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Botanical Sciences

versão On-line ISSN 2007-4476versão impressa ISSN 2007-4298

Bot. sci vol.100 no.3 México Jul./Set. 2022  Epub 16-Maio-2022

https://doi.org/10.17129/botsci.2960 

Phytochemistry

Molecular weight distribution of fructans extracted from Agave salmiana leaves

Distribución de tamaños de fructanos extraídos a partir de hojas de Agave salmiana

Arizbeth Plascencia1  2 
http://orcid.org/0000-0001-6061-3704

Antonia Gutiérrez-Mora1 
http://orcid.org/0000-0001-9279-8985

José Manuel Rodríguez-Domínguez1 
http://orcid.org/0000-0003-1148-4912

José Juvencio Castañeda-Nava1 
http://orcid.org/0000-0002-1825-8240

Juan Gallardo-Valdez1 
http://orcid.org/0000-0003-4077-5233

Harumi Shimada2 
http://orcid.org/0000-0002-8391-9324

Rosa María Camacho-Ruíz1  * 
http://orcid.org/0000-0001-9264-5655

1 Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, Guadalajara, México.

2 Universidad Nacional Autónoma de México, Escuela Nacional Superior de Estudios de León, León, México.


Abstract

Background:

In mezcal industry, Agave salmiana leaves are a crop residue and a potential source of fructans. Agave fructans are a soluble fiber that can act as prebiotic in gut microbiota.

Questions and Hypotheses:

The molecular weight distribution of agave fructans extracted from leaves of Agave salmiana depends on the region where agaves growth and on the subspecies.

Studied species:

Agave salmiana Otto ex Salm Dyck, spp. salmiana and Agave salmiana spp. crassispina.

Study site:

Twelve municipalities from Guanajuato México were sampled: Ocampo, San Felipe, San Diego de la Unión, Victoria, Xichú, Atarjea, Dolores Hidalgo, Doctor Mora, Santa Catarina, Tierra Blanca, San Miguel Allende and Comonfort.

Methods:

The base of the leaf close to the stem of Agave salmiana ssp. salmiana and A. salmiana ssp. crassispina plants, six years old, were harvested. Water soluble carbohydrates were extracted, the content and molecular weight distribution of fructans was determined by HPLC-SEC.

Results:

An average of 0.7 % of soluble carbohydrates was recovered from the leaves. The molecular weight distribution of Agave salmiana fructans was: number average molecular weight: 3,209 g/mol; average molecular weight: 5,046 g/mol; number average degree of polymerization: 19; weight average degree of polymerization: 30. High polymerization degree fructans content was greater in Agave salmiana spp. salmiana (55 %) than in Agave salmiana spp. crassispina (47 %).

Conclusions:

The leaves of Agave salmiana contain a low proportion of high-molecular-weight fructans, compared to commercial agave fructans. The results demonstrated the technical feasibility to obtain fructans from Agave salmiana leaves.

Keywords: Agave Phytochemicals; Polysaccharide characterization; Size Exclusion chromatography; soluble fiber

Resumen

Antecedentes:

Las hojas de Agave salmiana, como residuo de la industria mezclaera, podrían ser fuente de fructanos. Los fructanos de agave son fibras solubles prebióticas.

Hipótesis:

La distribución de tamaños de fructanos extraídos de hojas de Agave salmiana será distinta en función de la región y de la subespecie.

Especies estudiadas:

Agave salmiana Otto ex Salm Dyck, spp. salmiana and Agave salmiana spp. crassispina.

Lugar de estudio:

Doce municipios de Guanajuato: Ocampo, San Felipe, San Diego de la Unión, Victoria, Xichú, Atarjea, Dolores Hidalgo, Doctor Mora, Santa Catarina, Tierra Blanca, San Miguel Allende and Comonfort.

Métodos:

Se muestreó la base de hojas de Agave salmiana ssp. salmiana y A. salmiana ssp. crassispina de seis años. Se extrajeron los carbohidratos solubles en agua y se determinó contenido y distribución de tamaños de los fructanos por HPLC-SEC.

Resultados:

Se extrajeron en promedio 0.7 % de carbohidratos solubles. La distribución de tamaños de los fructanos de Agave salmiana fue: peso molecular promedio en número: 3,209 g/mol; peso molecular promedio en masa: 5,046 g/mol; grado de polimerización promedio en número: 19; grado de polimerización promedio en masa: 30. El contenido de fructanos con alto grado de polimerización fue mayor en Agave salmiana spp. salmiana (55 %) que en Agave salmiana spp. crassispina (47 %).

Conclusiones:

Las hojas de Agave salmiana contienen baja cantidad de fructanos de alto peso molecular comparados con fructanos de agave comerciales. Se demostró la factibilidad técnica de obtener fructanos a partir de hojas de Agave salmiana.

