Isolation and Identification of LAB Strains from Grains
Bacterial strains were isolated from four types of grains collected in the Republic of Korea and a total of 187 LAB strains were obtained through 16S rRNA gene sequencing followed by identification. From the 16S rRNA gene sequence analyses of the LAB strains, 20 strains had the closest similarities to the type strain of Levilactobacillus (previously Lactobacillus) brevis, 3 strains had the closest similarities tothe type strain of Latilactobacillus (previously Lactobacillus) curvatus, 22 strains had the closest similarities to the type strain of Lactiplantibacillus (previously Lactobacillus) plantarum, 17 strains had the closest similarities tothe type strain of Lactococcus taiwanesis, 82 strains had the closest similarities tothe type strain of Pediococcus pentosaceus, and 43 strains had the closest similarities tothe type strain of Weissella paramesenteroides. The genus Lactobacillus has been recently reclassified as 25 genera including Levilactobacillus, Latilactobacillus,and Lactiplantibacillus[31]. Levilactobacillus brevis, Latilactobacillus curvatus, Lactiplantibacillus plantarum, Pediococcus pentosaceus, and Weissella paramesenteroides have been shown to be isolated from grains [32, 33].
In RAPD-PCR analysis, six different band patterns were assigned to 82 strains with the closest 16S rRNA gene sequence similarities to Pediococcus pentosaceus, and four different band patterns were assigned to 43 strains with the closest 16S rRNA gene sequence similarities to Weissella paramesenteroides (Supplementary Fig. 1). The strains assigned as Levilactobacillus brevis, Latilactobacillus curvatus, Lactiplantibacillus plantarum, and Lactococcus taiwanesis each showed only one type of band pattern (Supplementary Fig. 1). Finally, two representative strains from each group, except for the three groups having only one strain, were randomly selected, and used for further functional characterization (Supplementary Fig. 1, Table 1).
Screening of Strains with Anti-Adipogenic Effects
Inhibitory effects of lipid accumulation were tested by treating the LAB cell extract on C3H10T1/2 cells. Because the strains assigned to Lactococcus taiwanensis and Weissella paramesenteroides caused cell damage during the treatment process, they were excluded from the test. A wide range of inhibitory effects of lipid accumulation was observed in the strains selected. Among the strains tested, five strains (Pediococcus pentosaceus K28, Levilactobacillus brevis RP20 and RP21, Lactiplantibacillus plantarum RP11 and RP12) reduced lipid accumulation by more than 20% compared with the control, indicating that they have anti-adipogenic effects (Fig. 1). The two strains (RP20 and RP21) assigned to Levilactobacillus brevis and the two strains (RP11 and RP12) of Lactiplantibacillus plantarum showed similar results, respectively (Fig. 1). Thus, one strain from RP20 and RP21 and one strain from RP11 and RP12 were selected, and the three strains (K28, RP21 and RP12) were used for further experiments. The above results indicate that the components of LAB cell extract might influence the adipocyte differentiation process, thereby suppressing fat production.
Significant diversity exists among LAB strains regarding functional characteristics that benefit health, such as antioxidant, antitumor, immunomodulatory, and hypocholesterolemic activities [34-40]. In several studies, the cellular components of LAB have shown beneficial effects on improving health [38, 41, 42]. It is not clear which substance(s) in LAB cell extract induce the anti-adipogenic effects. Exopolysaccharide (EPS) has been known to have anti-adipogenic effects [23]. The EPS, a cell wall component of LAB cells, is loosely associated with the cell envelope and easily released into the surrounding environment [43, 44].
Effects of LAB Strains on Cell Viability of C3H10T1/2
The cytotoxicity at various concentrations of strains LGG, K28, RP21, and RP12 on C3H10T1/2 cells was investigated by measuring cell viability using the MTS assay. C3H10T1/2 cells were found viable at all treatment concentrations of the four strains (Fig. 2).
Inhibition of Adipogenic Gene Expression by LAB Extract during Adipocyte Differentiation
Inhibition of adipogenesis by strains K28, RP21 and RP12 was investigated by measuring expression of six adipogenic genes using quantitative RT PCR (Fig. 3). PPARγ and C/EBPα are transcription factors that regulate the process of adipocyte differentiation [45, 46]. In addition, the activation of PPARγ promotes the expression of adipogenic genes, such as CD36 and LPL, which are important for the uptake and storage of triglycerides [47]. The down regulation of these adipogenic genes may affect decreased lipid accumulation in cells. Fatty acid synthase (FAS) gene is a downstream adipocyte gene that contributes to fatty acid synthesis [48]. Acetyl-coenzyme A carboxylase (ACC) is another key enzyme for fatty acid synthesis that catalyzes the synthesis of malonyl-CoA [49].
