Enhanced ceramides production by Lactobacillus rhamnosus IDCC 3201 and its proposed mechanism

The use of probiotics has been applied for a variety of fields (e.g., immune system, mental health, and heart). In this study, the feasibility of lysates from L. rhamnosus IDCC 3201 for cosmetic ingredients was evaluated. More specifically, enhanced ceramides production in human epidermal keratinocytes by the lysates and its proposed machanism were investigated through in vitro and genome analysis. In results, enhanced spingomyelinase activity and thereby increased ceramides production by the lysates from L. rhamnosus IDCC 3201 was observed. Furthermore, it was found that the existence of glucosylceramdase in L. rhamonsus IDCC 3201 was attributed to enhanced ceramides production. Finally, it was verified that the lysates from L. rhamonsus IDCC 3201 was regarded as safe for its use as cosmetic materials. Thus, these findings have significant implications that might lead to the development of functional and safe cosmetic products from probiotics.


Introduction
Recently, interest of skin health has been increased due to the escalating environmental stresses [1]. Especially, the most outer layer of the skin (stratum corneum) is thought to be a frotier of skin protection, and it is mainly composed of corneocytes and lipids [2]. These lipids mainly include several types of cholesterols, fatty acids, and ceramides [3]. Among them, ceramides, a member of sphingolipid family, play an important role in skin barrier function (e.g., skin homeostasis). Typically, ceramides are converted from glucosylceramides and sphingomyelins by glucosylceramidase (GCase) and sphingomyelinase (SMase), respectively [4]. Many clinical studies showed that deficiency of ceramides is involved in atopic dermatitis (AD) and psoriasis directly or indirectly [5,6]. Thus, one of the stategies for heathy skin should be to strengthen skin barrier through increased ceramides level [7]. For example, general approach is to regulate exogenous and endogenous factors involved in ceramides synthesis such as vitamin C and vitamin D [8,9].
Currently, probiotics have received considerable attention in cosmetic industry based on long-term uses of fermented broth for skin care [10,11]. Consistent with this notion, lysates of Streptococcus thermophilus S244 not only increased ceramides level of healthy elderly women but also alleviated symptoms of AD patients [12,13]. In addition, Lactobacillus rhamnosus strain promoted epidermal barrier formation in human skin model [14]. However, there was no direct evidence which components of probiotics improve ceramides production in human skin and thereby skin barrier function.
In this study, enhanced ceramides production by lysates from L. rhamnosus IDCC 3201 and its machanism Open Access were investigated through in vitro and genome analysis. For this, firstly, the lysate from a variety of bacteria that exhibited the highest SMase activity was screened. Secondly, ceramides production by the screened probiotic strain was evaluated in human epidermal keratinocytes (HEK). Then, genome and metabolome analysis of the selected strain was perfomed to propose the mechanism. Finally, safety of the strain was assessed to determine whether it produces toxic compounds in human skin. Thus, this study contributes to the development of functional and safe cosmetic products from probiotics.

Bacterial strains and preparation of bacteral lysates
L. rhamnosus IDCC 3201 from breast-fed infant's feces [15] and Streptococcus thermophilus IDCC 2201 from home-made yogurt [16] were industrial strains, while the other bacterial strains were isolated from kimchi (Korean fermented food). All the strains were incubated in an in-house medium designed by Ildong Bioscience, centrifuged, and freeze-dried to obtain cell density at > 10 10 colony forming units (CFU)/g. Then, lyophilized strains were suspended in 500 mL of phosphate buffered solution (PBS) and was lysised physically by using a microfluidizer (PicoMax MN400BF; Micronox, Seongnam, Korea) to preprare bacterial lysates.

Sphingomyelinase (SMase) activity
SMase activity was measured using a SMase assay kit (Abcam; # ab138876, Cambridge, UK). The principle of this assay kit was to quantify phosphocholine produced by the hydrolysis of sphingomyelin using colorimetric analysis at 655 nm. Briefly, 50 μL of sphingomyelin was incubated with lysates from five bacterial strains at 37 °C for 1 h. Then, color changes were detected using a microplate reader (SpectraMax iD3; Molecular Devices, CA, USA). Then, SMase activity was calculated according to calibration curves with the absorbance values of known amounts of SMase standards.

