extraction and characterization of mucilage from wild

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Journal of Food Process Engineering ISSN 1745-4530

EXTRACTION AND CHARACTERIZATION OF MUCILAGE FROM WILD SPECIES OF OPUNTIA SARAHI RODRÍGUEZ-GONZÁLEZ1, HECTOR E. MARTÍNEZ-FLORES2,5, CARLA K. CHÁVEZ-MORENO2, LOURDES. I. MACÍAS-RODRÍGUEZ3, EDER ZAVALA-MENDOZA1, M.G. GARNICA-ROMO4 and LUIS CHACÓN-GARCÍA3 1 Programa Institucional de Maestría en Ciencias Biológicas, 2Facultad de Químico Farmacobiología, 3Instituto de Investigaciones en Químico Biológicas and 4Facultad de Ingeniería Civil, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacan 58240, Mexico

5

Corresponding author. TEL: +52(443)-3142152, Ext. 201; FAX: +52(443)-3142152, Ext. 201; EMAIL: [email protected] Received for Publication December 6, 2013 Accepted for Publication February 7, 2014 doi:10.1111/jfpe.12084

ABSTRACT The aim of this work was to characterize the mucilage extracted from six species of Opuntia. The species studied were as follows: O. atropes, O. tomentosa, O. hyptiacantha, O. streptacantha y O. joconostle and O. ficus-indica. The first step was to find the best extraction conditions to obtain an unaltered chemical structure of mucilage. The mucilages were characterized using high-performance liquid chromatography and Fourier transform infrared spectroscopy techniques. The optimal conditions employed to obtain the mucilage were: ratio of cladodes : ethanol solution at 50% of 1:1 (w/v), temperature of 22C and precipitation of mucilage with a solution of ethanol 96%, at a ratio of 1:4 (v/v). Mucilage from all species studied had a high content of soluble dietary fiber, ranging from 51.79 to 67.51%. In decreasing order, sugars found in the mucilages were: L-arabinose (26.83–35.36%), D-galactose (21.59–45.48%), D-xylose (12.23– 17.05%), uronic acids (5.59–13.91%), D-glucose (5.18–16.21%) and L-rhamnose (1.41–5.40%).

PRACTICAL APPLICATIONS This research presents an optimization method for the extraction of mucilage five wild species of cactus based on temperature and time of extraction and solvent ratio: sample used. Mucilages were characterized chemically and by chromatographic and Fourier transform infrared spectroscopy techniques and were compared with the species mucilage Opuntia ficus-indica, the species most commercially studied. The mucilage obtained can be used for commercial purposes as additives in the food industry.

INTRODUCTION Food characteristics depend on their chemical components and sometimes, polysaccharides – such as hydrocolloids – are those molecules responsible for some physiochemical and mechanical properties in foods (Medina-Torres et al. 2003). Mucilages from different sources are compounds of great potential to be applied in food, pharmaceutical and other industries (Forni et al. 1994; Majdoub et al. 2001; Ribeiro et al. 2010; Hwan et al. 2013). Journal of Food Process Engineering 37 (2014) 285–292 © 2014 Wiley Periodicals, Inc.

Mucilage from Opuntia ficus-indica is an interesting ingredient for the food industry because of its viscosity (Sepúlveda et al. 2007). It has the capacity to create gels that retain great quantities of water (Medina-Torres et al. 2003; Sáenz et al. 2004) and also has good emulsifying properties (MedinaTorres et al. 2003; Sáenz et al. 2004; Bernardino-Nicanor et al. 2013) – being used in the production of edible films (Del-Valle et al. 2005; Espino-Díaz et al. 2010) – and applied to encapsulate both Saccharomyces boulardii (Zamora-Vega et al. 2012) and gallic acid (Medina-Torres et al. 2013). 285

MUCILAGE FROM WILD SPECIES OF OPUNTIA

S. RODRÍGUEZ-GONZÁLEZ ET AL.

TABLE 1. CHARACTERIZATION BY CHROMATOGRAPHY OF THE MUCILAGE FROM SIX SPECIES OF OPUNTIA Authors

Gal

Ara

Xyl

Rha

McGarvie and Parolis (1981)a Trachtenberg and Mayer (1981)a Nobel et al. (1992)a Medina-Torres et al. (2000)a Matsuhiro et al. (2006) Cárdenas et al. (2008)a Ginestra et al. (2009) Ribeiro et al. (2010)

18.4 40.1 21 17.92 + 7 + +

42.4 24.6 42 46.68 + 6 + +

24.5 22.2 22 23.45 + 1 + +

6.4 13.1 7 6.76 + 0.6 +

Uronic acids

Glu

Fru

+ +

+

Fuc

Man

+

+

8.4 8 5.19 + 85.4 + +

Ara, arabinose; Fru, fructose; Fuc, fucose; Gal, galactose; Gluc, glucose; Man, manose; Ram, rhamnose; Xyl, xylose. a Expressed as percentage.

