Symbiotic Supplementation (E. faecium and Agave Inulin) Improves Spatial Memory and Increases Plasticity in the Hippocampus of Obese Rats: A Proof-of-Concept Study.

Obesity has been linked to cognitive impairment through systemic low-grade inflammation. High fat and sugar diets (HFSDs) also induce systemic inflammation, either by induced Toll-like receptor 4 response, or by causing dysbiosis. This study aimed to evaluate the effect of symbiotics supplementation on spatial and working memory, butyrate concentration, neurogenesis, and electrophysiological recovery of HFSD-fed rats. In a first experiment, Sprague-Dawley male rats were given HFSD for 10 weeks, after which they were randomized into 2 groups (n = 10 per group): water (control), or Enterococcus faecium + inulin (symbiotic) administration, for 5 weeks. In the fifth week, spatial and working memory was analyzed through the Morris Water Maze (MWM) and Eight-Arm Radial Maze (RAM) tests, respectively, with 1 week apart between tests. At the end of the study, butyrate levels from feces and neurogenesis at hippocampus were determined. In a second experiment with similar characteristics, the hippocampus was extracted to perform electrophysiological studies. Symbiotic-supplemented rats showed a significantly better memory, butyrate concentrations, and neurogenesis. This group also presented an increased firing frequency in hippocampal neurons [and a larger N-methyl-d-aspartate (NMDA)/α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) current ratio] suggesting an increase in NMDA receptors, which in turn is associated with an enhancement in long-term potentiation and synaptic plasticity. Therefore, our results suggest that symbiotics could restore obesity-related memory impairment and promote synaptic plasticity.

Cranberry polyphenols and agave agavins impact gut immune response and microbiota composition while improving gut barrier function, inflammation, and glucose metabolism in mice fed an obesogenic diet.

The consumption of plant-based bioactive compounds modulates the gut microbiota and interacts with the innate and adaptive immune responses associated with metabolic disorders. The present study aimed to evaluate the effect of cranberry polyphenols (CP), rich in flavonoids, and agavins (AG), a highly branched agave-derived neo-fructans, on cardiometabolic response, gut microbiota composition, metabolic endotoxemia, and mucosal immunomodulation of C57BL6 male mice fed an obesogenic high-fat and high-sucrose (HFHS) diet for 9 weeks. Interestingly, CP+AG-fed mice had improved glucose homeostasis. Oral supplementation with CP selectively and robustly (five-fold) increases the relative abundance of Akkermansia muciniphila, a beneficial bacteria associated with metabolic health. AG, either alone or combined with CP (CP+AG), mainly stimulated the glycan-degrading bacteria Muribaculum intestinale, Faecalibaculum rodentium, Bacteroides uniformis, and Bacteroides acidifaciens. This increase of glycan-degrading bacteria was consistent with a significantly increased level of butyrate in obese mice receiving AG, as compared to untreated counterparts. CP+AG-supplemented HFHS-fed mice had significantly lower levels of plasma LBP than HFHS-fed controls, suggesting blunted metabolic endotoxemia and improved intestinal barrier function. Gut microbiota and derived metabolites interact with the immunological factors to improve intestinal epithelium barrier function. Oral administration of CP and AG to obese mice contributed to dampen the pro-inflammatory immune response through different signaling pathways. CP and AG, alone or combined, increased toll-like receptor (TLR)-2 (Tlr2) expression, while decreasing the expression of interleukin 1ß (ILß1) in obese mice. Moreover, AG selectively promoted the anti-inflammatory marker Foxp3, while CP increased the expression of NOD-like receptor family pyrin domain containing 6 (Nlrp6) inflammasome. The intestinal immune system was also shaped by dietary factor recognition. Indeed, the combination of CP+AG significantly increased the expression of aryl hydrocarbon receptors (Ahr). Altogether, both CP and AG can shape gut microbiota composition and regulate key mucosal markers involved in the repair of epithelial barrier integrity, thereby attenuating obesity-associated gut dysbiosis and metabolic inflammation and improving glucose homeostasis.

Hunting Bioactive Molecules from the Agave Genus: An Update on Extraction and Biological Potential.

Agaves are plants used in the production of alcoholic beverages and fibers. Ever since ancient times, pre-Hispanic cultures in Mexico have used them in traditional medicine to cure different ailments. Over the years, studies of the active principles responsible for the therapeutic benefits of agaves have increased. Leaves and fibers are the main agro-wastes generated in tequila and mezcal production, while fibers are the main waste product in the textile sector. Different investigations have referred to the agro-waste from agave processing as a source of bioactive molecules called secondary metabolites (SM). Among them, phenols, flavonoids, phytosterols, and saponins have been extracted, identified, and isolated from these plants. The role of these molecules in pest control and the prospect of metabolites with the biological potential to develop novel drugs for chronic and acute diseases represent new opportunities to add value to these agro-wastes. This review aims to update the biological activities and recent applications of the secondary metabolites of the genus Agave.

