CHENG JIYE (US)
BARRATT MICHAEL (US)
GORDON JEFFREY (US)
US20200297784A1 | 2020-09-24 |
HAN NATHAN D.; CHENG JIYE; DELANNOY-BRUNO OMAR; WEBBER DANIEL; TERRAPON NICOLAS; HENRISSAT BERNARD; RODIONOV DMITRY A.; ARZAMASOV : "Microbial liberation of N-methylserotonin from orange fiber in gnotobiotic mice and humans", CELL, ELSEVIER, AMSTERDAM NL, vol. 185, no. 14, 27 June 2022 (2022-06-27), Amsterdam NL , pages 2495, XP087113227, ISSN: 0092-8674, DOI: 10.1016/j.cell.2022.06.004
WHAT IS CLAIMED IS: 1. A synbiotic composition comprising at least one type of plant fiber and at least one microbial strain. 2. The synbiotic composition of claim 1, wherein the at least one type of plant fiber comprises a Rutaceae family plant fiber. 3. The synbiotic composition of claim 2, wherein the Rutaceae family plant fiber comprises citrus fiber. 4. The synbiotic composition of claim 3, wherein the citrus fiber comprises orange fiber. 5. The synbiotic composition of claim 1, wherein the at least one microbial strain comprises at least one bacterial strain. 6. The synbiotic composition of claim 5, wherein the at least one bacterial strain is selected from Bacteroides, Parabacteroides, Collinsella, and combinations thereof. 7. The synbiotic composition of claim 6, wherein the at least one bacterial strain comprises at least one strain of Bacteroides ovatus, Bacteroides finegoldii, Parabacteroides distasonis, Collinsella aerofaciens, or combinations thereof. 8. The synbiotic composition of claim 7, wherein the at least one bacterial strain comprises Bacteroides ovatus TSDC 17.2. 9. The synbiotic composition of claim 1, further comprising an iron-containing porphyrin. 10. The synbiotic composition of claim 9, wherein the iron-containing porphyrin is hemin. 11. A method for locally delivering a bioactive compound to a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the synbiotic composition of claim 1. 196 12. The method of claim 11, wherein the at least one microbial strain is a source of at least one CAZyme. 13. The method of claim 12, wherein the at least one CAZyme is selected from PL9, GH5_37, GH5_8, GH59, GH30_5, GH26, GH5_4, GH25, GH13_31, GH123, GH13_19, GH13_28, and combinations thereof. 14. The method of claim 13, wherein the at least one CAZyme comprises PL9. 15. The method of claim 11, wherein the bioactive compound is N-methylserotonin. 16. A method for increasing liver glycogen in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the synbiotic composition of claim 1. 17. A method for increasing tissue glutamate levels in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the synbiotic composition of claim 1. 18. A method for reducing adiposity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the synbiotic composition of claim 1. 19. A method for reducing high fat diet induced obesity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the synbiotic composition of claim 1. 20. A method for increasing fatty acid metabolism in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the synbiotic composition of claim 1. 197 21. A method for decreasing gastrointestinal transit time in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the synbiotic composition of claim 1. 22. A method for increasing colonic motility in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the synbiotic composition of claim 1. 23. A method for treating irritable bowel syndrome in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the synbiotic composition of claim 1. 24. A prebiotic composition comprising an iron-containing porphyrin and at least one type of plant fiber. 25. The prebiotic composition of claim 24, wherein the iron-containing porphyrin is hemin. 26. The prebiotic composition of claim 24, wherein the at least one type of plant fiber comprises a Rutaceae family plant fiber. 27. The prebiotic composition of claim 26, wherein the Rutaceae family plant fiber comprises citrus fiber. 28. The prebiotic composition of claim 27, wherein the citrus fiber comprises orange fiber. 29. A method for locally delivering a bioactive compound to a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the prebiotic composition of claim 24. 30. The method of claim 29, wherein the bioactive compound is N-methylserotonin. 31. A probiotic composition comprising an iron-containing porphyrin and at least one microbial strain. 198 32. The probiotic composition of claim 31, wherein the iron-containing porphyrin is hemin. 33. The probiotic composition of claim 32, wherein the at least one microbial strain comprises at least one bacterial strain. 34. The probiotic composition of claim 33, wherein the at least one bacterial strain is selected from Bacteroides, Parabacteroides, Collinsella, and combinations thereof. 35. The probiotic composition of claim 34, wherein the at least one bacterial strain comprises at least one strain of Bacteroides ovatus, Bacteroides finegoldii, Parabacteroides distasonis, Collinsella aerofaciens, or combinations thereof. 36. The probiotic composition of claim 35, wherein the at least one bacterial strain comprises Bacteroides ovatus TSDC 17.2. 37. A method for locally delivering a bioactive compound to a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the probiotic composition of claim 31. 38. The method of claim 37, wherein the at least one microbial strain is a source of at least one CAZyme. 39. The method of claim 38, wherein the at least one CAZyme is selected from PL9, GH5_37, GH5_8, GH59, GH30_5, GH26, GH5_4, GH25, GH13_31, GH123, GH13_19, GH13_28, and combinations thereof. 40. The method of claim 39, wherein the at least one CAZyme comprises PL9. 41. The method of claim 37, wherein the bioactive compound is N-methylserotonin. 199 |
[0074] Table 1B. Reagents and Resources
[0075] RESULTS [0076] A gnotobiotic mouse model reveals human gut bacterial liberation of N- methylserotonin from orange fiber. As a starting point for characterizing liberation of fiber- associated bioactive constituents by gut bacterial taxa, a commercial, food-grade source of orange fiber was selected (see Methods), derived from the byproducts of the juicing process; these byproducts include pulp cells, juice vesicles, segment membranes, rag/core and peel that are mechanically processed (washed with water, heated, dewatered, sheared) prior to drying. Importantly for the purpose of experiments for the present disclosure, the preparation had not been subject to chemical treatment or extraction; therefore, any proteins, lipids, and small molecules that are not removed by washing with water are retained in the preparation (see Table S1A,B for composition and glycosidic linkage analysis of constituent polysaccharides). [0077] Two groups of adult C57BL/6J germ-free mice were colonized with a 14-member consortium of sequenced human gut bacterial strains and monotonously fed the HiSF-LoFV diet ad libitum, with or without supplementation with 10% (w/w) orange fiber, for 21 days. Two other groups of mice were maintained as germ-free; mice