Cell extracts from strains K28, RP21, and RP12 decreased the expression of adipocyte-related genes in C3H10T1/2 cells (Fig. 3). The expression of the six genes decreased proportionally with increasing concentrations of the extracts of the three strains (Fig. 3). The three strains significantly reduced (p < 0.01 or 0.001) the expression of PPARγ and C/EBPα in all concentrations tested. In addition, expression of four other genes associated with adipogenesis, was significantly reduced (p < 0.05 or 0.01) in the three strains, except for LPL expression in 25 mg/mL treatment of strains K28 and RP12. Strain K28, which showed the lowest lipid accumulation, was analyzed to have the lowest values in expressions of the six genes after 100 mg/mL treatment (Fig. 3). These results indicate that the three strains may have anti-adipogenic effects by inhibiting the expression of adipogenesis-related genes.
Tolerance against Acid and Bile Salts
To have specific functionality, a probiotic must reach the intestines alive with resistance to acid and bile salts [ 50]. The acid tolerance of the selected strains and LGG as a reference strain was examined after incubation for 3 h in pH 3.0, 2.5, 2.0, and 1.0 (Table 2). The three strains and LGG maintained the values of more than 9 log CFU/mL at pH 3. Under pH 2.5 condition, the survival rates of strains K28 and RP21 decreased more than 2 log and approximately 1 log, respectively, whereas strains RP12 and LGG showed decreases of more than 3 log and 2 log, respectively (Table 2). Under pH 2 condition, the survival rate of strains K28 and RP21 decreased approximately 3 log and 2 log, respectively, and the survival rate of strains RP12 and LGG decreased approximately 6 log (Table 2). The three strains, except RP21 with approximately 3 log CFU/mL, showed low viability of less than 2 log CFU/mL at pH 1 (Table 2). Strain RP21 was also found to have higher acid resistance, as a strain of Levilactobacillus (Lactobacillus) brevis was shown highly acid-resistant in a previous study [51].The pH of gastric fluid in the body is maintained at approximately 3.0, and probiotics are generally known to be highly acid-resistant if they are maintained at pH 3 for approximately 3 h [52]. Thus, the three strains were concluded to be highly tolerant to acid. Because food matrix can help the survival of LAB in the gastrointestinal tract due to its buffering capacity, the strains are expected to have stronger viability when used with carrier foods [53].
Bile salts are another factor that can reduce bacterial survival in the gastrointestinal tract by destroying cell membranes [50]. Strains K28, RP21 and RP12 were found to survive after 6 h exposure to 0.3, 0.5, 1.0, and 2.0% bile salts, similar to LGG which is known to be highly resistant to bile salts (Table 2). Although the in vitro assaycannot provide the same conditions as the gastrointestinal tract, it is recognized as an effective evaluation method to select potential strains when using proper criteria [27].
Adherence to Caco-2 Cells
The adhesion ability of probiotics is a main factor that can increase the possibility of their survival and colonization in the gastrointestinal tract [54]. Adhesion is also required to prevent attachment of pathogenic bacteria through competition in intestinal epithelium [55]. Thus, the adherence ability has been considered an important biological property for the selection of useful probiotic strains [56]. In the present study, the adhesion ability of the three strains was evaluated using Caco-2 cells, which have morphological and physiological properties of human enterocytes, and their adhesion abilities were compared with that of the reference strain LGG (Fig. 4). Strain K28 had stronger adhesion ability than those of LGG and the two other strains (Fig. 4). The adhesion ability of strain K28 was highest at 1.95%, followed by LGG (1.79%), RP12 (1.67%), and RP21 (1.46%).
Antibiotic Susceptibility
Probiotics have been widely used in various fields including food and medical industries. Antimicrobial sensitivity for evaluation of probiotics is considered important for safety, because the resistant genes can be horizontally transferred to pathogenic bacteria, which can become a serious threat [57]. Sensitivity results of the strains for nine antibiotics used in this study are listed in Supplementary Table 1. For the nine antibiotics tested, strains K28, RP21 and RP12 showed sensitivity patterns similar to strain LGG. In this study, all four strains were equally sensitive to chloramphenicol and rifampicin, whereas strains K28 and RP21 were intermediate sensitive to tetracycline and strains RP12 and LGG were sensitive to tetracycline (Supplementary Table 1). Sensitivity or intermediate sensitivity of Lactobacillus species and Pediococcus species to chloramphenicol and tetracycline has been previously reported [58, 59]. Strains K28, RP21, RP12 and LGG were resistant to gentamycin, kanamycin, and streptomycin, which are known to inhibit protein synthesis targeting Gram-negative bacteria. The resistance to aminoglycoside antibiotics is an intrinsic property among Lactobacillus species and Pediococcus species [60]. Therefore, the three strains are unlikely to cause safety problems based on antibiotic susceptibility profile tested.
Enzyme Production
For the safety of probiotic strains, it may be required to assess whether the strains produce harmful enzyme. β-glucuronidase is known as the carcinogen enzyme, which may increase the likelihood of tumor induction in the colon [61, 62]. When the three strains were evaluated using API ZYM kit, strains RP12 and K28 did not produce any harmful enzymes such as β-glucuronidase, but strain RP21 was observed to produce β-glucuronidase (Supplementary Table 2).