Palabras clave: Caracterización de polisacáridos; Cromatografía de exclusión; Fibra soluble; Fitoquímicos de agave

Mezcal production is increasing in the last decade in México, Guanajuato state have been looking for an inclusion of norther municipalities in mezcal origin appellation. During the processes of tequila and mezcal production, the agave leaves are discarded and become a crop residue. To date, efforts have been made to exploit this residue as bioethanol and in the production of enzymes. Several studies have been focused on the recovery of fermentable sugars by chemical, enzymatic, and physical treatments followed by the use of sugars recovered for bioethanol or the production of enzymes (Close et al. 2017, Avila-Gaxiola et al. 2018, Contreras-Hernández et al. 2018, González-Llanes et al. 2018). In these cases, soluble carbohydrates extracted from leaves were hydrolyzed by thermal or enzymatic processes and transformed into fermentable reducing sugars. Fructans can also be obtained from agave leaves (Praznik et al. 2013). The agave fructans recovered from Agave salmiana leaves are a potentially valuable product.

Agave fructans are branched fructose polymers, their molecular structure is composed of fructose-fructose glycosidic bonds containing β(2-1) and β(2-6) linkages, with internal or external glucose units (López et al. 2003). Fructans are a soluble fiber that can act as a prebiotic in the gut microbiota. Fructans are a non-digestible fiber and are not hydrolyzed by human enzymes. Instead, fructans are used as a carbon source by microorganisms in the colon (Gomez et al. 2010). Besides their prebiotic effects, agave fructans have been reported to be immune system activators, body weight reducers, mineral absorption promoters, and microencapsulation agents (García-Vieyra et al. 2014, Moreno-Vilet et al. 2014, Ortiz-Basurto et al. 2017, Castillo-Andrade et al. 2018).

The degree of polymerization and the molecular weight distribution have an effect on the biological properties of agave fructans. For example, low- and medium-molecular-weight fructans extracted from Agave salmiana and A. tequilana presented greater prebiotic bacteria growth than high-molecular-weight fructans (García-Gamboa et al. 2018). The growth of some probiotic strains was greater when agave fructans with a low polymerization degree were used as the carbon source (Mueller et al. 2016). Moreover, agave fructans with a low degree of polymerization reduced the body weight and fat accumulation in mice fed high-fat diets (Márquez-Aguirre et al. 2013). Furthermore, the addition of low-polymerization-degree A. tequilana fructans reduced liver steatosis and hyperglycemia in mice fed high-fat diets (Márquez-Aguirre et al. 2016).

Agave tequilana fructans have been extensively studied because commercial agave fructans are extracted from this agave heads. However, few studies on A. salmiana fructans have since been published. The species A. salmiana is characterized by its broad, strong, and succulent leaves featuring long acuminate and sigmoid apices, short and solid stems with rosette shapes, and heights ranging from 1.5 to 3.4 m. This species has several different variants (Mora-López et al. 2011), including A. salmiana subspecies crassispina, which is characterized by its green color and a more developed apical spine compared to the A. salmiana subspecies salmiana, which has a larger size, wider leaves, and a cinder color. Studies on A. salmiana fructans focused on purification of the fructans by nanofiltration to remove low-molecular-weight sugars, thus enabling better drying (Reynoso-Ponce et al. 2017). On the other hand, Agave salmiana fructans have been tested in vitro as prebiotics and immune system activators (Moreno-Vilet et al. 2014, Martinez-Gutierrez et al. 2017) and as dietary supplement in rats (Castillo-Andrade et al. 2018).

With the aim to describe the molecular weight distribution of soluble carbohydrates contained in the leaves of Agave salmiana species, we extracted soluble carbohydrates and characterized them by size exclusion chromatography (SEC).

Materials and methods

Plant materials. The bases of the leaves close to the stems of Agave salmiana ssp. salmiana and Agave salmiana ssp. crassispina plants, approximately six years old, were harvested from northern Guanajuato in México (Figure 1). Twelve municipalities, that could be included in the appellation of origin Mezcal, were sampled: Ocampo (O), San Felipe (SF), San Diego de la Unión (SDU), Victoria (V), Xichú (X), Atarjea (A), Dolores Hidalgo (DH), Doctor Mora (DM), Santa Catarina (SC), Tierra Blanca (TB), San Miguel Allende (SMA), and Comonfort (C). Two agaves of each specie were sampled in each locality. The tissues were weighed before and after the cuticle and upper epidermis of the leaf were removed and the tissues were then fragmented into small pieces, washed with distilled water, and maintained at 4 °C for further use.

Fuente: INEGI / CONABIO (2010)

www.mapasparacolorear.com

Figure 1 Map of sampled sites in Guanajuato, México. O: Ocampo; SF: San Felipe; SDU: San Diego de la Unión; V: Victoria; X: Xichú; A: Atarjea; DH: Dolores Hidalgo; DM: Doctor Mora; SC: Santa Catarina; TB: Tierra Blanca; SMA: San Miguel Allende; C: Comonfort. 