in one of these groups were fed the unsupplemented HiSF-LoFV diet while those in the other group consumed the 10% orange fiber- supplemented diet (n=5 animals/treatment group). [0078] Untargeted liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Qtof-MS) of cecal contents harvested at the time of euthanasia revealed 116 features (m/z) that were increased at least 3-fold in colonized mice consuming the orange fiber-supplemented diet compared to the other three experimental groups (Table S2). A prominent feature with an m/z of 191.1186 was present at high abundance only in colonized mice fed the orange fiber- supplemented diet (FIG. 1A); it was tentatively identified as methylserotonin, with its major fragment (m/z 160.0760) consistent with methylation of its alkyl amine. Subsequent LC- Qtof/MS/MS co-characterization with a known standard confirmed that this compound was N- methylserotonin (FIG. 1B). N-methylserotonin has been previously identified in several plants, including black cohosh, Japanese pepper, and citrus fruits. There is very limited information on whether this compound has beneficial or potentially detrimental physiologic effects. [0079] The experiment was repeated and further compared different groups of mice colonized with the 14-member consortium and fed the HiSF-LoFV diet supplemented with 10% orange fiber or with 10% pea fiber. The latter was a natural food-grade commercial preparation consisting of insoluble and soluble fibers as well as resistant starch ( e Methods and Table S1A for composition). Targeted liquid chromatography-triple quadrupole mass spectrometry (LC- QqQ-MS) revealed that N-methylserotonin was present in significantly higher amounts in both the cecal and colonic contents and tissues of mice consuming the orange fiber-supplemented diet [173 ± 14 ng/g and 130 ± 13 ng/g (mean ± SD) in cecal contents and cecal tissue, respectively and 139 ± 13 ng/g and 164 ± 25 ng/g in colonic contents and tissue, respectively] compared to their small intestine, liver, gastrocnemius muscle and kidney (<1 ng/g) or plasma (1.22 ± 0.76 ng/mL). Non-targeted LC-Qtof-MS and targeted LC-QqQ-MS analysis revealed no statistically significant differences (p>0.05; t-test) in the levels of serotonin, dimethylserotonin, trimethylserotonin, 5-hydroxyindoleacetic acid, tryptamine, N-methyltryptamine, N,N- dimethyltryptamine, tryptophan, bufotenin or melatonin in small intestinal, cecal and colonic tissue or liver obtained from animals consuming the unsupplemented versus orange fiber- supplemented HiSF-LoFV diet, indicating that the host is unable to metabolize N-methylserotonin to these products. N-methylserotonin was below the limits of detection (<0.5 ng/g) in cecal or colonic contents or any of these intestinal and extra-intestinal tissues harvested from mice consuming the pea-fiber supplemented diet. [0080] Host effects of N-methylserotonin. To characterize the effects of N- methylserotonin on host physiology and metabolism, 12-week-old germ-free C57BL/6J mice were fed the unsupplemented HiSF-LoFV diet and administered N-methylserotonin via their drinking water at doses of 1 mg/kg/day or 50 mg/kg/day for 21 days (FIG. 2A). The 1 mg/kg/day dose was experimentally determined to result in fecal N-methylserotonin levels that were equivalent to those documented in mice colonized with the 14-member community consuming the orange fiber-supplemented HiSF-LoFV diet (133.5 ± 15 ng/g versus 131 ± 19 ng/g feces, respectively; p=0.92, unpaired t-test). The 50 mg/kg/d dose was equivalent to the estimated total amount of N-methylserotonin consumed each day in the 10% orange fiber-containing diet (based on the yield obtained after in vitro enzymatic digestion of the fiber with T. reesei cellulase). A control group of germ-free animals did not receive any N-methylserotonin. Food and water intake were measured daily and remained consistent throughout the experiment among all three groups of mice. [0081] While a statistically significant decrease in weight gain was observed with N- methylserotonin treatment (FIG. 2B), interpreting this result is confounded by the abnormally large contribution of the cecum to body weight in germ-free mice. However, compared to untreated controls, oral administration of N-methylserotonin resulted in a statistically significant reduction in epididymal fat mass at the higher dose but not the lower dose (FIG. 2C). [0082] The higher dose also produced statistically significant increases in liver glycogen (FIG. 2D) and statistically significant decreases in its metabolic precursors, uridine and uridine monophosphate (FIG. 2E,F), which the lower dose did not. Administration of the higher dose resulted a statistically significant decrease in liver glucose-6-phosphate (FIG. 2G), a key metabolic intermediate formed from either glycogenolysis or gluconeogenesis that is known to directly impact levels of glycogen in the liver, while the lower dose did not. Based on these results, untreated control animals and those that received 50 mg/kg/d of N-methylserotonin were used to perform RNA-Seq on liver and colon. [0083] A total of 716 genes exhibited statistically significant differences in their expression in the livers of N-methylserotonin-treated compared to untreated mice (FDR adjusted p-value <0.05; see Methods and Table S3A,B). Gene-set enrichment analysis and over- representation analysis (Methods) of all statistically significant differentially expressed genes (FDR adjusted p-value <0.05) revealed that GO Biological Pathway terms pertaining to circadian rhythm and fatty acid metabolism were the most significantly enriched (FIG. 6). Effects of N- methylserotonin on circadian rhythm-related genes included significantly decreased expression of Arntl and Clock [see Table S3 for loge-fold change and FDR adjusted p-values], both of which have been linked to suppressed gluconeogenesis and lipogenesis. Consistent with this observation, there were significant increases in expression of their regulators Per2, Per3, and Nr1d2. Per2 promotes glycogen synthesis. Moreover, genetically engineered disruption of Per3 is associated with resistance to leptin with resulting weight gain, while its deletion directly leads to increased adipogenesis. Nr1d2 acts a repressor of Nfil3; its statistically significant increased expression with N-methylserotonin treatment is associated with statistically significantly decreased hepatic levels of Nfil3 mRNA. Nfil3 serves as an important link between the gut microbiota, intestinal epithelial lipid metabolism and body composition. Nfil3 expression exhibits microbiota-modulated diurnal oscillation in epithelial cells via group 3 innate lymphoid cells, Stat3 and epithelial clock components, with accompanying changes in epithelial lipid absorption and export. Moreover, genetic ablation of Nfil3 attenuates high fat diet-induced obesity in mice. [0084] Seven hundred and forty-eight genes exhibited statistically significant differences in their expression