Glucosylceramide Changes Bacterial Metabolism and Increases Gram-Positive Bacteria through Tolerance to Secondary Bile Acids In Vitro

Glucosylceramide is present in many foods, such as crops and fermented foods. Most glucosylceramides are not degraded or absorbed in the small intestine and pass through the large intestine. Glucosylceramide exerts versatile effects on colon tumorigenesis, skin moisture, cholesterol metabolism and improvement of intestinal microbes in vivo. However, the mechanism of action has not yet been fully elucidated. To gain insight into the effect of glucosylceramide on intestinal microbes, glucosylceramide was anaerobically incubated with the dominant intestinal microbe, Blautia coccoides, and model intestinal microbes. The metabolites of the cultured broth supplemented with glucosylceramide were significantly different from those of broth not treated with glucosylceramide. The number of Gram-positive bacteria was significantly increased upon the addition of glucosylceramide compared to that in the control. Glucosylceramide endows intestinal microbes with tolerance to secondary bile acid. These results first demonstrated that glucosylceramide plays a role in the modification of intestinal microbes.


Introduction
The importance of diet in health has long been recognized worldwide. The Chinese ancient document, the Shurai, written in 800 BC, describes medical doctors who specialized in foods, and the ancient Greek medical doctor, Hippocrates (460-370 BC), described the importance of food in medicine. After the scientific approach was established, the relationship between health and intestinal microbes was studied by Mechnikov, a doctor who, in 1907, proposed that intestinal microbes lower the pH of the intestinal environment and prevent intestinal decay and aging [1]. These microbes were later classified as probiotics. In 1998, Gibson proposed that some substances promote the growth of preferable intestinal bacteria and termed them prebiotics [2]. Fructooligosaccharides [3], galactooligosaccharides [4], beta-glucan [5], polyunsaturated fatty acids [6], catechin [7][8][9], anthocyanins [10], pectin [11], alginate [3], and enzymes [12][13][14] have been proposed as prebiotics.
Sphingolipids are lipids composed of a sphingoid base, an amide-linked fatty acid moiety, and other sensory bases. Since the discovery by Thudichum in 1874, sphingolipids Technology and Evaluation. Blautia coccoides (JCM1395) was commercially purchased from the Japan Collection of Microorganisms, RIKEN BRC (Wako, Kyoto, Japan).
A glass vial (10 mL, 45 mm × 22 mm, Agilent Technologies Inc., Santa Clara, CA, USA) was used for anaerobic culture. Nitrogen gas infusion for 5 min was used to purge air. The vial was then locked using a PTFE/butyl cap and autoclaved. All bacteria were grown at 37 • C for 24 h under anaerobic conditions using resarulin (final concentration 1 µg/mL) as an indicator of an anaerobic environment.

Coculture of Glucosylceramide and Microbes
Microbes were inoculated into appropriate media and incubated at 37 • C for total 48 h. Cell-mock-1 was incubated as a mixture of 15 bacteria. Other bacteria were incubated individually. Considering that the deoxycholic acid concentration in the colon is 0.046-0.21 mM [40], the final concentrations of deoxycholic acid in the broths were adjusted to 0-2.0 mM. Deoxycholic acid (2 mM in distilled water) was diluted to the appropriate concentration, according to the experimental conditions. Using a syringe, 100 µL of pre-configured deoxycholic acid (24 h from the start of the culture), 25 µL of the glucosylceramide solution (20 µg/mL dissolved in ethanol or DMSO), vitamin solution (10 µL/mL) and a certain volume of the bacterial culture broth (OD 600 = 0.1) were added individually to the sterilized broth. The total volume of the liquid in the vial was 5 mL.

Analysis of Metabolites of Cultured Broth Using Gas Chromatography
The cultured broth was freeze-dried, and water-soluble metabolites were extracted and analyzed using gas chromatography with flame ionization detection (GC-FID), as previously described [45].

Analysis of Lactic Acid
The lactic acid concentration in the cultured broth was analyzed using high-performance liquid chromatography (HPLC), as previously described [46].