Reagents. Analytical standards were used for the HP-SEC analysis. Maltopentaose DP5, maltohexaose DP6, maltoheptaose DP7, nystose DP4, 1-kestose DP3, sucrose, fructose and blue dextran were obtained from Sigma-Aldrich (Sigma-Aldrich, Saint Louis, Misuri, USA). Dextran 5000 Da and Dextran 12000 Da were obtained from Fluka Analytical (Fluka, Buchs, Switzerland). Commercial Agave tequilana fructans (Olifructine®) were obtained from Nutriagaves (Nutriagaves, Ayotlan, Jalisco, México). Degasified and distilled water pH 5.4, filtered through a 0.45 µm polyethersulfone (PES) membrane was used as the mobile phase, and 10 % acetonitrile was used as washing solution.

Soluble carbohydrate extraction. Extraction of concentrated agave juice from plant tissues was performed using a CARVER hydraulic press (Carver, Inc. Wabash, Indiana, USA). The fragmented leaf tissues were subjected once to a pressure of up to 20 tonnes until all juice was extracted. Once the agave juice was recovered, soluble solids (°Bx) were measured with a digital refractometer (Atago, Tokyo, Japan). To avoid enzymatic activity and hydrolysis of the agave fructans, the juice was subjected to heating at 70 °C on an IKA (Ika, Cincinnati, Ohio, USA) stirring plate at 800 rpm, for one hour. To obtain a clear liquid without sediments, the juices were centrifuged (Eppendorf 5810R, Hamburg, Germany) at 4,000 rpm for 10 minutes at 4 °C. The soluble carbohydrates were measured again after centrifugation. Extracts were then maintained at -80 °C for further use.

Sample conservation by spray drying. After extraction, enzymatic inactivation, and centrifugation, the samples were filtered through an 80-micron mesh and then spray dried. The spray dryer (Yamato model ADL311S, Yamato, Tokyo, Japan) was stabilized with sterile distilled water running for 10 minutes under the following conditions: pressure of 0.2 MPa and an inlet temperature of 150 °C. The agave juice was then diluted with distilled water in a 1:1 ratio and passed through the spray dryer. The resulting whitish powder was passed through the cyclone system and collected at the bottom of a container. Subsequently, the white powder recovered with a spatula was deposited in 50 mL centrifuge tubes and stored in a drying chamber for further analysis. Some samples were not susceptible to being dried and were instead frozen until their characterization.

Molecular weight distribution of agave fructans. High Performance Size Exclusion Chromatography (HP-SEC) was used to estimate the molecular weight distribution of the extracted agave fructans. The pulverized samples were diluted to 25 g/L in distilled water in triplicate within volumetric flasks of 5 and 10 mL and then filtered through a polyethersulfone membrane (PES) of 0.45 μm. Next, the solution was placed in 1.5 mL vials. The HP-SEC system consisted of a high-resolution liquid chromatograph, Waters model E2695, coupled with a refractive index detector. An Ultrahydrogel DP column (7.8 mm d.i. x 300 mm, Waters, Milford, MA, USA) was used as the stationary phase. The mobile phase was prepared with tridistilled water and filtered on a polyethersulfone membrane (PES) of 0.45 μm; then, the pH was adjusted to 4.5-5. The conditions were as follows: Pump A, 100 % tridistilled water, pH = 4.5; mobile phase at flow: 0.36 mL/min; temperature of the column: 61.7 °C; IR detector temperature: 50 °C, running time: 32 min; and amount of injected sample: 20 µL. A calibration curve was then made, with sugars ranging from 180 Da to 12,000 Da used as the standards. The calibration curve and number average molecular weight (Mn), weight average molecular weight (Mw), dispersity index (D), number average degree of polymerization (DPn), and weight average degree of polymerization (DPw) were determined; mono and disaccharides were not considered in calculations (Moreno-Vilet et al. 2017). The equations used to calculate these parameters are classical for polymers and are described below:

Mn=niMini ; Mw=niMi2niMi ; D=MwMn ; DPn=Mn-18162.1 ; DPw=Mw-18162.1

Statistical analysis. Statistical analysis was performed by one way ANOVA using the Fisher LSD method for means comparison at 95 % of confidence level. Minitab software was used for calculations.

Results

Sampling of Agave salmiana leaves. We performed a sampling of Agave salmiana leaves from twelve municipalities in the north of Guanajuato (Figure 1). Two subspecies of A. salmiana were sampled in each municipality: A. salmiana spp. crassispina (known locally as agave “verde”) and A. salmiana spp. salmiana (known as agave “cenizo”). The site with the greatest diversity of agave species was Victoria. However, in Atarjea and Xichu, A. salmiana spp. crassispina was more abundant than A. salmiana spp. salmiana. Meanwhile, in Ocampo and San Francisco, A. salmiana spp. salmiana was more abundant than A. salmiana spp. crassispina.