in the colonic tissue of N-methylserotonin-treated versus untreated mice (FDR- adjusted p-value <0.05; Table S3C,D); they include Nr1d2, Per3, Per2, Arntl and Clock. In vitro studies have indicated that N-methylserotonin binds to various serotonin (5-hydroxytryptamine) G protein-coupled receptors, including 5-Htr7 and 5-Htr2A. RNA-Seq analysis of colon did not reveal any statistically significant effects of N-methylserotonin administration on colonic expression of its known (Htr7, Htr2A) or related (Htr3, Htr4, Htr5 and Htr6) receptors. [0085] Glutamate levels were significantly higher in colonic tissue harvested from germ- free mice receiving 50 mg/kg/day of N-methylserotonin compared to untreated controls (21 ± 1.4 versus 11 ± 0.9 ng/mg tissue, respectively; p<0.01, unpaired t-test). Glutamate is known to degrade Arntl when directly applied to tissue slices. Knockout of the Per3 homolog Per2 reduces expression of the glutamate transporter (Eaatl, Slc1A3) and uptake of glutamate in the brain. These observations raise the possibility that one way that N-methylserotonin might influence colonic circadian regulators is through its effects on glutamate levels in this tissue. [0086] Circadian rhythm-related genes are known to be expressed in the myenteric plexus which coordinates colonic motility. Orally administered carmine red was used to determine the gastrointestinal transit time on day 17 of the 21-day experiment in germ-free mice whose drinking water was supplemented with 1 mg/kg/day or with 50 mg/kg/day of N-methylserotonin. The assay revealed that both doses of N-methylserotonin produced equivalent reductions in transit time (i.e., increased motility) compared to the untreated control group (p<0.0001, one-way ANOVA; FIG. 2H). [0087] Bacterial strains capable of mining of N-methylserotonin from orange fiber in vitro. Given that detection of N-methylserotonin was dependent on colonization with the bacterial consortium and consumption of orange fiber, it was next investigated which community members were responsible for its appearance. Each of the 14 community members was grown in monoculture to stationary phase in TYG medium; 10 5 cells of each organism were incubated in 10 mL of a 5 mg/mL suspension of orange fiber for 8, 24, 48, 72 and 168 hours. N- methylserotonin was quantified using targeted LC-QqQ-MS. N-methylserotonin rose from levels that were not significantly above background at the 8h time point (background determined by measurements of control incubations containing sterile TYG medium), to levels that reached a maximum at the 72-hour time point. At this time point, Bacteroides ovatus strain TSDC 17.2 and a strain of Parabacteroides distasonis yielded similar quantities of product (29 ± 1 and 24 ± 2 ng N-methylserotonin/10 6 cells, respectively). In contrast, the 12 other strains yielded <1.5 ng/10 6 cells - an amount that was not appreciably higher than background levels (triplicate incubations/organism; FIG. 1C). Cultures grown in Wilkins-Chalgren anaerobe broth yielded results that were similar to those obtained with TYG medium (Table S4A), while testing each of these 14 organisms in an another nutrient rich medium (Mega medium 2.0) resulted in N- methylserotonin levels ranging from 5-25 ng/10 6 cells for P. distasonis, Bacteroidesfinegoldii and Collinsella aerofaciens (Table S4A). Given its capacity to support the growth of a number of cultured anaerobic gut bacterial taxa, 24 other phylogenetically diverse human gut bacterial strains were screened in Wilkins-Chalgren anaerobe broth containing 5 mg/mL of orange fiber; none yielded amounts of N-methylserotonin significantly above background (< 0.5 ng/10 6 cells) (Table S4B). [0088] Several other experiments were performed to characterize in vitro N- methylserotonin liberation by members of the 14 strain consortia. Adding either (i) 10 8 heat-killed cells of either B. ovatus TSDC 17.2 or the P. distasonis strain that had been grown to stationary- phase in TYG, or (ii) lysates prepared by bead-beating of 10 8 cells harvested from monocultures of each organism in TYG, or (iii) conditioned medium harvested from stationary phase TYG monocultures of each organism, to fresh TYG with orange fiber for 72 hours failed to yield levels of N-methylserotonin above background (Table S4C). These experiments indicate that mining requires intact viable cells. When N-methylserotonin was added at a concentration of 5 ng/mL to monocultures of the 14 strains that had been grown to stationary phase in TYG medium without orange fiber, no appreciable degradation was observed over a 72-hour period (3 replicate assays/organism/experiment; 97 ± 2% of input N-methylserotonin remaining intact/unmodified; triplicate incubations/condition; Table S4D). Evidence that B. ovatus TSDC 17.2 was not capable of synthesizing N-methylserotonin was also obtained. A homology-based search of the bacterial genome failed to reveal gene candidates involved in serotonin biosynthesis and metabolism. Moreover, when the organism was cultured in TYG medium supplemented with either tryptophan, tryptamine, serotonin, dimethylserotonin, trimethylserotonin, methyltryptamine, or S-adenosyl methionine (see Methods), N-methylserotonin was not detected above background after either 24 or 72 hours. Incubating this strain in TYG medium supplemented with either pea fiber, or two other commercial dietary fiber preparations (apple pectin or oat beta glucan) also did not yield levels of N-methylserotonin above background. [0089] Based on these in vitro findings, adult C57BL/6J germ-free mice were colonized with (i) a 4-member consortium comprised of the B. ovatus, P. distasonis, B. finegoldii and C. aerofaciens strains with in vitro mining activity, or (ii) the full 14-member consortium, or (iii) the 10 remaining strains from the 14-member consortium. Three days after gavage, animals (n=5/group) were switched from the unsupplemented HiSF-LoFV diet to a HiSF-LoFV diet supplemented with 10% (w/w) orange fiber. This diet was then administered ad libitum for 21 days (FIG. 3A,B). Short-read shotgun sequencing of DNA isolated from fecal samples collected at the time of euthanasia revealed that all strains in each consortium were able to colonize recipient animals (see Table S5 for their absolute abundances). The total biomass (bacterial genome equivalents/g feces) in mice harboring the 4-member consortium was 2-fold lower than in animals colonized with the 14-member consortium (p<0.01; one-way ANOVA) while there was no significant difference in bacterial load between animals hosting the 10- and 14-member communities (p=0.81, FIG. 3C). Targeted LC-QqQ-MS analysis of fecal samples obtained at the time of euthanasia revealed that levels of N-methylserotonin in mice colonized with the 4-strain consortium were equivalent to those in animals harboring the full 14-member community and significantly higher than in mice colonized with the 10-member consortium (p=0.002; one-way ANOVA; FIG. 3D). Animals