African Journal of
Plant Science

  • Abbreviation: Afr. J. Plant Sci.
  • Language: English
  • ISSN: 1996-0824
  • DOI: 10.5897/AJPS
  • Start Year: 2007
  • Published Articles: 692

Full Length Research Paper

Characterization of fructans from Agave durangensis

Alma D. Orozco-Cortes
  • Alma D. Orozco-Cortes
  • Department of Chemical and Biochemical Engineering, Instituto Tecnológico de Durango, Blvd. Felipe Pescador 1830 Ote., 34080, Durango, Dgo, Mexico.
  • Google Scholar
Gerardo Alvarez-Manilla
  • Gerardo Alvarez-Manilla
  • Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, United States.
  • Google Scholar
Gerardo Gutierrez-Sanchez
  • Gerardo Gutierrez-Sanchez
  • Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, United States.
  • Google Scholar
Olga M. Rutiaga-Quinones
  • Olga M. Rutiaga-Quinones
  • Department of Chemical and Biochemical Engineering, Instituto Tecnológico de Durango, Blvd. Felipe Pescador 1830 Ote., 34080, Durango, Dgo, Mexico.
  • Google Scholar
Javier Lopez-Miranda
  • Javier Lopez-Miranda
  • Department of Chemical and Biochemical Engineering, Instituto Tecnológico de Durango, Blvd. Felipe Pescador 1830 Ote., 34080, Durango, Dgo, Mexico.
  • Google Scholar
Nicolas O. Soto-Cruz*
  • Nicolas O. Soto-Cruz*
  • Department of Chemical and Biochemical Engineering, Instituto Tecnológico de Durango, Blvd. Felipe Pescador 1830 Ote., 34080, Durango, Dgo, Mexico.
  • Google Scholar


  •  Received: 23 January 2013
  •  Accepted: 12 August 2015
  •  Published: 30 September 2015

 ABSTRACT

Agave plants are members of the Agavaceae family and utilize crassulacean acid metabolism (CAM) for CO2 fixation. Fructans are the main photosynthetic products produced by Agave plants, and are their principal source of storage carbohydrates. The aim of this work was to determine the chemical and molecular characterization of fructans from Agave durangensis. Fructans were extracted from 10 year old A. durangensis plants. Trimethylsilyl derivatization was employed to determine the monomer composition. The linkage types in these carbohydrates were determined by methylation followed by reduction and O-acetylation, and finally analysis by gas chromatography-mass spectrometry (GC-MS). Samples were shown to contain t-β-D-Fruf, t-α-D-Glup, i-α-D-6-Glup and 1,6-di-β-D-Fruf linkages. The analysis of the degree of polymerization (DP) was confirmed by MALDI-TOF-MS, showing a wide DP ranging from 2 to 29 units. The analyses performed revealed that fructans from A. durangensis are formed of 97.11% fructose and 2.89% glucose, and are a complex mixture of fructooligosaccharides of the neo-fructan type containing principally β(2-1) and β(2-6) linkages, with branch moieties.

 

Key words: Degree of polymerization (DP), GC-MS, MALDI-TOF-MS.

Abbreviation: DMSO, Dimethyl sulfoxide; TFA, trifluoroacetic acid; EtOH, ethanol; HMDS, hexamethyldisilazane; NaOH, sodium hydroxide; CH3I, iodomethane; NaBD4, sodium borodeuteride; NH4OH, ammonium hydroxide; N2, nitrogen; CO2, carbon dioxide; H2O, water; CAM, crassulacean acid metabolism; PAAMs, partially methylated alditol acetates; WSC, water soluble carbohydrates; DP, degree of polymerization; GC-MS, gas chromatography-mass spectrometry; MALDI-TOF-MS, matrix-assisted laser desorption time-of-flight mass spectrometry.