Next-Generation Sequencing Analysis of Cell-Mock-1 Incubated with Glucosylceramide
Cultured broth (5 mL) was collected and frozen at −27 • C. Next-generation sequencing (NGS) analysis was performed using the Techno Suruga Laboratory (Shimizu, Japan). Briefly, bacterial DNA was extracted using the MORA-EXTRACT kit (Kyokuto Pharmaceutical, Tokyo, Japan) and FastPrep-24 5G (MP Biomedicals, Irvine, CA, USA). Conditions for PCR amplification of 16S rDNA including primers are described by Takahashi et al. [47]. The PCR products were quantified, and deletion of primer sequences and determination of the sequences was performed using the MiSeq system (Illumina, San Diego, CA, USA) and MiSeq Reagent Kit v3 (600 cycles) (Illumina, San Diego, CA, USA). Fastq-join [48] was used to join pair-ends, and the FASTX-Toolkit [49] was used to filter sequences with more than 99% quality values (>20). QIIME ver. 1.8.0 [50] was used for the deletion of chimeric sequences detected by Usearch6.1.544_i86. The RDP MultiClassifier ver. 2.11 was used to search for homologous sequences with a confidence of more than 0.8.

Statistical Analysis
IBM SPSS Statistics (ver. 23.0; IBM Inc., Armonk, NY, USA) and SIMCA-P+, ver. 13.0 (Umetrics, Umeå, Sweden) were used for statistical analyses. Normality of the data was verified using the Shapiro-Wilk test. Significance of differences between the means was verified using a one-sided unpaired Student's t-test. Hierarchical clustering and principal component analysis were used to group the operational taxonomic units (OTUs) of the NGS analyses. Hierarchical clustering, principal component analysis, and partial least squares-discriminant analysis (PLS-DA) were used to analyze the data obtained by GCmass spectrometry (MS). Hierarchical clustering was performed using MetaboAnalyst ver. 5.0 (https://www.metaboanalyst.ca/home.xhtml, accessed on 5 May 2022) [51]. The OTU counts of the 16 bacterial species were divided by the total OTU count. IC 50 values were calculated using the ImageJ software. Curve fitting (four-variable logistic curve) with the ImageJ software was used to create approximate dose-response curve equations. The concentration of deoxycholic acid at a 50% reduction in bacterial concentration was considered the semi-inhibitory concentration of the bacteria. Each experiment was repeated three times, and the average value was calculated.

Glucosylceramide Addition Alters Metabolism of Blautia coccoides
As most glucosylceramide reaches the colon without enzymatic degradation, glucosylceramide was directly added to the in vitro culture of intestinal microbes and analyzed as a model large intestine. The effect of culturing Blautia coccoides, a major intestinal microbe involved in the decrease in visceral fat accumulation [52], with purified glucosylceramide was analyzed in vitro. To determine the effect of glucosylceramide, metabolites of B. coccoides were incubated with or without the addition of glucosylceramide. Considering that glucosylceramide is contained in crops and fermented foods at a concentration of 0.94-2.2 mg/g [19,24,25,53], the concentration was set at a final concentration of 20 µg/mL, since it can be hypothesized that 25 g of crops or fermented foods reach the large intestine, which has an average capacity of 1065 cc [54] (corresponds to 21 µg/mL in the large intestine if the food contains 10 % water). The cocultured metabolites were analyzed and compared (n = 8, Supplementary Table S1). Consequently, 32 peaks were identified (Table 1). Based on these data, PLS-DA was performed and corrected using an internal standard approach ( Figure 1A), which revealed that the control and glucosylceramide groups were separated ( Figure 1C). The permutation test results demonstrated that this was not an overfitting model ( Figure 1D). Important components that led to the separation of the two groups, as indicated by a VIP score greater than 1.0, were chosen. Substances, such as citric acid, threonine, lysine, lactic acid, valine, proline, and isoleucine, contributed to the separation of the two groups (Table 2). A one-sided unpaired Student's t-test revealed a substantial increase in citric acid content in the glucosylceramide group compared with that of the control group (p < 0.01). To further elucidate the effect of glucosylceramide on the physiology of B. coccoides, the short-chain fatty acids produced by B. coccoides were analyzed. Lactic acid levels were significantly increased in the broth supplemented with glucosylceramide compared with that of the control broth ( Figure 1E). Taken together, these results indicate that glucosylceramide addition alters the metabolism of B. coccoides.