Water-soluble carbohydrate extraction. Water-soluble carbohydrates were extracted from the bottoms of the leaves (near the stems). Between 0.32 % and 2.22 % of the total leaf weight (wet basis) corresponded to water-soluble carbohydrates. The carbohydrate content was related to the place from which the samples were collected. Agaves collected from Doctor Mora (DM) had the greatest soluble sugar content, as shown in Figure 2. Meanwhile, agaves collected from Atarjea (A) contained six times fewer carbohydrates than agaves from DM (Figure 2). Notably, in almost all sampled sites, A. salmiana spp. crassispina contained a slightly greater content of soluble carbohydrates (0.74 %) compared to the A. salmiana spp. salmiana subspecies (0.68 %).

Figure 2 Content of water-soluble carbohydrates. The percentage refers to the grams of carbohydrate per 100 grams of sample (wet basis). The results are the average of three replicate samples. O: Ocampo; DH: Dolores Hidalgo; SDU: San Diego de la Unión; V: Victoria; SC: Santa Catarina; A: Atarjea; TB: Tierra Blanca; C: Comonfort; SMA: San Miguel Allende; X: Xichú; SF: San Felipe; DM: Doctor Mora. Means comparison was performed using Fisher LSD. Lower case letters: means that do not share a letter are significantly different. 

Molecular weight distribution of agave fructans. The molecular weight distribution of fructans extracted from Agave salmiana was determined by HP-SEC, as described in the Materials and Methods section (Table 1). The average molecular weight in number (Mn) fluctuated from 1,156 to 4,933 g/mol, with an average of 3,209 g/mol. The average molecular weight in weight ranged from 1,975 to 6,432 g/mol, with an average of 5,046 g/mol. The number average degree of polymerization (DPn) fluctuated between 7 and 30, with an average of 19. The weight average degree of polymerization (DPw) ranged from 12 to 39, with an average of 30. In general, A, salmiana spp. salmiana presented a lower molecular weight and DP than A, salmiana spp. crassispina. The content of lower degree of polymerization fructans was also greater in A, salmiana spp. salmiana (55 %) than in A, salmiana spp. crassispina (47 %) (Figure 3).

Table 1 Molecular weight distribution of fructans extracted from Agave salmiana 

Sampled site ssp. Mn
g/mol
Mw
g/mol
DPn DPw D DP>10
(%)
DP<10
(%)
O salmiana 4620 ± 52ab 5652 ± 5cdefg 28 ± 1ab 34 ± 1bcdef 1.2 ± 0.1k 80 ± 1b 20 ± 1j
crassispina 4568 ± 45abc 6432 ± 27a 28 ± 1abc 39 ± 1a 1.4 ± 0.1ij 64 ± 1cd 36 ± 1hi
DH salmiana 2078 ± 23op 4703 ± 8jk 13 ± 1m 29 ± 1jk 2.2 ± 0.1cd 29 ± 1i 73 ± 2c
crassispina 3933 ± 52efg 6074 ± 57abcde 24 ± 1efgh 37 ± 1abcd 1.5 ± 0.1ghi 55 ± 1efg 45 ± 1fg
SDU salmiana 4096 ± 23de 4765 ± 21jk 25 ± 1de 29 ± 1jk 1.1 ± 0.1k 88 ± 1a 12 ± k
crassispina 2627 ± 238mn 5533 ± 282efgh 16 ± 2kl 34 ± 2defg 2.1 ± 0.1d 32 ± 3i 68 ± 3c
TB salmina 1293 ± 15q 3436 ± 17l 8 ± 1n 21 ± 11 26 ± 0.1a 10 ± 1k 90 ± 1a
crassispina 3551 ± 50ghi 5492 ± 23fgh 22 ± 1hi 34 ± 2efgh 1.5 ± 0.1ghi 58 ± 3de 42 ± 3gh
A salmiana 3286 ± 15ijk 5552 ± 14defgh 20 ± 1ij 34 ± 1cdefg 1.7 ± 0.1efgh 45 ± 1h 55 ± 1de
crassispina 4065 ± 262de 4802 ± 229ijk 25 ± 2def 30 ± 2ijk 1.1 ± 0.2k 84 ± 7ab 16 ± 7jk
SC salmiana 2344 ± 36no 3489 ± 141l 14 ± 1lm 21 ± 1l 1.4 ± 0.1ij 43 ± 1h 57 ± 1de
crassispina 4348 ± 225bcd 5007 ± 5hij 27 ± 2bcd 31 ± 1ghijk 1.1 ± 0.1k 86 ± 4ab 17 ± 1jk
C salmiana 1902 ± 9p 4448 ± 180k 12 ± 1m 27 ± 1k 2.3 ± 0.1bc 20 ± 2j 80 ± 2b
crassispina 3002 ± 533klm 5363 ± 663fghi 18 ± 3jk 33 ± 4efghi 1.7 ± 0.1ef 43 ± 4h 58 ± 4d
SMA salmiana 4933 ± 248a 5868 ± 285bcdef 30 ± 2a 36 ± 2abcde 1.1 ± 0.1k 82 ± 1ab 18 ± 1jk
crassispina 3683 ± 1fgh 6120 ± 51abc 23 ± 1fghi 37 ± 1abcd 1.6 ± 0.1ghi 50 ± 1fgh 50 ± 1ef
X salmiana 3623 ± 114fghi 6208 ± 174ab 22 ± 1ghi 38 ± 1ab 1.7 ± 0.1efg 50 ± 2fgh 50 ± 1de
crassispina 4226 ± 12cde 6122 ± 41abc 26 ± 1cde 38 ± 1abc 1.4 ± 0.1i 62 ± 1de 39 ± 1gh
SF salmiana 2036 ± 133op 4895 ± 217ijk 12 ± 1m 30 ± 2hijk 2.4 ± 0.1ab 21 ± 2j 79 ± 2b
crassispina 3404 ± 161hij 5766 ± 138bcdef 21 ± 1ij 35 ± 1bcdef 1.6 ± 0.1efgh 47 ± 2h 53 ± 2de
V salmiana 3981 ± 42def 6100 ± 187abcd 24 ± 1defg 38 ± 1abcd 1.5 ± 0.1hi 57 ± 2ef 44 ± 2fg
crassispina 3060 ± 252jkl 5209 ± 11ghij 19 ± 2j 32 ± 1fghij 1.7 ± 0.1efg 48 ± 5gh 54 ± 2de
DM salmiana 1156 ± 21q 2113 ± 25m 7 ± 1n 13 ± 1m 1.8 ± 0.1e 8 ± 1k 92 ± 1a
crassispina 1211 ± 42q 1975 ± 187m 7 ± 1n 12 ± 1m 1.6 ± 0.1fgh 8 ± 1k 92 ± 1a
R A. tequilana 2701 ± 200lmn 3416 ± 184l 16 ± 1kl 20 ± 1l 1.26 ± 0.1jk 70 ± 3c 30 ± 2i