colonized with the 4-member consortium had a significantly lower epididymal fat pad mass compared to mice colonized with the 10-member consortium (p=0.007 and p=0.001, respectively; one-way ANOVA). No significant differences in adiposity were noted between mice harboring the 4- and 14-member communities (FIG. 3E,F). Mice colonized with the 4-member consortium also had a statistically significant reduction in gut transit time compared to animals containing the 10-member community [184 ± 32 minutes (mean ± SD) versus 319±8 minutes, respectively; p<0.0001, one-way ANOVA). The 14-member community was associated with transit times (278 ± 14 minutes), that were also significantly shorter compared to mice with the 10-member community (p=0.025) yet were still significantly longer compared to mice harboring the 4-member community (p<0.0001) (FIG. 3G). [0090] Identifying genes involved in release of N-methylserotonin from orange fiber. Reasoning that microbial disruption of complex polysaccharides in fibers might be needed to liberate sequestered N-methylserotonin, a set of experiments was performed where 50 mg of orange fiber was incubated separately with 13 commercially available glycoside hydrolase preparations (see Methods). The greatest amount of N-methylserotonin was recovered when orange fiber was incubated with a preparation containing endoglucanases and cellulases from Trichoderma reesei (total yield; 2728 ± 26 ng/50 mg orange fiber) (Table S4E). When orange fiber (50 mg) was subjected to repeated rounds of extraction with methanol, small quantities of N-methylserotonin were released after each round (31 ng after two rounds; 171 ng in total after 15 rounds; Table S4F). Similar yields were obtained in separate experiments using acetonitrile or acetone (30-31 ng after two rounds of extraction). A comparison of the results of serial methanol extractions of N-methylserotonin against a spike-in compound added to orange fiber whose structure was similar to N-methylserotonin (2-methylserotonin) showed that 95% of 2- methylserotonin was removed by the first cycle of extraction and all of the remaining by the third cycle (Table S4F). These latter observations additionally supported the discovery that N- methylserotonin is "trapped" within orange fiber. [0091] A comparative genomic and functional genomics approach was taken to further characterize the mechanisms underlying release of N-methylserotonin from orange fiber. The N- methylserotonin `mining' activity of B. ovatus TSDC 17.2 was first compared to 11 other human gut-derived strains of B. ovatus. All strains were grown on TYG medium and subjected to the same protocol for assaying N-methylserotonin release from orange fiber as described herein (i.e., 10 5 input bacterial cells/incubation containing 5mg/mL orange fiber; 72-hour incubation; 3 replicate assays/strain). Compared to control incubations lacking orange fiber where the yield of N-methylserotonin was 1.5 ± 0.2 ng/10 6 cells (mean ± SD), it was found that all strains were able to release this compound. The amounts released varied between strains, however all strains had mining activities that were significantly lower than TSDC 17.2 (triplicate assays/strain; one-way ANOVA, all P-values <0.0001). B. ovatus 115 had the lowest activity [2.8 ± 0.1 ng (mean ± SD) N-methylserotonin released/10 6 cells compared to 33.3 ± 2.8 ng/10 6 cells for TSDC 17.2 (FIG. 4A, Table S4G)]. [0092] As noted herein, several of the bacterial taxa tested exhibited mining activity that was dependent upon the growth medium used. This observation led to a search for components in TYG whose presence was essential for N-methylserotonin release. This search yielded hemin, a known regulator of gene expression in Bacteroides species. While all strains grew to comparable densities in TYG with or without hemin, no microbe-dependent N-methylserotonin release occurred when these cells were added to reactions containing fresh hemin-deficient TYG plus orange fiber (controls; incubations containing TYG ± hemin but lacking orange fiber; FIG. 4B, Table S4H). [0093] To identify the genes likely to contribute to N-methylserotonin release, the genomes of all 12 strains were sequenced, annotated all of their known or predicted encoded proteins, and performed microbial RNA-Seq analysis of gene expression in B. ovatus TSDC 17.2 and B. ovatus 115 grown under conditions identical to those used during assays for N- methylserotonin release activity (72-hour incubation with or without orange fiber in TYG medium with or without hemin, or in MEGA medium). Gene expression in strain TSDC 17.2 under the permissive N-methylserotonin releasing condition (TYG medium ± orange fiber) was first compared with gene expression under non-permissive conditions (TYG minus hemin with and without orange fiber).133 genes were identified that (i) exhibited a statistically significant >1 log2-fold increase in expression under releasing conditions (Benjamini and Hochberg FDR- adjusted Wald test p-value <0.05), and (ii) were either not significantly differentially expressed (FDR-adjusted p-value > 0.1), or were significantly downregulated (FDR-adjusted p-value <0.05) under non-releasing conditions (see Table S6A for a list of these 133 genes plus FIG. 4C). [0094] Natural products are embedded/entrapped in dietary fiber through various chemical and physical interactions. As noted herein, the orange fiber preparation contained nearly 60% (w/w) uronic acid, with prominent representation of homogalacturonan, rhamnogalacturonan, xylan and arabinan structures (Table S1B). Bacteroides species possess multiple polysaccharide utilization loci (PULs). These PULs encode proteins (SusC and SusD homologs) involved in binding and import of various glycan structures as well as carbohydrate active enzymes (CAZymes) that catalyze their degradation [glycoside hydrolases (GH) and polysaccharide lyases (PL)]. Therefore, previously described methods were used to identify PULs and CAZyme gene clusters present in Bacteroides ovatus strains TSDC 17.2 and 115. The results revealed that PUL conservation and synteny between the two strains is very high (Table S7). [0095] Among the 133 genes with statistically significant differential expression in TSDC 17.2, those that manifested the most prominent induction under N-methylserotonin releasing conditions were concentrated in PUL27, PUL28 and PUL29, and to a lesser extent in several other PULs (e.g., PUL4 and PUL13). Proteins encoded by these PULs exhibit >95% amino acid sequence identity with those in strain 115 and share orthologs in the other strains tested (Table S6A, Table S7). Functional assignments for these proteins were made by identifying their best scoring alignments with the sequences of experimentally characterized CAZymes in the CAZy database (www.cazy.org) (Table S6B). The results disclosed members of CAZyme families with reported activities against the backbones of homogalacturonan [GH family 105 (unsaturated rhamnogalacturonyl