Transcriptome-based metabolic profiling of flavonoids in Agave lechuguilla waste biomass.

Agave lechuguilla is one of the most abundant species in arid and semiarid regions of Mexico, and is used to extract fiber. However, 85 % of the harvested plant material is discarded. Previous bioprospecting studies of the waste biomass suggest the presence of bioactive compounds, although the extraction process limited metabolite characterization. This work achieved flavonoid profiling of A. lechuguilla in both processed and non-processed leaf tissues using transcriptomic analysis. Functional annotation of the first de novo transcriptome of A. lechuguilla (255.7 Mbp) allowed identifying genes coding for 33 enzymes and 8 transcription factors involved in flavonoid biosynthesis. The flavonoid metabolic pathway was mostly elucidated by HPLC-MS/MS screening of alcoholic extracts. Key genes of flavonoid synthesis were higher expressed in processed leaf tissues than in non-processed leaves, suggesting a high content of flavonoids and glycoside derivatives in the waste biomass. Targeted HPLC-UV-MS analyses confirmed the concentration of isorhamnetin (1251.96 µg), flavanone (291.51 µg), hesperidin (34.23 µg), delphinidin (24.23 µg), quercetin (15.57 µg), kaempferol (13.71 µg), cyanidin (12.32 µg), apigenin (9.70 µg) and catechin (7.91 µg) per gram of dry residue. Transcriptomic and biochemical profiling concur in the potential of lechuguilla by-products with a wide range of applications in agriculture, feed, food, cosmetics, and pharmaceutical industries.

Metabolomic profiling of five Agave leaf taxa via UHPLC/PDA/ESI-MS inrelation to their anti-inflammatory, immunomodulatory and ulceroprotective activities.

Agave plants are popular for their myriad applications in traditional medicine attributed to their reported anti-inflammatory, immunomodulatory, cytotoxic and antifungal activities. The aim of this study was to examine the anti-inflammatory, immunomodulatory and ulceroprotective activity of Agave species in relation to their metabolite fingerprint via a metabolome based ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) approach coupled to chemometrics. The metabolomic differences among five examined Agave leaves viz. Agave americana L., A. americana var. marginata Trel, A. angustifolia Haw. cv. marginata, A. desmettiana Jacobi, A. pygmaea Gentry were determined via a total of 56 annotated metabolites. Identification based on MSn and UV spectra revealed 25 steroidal saponins and sapogenins, 6 flavonoids, 2 homoisoflavonoids, 7 phenolic acids, 6 fatty acids and 3 fatty acid amides, some of which are reported for the first time in Agave. Metabolites heterogeneity was assessed among leaf taxa via multivariate data analyses for samples classification, showing that saponins is the major metabolite contributing to their classification. The carrageenan induced acute inflammatory rat model was used to assess the anti-inflammatory activity of Agave extracts via monitoring of blood cytokine levels. Additionally, their effects on ethanol-induced gastric ulcer in rats were evaluated. A. pygmaea showed the most significant anti-inflammatory and immunomodulatory activity, while A. angustifolia var. marginata possessed the highest ulceroprotective activity, which could be attributable to the high abundance of various saponins and homoisoflavonoids in those taxa.

Natural sweetener agave inhibits gastric emptying in rats by a cholecystokinin-2- and glucagon like peptide-1 receptor-dependent mechanism.

Low-calorie sweeteners are considered to be beneficial in calorie control, but the impact of these sweeteners on gastric emptying is not well described. The purpose of this study was to compare the gastric emptying rate of agave nectar with those of glucose and fructose, and to evaluate the interaction of cholecystokinin (CCK)-1, CCK-2 and glucagon-like peptide-1 (GLP-1) receptors in agave-induced alterations in gastric emptying. Female Sprague-Dawley rats were fitted with gastric cannulas. Following the recovery, the gastric emptying rates of glucose, fructose and agave at 12.5%, 15% or 50% concentrations were measured and compared with that of saline. GLP-1 receptor antagonist exendin fragment 9-39 (30 µg kg-1), CCK-1 receptor antagonist devazepide (1 mg kg-1) or gastrin/CCK-2 receptor antagonist YM022 (1 mg kg-1) was injected subcutaneously 1 min before the emptying of glucose, fructose or agave at their 50% concentrations. When compared with saline emptying, gastric emptying of glucose was significantly delayed at its 25% and 50% concentrations, but the emptying of 12.5% glucose was not different from that of saline. Agave emptying, which was delayed with respect to saline emptying, was not altered by CCK-1 receptor blockade; but agave emptied from the stomach as rapidly as saline following the blockade of either CCK-2 or GLP-1 receptors. The findings demonstrate that the inhibitory effect of agave on gastric emptying is mediated by both CCK-2 and GLP-1 receptors, suggesting that natural sweeteners including agave may have satiating effects through the inhibition of gastric motility via enteroendocrine mechanisms.