Mono and disaccharides are not considered in calculations. O: Ocampo; DH: Dolores Hidalgo; SDU: San Diego de la Unión; V: Victoria; SC: Santa Catarina; A: Atarjea; TB: Tierra Blanca; C: Comonfort; SMA: San Miguel Allende; X: Xichú; SF: San Felipe; DM: Doctor Mora; R: commercial agave fructans from Agave tequilana; Mn: number average molecular weight; Mw: weight average molecular weight; D: dispersity index; DPn: number average degree of polymerization; DPw: weight average degree of polymerization; DP > 10: fructans with higher polymerization degree; DP < 10: fructans with lower polymerization degree. Means comparison was performed using Fisher LSD. Means that do not share a letter are significantly different.

Figure 3 Distribution of agave fructans with a lower and higher degree of polymerization (DP) according to the agave subspecies. 

The molecular weight distributions of the fructans extracted from A, salmiana and commercial agave fructans were also compared. Ten samples of commercial fructans (Olifructine®) extracted from A, tequilana were analyzed (Table 1, sample R). The molecular mass and degree of polymerization was calculated for the ten samples, and the averages obtained were as follows: Mn: 2,701 g/mol; Mw: 3,416 g/mol; DPn: 16; DPw: 20; DP > 10: 70; and DP < 10: 30. The molecular weight distribution averages from the A, salmiana fructans were as follows: Mn: 3,209 g/mol; Mw: 5,046 g/mol; DPn: 19; DPw: 30; DP > 10: 48; and DP < 10: 52. We observed a higher molecular weight and average degree of polymerization in the A, salmiana fructans. Analyzing the abundance of fructans according to their polymerization degree revealed a greater abundance of A, salmiana fructans of between 23 and 32 DP, whereas A, tequilana fructans had a lower abundance between 4 and 20 DP (Figure 4). On the other hand, the proportion of fructans with lower and higher degrees of polymerization were remarkably different between A. salmiana and A. tequilana. Fructans extracted from the leaves of A. salmiana featured a greater proportion of fructans with a lower degree of polymerization than that of the commercial fructans extracted from A. tequilana stems (Table 1).

Figure 4 Molecular weight distribution of agave fructans. Relative abundance of fructans according to their polymerization degree. A: commercial agave fructans from Agave tequilana Olifructine®; B: fructans from Agave salmiana spp. salmiana leaves; C: fructans from Agave salmiana spp. crassispina leaves. DP 1, 2 %: content of mono and disaccharides in the samples. 

Discussion

Agave leaves are a type of crop waste from the mezcal and tequila industries. In the last five years, research has focused on exploring options to exploit these leaves to produce high-value products. However, these studies have focused primarily on the recovery of fermentable sugars from agave leaves for use as carbon sources in the production of biofuels and enzymes (Close et al. 2017, Avila-Gaxiola et al. 2018, Contreras-Hernández et al. 2018, González-Llanes et al. 2018). However, agave leaves synthesize fructans that have been shown to offer digestive health benefits (Gomez et al. 2010, Praznik et al. 2013). Therefore, the extraction of fructans from leaves is of great interest especially fructans from agaves such as A. salmiana, which have been rarely studied. On the other hand, municipalities of northern Guanajuato are applying for Denomination of Origin of Mezcal (DOM) status, and an increase in agave crop residue generation is expected (Capilla-Vilchis 2017). Thus, the different potential processes for exploiting fructans represents a compelling avenue of research.