hydrolase/unsaturated glucuronyl hydrolase)] or rhamnogalacturonan [PL9, PL11 (rhamnogalacturonan lyase); GH28 (RGI-specific a-galacturonidase)]. These structures are prominently represented in pectin and in the orange fiber preparation of the present disclosure (>50% of glycosyl linkages, Table S1B). The PULs also included CAZymes with predicted activities directed at oligosaccharides linked to these backbone structures [arabinofuranosidase (GH4318), galactosidase (GH36), and apiosidase (GH140)]. [0096] Despite the high degree of PUL conservation between B. ovatus TSDC 17.2 and B. ovatus 115, almost none of their component genes are expressed in the latter strain under mining-permissive conditions (Table S7). In an attempt to define the origin of the observed differences in expression of these PUL genes, and by extrapolation, the discordant N- methylserotonin mining activities of strains TSDC 17.2 and 115, potential transcriptional regulons were reconstructed using comparative genomics (FIG. 7). PUL27, PUL28 and PUL29 form a large chromosomal cluster of 60-70 genes that encode 28 CAZymes, six SusC/SusD transport systems, and three paralogs of a previously characterized rhamnogalacturonan-specific regulator in Bacteroides thetaiotaomicron, HTCS_Rgu-2. Hybrid two-component systems (HTCS) are single polypeptide chains comprised of a transmembrane sensor histidine kinase, a DNA-binding response regulator, and a carbohydrate sensing domain. The reconstructed HTCS_Rgu-2 regulon in B. ovatus strains includes 42 genes from PUL27, PUL28, PUL29, and PUL30, of which 30 were significantly upregulated (FDR-adjusted p-value < 0.05) in the presence of orange fiber (Table S7). However, all identified HTCS_Rgu-2 binding sites are highly conserved between the 17.2 and 115 strains, and the orthologous pairs of HTCS regulators are 98¬99% identical to each other, indicating (i) conservation of this feature of regulation of rhamnogalacturonan-I utilization loci between the two strains of B. ovatus and (ii) that the observed difference in regulon expression is likely not ascribable to this HTCS alone. [0097] N-methylserotonin levels and microbiome CAZyme gene abundances in humans consuming fiber snack prototypes. To assess the translatability of results obtained from these in vitro analyses and mouse model to humans, two exploratory 10-week open-label, single group assignment studies were performed. The studies involved orange fiber- and pea fiber- supplemented snack food prototypes and dizygotic twins 36.6 ± 2.9 years old (mean ± SD) recruited from the Missouri Adolescent Female Twin Study (MOAFTS) cohort. The two studies had the same design (see Methods), with participants supplementing their normal, unrestricted diets with one or the other snack food prototype. In brief, consumption of the fiber snack prototypes escalated from none consumed during the first two weeks, to one snack per day during the third week, then two servings a day during week 4, and finally, beginning week 5, three snacks at which time the maximum daily dose of —25-30g per day of either pea fiber (Study 1; n=18 participants) or orange fiber (Study 2; n=24 participants, including all 18 from Study 1) was achieved. This dose level was then maintained for 4 weeks (see Table S8A for the composition of the snack prototypes and Table S8B for participant characteristics). Importantly, the orange and pea fiber preparations used for these human studies were obtained from the same commercial sources as those used in preclinical studies. Therefore, analysis of fecal samples collected during the course of these two studies, including from subjects who had participated in both, provided an opportunity to examine the relationship between features of the microbiome and fecal levels of N-methylserotonin as a function of fiber consumption. Specifically, the present disclosure enabled an assessment of whether mining was robust to different background diets, exhibited specificity for orange fiber and was dependent upon the amount of orange fiber consumed. [0098] N-methylserotonin it was present in 98% of the 48 samples obtained from participants consuming the orange fiber snack prototype (FIG. 5A and Table S8B). Fecal N- methylserotonin levels were significantly correlated with the number of orange fiber snacks consumed per day (Pearson's r=0.72; p<0.0001) with concentrations reaching 72.5 ± 38.4 µM (mean ± SD) at maximal dose. To put this concentration in context, the reported IC50 of binding to a known N-methylserotonin receptor, 5-HT1A, is —2nM. Fecal serotonin levels were 0-8.6% of that of N-methylserotonin (7±5.7 µM; mean + SD) and did not vary significantly as a function of the dose of orange fiber (Pearson's r = ¬0.105, p=0.381; one-way ANOVA p=0.62) (Table S8B). In contrast, N-methylserotonin was undetectable (<0.05 µg/g) in 87% of the 36 fecal samples collected from individuals consuming the pea fiber snack prototype during the supplementation period (at week 3 when one snack per day was being consumed, and at end of week 5, when the maximum dose was being administered) (see the legend to Table S8B regarding the four donors who had positive samples). [0099] Neither the relative abundance of B. ovatus nor of any of the bacterial taxa (Amplicon Sequence Variants, ASVs) that exhibited statistically significant changes in their relative abundances in the fecal microbiota after orange fiber and/or pea fiber snack consumption (Table S8C) had statistically significantly correlations with N-methylserotonin levels at the end of week 5 (Spearman correlation q>0.30) [A statistically significant loge-fold change in relative abundance of a taxon at week 5 compared to the pre-intervention period was defined by q-value <0.1 (linear mixed effect model) and, using higher order singular value decomposition, by positioning of that taxon at the tails (a<0.1) of the distribution of ASVs along tensor component 1; see Methods]. [0100] Using shotgun sequencing datasets generated from fecal DNA samples collected at the end of weeks 1 and 5 of the study, a Spearman correlation was performed between (i) the abundances of 213 annotated CAZyme genes [glycoside hydrolases (GH) and polysaccharide lyases (PL)] that were present in at least one study participant at these time points, and (ii) fecal levels of N-methylserotonin prior to fiber supplementation and at the end of week 5 (Table S8D). CAZyme genes whose loge fold-changes in abundance were significantly correlated with levels of N-methylserotonin (q-value <0.1) are shown in FIG. 5B. The strongest positive correlation was with PL9 (rhamnogalacturonan lyase; Spearman rho = 0.51, q-value = 0.025) (FIG. 5B, Table S8E) — a CAZyme whose expression was significantly upregulated in vitro under mining permissive conditions (loge-fold change 1.2, FDR adjusted p-value (q) = 2.2 x104, FIG. 4C, Table S6A and Table S7). The CAZyme gene with the second most positive correlation with levels of N-methylserotonin was GH5 37 (Spearman