Synthesis of silver nanoparticles using aqueous extracts of Heterotheca inuloides as reducing agent and natural fibers as templates: Agave lechuguilla and silk.

Silver nanoparticles (Ag NPs) were synthesized using a one-pot green methodology with aqueous extract of Heterotheca inuloides as a reducing agent, and the support of natural fibers: Agave lechuguilla and silk. UV-Vis spectroscopy, X-Ray photoelectron spectroscopy XPS and transmission electron microscopy TEM were used to characterize the resulting bionanocomposite fibers. The average size of the Ag NPs was 16nm and they exhibited low polydispersity. XPS studies revealed the presence of only metallic Ag in the nanoparticles embedded in Agave. lechuguilla fibers. Significant antibacterial activities against gram-negative Escherichia coli and gram-positive Staphylococcus aureus were determined. AgO as well as metallic Ag phases were detected when silk threads were used as a substrates hinting at the active role of substrate during the nucleation and growth of Ag NPs. These bionanocomposites have excellent mechanical properties in tension which in addition to the antibacterial properties indicate the potential use of these modified natural fibers in surgical and biomedical applications.

Prebiotic effect of Agave fourcroydes fructans: an animal model.

The use of prebiotics such as fructans has increased in human and animal nutrition because of their productive performance and health benefits. Agave fourcroydes has shown high concentrations of fructans in their stems; however, there is no information on new products derived from this plant that might enhance its added value. Therefore, we evaluated the prebiotic effect of Agave fourcroydes fructans in an animal model. Male mice (C57BL/6J) were fed on parallel form with a standard diet or diets supplemented with 10% of fructans from Cichorium intybus (Raftilose P95) and Agave fourcroydes from Cuba for 35 days. The body weight, food intake, blood glucose, triglycerides and cholesterol, gastrointestinal organ weights, fermentation indicators in cecal and colon contents and mineral content in femurs were determined. The body weight and food intake of mice were not significantly modified by any treatment. However, serum glucose, cholesterol and triglycerides decreased (P < 0.01) in the fructans groups with respect to the standard diet group; this decrement was higher in the A. fourcroydes group with respect to the Raftilose P95 group. Mice groups supplemented with fructans exhibited increased (P < 0.01) total and wall cecal and colon weights. The fermentation indicators, short-chain fatty acids (SCFAs) and pH decreased (P < 0.001) in the groups that consumed fructans in their diets with respect to the standard diet. The diets supplemented with fructans also increased the mineral concentrations of calcium (P < 0.01) and magnesium (P < 0.05) in the right femurs. In conclusion, the inclusion of fructans from Agave fourcroydes in the mice diet induced a prebiotic response, similar to or greater than the commercial product (Raftilose P95) and this constitutes a promising alternative with potential use not only in animal but also in human diets.

Prebiotic Effects of Agave salmiana Fructans in Lactobacillus acidophilus and Bifidobacterium lactis Cultures.

Agave salmiana is a fructan rich species that is widely distributed in Mexico. The aim of this investigation was to extract the fructans of A. salmiana and evaluate their prebiotic effect in 48 hours in vitro cultures of Bifidobacterium lactis and Lactobacillus acidophilus and to compare this effect with other available fructan sources. A significant difference in pH, optical density and biomass was found in the cultures depending on the source of fructans and the type of bacteria. It was possible to determine a dose-response effect of the A. salmiana fructans and the growth of the studied strains.

Agavins from Agave angustifolia and Agave potatorum affect food intake, body weight gain and satiety-related hormones (GLP-1 and ghrelin) in mice.