 INTRODUCTION

Mexico has been considered the center of origin and biodiversity of the Agave genus, due to the taxonomic diversity found within its borders. Of the 310 species reported, about 272 can be found in this country. Members of the Agavaceae family are distributed throughout Mexico, and are well adapted to, both arid and semiarid regions (García-Mendoza and Galván, 1995). They have undergone both morphological and physiological adaptations to survive in such adverse conditions (López et al., 2003). One such physiological adaptation of the plant is the use of crassulacean acid metabolism (CAM), which serves to minimize water loss (Santamaría et al., 1995) by opening stomata at night when the temperature is cooler (Nobel and Linton, 1997). The principal photosynthetic products of CAM in Agave plants are fructans (Sánchez-Marroquín and Hope, 1953), which are synthesized and stored in the stems, and whose primary function in such plants is storage. Nevertheless, agave plants also are source of saponins and polyphenols, which are compounds considered agents with several activities such as anticancer, antifungal, anti-inflammatory, antidiabetic, anti-inflam-matory, among others (Santos-Zea et al., 2012). Fructans may also act as osmo-protectants during drought (Wang and Nobel, 1998), which represents a secondary physiological adaptation. In plants, ~ 15% of higher species contain fructans, and in certain species fructans constitute the plant’s sole reserve of carbohydrates. Fructans are polymers or oligomers of fructofuranosyl residues, are commonly water soluble, and are synthesized from sucrose accumulated in the vacuole of the plant (Vijn and Smeekens, 1999). Agave fructans have been used as ingredients of a granola bar, resulting in a product which has a moderate glycemic index (Zamora-Gasga et al., 2014). Indigestible fraction of this granola bar showed potential prebiotic activity, since it affected anaerobic batch cultures inoculated with human gut flora, demonstrating that agave ingredients are good sources of fermentable dietary fiber (Zamora-Gasga et al., 2015). Another work (Crispín-Isidro et al., 2015) reported that agave fructans enhanced sensory attributes of a reduced milk-fat yogurt.

According to the manner that the β-fructofuranosyl units are linked, five main types of fructan can be identified: (i) linear inulins with β(2-1)-fructofuranosyl linkages, (ii) levans with β(2-6) linkages, (iii) graminans, which are mixed fructans containing type (i) and (ii) linkages, (iv) neoseries inulins, which contains a glucose residue between two fructofuranosyl units containing β(2-1) linkages, and (v) neoseries levans, formed by β(2-1) and β(2-6)-fructofuranosyl linkages (Mancilla-Margalli and López, 2006; Sims et al., 2001). Fructans are usually present in plants as a heterogeneous mixture with varying degrees of polymerization (DP) and structure as a result of both environmental conditions and the developmental stage of the plant (Sims, 2003).

The presence of fructans in Agave was first reported in 1888 (Suzuki, 1993), with Agave veracruz and Agave americana being the most studied species (Aspinall and Gupta, 1959; Bathia and Nandra, 1979). More recently, Sims et al. (2001) reported the presence of a similar fructan structure in members of the Asparagales order, in which the Agavaceae family is included. However, in Agave species more than one fructan structure has been reported.      Sánchez-Marroquín and Hope (1953) and Bathia and Nandra (1979) reported inulins the principal storage carbohydrate in Agave tequilana and Agave americana, respectively. Meanwhile, reports (Aspinall and Gupta, 1959; Dorland et al., 1977) on Agave veracruz showed the presence of a complex mixture of highly branched fructans with an internal glucose and containing both β(2-1) and β(2-6) linkages. More recently, Wang and Nobel (1998) reported the presence of a DP5 in Agave deserti, primarily in the vascular tissue. Therefore, different agaves contain fructans with a wide variety of structures, so it is necessary to characterize the fructans of each species of agave. Thus, the aim of this work was to determine the chemical and molecular characterization of fructans from Agave durangensis


 MATERIALS AND METHODS

Standard material

1-Kestose and Nystose standards (inulin series DP3 and DP4, respectively) were supplied by Sigma; 1, 1, 1-Kestopentaose (inulin DP5) was from Megazyme. Fructans from Agave durangensis were extracted and derivatized to trimethylsilyl (TMS) oximes as described below. Derivatization reagents were supplied by Sigma.

 

Plant materials

Ten year old Agave durangensis plants were harvested in the wild, in the zone of Nombre de Dios, Durango, Mexico.

 

Extraction of Agave fructans

The pines of Agave were cut off, the cuts were small and uniform (2×2×2 cm). Five kilograms of pine produced from mature A. durangensis heads were placed in a container with 10 L of distilled water at 75°C and heated for 3 h to extract the fructans content. The obtained juice was then filtered through a filter paper Whatman No. 4 (non.sterile) and stored at -20°C until further analysis (Waleckx et al., 2007).