rho=0.438, q=0.08) which has reported specificity for f3-glucan/cellulose. It is notable that in vitro enzymatic digestion experiments of the present disclosure revealed that a preparation enriched in endoglucanases and cellulases exhibited a high level of N-methylserotonin mining activity (Table S4E). Other CAZymes significantly correlated with fecal N-methylserotonin levels included GH30 5 (Spearman rho=0.44, q=0.08) and GH59 (Spearman rho=0.52, q=0.02) which possess homogalacturonan/rhamnogalacturonan processing functions or target pectin components such as galactans and arabinogalactans (FIG. 5B). As was the case with PL9, and GH5 37, the abundances of GH30 5 and GH59 increased significantly in the microbiomes of participants consuming the orange fiber snack (analogous to ASVs, a statistically significant loge-fold change for a CAZyme gene was defined by q-value < 0.1 (linear mixed-effects model) and, using higher order singular value decomposition, by the its positioning at the tails (a<0.1) of the distribution of CAZyme genes along tensor component 1). Taken together, the results of the human study of the present disclosure revealed an orange fiber specific, dose-dependent accumulation of N- methylserotonin in feces, where its concentration was positively correlated with the abundances of microbiome genes encoding CAZymes targeting pectic glycans. [0101] Screening of plant sources for N-methylserotonin. Seeking to identify whether the presence of N-methylserotonin is indeed ubiquitous in citrus plants, an array of 23 additional commercially available sources of citrus fibers , sourced from major citrus producing countries across the globe, was screened (Table S9). An orange fiber preparation from Fiberstar, served as control. All but two of the samples tested had significant quantities of N-methylserotonin upon enzymatic digestion, with the ones lacking being either a highly purified/processed pectin product or else a heavily processed unknown citrus fiber blend. The amount of N-methylserotonin present was highly variable between both sample type as well as between individual samples, with some samples being highly variable but approaching control levels (e.g. grounded orange peel, 67±21%) while others having low but more consistent levels (e.g. grounded lemon peel, 3±0.3%). Of particular note is that consistent with the observation of N-methylserotonin being entrapped within orange fiber, the “fine” preparation of the orange fiber used herein (i.e., more heavily refined & processed) yielded much less N-methylserotonin than its less processed counterpart (15±8%). [0102] To further confirm these results, a follow-up screen consisting of locally sourced citrus products were carried out (Table S10). As the orange fiber used herein was described by the manufacturers to be sourced from citrus juice processing, an attempt was made to delineate plausible components of citrus juice processing in the screen. Commonly found edible table orange, lemon, lime, and grapefruits were divided into components labeled “Skin,” “Fruit (with pulp),” “Pulp,” and “Juice” prior to being applied to the same enzymatic screen as described above (Table S10). N-methylserotonin was not found in grapefruit and found in low quantities in the lemon and lime. As expected, it is prominently found in the skin of orange fruits, followed by the fruit, pulp, and juice (161±65%, 122±52%, 35±4%, 36±1%; all relative to the orange fiber used herein). Entrapment of N-methylserotonin is further confirmed by the observation that relative to the enzymatic digestion, very low quantities of N-methylserotonin was released from the same orange peel using non-enzymatic processing methods such as mechanical disruption, methanol extraction, or liquid nitrogen freeze-thawing (Table S10). The presence of N- methylserotonin in the juice is likely due to residue fibrous components remaining in the orange juice during processing, where filtration of orange juice prior to the enzymatic assay is shown to reduce N-methylserotonin levels to negligible levels. [0103] Having confirmed the specificity of N-methylserotonin within citrus fruits and its abundance in oranges, the scope of the screen was broadened to confirm the specificity of plants containing N-methylserotonin. The same enzymatic digestion assay was applied to a broad range of various fruit, vegetable, and grain samples, in total reporting results from 133 different types of edible plants in Table S11(A-E). Samples selected included major global staples such as corn, wheat, rice, and cassava as reported by the Food and Agriculture Organization of the United Nations, as well as commonly consumed fruits and vegetables in America as reported by the USDA and FDA. N-methylserotonin was found in only three sample types, all of which are “peppers” in the Zanthoxylum genus which are in the Rutaceae family alongside citrus fruits. These include two types of the Japanese mountain pepper (Z. piperitum), which is reported to contain N-methylserotonin, and one preparation of the Chinese Sichuan pepper (Z. bungeanum). The otherwise evident absence of N-methylserotonin, including samples belonging to members of the Solanaceae family (various types of hot chili peppers as well as bell peppers) and the Piperaceae family (common black pepper) indicate that this compound is indeed specific to citrus fruits and other members in the Rutaceae family. [0104] DISCUSSION [0105] Gnotobiotic mice colonized with defined collections of human gut microbes were used together with in vitro assays to show that N-methylserotonin is a compound present in orange fiber that is directly released only by specific members of the gut community to produce significant effects on host physiology and metabolism. A short duration study of a small cohort of adult dizygotic twins disclosed dose-dependent, orange-fiber specific accumulation of N- methylserotonin in their feces. As described herein, orange fiber preparations and their releasable N-methylserotonin are naturally-based analogs of polysaccharide-based drug delivery systems. [0106] Many natural products are embedded in dietary fiber through various chemical and physical interactions, including hydrophobic interactions, hydrogen as well as covalent bonds, and/or physical entrapment. The term "celobiotic" (from the latin `conceal or disguise') is proposed herein to describe a bioactive compound that is liberated from fibers, rather than synthesized or further metabolized, through the actions of one or more microbial enzymes, and whose biological/pharmacologic activities are not dependent upon additional microbial biotransformation. [0107] Several key results aided in deciphering how N-methylserotonin is liberated from orange fiber. A switch was discovered for turning mining activity on and off: Bacteroides ovatus TSDC 17.2, a prominent miner in the preclinical gnotobiotic mouse model described herein, exhibited hemin-dependent release of N-methylserotonin in vitro. Pronounced