissolved in water, 0.167 mM p-nitrophenylpalmitate (PNP; Sigma, USA) solution, and 0.061 M (pH 8.5) Tris-HCl buffer were mixed in the well of a plate, and then 0.3 mg/mL of the lipase solution was added to initiate the reaction. After incubation at 25°C for 10 min, its absorbance was measured at 400 nm.

Cell differentiation and sample treatment
The cells were cultured as described in the "Cell Culture" section. To examine the effects of the isolated strains on adipocyte differentiation, the medium was treated every alternate day until the end of the experiment on day 8. For the positive control, cells were cultured in the same media containing a drug-free vehicle and baicalin (100 μM), reported to be an adipogenesis inhibitor [10].

Cell viability and oil red O staining
Cell viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. 3T3-L1 preadipocytes were treated with selected strains at concentrations of 0, 100, and 1,000 μg/mL. After 24 h, 20 μL of MTT solution was added, and cells were incubated at 37°C for 4 h. The supernatant was then discarded, and 200 μL of dimethyl sulfoxide was added. Absorbance was measured on a microplate reader (Bio-Rad Model 550; Hercules, CA, USA) at 546 nm to obtain the percentage of viable cells.
Eight days after inducing differentiation, the media was discarded, and the cells were washed twice with PBS and fixed with 3.7% formalin for 15 min. To visualize the lipid droplets, the fixed cells were washed twice with 60% isopropanol (in PBS) and then stained with a 0.5% Oil Red O solution for 40 min at room temperature. The Oil Red O solution was then removed, and the cells were washed twice with deionized water before being photographed. For quantification of Oil Red O uptake, cells were incubated with isopropanol, and the optical density of the solution was measured at 540 nm. The percentage of the cells stained with Oil Red O relative to the control wells containing the cell culture medium without compounds was calculated as sample OD/ Control OD (with baicalin) × 100.

Biochemical tests and identification
Selected isolates were identified by Gram staining, conventional biochemical tests [11], and sequencing of the 16S ribosomal RNA gene using universal primers (518F and 800R). PCR and 16s rRNA sequencing were outsourced to Macrogen Co. (Daejeon, Korea). Sequence similarity between strains was analyzed by nucleotide alignment using the Macrogen Alignment program, a web-based tool for identification based on 16S rRNA gene sequences [12]. Basic Local Alignment Search Tool (BLAST) analysis (http://blast.ncbi.nlm.nih. gov/Blast.cgi) was performed to compare the sequences obtained with available DNA sequences registered in the database of the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm. nih.gov). Sequences were aligned using the PHYDIT program (http:// plaza.snu.ac.kr/~jchun/phydit/), and alignments were manually corrected. A phylogenetic tree was constructed with the neighborjoining method using the MEGA 5.0 software [13].

Probiotic activity
Acid resistance and bile salt tolerance of selected strains: Strains that exhibited high anti-adipogenic activity in vitro were cultured for 18 h at 37°C. The culture was centrifuged at 12,000 × g for 10 min at 4°C. The pellet was washed twice with PBS buffer (pH 7.2) for the following experiments. The bile salt and pH tolerance of selected strains were investigated according to the methods described by Argyri et al. [14], with minor modifications. The cells were resuspended in PBS (pH 3.0 or 7.0 (control), adjusted with 1 N HCl) solution, and the cell density was adjusted to 10 8 CFU/mL. Then, the resuspensions were incubated at 37°C for 3 h, and viable cells were counted by standard plate counting. Measurements were performed in triplicates, and the mean values were shown.
The cells were resuspended in PBS solution (pH 8.0) containing 0.3% (w/v) bile salts (Difco, MI, USA), and the cell density was adjusted to 10 8 CFU/mL. Then, the resuspensions were incubated at 37°C for 3 h. Resistance to bile salts was assessed using the plated count method in triplicates.

Auto aggregation test:
The auto aggregation ability of the selected strains was determined as previously described [15]. Bacterial cells were cultured in MRS broth at 37°C for 18 h under anaerobic conditions. They were resuspended in PBS (pH 6.2) to a final concentration of 10 8 CFU/mL. The microbial suspension was dropped onto a glass slide and auto aggregation was observed with a light microscope. If the cells aggregated within 2 min, auto aggregation was considered positive.
Hemolysis: For determining the hemolysis ability, the selected strains were streaked on 5% sheep blood agar (Hardy Diagnostics, CA, and USA) and incubated at 37°C for 48 h [16]. Isolates that formed a green zone around the colony were designated as alpha hemolysis (α-hemolysis) and those that formed a clear zone were denoted as beta hemolysis (β-hemolysis), sometimes known as complete hemolysis. Isolates that did not produce any zone around the colony were reported to show no hemolysis or gamma hemolysis (γ-hemolysis) [17].

Inhibitory effect on lipase activity
Several approaches have been reported for the prevention and treatment of obesity [18]. Among these, both natural and synthetic pancreatic lipase inhibitors are effective in preventing obesity, which is likely due to the inhibition of intestinal lipid absorption [19]. Therefore, lipase inhibitors are used for designing drugs for the treatment of obesity and acne [20]. After incubating in MRS broth at 37°C for 18 h, 26 strains exhibited lipase inhibitory activity from the 221 strains tested. Anti-lipase activities of the selected strains were repeatedly measured in duplicates. Table 1 show that the MG2211 and MG2333 strains exhibited highest lipase inhibitory activity among the 26 selected strains.