Cell culture
Normal human epidermal keratinocyte (HEK) cells were purchased from PromoCell (Heidelberg, Germany). Cells were grown in growth medium 2, supplemented with bovine pituitary extract, epidermal growth factor, Insulin, hydrocortisone, epinephrine, transferin and 0.06 mM of CaCl 2 (PromoCell). After confluence for 3-5 days at 37 °C with 5% CO 2 , cells were maintained by sub-culturing the detached cells with a 0.25% trypsin-EDTA solution.

Ceramides production
Ceramide contents in the suspension from the HEK cells were measured using an ELISA kit (Human Ceramides ELISA Kit; MyBioSource, CA). Briefly, The HEK cell culture suspension with SMase was treated to a 96-well plate which coated by human monoclonal antibody. The plate was incubated with biotin-labeled polyclonal antibody which is conjugated with avidin-peroxidase. Next, a chromogen solution was added into the plate for ELISA colormetric detection at 450 nm. Finally, amounts of ceramide were calculated according to caribration curve prepared with ceramides standards.

Real-Time PCR
Gene expression for ceramides synthesis was investigated using real-time PCR. Total RNAs were isolated from HEK cells treated with either SMase (positive control) and bacterial lysates by a Trizol method. PCR reactions were performed using CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA). The housekeeping gene, GAPDH was used as a constitutive control for normalization. The specific primers used for real-time PCR are listed in Table 1.

Bacterial gene search for ceramides biosynthesis
The whole-genome sequencing of L. rhamnosus IDCC 3201 was performed by a PacBio RSII instrument with an Illumina platform (Macrogen, Seoul, Korea). A sequence of nucleotides was generated by single molecule realtime (SMRT) sequencing system. Contigs were constructed by pre-assembling seed reads, by generating a consensus sequence of the mapped reads, and by correcting and filtering the reads. Finally, a consensus sequence with higher quality was obtained after error-correction of the constructed contigs by Pilon (version 1.21).
To find bacterial enzymes involved in ceramides biosynthesis from probiotics, amino acid sequence of enzymes were collected: sphingomyelinase (EC 3.  [17] from UniProtKB database, and built hidden Markov models (HMMs), representing the conserved amino acid sequence patterns in these enzymes (Table 2). Then, genome of L. rhamnosus IDCC 3201 was targeted to search using the HMMsearch tool in HMMER package with the constructed profile of HMMs [18]. Finally, the candidate enzymes were verified using BLASTP tool in NCBI BLAST and HMMscan tool in HMMER package against SWISS PROT database and PFAM database, respectively.

Metabolites analysis
Bacterial lysates were diluted in ice-cold methanol to a final concentration of 80% with a vortex for 1 min on ice. After centrifugation at 13,000g for 10 min at 4 °C, the upper layer of the supernatant was collected, concentrated to dryness in a vacuum concentrator, and stored at − 80 °C prior to derivatization and analysis by GC-MS.
The extract was derivatized with 30 µL of a solution of 20 mg/mL methoxyamine hydrochloride in pyridine (Sigma, St. Louis, MO) at 30 °C for 90 min, and 50 µL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA; Sigma) was subsequently added at 60 °C for 30 min. A mixture of fatty acid methyl esters and fluoranthene was added to the extract as internal standards. The GC-MS analysis was conducted using a Thermo Trace 1310 GC (Waltham, MA) coupled to a Thermo ISQ LT single quadrupole mass spectrometer (Waltham, MA). A DB-5MS column with 60-m length, 0.2-mm i.d. and 0.25-µm film thickness (Agilent, Santa Clara, CA) was used for separation. For analysis, the sample was injected at 300 °C and split ratio 1:5 with 7.5 mL/min helium split flow. The metabolites were separated with 1.5 mL constant flow helium with an oven ramp of 50 °C (2 min hold) to 180 °C (8 min hold) at 5 °C/min, to 210 °C at 2.5 °C/min, and to 325 °C (10 min hold) at 5 °C/min. The mass spectra were acquired in a scan range of 35-650 m/z at an acquisition rate of 5 spectra per sec. The ionization mode was subjected to electron impact, and the temperature for the ion source was set to 270 °C. The spectra were processed by Thermo Xcalibur software using automated peak detection, and the metabolites were identified by matching the mass spectra and retention indices of the NIST Mass spectral search program (version 2.0, Gaithersburg, MD). The metabolite data were then normalized based on the intensity of the fluoranthene internal standard.