Glucosylceramide Addition Increases Gram-Positive Intestinal Microbes
These results clearly indicate that glucosylceramide alters the metabolism of the intestinal microbes in this system. Next, to determine which bacteria were increased in response to glucosylceramide, NGS analysis was performed after incubation of model intestinal microbes (cell-mock-1), consisting of known ratios of bacteria that reside within human beings with glucosylceramide (Supplementary Table S2). Cell-mock-1 is a composite of bacteria Bacillus subtilis, Bifidobacterium pseudocatenulatum, Clostridium butyricum, Corynebacterium striatum, Cutibacterium acnes subsp. acnes, Lactobacillus delbrueckii subsp. delbrueckii, Streptococcus mutans, Staphylococcus epidermidis, Acinetobacter radioresistens, Bacteroides uniformis, Clostridium clostridioforme, Comamonas terrigena, Escherichia coli, Parabacteroides distasonis, and Pseudomonas putida. These bacteria were incubated in YCFA medium as a mixed state anaerobically with or without glucosylceramide. From the incubated culture, genomes were extracted and analyzed using NGS. As a result, relative OTU counts of Clostridium sensu stricto and Escherichia/Shigella were dominant (Figure 2A), maybe because of the anaerobic condition. These dominant bacteria were not significantly affected by glucosylceramide. In contrast, relative OTU counts of minor Gram-positive bacteria, such as Lactobacillus delbrueckii ( Figure 2B), Staphylococcus epidermidis ( Figure 2C), Streptococcus mutans ( Figure 2D), Corynebacterium striatum ( Figure 2E), and Clostridium butyricum ( Figure 2F), were significantly increased in glucosylceramide-treated cultures compared with those in control cultures (p < 0.05). In contrast, the Gram-negative bacterium, Acinetobacter radioresistens ( Figure 2G), decreased in glucosylceramide-treated cultures compared with that in control cultures (p < 0.05). affected by glucosylceramide. In contrast, relative OTU counts of minor Gram-positive bacteria, such as Lactobacillus delbrueckii ( Figure 2B), Staphylococcus epidermidis ( Figure 2C), Streptococcus mutans ( Figure 2D), Corynebacterium striatum ( Figure 2E), and Clostridium butyricum ( Figure 2F), were significantly increased in glucosylceramide-treated cultures compared with those in control cultures (p < 0.05). In contrast, the Gram-negative bacterium, Acinetobacter radioresistens ( Figure 2G), decreased in glucosylceramide-treated cultures compared with that in control cultures (p < 0.05).
(A)  Data are expressed as means ± standard errors. Data normality was verified using the Shapiro-Wilk test. Statistical differences in means of data with normalities were analyzed using a one-sided unpaired Student's t-test (n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001).

Glucosylceramide Confers Tolerance of Intestinal Microbes to Secondary Bile Acids
Considering that certain intestinal bacteria have a tolerance to bile acids [55], we hypothesized that these bacteria increased growth in the existence of glucosylceramide because they acquired tolerance to deoxycholic acid. Therefore, tolerance of these bacteria towards deoxycholic acid was investigated.
With the addition of glucosylceramide, L. delbrueckii showed increased growth at 1.0-2.0 mM deoxycholic acid relative to that in the control ( Figure 3A). Glucosylceramidesupplemented L. delbrueckii displayed an increased half-maximal inhibitory concentration value compared with that of the control, although the difference was not statistically significant (Table 3).