Inhibitory effect of isolated strains on adipocyte differentiation and cell viability
Adipogenesis is a highly regulated, complex differentiation process, in which preadipocytes are transformed into differentiated adipocyte cells, and is triggered by the coordinated signaling of growth factors, cytokines, and hormones [21]. In this study, we screened the 26 selected strains to test their potential anti-adipogenic activity. 3T3-L1 adipocytes were cultured and differentiated in DMEM containing 10% FBS for 6-8 days in the absence or presence of the samples (at a final concentration of 1,000 μg/mL), according to the differentiation protocols. As shown in Table 1 and Salmonella spp. [22], and recent studies have provided evidence of the anti-obesity effects of LAB. Choi et al. [23] showed that oral administration of Bifidobacterium breve results in the fermentation of isoflavone aglycones, effectively suppressing the absorption of excess lipids and inhibiting the differentiation of 3T3-L1 adipocytes in a dosedependent manner [23].
The effect of the 26 selected strains on cell viability in preadipocytes was demonstrated by using the MTT assay Table 1

Identification
The aforementioned nine selected strains were further analyzed by sequencing their 16S rRNA genes Table 2, and the selected strains were identified as follows: Lactobacillus plantarum (five strains), L. fermentum (three strains), and L. gasseri. LAB provides complex nutritional requirements and is frequently used as probiotics and for the fermentation of food products. Probiotics that consist of one or more species of viable bacteria, such as Lactobacilli and Bifidobacteria, directly affect the intestinal flora and other organs by modulating immunological parameters and intestinal permeability and producing bioactive or regulatory metabolites [24].

Strains
Anti

Tolerance to low pH and bile salts
Stomach and execute their health effects as viable potent cells on reaching the colon [25]. The effects of highly acidic pH (3.0) on the survival of the nine selected strains over different incubation periods are shown in Figure 1. The population densities of eight selected strains were found to be <6 log CFU/mL after exposure to a pH of 3.0 for 3 h, except for MG4002. In particular, MG4001, MG2211, MG2311, MG2444, and MG4203 showed very high resistance to a pH of 3.0 with final viable populations exceeding 7 log CFU/mL. In fact, research has shown that Lactobacillus strains are resistant to pH varying between 2.5 and 4.0. The work of Maragkoudakis et al. [26] showed that the viability of the Lactobacillus strains of dairy origin was not affected by a pH of 3.0. However, in the study by Prasad et al. [27], only four LAB from the 200 strains isolated from dairy origin were resistant to a pH of 3.0 (≥ 80%) (Figure 1).
Resistance of LAB to bile salts is also an important factor for their colonization [28]. As shown in Figure 2, the viable counts of all selected strains were found to be >8 log CFU/mL after exposure to 0.3% (w/v) bile salts for 24 h, with low reduction in viable counts (<1 log CFU/ mL). However, MG4003, MG2211, and MG2700 exhibited a lower tolerance to bile salts, and their population densities reduced by more than 2 log CFU/mL after 24 h of exposure to bile salts ( Figure 2).

Auto aggregation
In general, the probiotic strains showed higher auto aggregation abilities. All nine selected strains tested showed higher percentages of aggregation after 5 h of incubation Figure 3. MG4002 and MG2855 showed the highest auto aggregation percentages at 37°C. The ability to adhere to epithelial cells and mucosal surfaces has been suggested to be an important property of many bacterial strains that are used as probiotics. Cell adhesion is a multistep process involving contact of the bacterial cell membrane and interacting surfaces. In most cases, aggregation ability is related to cell adherence properties [29] (Figure 3).

Antibiotic susceptibility assay and hemolysis
Antibiotic overuse results in the development and dispersal of antibiotic resistance genes in a species, and such resistance genes are then transferred to other microorganisms. Thus, the sensitivity of probiotics to conventional antibiotics is a fundamental health-promoting characteristic [30]. Table 3 shows the antibiotic susceptibility results of selected strains, which were resistant to CTT, GM, K, S, CIP, NA, and VA. However, the selected strains were interpreted to be susceptible to AM, CF, SXT, C, and E. Several authors have found that the strains of L. plantarum are resistant to various antibiotics [31]. Wang et al. [32] reported that different isolated LAB strains were resistant to different classes of antibiotics. According to Tulumoglu et al. [33], all isolated L. fermentum strains were resistant to VA and some strains were susceptible to clindamycin. Angmo et al. [34] indicated that most Lactobacillus strains isolates were almost susceptible to all investigated antibiotics, with VA being an exception. L. fermentum isolate was also resistant to norfloxacin, S, K, NA, and VA. In the present study,       none of the selected strains exhibited hemolytic activity against human blood. Therefore, all strains were considered as safe and could be used as potential probiotic strains. This result was concurrent with previous reports of Gao et al. [35].

Conclusion
In conclusion, we investigated whether LAB isolated from the human vagina exhibited any anti-obesity activity such as inhibition of adipocyte differentiation of the 3T3-L1 cells. In the present study, we isolated potential probiotic strains having functional properties of food. L. gasseri MG2855 showed essential probiotic properties, including complete tolerance to acid and bile salts and high auto aggregation activity. Furthermore, it was antibiotic resistant and did not cause human blood hemolysis. These results demonstrate that L. gasseri MG2855 could be an excellent anti-obesity probiotic.