Agavins act as a fermentable dietary fiber and have attracted attention due to their potential for reducing the risk of disease. Therefore, we evaluated the effect of supplementation using 10% agavins with a short-degree of polymerization (SDP) from Agave angustifolia Haw. (AASDP) or Agave potatorum Zucc. (APSDP) along with chicory fructans (RSE) as a reference for 5 weeks, on the energy intake, body weight gain, satiety-related hormones from the gut and blood (GLP-1 and ghrelin), blood glucose and lipids, and short-chain fatty acids (SCFAs) from the gut of ad libitum-fed mice. We evaluated the energy intake daily and weight gain every week. At the end of the experiment, portal vein blood samples as well as intestinal segments and the stomach were collected to measure glucagon-like peptide-1 (GLP-1) and ghrelin using RIA and ELISA kits, respectively. Colon SCFAs were measured using gas chromatography. The energy intake, body weight gain, and triglycerides were lower in the fructan-fed mice than in the STD-fed mice. The AASDP, APSDP, and RSE diets increased the serum levels of GLP-1 (40, 93, and 16%, respectively vs. STD) (P ≤ 0.05), whereas ghrelin was decreased (16, 38, and 42%, respectively) (P ≤ 0.05). Butyric acid increased significantly in the APSDP-fed mice (26.59 mmol g(-1), P ≤ 0.001) compared with that in the AASDP- and RSE-fed mice. We concluded that AASDP and APSDP are able to promote the secretion of the peptides involved in appetite regulation, which might help to control obesity and its associated metabolic disorder.

Effects of consuming diets containing Agave tequilana dietary fibre and jamaica calyces on body weight gain and redox status in hypercholesterolemic rats.

Dietary fibre (DF) obtained from Agave tequilana, which is rich in fructans and insoluble DF, and jamaica calyces (Hibiscus sabdariffa), which is rich in DF and phenolic compounds, were assessed as new potential functional ingredients using the hypercholesterolemic animal model. Wistar rats (200-250 g) were divided into 3 groups (n=8) and fed with cholesterol-rich diets supplemented with cellulose (CC, control), agave DF (ADF) or ADF with jamaica calyces (ADF-JC). After consuming the test diets for 5 weeks, weight gain in the ADF-JC group was significantly lower than in the other groups. The ADF and ADF-JC groups had a reduced concentration of cholesterol transporters in the caecum tissue, although no changes were observed in the plasma lipid profile. Both treatments improved the redox status by reducing the malondialdehyde serum levels and protein oxidative damage, compared to the CC group. DF from A. tequilana alone, or in combination with jamaica calyces, shows promising potential as a bioactive ingredient.

Cellular and molecular changes associated with somatic embryogenesis induction in Agave tequilana.

In spite of the importance of somatic embryogenesis for basic research in plant embryology as well as for crop improvement and plant propagation, it is still unclear which mechanisms and cell signals are involved in acquiring embryogenic competence by a somatic cell. The aim of this work was to study cellular and molecular changes involved in the induction stage in calli of Agave tequilana Weber cultivar azul in order to gain more information on the initial stages of somatic embryogenesis in this species. Cytochemical and immunocytochemical techniques were used to identify differences between embryogenic and non-embryogenic cells from several genotypes. Presence of granular structures was detected after somatic embryogenesis induction in embryogenic cells; composition of these structures as well as changes in protein and polysaccharide distribution was studied using Coomassie brilliant blue and Periodic Acid-Schiff stains. Distribution of arabinogalactan proteins (AGPs) and pectins was investigated in embryogenic and non-embryogenic cells by immunolabelling using anti-AGP monoclonal antibodies (JIM4, JIM8 and JIM13) as well as an anti-methyl-esterified pectin-antibody (JIM7), in order to evaluate major modifications in cell wall composition in the initial stages of somatic embryogenesis. Our observations pointed out that induction of somatic embryogenesis produced accumulation of proteins and polysaccharides in embryogenic cells. Presence of JIM8, JIM13 and JIM7 epitopes were detected exclusively in embryogenic cells, which supports the idea that specific changes in cell wall are involved in the acquisition of embryogenic competence of A. tequilana.

Fermentation of Agave tequilana juice by Kloeckera africana: influence of amino-acid supplementations.