In this study, we observed great differences between soluble carbohydrate content in sampled agave leaves, ranging between 0.32 and 2.22 % (Atarjea and Doctor Mora, respectively). These differences indicated a relationship with the place of sampling. It was previously reported that soil composition, climate, and other factors, such as agave age, are related to the carbohydrate composition and fructan structure in agave plants (Arrizon et al. 2010, Mellado-Mojica & López 2012). Although the agaves sampled were all A. salmiana, the climate differed greatly between the sampled locations. Atarjea is located in the “Sierra Gorda” of Guanajuato and contains several microclimates (tropical, warm-humid, and semi-warm), while the Doctor Mora climate is predominantly temperate-dry.

The fructan content in the agave leaves of some agave species has been determined. The content of fructans in A. durangensis leaves was reported as 8.9 % (Contreras-Hernández et al. 2018), which is greater than the 2.22 % from the A. salmiana leaves tested in the present study. The powder obtained from A. tequilana leaves presented fructan content of 37.5 % (dry matter) (Avila-Gaxiola et al. 2018). A recent study noted that the fructan content in agave leaves decreases according to the distance between the leaves and the stem, with higher levels observed closer to the bases of the leaves near the stem (González-Llanes et al. 2018). The fructan content at the bases of the A. salmiana leaves measured in this work was lower than that observed in the different agave species tested by Gonzalez-Llanes: A. salmiana spp. salmiana (6.8 %), A. salmiana spp. crassispina (6 %), and A. americana (3 %) (González-Llanes et al. 2018). The leaves collected from two-year-old Agave tequilana plants were dried, and agave fructans were extracted, detecting 70.9 g of fructans/kg of agave (dry weight) (Close et al. 2017).

It is known that the properties of fructans depend on their degree of polymerization. For example, fructooligosaccharides (DP < 10) and mildly polymerized fructans are effective as prebiotics (Biedrzycka & Bielecka 2004). Agave fructans with low and medium degrees of polymerization perform well as prebiotics (Mueller et al. 2016, García-Gamboa et al. 2018). On the other hand, agave fructans with large degrees of polymerization have been reported to function as fat replacers in baked products (Santiago-García et al. 2017). The fructans extracted from Agave salmiana leaves showed diverse average polymerization degrees ranging from 7 to 30, with an average DPn of 19 (mono and disaccharides were not considered in calculations). This diversity could be attributed to the soil composition and climate of the sampling locations, in addition to the ages of the agaves. In the present study, the ages of the samples were estimated to be six years old; however, the ages of wild agaves remain uncertain. It was previously reported that young agaves contain fructans with a lower degree of polymerization, while mature agaves feature fructans with a higher degree of polymerization. In addition, the branching of agave fructans increases with age (Arrizon et al. 2010, Mellado-Mojica & López 2012).

We next compared fructans extracted from A. salmiana leaves with commercial fructans. Commercial fructans showed the greatest proportion of high-molecular-weight fructans. On the other hand, fructans from A. salmiana leaves presented almost equal proportion of low- and high-molecular-weight fructans; indeed, some samples contained more than 90 % low-molecular-weight fructans. Agave tequilana synthesizes low-molecular-weight fructans in its leaves. Later, these fructans are transported to the stem and transformed into high-molecular-weight fructans (Praznik et al. 2013).

According to the results, all sampled agave leaves have the potential to be exploited, but the agave leaves collected from DM presented the greatest quantity of soluble carbohydrates, and the largest amount of short-chain agave fructans DP < 10. These characteristics make DM agave leaves an interesting subject for future studies.

The technical feasibility to obtain fructans from A. salmiana leaves was demonstrated in this study. Water-soluble carbohydrates were recovered from the bases of the leaves near the stem, and fructans represented 0.7 % of leaf weight on a wet basis. Fructans content in the leaves is low but it is proposed by other authors the use of the leaves in different applications, if we think in a biorefinery scheme where more than one single product is obtained the agave leaves exploitation could be successful. The fructans extracted were susceptible to be dried. Moreover, we determined the molecular weight distribution of the fructans extracted from the leaves. We found great diversity in molecular weight distributions, but, in general, the leaves of A. salmiana contained a low proportion of high-molecular-weight fructans compared to commercial agave fructans. However, the average DPn of high-molecular-weight fructans (DPn = 19) in the samples tested was greater than that of commercial fructans (DPn 16). Ultimately, leaves from Agave salmiana are a good source of fructans, but the commercial feasibility of these leaves must be explored further.