B. ovatus strain- specific differences in N-methylserotonin releasing activities were documented under in vitro mining permissive conditions; hemin-dependence was a feature of release in all strains. Taking advantage of this hemin-dependency and strain-specificity, microbial RNA-Seq analysis of gene expression in a strong versus weak B. ovatus mining strain incubated in the presence or absence of orange fiber and presence or absence of hemin, revealed a set of glycoside hydrolase and polysaccharide lyase genes associated with release; their known/predicted substrate specificities were consistent with prominent representation of pectic polysaccharides present in orange fiber. Moreover, these in vitro results translated to humans, where treatment with orange and pea fiber snack prototypes disclosed orange fiber-specific accumulation of N-methylserotonin in their feces, with levels of this compound correlating most significantly with the abundances of PL and GH genes involved in processing of glycan structures in pectins. In this respect, and although the specific means of small molecule entrapment differ, it is noteworthy that several members of Bacteroidetes have recently been shown to possess a polysaccharide utilization locus that encodes esterases capable of extracting ferulic acid, a well-documented component of multiple cereal grains. [0108] There have been a limited number of reports describing the biological effects of N-methylserotonin; most of these studies have been conducted in vitro. Similar to serotonin, N- methylserotonin is able to increase glucose uptake in cultured rat muscle via its agonist activity on the 5-HT2A receptor. A maleated form of methylserotonin enhanced insulin secretion in human and mouse beta cells via activation of the 5-HT2B receptor. The closely related compound alpha-methylserotonin, by means of its engagement of 5-HT1 and 5-HT2A, is able to increase glycogen synthesis in rat hepatocytes via a direct increase in glycogen synthase activity as well as cAMP-dependent inactivation of glycogen phosphorylase; binding of serotonin to 5-HT2B/C receptors has an opposing effect and decreases glycogen synthesis. [0109] It was found that oral administration of N-methylserotonin to germ-free mice consuming a high saturated fat, low fiber representative USA diet produced a number of phenotypic changes including reduced adiposity and alterations in hepatic energy (glucose) metabolism. Intriguingly, N-methylserotonin affected expression of regulators of circadian rhythm in both liver and colon, including Arntl, Clock, Pert, Per3, plus Nfil3 and its repressor, Nr1d2. Gut microbiota has been linked to microbiota-regulated diurnal oscillation of epithelial expression of clock components, and the effects of these components (e.g., Nfil3) on lipid absorption and export. RNA-Seq did not reveal significant effects of N-methylserotonin on intestinal or liver levels of mRNAs encoding its known (Htr7, Htr2A) or related (Htr3, Htr4, Htr5 and Htr6) receptors. However, the absence of changes in receptor expression does not preclude effects on their signal transduction pathways, or the possibility that N-methylserotonin exerts its effects on circadian regulators through other metabolites, such as glutamate, whose colonic levels increased after N-methylserotonin administration to germ-free animals. [0110] The ability to manipulate luminal levels of N-methylserotonin in gnotobiotic mice fed orange fiber by including or excluding N-methylserotonin-releasing bacterial species in their gut community illustrates a synbiotic design strategy where fibers containing concealed celobiotics are administered together with probiotic `miners' to enhance/expand the biological effects of fibers to the benefit of the host. For example, administration of free, unbound N- methylserotonin to germ-free mice was found to produce a dose-dependent increase in gastrointestinal transit time, indicating that a synbiotic composed of orange fiber plus a N- methylserotonin miner such as B. ovatus represents an approach for treatment of certain forms of irritable bowel syndrome (IBS-C). Moreover, the systemic effects observed on metabolism in mice exposed to N-methylserotonin indicate the potential for additional beneficial pharmacological properties. [0111] As disclosed herein, celobiotics provide valuable analytic opportunities to both food and microbiome scientists, as well as for synbiotic compositions and therapeutic methods. Liberation of celobiotics from fiber preparations during in vitro incubations of intact uncultured (fecal) microbiota samples, defined consortia of cultured microbes, or single microbial strains operationally define the compositional `equivalence' of different lots of a fiber preparation and/or a comparative assessment of the impact of different food processing methods. As disclosed herein, knowledge of whether a consumer of a fiber preparation harbors a gut microbiota with miners of a specific celobiotic enables analysis of interpersonal variations in responses to that fiber in longer duration clinical studies. A corollary is that more personalized dietary recommendations are enabled by the present disclosure about the types of fiber preparations providing specific health benefits based on a given consumer's known microbiota/microbiome composition. [0112] SUPPLEMENTAL TABLES [0113] Table S1(A-B) — Chemical analysis of fiber preparations; related to FIG. 1(A- C), FIG. 2(A-H), and FIG. 5(A-B). Table S1A Composition of orange and pea fibers. Table S1B Linkage analysis of orange fiber. [0114] Table S2(A-B) — Cecal analytes/features identified by LC-Qtof-MS; related to FIG. 1(A-C). Table S2A Features that are colonization and fiber-dependent; Table S2B Other features. All features shown have peak areas ≥ 3-fold higher in colonized mice consuming the HiSF-LoFV + orange fiber diet relative to the other groups. Features are organized by M/Z. Data are presented as peak area. [0115] Table S3(A-D) — Differentially expressed genes in the livers and colons of germ- free mice treated with 50mg/kg/d N-methylserotonin compared to untreated germ-free controls; related to FIG. 2(A-H). Table S3A Upregulated genes in liver. Table S3B Downregulated genes in liver. Table S3C Upregulated genes in colon. Table S3D Downregulated genes in colon. [0116] Table S4(A-H) — In vitro screening of bacterial strains for N-methylserotonin releasing activity; related to FIG. 1(A-C) and FIG. 4(A-C). Table S4A Levels of N- methylserotonin (ng) released by cultured bacterial strains in TYG with and without hemin, Wilkins-Chalgren anaerobe broth or MEGA medium 2.0 containing orange fiber. Table S4B Screening of additional bacterial strains for N-methylserotonin release from orange fiber. Table S4C Additional control experiments of microbial N-methylserotonin release. Table S4D N- methylserotonin degradation test. Table S4E Release of N-methylserotonin from orange fiber via enzymatic reaction after 72 hours. Table S4F