 

Isolation of fructans

Four fructan fractions were obtained from the supernatant and four from the pellet by precipitation of individual samples with different amounts of EtOH (final concentrations: 100% v/v, 80% v/v, 60% v/v and 40% v/v) at 4°C overnight. The fructan fractions were collected by centrifugation (5000 g; 10 min), washed twice with the respective EtOH concentration and freeze-dried to give a white product (Wack and Blaschek, 2006).

 

TMS Derivatization

A test tube containing 500 µL of extract, 57 µL of 2 M acetic acid and 20 µL of inositol (as internal standard) was placed on a heating block for 45 min at 75°C and the solvent evaporated to dryness with a stream of dry nitrogen. Sugars were initially converted to their oximes by the addition of 500 µL of methoxyamine hydrochloride (25 mg/mL in pyridine) and heated for 30 min at 70°C. Sugars were then trimethylsilylated with a mixture of 900 µL HMDS (hexamethyldisilazane) and 100 µL TFA (trifluoroacetic acid), and the resultant mixture was heated for 1 h at 100°C (5, 8, 12). One microliter was injected on a gas chromatograph and separated on a 15 m x 0.25 mm x 0.25 μm DB-1 column (Hewlett-Packard) with an initial temperature of 150°C for 4 min followed by a temperature program: 4°C/min until 192°C for 0.5 min, 10°C/min until 240°C for 7 min, and then 10 °C/min until 300°C held for 10 min. The injector and detector temperatures were 260 and 310°C, respectively (Total elution time: 42.80 min).

Standard substances (1 mg/mL): fructose, glucose, inositol, sorbitol, mannitol, mannose, xylose and galactose (Sigma). The ionization spectra of all compounds were compared with those from derivatized standards.

 

Glycosyl linkage analysis by methylation

One milligram of Agave durangensis fructans were dissolved in 20 drops of DMSO, stirred on low speed overnight or until complete dissolution. Derivatization to PAAMs was carried out using the method of Ciucanu and Kerek with some modifications (Ciucanu and Kerek, 1984). Methylation was carried out by subsequent additions of NaOH (prepared from 50% aqueous NaOH in DMSO by sonication and washing twice the precipitate with DMSO) and CH3I (2 M stabilized by copper, Sigma). Permethylated carbohydrates were extracted once with methylene chloride, washed with water, and dried under a stream of nitrogen. Those derivatives were hydrolyzed under acidic conditions with 2 M TFA at 121°C for 2 h. Reduction was carried out with NaBD4 dissolved in 1 M NH4OH at room temperature for 3 h. Excess borate was neutralized with acetic acid, and the products were taken to complete dryness with repeated addition of 9:1 acetic acid in a methanolic solution. Acetylation was performed at 50°C for 20 min using 250 μL of acetic anhydride and 250 μL of concentrate TFA. The products were extracted with methylene chloride; the organic phases were washed with water and dried under a stream of N2. The derivatized carbohydrates were separated and identified by GC-MS. Samples were dissolved in 100 μL of methylene chloride, and 1 μL was injected into the GC-MS. Derivatized mono-saccharides were separated on a 30 m x 0.25 mm i.d. x 0.25 μm SP-2330 column (Supelco, Bellefonte, PA), using helium as the carrier gas at 2.5 mL/min. The oven temperature was 80°C for 2 min and then ramped at a rate of 30°C/min to 170°C and then at 4°C/min to 240°C and held for 20 min. Injector and detector temperatures were 300°C, and column head pressure was kept at 5 psi.

 

MALDI-TOF-MS Analysis

Five hundred microliters of fructans from agave were dissolved in 200 μL of DMSO, purged with dry nitrogen, and sonicated for 10 min. After this time, 300 μL of NaOH and 150 μL of CH3I were added to the sample, stirred and sonicated again for 15 min. The products were extracted with methylene chloride; the organic phases were washed with water and dried under a stream of N2. Permethylated glycans were dissolved in 25 μL of 100% methanol, and the matrix was 2,5-dihydroxybenzoic acid; sample mixtures from 0.5 to 1 μL were applied onto the plate and quickly dried under N2. The sample solution was serially dried with matrix to obtain optimal sensitivity. A mixture of oligosaccharides was used as the calibration standard. MALDI-TOF-MS measurement was performed using a Hewlett-Packard (Cupertino, CA) LDI AOOXP MS in the positive ion mode. The instrument was operated at an accelerating voltage of 30 kV and an extractor voltage of 9 kV. The pressure was ~2.1 x 10-6 Torr (Stahl et al., 1997). 