Acknowledgments

This work was supported by the National Council of Science and Technology CONACYT, FOMIX-GUANAJUATO [grant number 285454]. Within the project “Estudio técnico científico de las características de la región Norte del Estado de Guanajuato para determinar la viabilidad, factibilidad y potencialidad del agave mezcalero, la producción de mezcal y otros productos derivados”. Arizbeth Plascencia thanks Fomix-Guanajuato for her scholarship. The authors are grateful to Flor Isela García for her technical assistance with HPLC-SEC.

Literature cited

Arrizon J, Morel S, Gschaedler A, Monsan P. 2010. Comparison of the water-soluble carbohydrate composition and fructan structures of Agave tequilana plants of different ages. Food Chemistry122: 123-130. DOI: https://doi.org/10.1016/j.foodchem.2010.02.028 [ Links ]

Avila-Gaxiola J, Velarde-Escobar OJ, Millan-Almaraz JR, Ramos-Brito F, Atondo-Rubio G, Yee-Rendon C, Ávila-Gaxiola E. 2018. Treatments to improve obtention of reducing sugars from agave leaves powder.Industrial Crops and Products112: 577-583. DOI: https://doi.org/10.1016/j.indcrop.2017.12.039 [ Links ]

Biedrzycka E, Bielecka M. 2004. Prebiotic effectiveness of fructans of different degrees of polymerization.Trends in Food Science & Technology15: 170-175. DOI: https://doi.org/10.1016/j.tifs.2003.09.014 [ Links ]

Capilla-Vilchis R. 2017. Denominación de origen del Mezcal en Guanajuato. Agencia Informativa Conacyt. http://www.cienciamx.com/index.php/ciencia/economia/17904-denominacion-origen-mezcal-guanajuato (Accessed June 4, 2021). [ Links ]

Castillo-Andrade AI, Rivera-Bautista C, Godínez-Hernández C, Ruiz-Cabrera MA, Fuentes-Ahumada F, García-Chávez E, Grajales-Lagunes A. 2018. Physiometabolic effects of Agave salmiana fructans evaluated in Wistar rats.International Journal of Biological Macromolecules 108: 1300-1309. DOI: https://doi.org/10.1016/j.ijbiomac.2017.11.043 [ Links ]

Close D, Rodriguez M, Hu R, Yang X. 2017. Disposition and bioavailability of inulin and free sugar in untreated and dilute acid pretreated Agave tequilana leaves.Biomass & Bioenergy106: 176-181. DOI: https://doi.org/10.1016/j.biombioe.2017.08.032 [ Links ]

Contreras-Hernández MG, Ochoa-Martínez LA, Rutiaga-Quiñones JG, Rocha-Guzmán NE, Lara-Ceniceros TE, Contreras-Esquivel JC, Rutiaga-Quiñones OM. 2018. Effect of ultrasound pre-treatment on the physicochemical composition of Agave durangensis leaves and potential enzyme production.Bioresource Technology249: 439-446. DOI: https://doi.org/10.1016/j.biortech.2017.10.009 [ Links ]

García-Gamboa R, Ortiz-Basurto RI, Calderón-Santoyo M, Bravo-Madrigal J, Ruiz-Álvarez BE, González-Ávila M. 2018. In vitro evaluation of prebiotic activity, pathogen inhibition and enzymatic metabolism of intestinal bacteria in the presence of fructans extracted from agave: A comparison based on polymerization degree.LWT-Food Science and Technology92: 380-387. DOI: https://doi.org/10.1016/j.lwt.2018.02.051 [ Links ]

García-Vieyra MI, Del Real A, López MG. 2014. Agave fructans: their effect on mineral absorption and bone mineral content.Journal of Medicinal Food17: 1247-1255. DOI: https://doi.org/10.1089/jmf.2013.0137 [ Links ]

Gomez E, Tuohy KM, Gibson GR, Klinder A, Costabile A. 2010. In vitro evaluation of the fermentation properties and potential prebiotic activity of Agave fructans.Journal of Applied Microbiology108: 2114-2121. DOI: https://doi.org/10.1111/j.1365-2672.2009.04617.x [ Links ]

López MG, Mancilla-Margalli NA, Mendoza-Díaz G. 2003. Molecular structures of fructans from Agave tequilana Weber var. Azul. Journal of Agricultural and Food Chemistry 51: 7835-7840. https://doi.org/10.1021/jf030383v [ Links ]

González-Llanes MD, Hernández‐Calderón OM, Rios‐Iribe EY, Alarid‐García C, Castro Montoya AJ, Escamilla‐Silva EM. 2018. Fermentable sugars production by enzymatic processing of agave leaf juice.Canadian Journal of Chemical Engineering96: 639-650. DOI: https://doi.org/10.1002/cjce.22959 [ Links ]