Recovery of N-methylserotonin from orange fiber after repeated rounds of methanol extraction, compared to recovery of a closely related spike-in compound (2-methylserotonin) under the same conditions. Table S4G Screening of additional Bacteroides ovatus strains for N-methylserotonin release from orange fiber using TYG medium with hemin. Table S4H Screening of additional Bacteroides ovatus strains for N-methylserotonin release from orange fiber using TYG medium without hemin. N-methylserotonin levels are shown at the time point specified, tested using a concentration of 5 mg/ml orange fiber, normalized to 10 6 microbes where applicable, and n = 3. ND (not detected) = N-methylserotonin levels below 0.02 ng. [0117] Table S5 - Absolute abundances of fecal bacterial community members measured at experimental day 21 (mean ± SD); related to FIG. 3(A-G). [0118] Table S6(A-B) - List of "mining" (N-methylserotonin releasing) candidate genes in B. ovatus TSDC 17.2, related to FIG. 4(A-C). Table S6A B. ovatus TSDC 17.2 genes, arranged by log2fold-change under the permissive releasing condition (TYG medium containing hemin, with versus without orange fiber). Genes shown exhibit >1 log2-fold increased expression under the permissive condition, but are not significantly upregulated or downregulated under non- releasing conditions. Highlighted in blue are statistically significant decreases in expression of the same gene under conditions where there is no N-methylserotonin release. Table S6B Functional predictions of CAZymes deemed to be candidate mediators of N-methylserotonin release. [0119] Table S7(A-B) —PUL map of B. ovatus TSDC 17.2; related to FIG. 4(A-C). B. ovatus TSDC 17.2 genes designated as candidates for involvement in N-methylserotonin release from orange fiber (OF) are highlighted in bold font. Table S7A B. ovatus TSDC 17.2; Table S7B Corresponding PUL (if present) in B. ovatus 115. B. ovatus TSDC 17.2 genes designated as candidates for involvement in N-methylserotonin release from orange fiber (OF) are highlighted in bold font and x = present. [0120] Table S8(A-E) — Levels of N-methylserotonin and serotonin in feces collected from adult dizygotic twins consuming orange fiber- or pea fiber-containing snack food prototypes; related to FIG. 5(A-B). Table S8A Composition of the snack food prototypes. Table S8B Ages and BMIs of participants plus levels of N-methylserotonin and serotonin in their fecal samples collected at the end of study weeks 1, 3 and 5. Table S8C ASVs with statistically significant loge-fold changes in relative abundances in the fecal microbiota of participants between the pre-intervention and 5-week time points of the pea and orange fiber snack studies. Table S8D-Week 1 and Table S8D-Week 5 CAZyme (GH and PL) gene representation in the fecal microbiomes of participants consuming orange fiber snacks. Table S8E Spearman correlations of abundances of GH and PL genes and levels of N-methylserotonin in fecal samples collected from study participants at week 5.
[0121] Table S9. Assessment of commercially available orange fibers for the presence of N-methylserotonin. [0122] Table S10. Assessment of locally commercially available citrus fruits for the presence of N-methylserotonin. [0123] Table S11(A-E) — N-Methylserotonin screening in other plants, relative to orange fiber (OF). Samples containing N-methylserotonin, where applicable, are mean±SD; "-" indicate not found. Table S11A cash crops and staples. Table S11B common prebiotic supplements. Table S11C spices. Table S11D fruits. Table S11E vegetables. Table S1A. Composition of orange and pea fibers.
Table S2A. Features that are colonization and fiber-dependent. Features that are colonization and fiber-dependent Colonized M/ 191.1 nin 362.1 372. 385.0 416. 416. 427.0 452. 459.2 462. 466.1 473.2 561.3 633.3
408.2423 10.38 64731 7437 8812 413.1975 10.39 132575 18385 30995 4143342 87 90818 0 16476 416 416 416 416 416 416 418 424 424 428 430 430 432 433 434 434 435 436 442 444 448 451 454 454 468 468 471 478 479 496 497 503 507 507 515 522 533 533 539 545 547 547 547 549 579 619 653.1755 6.57 54859 14085 0 1079.6506 6.29 36667 9593 12101 Table S3A. Upregulated genes in liver. 11 33 31 27 62 18 69 53 67 76 23 10 76 10 67 14 27 66 18 15 24 64 67 13 66 70 54 72 74 22 13 10 10 66 26 23 24 59 75 13 66 50 22 23 67 22 20
211389 Suox sulfite oxidase -0.27 4.53E-02 1 2 2 1 1 1
Table S3C. Upregulated genes in colon. G 3 3 2 4 3 1 2 2 4 2 2 2 2 2 6 6 2 2 3 3 2 7 1 2 1 3 2 2 3 2 9 1 1 2 5 3 1 2 1 1 4 3 1 2 3 1 5 2 2 2 7 6 5 1 2 1 1 5 2 2 1 1 1 1 2 6 2 6 6 1 1 1 1
Table S3D. Downregulated genes in colon. G 10 10 1
T B B B B
Table S4D. N-methylserotonin degradation test. Ending N-methylserotonin level Bacterial strain Starting N-methylserotonin level after 72h incubation (ng) B
Table en en endo
Table S4G. Screening of additional Bacteroides ovatus strains for N-methylserotonin release from orange fiber using TYG media with hemin. B B
Table S4H. Screening of additional Bacteroides ovatus strains for N-methylserotonin release from orange fiber using TYG media without hemin.
[0124] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. [0125] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters are be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. [0126] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) are construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or to refer to the alternatives that are mutually exclusive. 193 [0127] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and may also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and may cover other unlisted features. [0128] All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure. [0129] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member is referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group are included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims. [0130] To facilitate the understanding of the embodiments described herein, a number of terms are defined herein. The terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present disclosure. Terms such as "a," "an," and "the" are not intended to refer to only a singular entity, but rather include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the disclosure, but their usage does not delimit the disclosure, except as outlined in the claims. 194 [0131] All of the compositions and/or methods disclosed and claimed herein may be made and/or executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of the embodiments included herein, it will be apparent to those of ordinary skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the disclosure as defined by the appended claims. [0132] This written description uses examples to disclose the disclosure, including the best mode, and also to enable any person skilled in the art to practice the disclosure, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the disclosure is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims. 195