Genome analysis
The VFDB database was searched for virulence genes [19], and ResFider software (ver. 3.2) with the CARD database was searched for antibiotic resistance genes [20]. The search parameters were set to the identity of > 80% and coverage of > 80% for gene identification. Transposases and transferases were annotated using the protein-protein basic local search program (BLASTP) against the NCBI GenBank proteins. Prophage regions were identified using PHASTER web-based program [21].

In vitro analysis
Lactobacillus rhamnosus IDCC 3201 was evaluated for susceptibility to antimicrobials including ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol (Sigma-Aldrich, St. Louis, MO), which are typically used to treat enterococcal infections [22]. Briefly, a single colony from plate was inoculated to MRS broth and preincubated for 18 h. The cultured cells and antibiotic solution were mixed in 96-well plate to obtain the cell density of 5 × 10 5 CFU/mL, and the plate was then incubated at 37 °C anaerobically for 18 h. The optical density was observed using a microplate reader (BioTek, Winooski, VT). The minimal inhibitory concentrations (MICs) were determined compared to the cut-off values suggested by European Food Safety Authority (EFSA) [23].

Screening of lactic acid bacteria with higher sphingomyelinase (SMase) acitivity
SMase is the key enzyme of sphingomyelin catabolism, converting into ceramides and phosphorylcholine [24]. Thus, decreased sphingomyelinase activity often leads to decreased ceramides level, resulting in skin troubles (e.g., atopic dermatitis) [25]. Here, various lactic acid bacteria were screened to obtain the strain(s), producing higher concentration of ceramides. For this, lysates from various lactic acid bacteria were prepared to observe whether the SMase increased. In particular, the lysates of tested strains used in this experiments were prepared from cell desity at 5 × 10 7 CFU/mL. As a result, L. rhamnosus IDCC 3201 exhibited highest SMase activity 1.85 ± 0.03 mU/mL, while the other strains exhibited only at 0.28-0.43 mU/mL (Fig. 1a). The value of L. rhamnosus IDCC 3201 was 3.4 times higher than those of other strains, such as L. mesenteroides, L. plantarum, L. casei, and S. thermophilus. Consequently, L. rhamnosus IDCC 3201 strain was selected as the best performer with regard to SMase activity. Next, optimal cell density of L. rhamnosus IDCC 3201 was evaluated to determine economically feasible concentration. The lysates from 1 × 10 7 to 5 × 10 10 CFU/mL were loaded onto the substractes. In results, SMase activity was observed to increase up to 1.65 ± 0.09 mU/mL, as cell density increased (Fig. 1b). In contrast, in the view of efficiency of enzymatic activity (enzymatic unit/cell density), when cell density was the lowest as 1 × 10 7 CFU, enzymatic efficiency of SMase was highest (Fig. 1b). Thus, cell amount of 1 × 10 7 CFU/mL of L. rahmonsus IDCC 3201, showing the highest SMase efficiency was selected as optimal cell density for further study.

Enhanced ceramides production by L. rhamnosus IDCC 3201
Ceramides, the major lipid consitituents in the upper epidermal layer play a role in maintaining skin barrier integrity [26]. Thus, this layer prevents the skin from excess water loss and protects skin against various stimuli, including bacterial or microbial infection [8]. Here, ceramides production was investigated whether enhanced SMase activity by L. rhamnosus IDCC 3201 affects ceramides production in HEK cells. Prior to investigating the impact of lysates of L. rahmonsus IDCC 3201, cytotoxicity of HEK by the lysates was assessed. As a result, no significant reduction of cell proliferation was observed at cell density, ranging from 1 × 10 7 to 5 × 10 8 CFU/mL for 24 h (Fig. 2). Furthermore, to substantiate that increased SMase activity is responsible for enhanced ceramides production, lysates of L. rhamnosus IDCC 3201 and lactic acid bacteria tested in Fig. 1 were treated in HEK cells. Overall, increased ceramides production is correlated with increased SMase activity (R 2 = 0.77, Fig. 3) to some degree. Ceramides concentration by L. rhamnosus IDCC 3201 was 1.7-5.2 times higher than those by other lactic acid bacteria. More specifically, 1153.5 ± 334.5 pg/mL of ceramides was measured in HEKs treated by L. rhamnosus IDCC 3201, while 651.1 ± 72.9, 417.9 ± 55.8, 216.8 ± 69.3, and 626.2 ± 216.5 pg/mL of ceramides were measured in HEKs by L. mesenteroides, L. plantarum, L. casei, and S. thermophilus, respectively (Fig. 3). The observation that HEK cells treated with the lysates of L. rhamnosus showed higher production of ceramides, might be due to the induction of higher SMase activity (Fig. 1). To validate this notion, real-time PCR was performed to analyze the expression levels of SMPD3, CERS1 and CERS2 responsible for ceramides synthesis: SMPD3 coding for sphingomyelin phosphodiesterase 3; CERS1 for ceramide synthase 1; CERS 2 for ceramide synthase 1. In results, the expression level of SMPD3 was significantly increased in response to L. rhamnosus IDCC 3201, but not in L. plantarum. More specifically, each strain exhibited 35% and 11.4% increase of SMPD3 expression, respectively   (Fig. 4a). Based on the results, it was concluded that comparative higher production of cermides by L. rhamnosus IDCC 3201 was due to the up-regulation of SMPD3 rather than CERS1 or CERS2. As a conclusion, it is expected that lysates of L. rhamnosus IDCC 3201 contribute to the improvement of the skin barrier through the increased level of ceramides.