 RESULTS AND DISCUSSION

Composition of Fructans from Agave durangensis

The content of fructose and glucose was determined by gas chromatography coupled to mass (GC-MSof their TMS derivatives Carbohydrates are commonly analyzed by gas chromatography (GC) as their trimethylsilyl (TMS) derivatives. Because tautomeric forms of reducing sugars can produce multiple peaks, approaches have been taken in order to suppress the anomeric center before silylation, the most popular being the formation of oximes from the carbonyl group. The major peak, which always eluted first (Figure 1) was assigned to syn (E) isomer and the minor to the anti-oxime (Z) isomer, according to the results found by Sanz et al. (2003). The syn (E) and anti-oxime (Z) isomers produced in the reaction can be separated by GC.

 

 

This method has been used to analyze different monosaccharides (Scanlon and Willis, 1985; Willis, 1983). Although several disaccharides have been analyzed as their TMS oximes, GC retention data for these derivatives are relatively scarce. This methodology was used to analyze the four fractions of fructans (100, 80, 60 and 40% in EtOH-H2O), obtained from the supernatant, pellet and fructan raw extract. The analysis revealed only the presence of glucose and fructose.

The chromatograms of certain fractions showed only the presence of fructose and were discarded from the analysis. These included the 40 and 100% EtOH fractions of the supernatant, and the 40, 60, and 100% fractions of the pellet. The percentages of glucose and fructose in the remaining fractions are shown in Table 1, these results show that fructans from A. durangensis contain fructose in a proportion higher than 90%. This table also shows that the fraction of raw fructan, showed the highest content of fructose (98.63 ± 0.30%), because this fraction did not undergo any modification, unlike the rest, which were precipitated with EtOH at different concentrations. Figure 1 shows the different sugars present in the samples, and the fructose and glucose each yielded two peaks, possibly α and β isomers (Chapman and Horvat, 1989), in a 1:1 ratio for fructose and 6:1 for glucose.

 

 

Glycosyl linkage types

The results of the methylation analysis (Ciucano and Kerek, 1989) of agave fructans provided highly valuable information on the linkage types presents in A. durangensis. Figure 2 shows a typical chromatogram of the reductive cleavage of agave fructans. A good separation of all methylated compounds can be observed.

 

 

Table 2 shows all derivatized compounds found. 2,5-di-O-acetyl-(2-deuterio)-1,3,4,6-tetra-O-methyl-D-mannitol (Peak 1) and 2,5-di-O-acetyl-(2-deuterio)-1,3,4,6-tetra-O-methyl-D-glucitol (Peak 2) were the products of a terminal fructose (t-β-D-Fruf). 1,5-di-O-acetyl-(1-deuterio)-2,3,4,6-tetra-O-methylglucitol (Peak 3) resulted from the presence of a terminal glucose (t-α-D-Glup). The compound 2,5,6-tri-O-acetyl-(2-deuterio)-1,3,4-tri-O-methylglucitol and 1,2,5-tri-O-acetyl-(2-deuterio)-3,4,6-tri-O-methylglucitol (Peak 4) indicated the existence of branches in the fructans, while, the presence of internal glucose was confirmed by the compound 1,5,6-tri-O-acetyl-(1-deuterio)-2,3,4-tri-O-methylglucitol (Peak 6). Finally the compound 1,2,5,6-tetra-O-acetyl-(2-deuterio)-3,4-di-O-methylhexitol (Peaks 7 and 8) was due to the presence of a 1,6-di-β-D-fructofuranose. The derivatization products of Agave durangensis were compared with those from well-studied Agave tequilana Weber var. azul (López et al., 2003; Mancilla-Margalli and López, 2006). The fructan structural characteristics determined for A. durangensis coincided with those reported for other Agave species; the linkages, the fragmentation patterns and degree of polymerization were similar. The identity of each compound derivative was determined by comparison with standards and fragmentation patterns of spectra generated by gas chromatography coupled to mass spectrometry, and can be seen in Table 2.

 

 

In this study the fractions of fructans (80, 60 and 40% from the supernatant, and 80% from the pellet) analyzed that contained some glucose all had the same number of peaks (eight peaks), and each had the same retention time compared with other fractions, the differences were only in the abundance of each peak.