Glucosylceramide Confers Tolerance of Intestinal Microbes to Secondary Bile Acids
Considering that certain intestinal bacteria have a tolerance to bile acids [55], we hypothesized that these bacteria increased growth in the existence of glucosylceramide because they acquired tolerance to deoxycholic acid. Therefore, tolerance of these bacteria towards deoxycholic acid was investigated.
With the addition of glucosylceramide, L. delbrueckii showed increased growth at 1.0-2.0 mM deoxycholic acid relative to that in the control ( Figure 3A). Glucosylceramidesupplemented L. delbrueckii displayed an increased half-maximal inhibitory concentration value compared with that of the control, although the difference was not statistically significant (Table 3). Values are mean ± standard errors of triplicate independent cultures. Statistical differences of the means were evaluated by one-sided unpaired Student's t-test (n = 3, * p < 0.05, ** p < 0.01).  The tolerance of another bacterium, S. epidermidis, to deoxycholic acid was also investigated. There was no significant difference in the growth or half-maximal inhibitory concentration between the glucosylceramide-treated and control S. epidermidis ( Figure 3B and Table 3). Growths of S. mutans, C. striatum and C. butyricum in the presence of deoxycholic acid were also partially rescued with glucosylceramide ( Figure 3C-E). IC 50 of S. mutans was significantly increased (p < 0.01, Table 3). The growth of E. coli was not inhibited by deoxycholic acid ( Figure 3F) and it was difficult to measure the effect of glucosylceramide. The glucosylceramide effect was not observed in Bacteroides uniformis ( Figure 3G). Furthermore, the tolerances of other major intestinal microbes, including Blautia coccoides and Enterococcus faecalis, against deoxycholic acid were investigated. The growth of B. coccoides was rescued with glucosylceramide at 1 mM deoxycholic acid concentration ( Figure 3H). Similarly, the growth of E. faecalis was partially rescued with glucosylceramide ( Figure 3I). The IC 50 value for E. faecalis was also significantly higher after glucosylceramide addition than that in the control (Table 3). These results suggest that glucosylceramide increased the tolerance of certain intestinal microbes to deoxycholic acid.