This study aimed to improve the fermentation efficiency of Kloeckera africana K1, in tequila fermentations. We investigated organic and inorganic nitrogen source requirements in continuous K. africana fermentations fed with Agave tequilana juice. The addition of a mixture of 20 amino-acids greatly improved the fermentation efficiency of this yeast, increasing the consumption of reducing sugars and production of ethanol, compared with fermentations supplemented with ammonium sulfate. The preference of K. africana for each of the 20 amino-acids was further determined in batch fermentations and we found that asparagine supplementation increased K. africana biomass production, reducing sugar consumption and ethanol production (by 30, 36.7 and 45%, respectively) over fermentations supplemented with ammonium sulfate. Therefore, asparagine appears to overcome K. africana nutritional limitation in Agave juice. Surprisingly, K. africana produced a high concentration of ethanol. This contrasts to poor ethanol productivities reported for other non-Saccharomyces yeasts indicating a relatively high ethanol tolerance for the K. africana K1 strain. Kloeckera spp. strains are known to synthesize a wide variety of volatile compounds and we have shown that amino-acid supplements influenced the synthesis by K. africana of important metabolites involved in the bouquet of tequila. The findings of this study have revealed important nutritional limitations of non-Saccharomyces yeasts fermenting Agave tequilana juice, and have highlighted the potential of K. africana in tequila production processes.

Effect of dilution rate and nutrients addition on the fermentative capability and synthesis of aromatic compounds of two indigenous strains of Saccharomyces cerevisiae in continuous cultures fed with Agave tequilana juice.

Knowledge of physiological behavior of indigenous tequila yeast used in fermentation process is still limited. Yeasts have significant impact on the productivity fermentation process as well as the sensorial characteristics of the alcoholic beverage. For these reasons a better knowledge of the physiological and metabolic features of these yeasts is required. The effects of dilution rate, nitrogen and phosphorus source addition and micro-aeration on growth, fermentation and synthesis of volatile compounds of two native Saccharomyces cerevisiae strains, cultured in continuous fed with Agave tequilana juice were studied. For S1 and S2 strains, maximal concentrations of biomass, ethanol, consumed sugars, alcohols and esters were obtained at 0.04 h⁻¹. Those concentrations quickly decreased as D increased. For S. cerevisiae S1 cultures (at D=0.08 h⁻¹) supplemented with ammonium phosphate (AP) from 1 to 4 g/L, concentrations of residual sugars decreased from 29.42 to 17.60 g/L and ethanol increased from 29.63 to 40.08 g/L, respectively. The S1 culture supplemented with AP was then micro-aerated from 0 to 0.02 vvm, improving all the kinetics parameters: biomass, ethanol and glycerol concentrations increased from 5.66, 40.08 and 3.11 g/L to 8.04, 45.91 and 4.88 g/L; residual sugars decreased from 17.67 g/L to 4.48 g/L; and rates of productions of biomass and ethanol, and consumption of sugars increased from 0.45, 3.21 and 7.33 g/L·h to 0.64, 3.67 and 8.38 g/L·h, respectively. Concentrations of volatile compounds were also influenced by the micro-aeration rate. Ester and alcohol concentrations were higher, in none aerated and in aerated cultures respectively.

Ethanol production from henequen (Agave fourcroydes Lem.) juice and molasses by a mixture of two yeasts.

In the fermentation process of henequen (Agave fourcroydes Lem.) leaf juice, complemented with industrial molasses, the use of an inoculum comprising two yeasts: Kluyveromyces marxianus (isolated from the henequen plant) and Saccharomyces cerevisiae (commercial strain) was studied. An ethanol production of 5.22+/-1.087% v/v was obtained. Contrary to expected, a decrease on ethanol production was observed with the use of the K. marxianus strain. The best results were obtained when a mixture of 25% K. marxianus and 75% S. cerevisiae or S. cerevisiae alone were used with an initial inoculum concentration of 3x10(7)cellmL(-1). Furthermore, it was possible to detect a final concentration of approximately 2-4gL(-1) of reducing sugars that are not metabolized by the yeasts for the ethanol production. These results show that although the use of a mixture of yeasts can be of interest for the production of alcoholic beverages, it can be the opposite in the case of ethanol production for industrial purposes where manipulation of two strains can raise the production costs.

Effect of Agave tequilana juice on cell wall polysaccharides of three Saccharomyces cerevisiae strains from different origins.

In this study, a characterization of cell wall polysaccharide composition of three yeasts involved in the production of agave distilled beverages was performed. The three yeast strains were isolated from different media (tequila, mezcal and bakery) and were evaluated for the beta(1,3)-glucanase lytic activity and the beta-glucan/ mannan ratio during the fermentation of Agave tequilana juice and in YPD media (control). Fermentations were performed in shake flasks with 30 g l(-1) sugar concentration of A. tequilana juice and with the control YPD using 30 g l(-1) of glucose. The three yeasts strains showed different levels of beta-glucan and mannan when they were grown in A. tequilana juice in comparison to the YPD media. The maximum rate of cell wall lyses was 50% lower in fermentations with A. tequilana juice for yeasts isolated from tequila and mezcal than compared to the bakery yeast.