Genome and metabolome analysis for elucidating a proposed mechanism
To elucidate a mechanism of enhanced ceramides production by L. rhamnosus IDCC 3201, information of enzymes that can synthesize ceramides in microorganisms was collected (Table 1). Among 13 genes, L. rhamnosus IDCC 3201 harbored galc and gba, which encodes for galactosylceramidase and glucosylceramidase, respectively. According to the gene annotation analysis using homology search, only gba gene was determined to be responsible for ceramides production from glucosylceramide, that is abundant in human keratinocytes [4]. Previous studies reported that the lack of glucosylceramidase encoded by the gba gene disturbed ceramides production and caused the failure of formation of competent skin barrier [9]. Thus, the increased ceramides in HEK cells was due to the expression of glucosylceramidase or increased SMase activity in the lysates of L. rhamonsus IDCC 3201. In order to specify the possible effects of lysate components on the enhanced ceramides production, we analyzed the metabolites present in the culture supernatants of L. rhamnosus and L. plantarum using GC-TOF mass spectroscopy. As shown in Fig. 4b, we found that abundances of glycerate and serine increased in the L. rhamnosus culture compared to L. plantarum. The first step of de novo synthesis of ceramides begins with the condensation of palmitate and serine, producing 3-keto-dihydrosphingosine. In this process, serine metabolism regulates ceramides and sphingolipid synthesis [27]. In addition, 3-phosphoglycerate, as one of the glycerate derivatives, is a precursor for the serine synthesis, supporting the notion that the increased glycerate and serine in the L. rhamnosus culture would induce the ceramides synthesis [28]. Finally, the reasons of enhanced ceramides production can be summarized by 1) indirect effect of components of the lysates (i.e., glycerate and serine) and 2) direct effect of glucosylceramidase on cermides production (Fig. 5).

Safety evaluation of L. rhamnosus IDCC 3201
Genome analysis of L. rhamnosus IDCC 3201 indicated that the size of the genome was approximately 3.05 Mbps Fig. 5 Proposed mechanism for enhanced ceramides production by the lysates from L. rhamnosus IDCC 3201. Preparation of unanimated L. rhamnosus IDCC 3201 and its components consist of exopolysaccharides, supernatant, cell wall fragments, bacterial lysates, enzymes, and various metabolites. Among them, an enzyme, glucosylceramidase converts glucosyceramides into ceramides in HEKs. In addition, the metabolites such as glycerate and serine promote ceramide synthesis in HEKs with a GC content of 46.71% and 2,821 functional genes (Additional file 1: Tables S1 and S2) (Fig. 6). Furthermore, L. rhamnosus IDCC 3201 does not have any toxigenic gene or virulence factor. According to the MIC tests, L. rhamnosus IDCC 3201 was susceptible to all of the antibiotics except for gentamycin and kanamycin (  6 Circular map of the chromosome of L. rhamnosus IDCC 3201. The whole genome was used for searching the genes responsible for ceramides production, antibiotic resistance, virulence, mobile elements, and prophage regions. Marked characteristics are shown from outside to the center, mobile elements (transposases, dark red; intact prophage region, dark blue), CDS on forward strand, CDS on reverse strand, RNA genes (tRNAs orange; rRNAs, red; other RNAs, green), GC content (black) and GC skew (light green/orange)