Discussion
Although we have long known that glucosylceramide is present in foods such as crops and fermented foods at 0.94-2.2 mg/g [19,24,25,53], and the majority of the molecule passes through the small intestine and reaches the colon, the mechanism of action of glucosylceramide on intestinal microbes has not yet been reported. In this study, we first report the in vitro prebiotic effects of glucosylceramide. Using metabolome analysis followed by PLS-DA, we demonstrate that adding glucosylceramide to the major intestinal bacterium B. coccoides has a significant metabolic effect. Furthermore, the addition of glucosylceramide increased the number of Gram-positive bacteria through tolerance to deoxycholic acid. These results provide the first evidence of a mechanism underlying the action of glucosylceramide on intestinal microbes. This research has certain limitations and weaknesses. Adding pure components to monocultures does not necessarily reflect what occurs in the complex microbiota of the gut, since the actual gut is filled with various substances and microbes. Moreover, a certain amount of glucosylceramide, although very small [38], might be degraded and absorbed in the small intestine. This research did not consider the absorption of glucosylceramide into the epithelial cells or mucosa in the intestine into account. However, it has a strong aspect as relative to in vivo studies. During the passage through the intestine, glucosylceramide might interact with many bacteria. Some bacteria might attach to the epithelial cells of the intestine, and might not be detected in the feces. On the contrary, in vitro studies generates the direct interaction knowledge between glucosylceramide and bacteria. Therefore, the knowledge obtained through in vitro study should mutually complement in vivo studies to reach a comprehensive understanding of the interaction between substances and intestinal microbes.
Secondary bile acids are formed in the large intestine by intestinal microbes and disrupt the membranes of bacteria, killing them [41]. In the present study, glucosylceramide attenuated these effects. Therefore, glucosylceramide that reaches the large intestine may protect the intestinal microbes from secondary bile acids.
Blautia species are one of the most dominant intestinal epithelial barrier-associated bacteria, which belongs to the Lachnospiraceae family and Firmicutes phylum [56]. Blautia species are strictly anaerobic, non-motile bacteria with spherical or oval morphologies. They can assimilate various carbohydrates such as glucose, fructose, lactose, mannose, arabinose and xylose, CO and H 2 /CO 2 , and produces acetic acid, succinic acid, lactic acid and ethanol. It is suggested from gene expression analysis that the tendency of assimilation of carbohydrates of Blautia species is lower than that of lactic acid bacteria. There is a negative correlation between the abundance of Blautia and the markers of obesity-related metabolic disorders when fed with maize-derived non-digestible feruloylated oligo-and polysaccharides [57], potato fibers [58] and soy milk [59]. Intestinal microbes of Japanese people contained a high abundance of Blautia and Bifidobacterium and a low abundance of Bacteroides [60,61]. Considering that Blautia has a strong taxonomic association with twin inheritance [62], there might be a genetic preference of Blautia towards the intestine of specific genetic background. Blautia species are also linked to abnormal Paneth cell counts [63], Crohn's disease and primary sclerosing cholangitis [64]. B. coccoides in the intestine increases with glucosylceramide in an animal model [23], but decreases with pectins and flavanones in humans [65]. Blautia species produce useful secondary metabolites such as chrysin, apigenin, desmethyllicaritin, 3 desmethyllarctigenin, bisdemethylcurcmin, demethyldemethoxycurcumin and ceramide in the intestine [54].
Promoted survival of Gram-positive bacteria elucidated in this study suggests the clinical benefits of glucosylceramide. For example, feeding of L. brueckii subsp. delbrueckii improves immune health in an animal model [66] and inhibits the growth of Klebsiella pneumonia [67]. C. butyricum also functions as probiotics by inducing interleukin-10-producing macrophages in inflamed mucosa via the Toll-like receptor 2/myeloid differentiation primary response gene 88 pathway [68] and has many clinical applications [69]. Although pathogenicity is reported for C. striatum [70], these events are not reported in the intestine. Since S. mutans [71] and S. epidermidis [72] form biofilms and are opportunistic pathogens, the health benefits of the modulation of intestinal microbes through the ingestion of glucosylceramide should be considered comprehensively. Still, this study suggests that the ingestion of glucosylceramide, on the whole, might promote the survival of beneficial intestinal microbes, which is a target of future studies.
This study did not elucidate how or why glucosylceramide increased the ratio of Gram-positive bacteria. Since Gram-positive bacteria have a thick peptidoglycan layer, they may be better suited for the attachment of glucosylceramide and its incorporation into their structures as compared with that of Gram-negative bacteria. Consistent with this hypothesis, Lacticaseibacillus casei was reported to bind glucosylceramide [73]. In addition, several other lactic acid bacteria have been shown to bind sphingolipids containing carbohydrate moieties, including Lactobacillus johnsonii to asialoganglioside [74] and Propionibacterium freudenreichii to lactosylceramide [75]. Therefore, it can be hypothesized that glucosylceramide bound to the surface of Gram-positive bacteria and endowed tolerance to deoxycholic acid. The effect of glucosylceramide on other intestinal bacteria should also be investigated.
This study proposes a potential mechanism for the observed effects in vivo [26][27][28][29][30]. Our results suggest that glucosylceramide protects intestinal microbes, especially Grampositive bacteria, increases the production of substances such as citric acid, and as a result, might exert various health effects. Consistent with this hypothesis, citric acid improves several nutritional profiles in quails [76]. However, this hypothesis requires further investigation.
Our analysis of bacterial tolerance to deoxycholic acid was generally consistent with our results obtained from NGS analysis. However, S. epidermidis did not show significant recovery of tolerance to deoxycholic acid with glucosylceramide, which was inconsistent with the NGS analysis. The tolerance analysis may have lacked sufficient sensitivity to detect the difference in S. epidermidis tolerance towards deoxycholic acid, which can be elucidated using a more precise analysis tool in future studies.
Several substances have been proposed as potential prebiotics. However, there are no reports of the effects of glucosylceramide on intestinal microbes. Based on our results, glucosylceramide can be hypothesized as a type of prebiotic.
Feeding of fructooligosaccharide and galactooligosaccharide decreased butyrate, increased Actinobacteria, Bifidobacterium, Acidaminococcosu and decreased Salmonella, Coprococcus, Turicbacter, Enterobacter and Phascolactobacterium in vivo [77]. Therefore, it could be hypothesized that glucosylceramide might function differently from oligosaccharides, which is a target of future studies.
Sphingolipid synthesis was shown to improve the survival of intestinal Bacteroides [78]. Therefore, it is suggested that exogenous glucosylceramide also improves the survival of these intestinal bacteria, which is a target of future studies.
Glucosylceramide in the luminal side of the intestine, which was investigated in this study, might affect that in the epithelial cells. Glucosylceramide is shown to protect CaCO 2 cells treated with lipopolysaccharide in an in vitro intestinal tract model [79]. Furthermore, glucosylceramide production maintains colon integrity when challenged with Bacteroides fragilis toxin [80]. Together with the results shown in this study that glucosylceramide protects intestinal microbes, glucosylceramide exerts its effects both in the luminal space of the intestine and the epithelial cells.
In conclusion, we demonstrate that the addition of glucosylceramide induces changes in the metabolism of B. coccoides and increases the number of Gram-positive bacteria in an intestine-simulating environment through tolerance to deoxycholic acid. These results first indicate the prebiotic-like function of glucosylceramide.