Reduced fructose produces mannitol and glucitol epimers, in the case of the terminal β-D-fructofuranose (t-β-D-Fruf), both epimers were resolved well in the column used (SP-2330) and correspond to peaks 1 and 2 (Figure 2), indicating the presence of short chain fructans [~DP3-10 (14)]. These molecules are characterized by the presence of a doublet at m/z 161 and 162 as primary fragments and doublets at m/z 145 and 146, and m/z 101 and 102 as secondary fragments, which can be observed in the spectra of Figure 4.

The elution of peak 3 corresponds to the terminal α-D-glucopyranoside (t-α-D-Glcp) with a primary fragment at m/z 205. (2-1)-β-D-fructofuranosyl and (2-6)-β-D-fructofuranosyl linkages were found in peak 4, with primary fragments at m/z 161 and 189 respectively, these linkages indicate the presence in A. durangensis of the Neo-fructans class.

The fragmentation pattern of an aditional peak (Peak 6) indicates the presence of internal α-D-6-glucopyranose (i-α-D-6-Glup), with a fragment at m/z 162, indicative of an additional acetyl group in the C6 position (Figure 3). Finally, 1-6-di-β-D-fructofuranosyl linkage was identified in peak 7 and 8, meaning the presence of branched fructans. The six different linkages above were found in all fructan fractions differing only in the abundance of each peak. These linkages types are characteristic of species included in the Asparageles order and A. tequilana (López et al., 2003).

 

 

Degree of polymerization (DP) of fructans

Among all of the different analytical measurements performed with agave fructans, MALDI-TOF-MS proved to be the best option for establishing the DP distribution of these types of carbohydrates. Since 1991, MALDI has also been successfully applied to glycan analysis and is a superior technique if complex mixtures of oligo- or polysaccharides are to be analyzed as such (Stahl et al., 1997). The spectrum of masses of A. durangensis fructans is shown in Figure 4. It can clearly be seen that the extract displayed a complex mixture of fructans molecules; this mixture presented a molecular weight distribution of 273-5936 Da, which corresponds to a range of degree of polymerization (DP) from 2 to 29 units.

 

 

Finally, it is important to mention that the physiological functions of fructan metabolism in agave plants need to be studied carefully, because they could point to many other relevant functions such as resistance under the adverse conditions where most agave plants grow.


 CONCLUSIONS

The fructans from A. durangensis are constituted by: 82% of water soluble carbohydrates (WSC), only fructose (97.11 ± 1.17%) and glucose (2.89 ± 1.31%) sugars; the presence of the β(2-1) and β(2-1/2-6) linkages and a molecular weight distribution of 273-5936 Da, which corresponds to a range of degree of polymerization (DP) from 2 to 29 units. These fructans are a complex mixture of oligosaccharides from Neo-fructans type. The presence of different linkages, including β(2-1) and β(2-1/2-6), the former being the most abundant, as well as GC-MS data allowed the establishment of the fructan type present in A. durangensis


 CONFLICT OF INTEREST

The authors have not declared any conflict of interest.


 ACKNOWLEDGEMENT

This work was supported in part by the DOE Center for Plant and Microbial Complex Carbohydrates (DE-FG05- 93ER20097).



 REFERENCES

Aspinall GO, Das Gupta PC (1959). The structure of the fructosan from Agave veracruz. Mill. J. Am. Chem. Soc. 81:718-722.
 
Bathia IS, Nandra KS (1979). Studies on fructosyl transferase from Agave americana. Phytochem. 18:923-927.
Crossref
 
Chapman GW, Horvat RJ (1989). Determination of nonvolatile acids and sugars from fruits and sweet potato extracts by capillary GLC and GLC/MS. J. Agric. Food Chem. 37:947-950.
Crossref
 
Ciucanu I, Kerek FA (1984). A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res. 131:209-217.
Crossref
 
Crispín-Isidro G, Lobato-Calleros C, Espinosa-Andrews H, Alvarez-Ramirez J, Vernon-Carter EJ (2015). Effect of inulin and agave fructans addition on the rheological, microstructural and sensory properties of reduced-fat stirred yogurt. LWT - Food Sci. Technol. 62:438-444.
 