Márquez-Aguirre AL, Camacho-Ruiz RM, Arriaga-Alba M, Padilla-Camberos E, Kirchmayr MR, Blasco JL, González-Avila M. 2013. Effects of Agave tequilana fructans with different degree of polymerization profiles on the body weight, blood lipids and count of fecal Lactobacilli/Bifidobacteria in obese mice.Food & Function4: 1237-1244. DOI: https://doi.org/10.1039/C3FO60083A [ Links ]

Márquez-Aguirre AL, Camacho-Ruíz RM, Gutiérrez-Mercado YK, Padilla-Camberos E, González-Ávila M, Gálvez-Gastélum FJ, Díaz-Martínez NE, Ortuño-Sahagún D. 2016. Fructans from Agave tequilana with a lower degree of polymerization prevent weight gain, hyperglycemia and liver steatosis in high-fat diet-induced obese mice.Plant Foods for Human Nutrition71: 416-421. DOI: https://doi.org/10.1007/s11130-016-0578-x [ Links ]

Martinez-Gutierrez F, Ratering S, Juárez-Flores B, Godinez-Hernandez C, Geissler-Plaum R, Prell F, Zorn H, Czermak P, Schnell S. 2017. Potential use of Agave salmiana as a prebiotic that stimulates the growth of probiotic bacteria.LWT-Food Science and Technology84: 151-159. DOI: https://doi.org/10.1016/j.lwt.2017.05.044 [ Links ]

Mellado-Mojica E, López MG. 2012. Fructan metabolism in A. tequilana Weber blue variety along its developmental cycle in the field.Journal of Agricultural and Food Chemistry60: 11704-11713. DOI: https://doi.org/10.1021/jf303332n [ Links ]

Mora-López JL, Reyes-Agüero JA, Flores-Flores JL, Peña-Valdivia CB, Aguirre-Rivera JR. 2011. Variación morfológica y humanización de la sección Salmianae del género Agave.Agrociencia45: 465-477. [ Links ]

Moreno-Vilet L, Bostyn S, Flores-Montaño JL, Camacho-Ruiz RM. 2017. Size-exclusion chromatography (HPLC-SEC) technique optimization by simplex method to estimate molecular weight distribution of agave fructans.Food Chemistry237: 833-840. DOI: https://doi.org/10.1016/j.foodchem.2017.06.020 [ Links ]

Moreno-Vilet L, Garcia-Hernandez MH, Delgado-Portales RE, Corral-Fernandez NE, Cortez-Espinosa N, Ruiz-Cabrera MA, Portales-Perez DP. 2014. In vitro assessment of agave fructans (Agave salmiana) as prebiotics and immune system activators.International Journal of Biological Macromolecules63: 181-187. DOI: https://doi.org/10.1016/j.ijbiomac.2013.10.039 [ Links ]

Mueller M, Reiner J, Fleischhacker L, Viernstein H, Loeppert R, Praznik W. 2016. Growth of selected probiotic strains with fructans from different sources relating to degree of polymerization and structure.Journal of Functional Foods24: 264-275. DOI: https://doi.org/10.1016/j.jff.2016.04.010 [ Links ]

Ortiz-Basurto RI, Rubio-Ibarra ME, Ragazzo-Sanchez JA, Beristain CI, Jiménez-Fernández M. 2017. Microencapsulation of Eugenia uniflora L. juice by spray drying using fructans with different degrees of polymerization.Carbohydrate Polymers175: 603-609. DOI: https://doi.org/10.1016/j.carbpol.2017.08.030 [ Links ]

Praznik W, Löppert R, Cruz-Rubio JM, Zangger K, Huber A. 2013. Structure of fructo-oligosaccharides from leaves and stem of Agave tequilana Weber, var. azul.Carbohydrate Research381: 64-73. DOI: https://doi.org/10.1016/j.carres.2013.08.025 [ Links ]

Reynoso-Ponce H, Grajales-Lagunes A, Castillo-Andrade A, González-García R, Ruiz-Cabrera MA. 2017. Integration of nanofiltration and spray drying processes for enhancing the purity of powdered fructans from Agave salmiana juice.Powder Technology322: 96-105. DOI: https://doi.org/10.1016/j.powtec.2017.09.003 [ Links ]

Santiago-García PA, Mellado-Mojica E, León-Martínez FM, López MG. 2017. Evaluation of Agave angustifolia fructans as fat replacer in the cookies manufacture.LWT-Food Science and Technology77: 100-109. DOI: https://doi.org/10.1016/j.lwt.2016.11.028 [ Links ]

Received: June 09, 2021; Accepted: October 30, 2021; Published: March 22, 2022

*Author for correspondence: rcamacho@ciatej.mx

Associate editor: Juan Rodrígo Salazar

Author contributions: AP Orcid, writing, data analysis, laboratory work; AGM Orcid, field work, review; JMRD Orcid field work, specimen selection; JJCN Orcid, field work, writing; JGV Orcid, funding acquisition, review; HS Orcid, review, writing; RMCR Orcid, conceptualization, writing, field work, data analysis.

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