Dorland L, Kamerling JP, Vliegenthart JFG, Satyanarayana MN (1977). Oligosaccharides isolated from Agave veracruz. Carbohydr. Res. 54:275-284.
Crossref
 
López MG, Mancilla-Margalli NA, Mendoza-Díaz G (2003). Molecular structures of fructans from Agave tequilana Weber var. azul. J. Agric. Food Chem. 51:7835-7840.
Crossref
 
Mancilla-Margalli NA, López MG (2006). Water-soluble carbohydrates and fructan structure patterns from Agave and Dasylirion species. J. Agric. Food Chem. 54:7832-7839.
Crossref
 
Nobel P, Linton MJ. (1997). Frequencies microclimate and root properties for three codominant perennials in the northwestern Sonora desert on north -vs south- facing slopes. Annals of Botany. 80: 731-739.
Crossref
 
Sánchez-Marroquín A, Hope PH (1953). Agave juice: Fermentation and chemical composition studies of some species. J. Agric. Food Chem. 1:246-249.
Crossref
 
Santamaría JM, Herrera JL, Robert ML (1995). Stomatal physiology of a micropropagated CAM plant, Agave tequilana Weber. Plant Growth Regul. 16:211-214.
Crossref
 
Santos-Zea L, Leal-Diaz MA, Cortes-Ceballos E, Gutierrez-Uribe AJ (2012). Agave (Agave spp.) and its traditional products as a source of bioactive compounds. Curr. Bioact. Compd. 8:2018-231.
Crossref
 
Sanz ML, Diez-Barrio MT, Sanz J, Martínez-Castro I (2003). GC behavior of disaccharide trimethylsilyl oximes. J. Chromatogr. Sci. 41:205-208.
Crossref
 
Scanlon JT, Willis DE (1985). Calculation of flame ionization detector relative response factors using the effective carbon number concept. J. Chromatogr. Sci. 23:333-340.
Crossref
 
Sims IM, Cairns AJ, Furneaux RH (2001). Structure of fructans from excised leaves of New Zealand flax. Phytochem. 57:661-668.
Crossref
 
Sims M (2003). Structural diversity of fructans from members of the order Asparagales in New Zealand. Phytochem. 63:351-359.
Crossref
 
Stahl B, Linos A, Karas M, Hillenkamp F, Steups M (1997). Analysis of fructans from higher plants by Matriz Assisted Laser Desorption/Ionization Mass Spectrometry. Anal. Biochem. 246:195-204.
Crossref
 
Suzuki M (1993). History of fructan research: Rose to Edelman. In Science and technology of fructans. Suzuki, M. Chatterton, N. J. Eds. CRC Press: Boca Raton, FL. pp. 21-39.
 
Vijn I, Smeekens S (1999). Fructan: more than a reserve carbohydrate? Plant Physiol. 120:351-359.
Crossref
 
Wack M, Blaschek W (2006). Determination of the structure and degree of polymerisation of fructans from Echinacea purpurea roots. Carbohydr. Res. 341:1147-1153.
Crossref
 
Waleckx E, Gschaedler A, Colonna-Ceccaldi B, Monsan P (2007). Hydrolysis of fructans from Agave tequilana Weber var. azul during the cooking step in a traditional tequila elaboration process. Food Chem. 108:40-48.
Crossref
 
Wang N, Nobel P (1998). Phloem transport of fructans in the crassulacean acid metabolism species Agave deserti. Plant Physiol. 116:709-714.
Crossref
 
Willis DE (1983). GC analysis of C2– C7 carbohydrates as the trimethylsilyloxime derivatives on packed and capillary columns. J. Chromatogr. Sci. 21:132-38.
Crossref
 
Zamora-Gasga VM, Bello-Pérez LA, Ortíz-Basurto RI, Tovar J, Sáyago-Ayerdi SG (2014). Granola bars prepared with Agave tequilana ingredients: Chemical composition and in vitro starch hydrolysis. LWT - Food Sci. Technol. 56: 309–314.
Crossref
 
Zamora-Gasga VM, Loarca-Pi-a G, Vázquez-Landaverde PA, Ortiz-Basurto RI, Tovar J, Sáyago-Ayerdi SG (2015). In vitro colonic fermentation of food ingredients isolated from Agave tequilana Weber var. azul applied on granola bars. LWT - Food Sci. Technol. 60:766-772.

 




          */?>