A molecule of mucilage from O. ficus-indica can contain more than 30,000 sugar subunits or residues (McGarvie and Parolis 1981; Gibson and Nobel 1990; Medina-Torres et al. 2000). It is a polysaccharide with a reported molecular weight of 2.3 × 104 to 4.3 × 106 Daltons (Trachtenberg and Mayer 1982; Medina-Torres et al. 2000). Mucilage represents about 14% dry basis in nopal cladodes (Ginestra et al. 2009). The sugars found in mucilage are arabinose (35– 40%), galactose (20–25%), rhamnose (7–8%), xylose (20– 25%) and uronic acids (7–8%) (Gibson and Nobel 1990). According to McGarvie and Parolis (1981), a hypothetical model of the mucilage from O. ficus-indica consists of alternating rhamnose and galacturonic acid residues, attached to the side chains composed of three galactose residues. Arabinose and xylose sugars are branches on the galactose side chains. In general, xylose appears to be joined to arabinose, which is then bonded to galactose. Some galactose side chains have arabinose but not xylose, and some have one xylose with two arabinose residues. The mucilage from other species of the Opuntia is composed of the same sugar residues. However, the residues are present in different ratios than for the O. ficus-indica. Mucilage molecules tend to be negatively charged, because hydrogen ions can dissociate from their carboxylic part of the galacturonic acids (Gibson and Nobel 1990). This is important, because some physiochemical properties of the mucilage are dependent on the ionized form of the carboxylic group. According to Cárdenas et al. (2008), the rheological properties, mainly the intrinsic viscosity of mucilage, are dependent on their chemical composition, pH, sterification degree and the presence of calcium ions. Mucilage is a compound of a low degree of esterification (DE) (Cárdenas et al. 2008). Some authors studied the sugar composition of mucilage from O. ficus-indica normally using chromatography techniques (Table 1). The objective of this work was to characterize the profile of the sugar composition of mucilage from six species of Opuntia collected in Michoacán, México. Five of the specimens studied were from wild species and were compared with the species most studied in the world, 286

O. ficus-indica. The mucilages from the wild species of Opuntia could be used as potential ingredients in the food industry.

MATERIAL AND METHODS Materials The studied species O. atropes, O. hyptiacantha, O. joconostle, O. streptacantha, O. tomentosa and O. ficus-indica grown in the Morelia area (Michoacán State, México) were harvested in May of 2011. Cladodes (pads) had two ages at the time of collection.

Mucilage Extraction Procedures Three methods were evaluated for mucilage extraction. The objective of testing by three methods was to find the best extraction conditions without causing damage to the native structure of the mucilage. The methods are described as follows: (1) Fresh cladodes were mixed for 1 min in a blender with water in a ratio of 1:8 (w/v). Then, suspension was maintained at 83C for 2 h. Afterwards suspension was centrifuged at 3,500 rpm for 15 min. The supernatant was added with ethanol 96% in a ratio of 1:4 (v/v). Mucilage mass was separated by centrifugation at 3,500 rpm for 15 min (a method proposed by Zavala 2012). (2) Fresh cladodes were mixed for 1 min in a blender with water in a ratio of 1:2 (w/v). Then, suspension was maintained at 83C for 1 h. Following that, suspension was centrifuged at 3,500 rpm for 15 min. The supernatant was added with ethanol 96% in a ratio of 1:4 (v/v). Mucilage mass was separated by centrifugation at 3,500 rpm for 15 min (a method proposed by Rodríguez 2010); and (3) Fresh cladodes were mixed for 1 min in a blender with ethanol solution 50% in a ratio of 1:1 (w/v). Then, suspension maintained at 22C was centrifuged at 3,500 rpm for 15 min. The supernatant was added with ethanol 96% in a ratio of 1:4 (v/v). Mucilage mass was separated by centrifugation at 3,500 rpm for Journal of Food Process Engineering 37 (2014) 285–292 © 2014 Wiley Periodicals, Inc.



15 min (a modified method proposed by our group research). The mucilage mass obtained by the three methods was dried and stored at 4C until their analysis.

Chemical Composition Moisture, ash, protein and fat content of mucilages were determined by methods 925.45, 945.18, 955.04 and 920.39 of the AOAC (2005), respectively. The protein quantity was calculated by multiplying the nitrogen content using the Kjeldahl method by the coefficient 5.73. Total dietary fiber (TDF), soluble fiber (SF) and insoluble fiber (IF) was carried out using the method of Prosky et al. (1998). Total available carbohydrates (Anthrone method) were determined according to the method reported in AOAC (1990).

Thin Layer Chromatography Mucilage was evaluated by thin layer chromatography (TLC) according to the method proposed by Ribeiro et al. (2010). Dried mucilage of each species of Opuntia was suspended in deionized water and placed on silica gel plates that have been immersed in a TLC chamber. The elution system used for monosaccharides was acetone/n-butanol/ water (80:10:10 v/v/v), and revealed with 2% diphenylamine prepared in acetone, 2% aniline prepared in acetone and 85% orthophosphoric acid (5:5:1 v/v/v). The standards employed were D-galactose, D-xylose, L-arabinose, D-glucose, fructose L-rhamnose and sucrose. For the acid sugars (glucuronic acid and galacturonic acid), the elution system used was chloroform/methanol/acetic acid/water (40:40:10:10 v/v/v/v) and revealed with 2% diphenylamine prepared in acetone, 2% aniline prepared in acetone and 85% orthophosphoric acid (5:5:1 v/v/v).

FIG. 1. PROCEDURE OF ANALYSIS OF GAS CHROMATOGRAPHY TO DETERMINE THE SUGAR COMPOSITION OF MUCILAGES EXTRACTED FROM OPUNTIA FICUS-INDICA

Gas Chromatography

(Bruker Optics, Inc., Billerica, MA). The frequency range used was between 400 and 4,000 cm−1.

Gas chromatography (GC) was carried out using an Agilent 6859 Series II provided with a detector MS Agilent 5973 (Palo Alto, CA). The GC procedure of the extracts is described in Fig. 1, based on the method proposed by Macías-Rodríguez et al. (2002), Habibi et al. (2004) and Ribeiro et al. (2010). The standards used were D-galactose, D-xylose, L-arabinose, D-glucose, fructose, L-rhamnose and sucrose.

Fourier transform infrared spectroscopy Fourier transform infrared spectroscopy (FTIR) was performed on the mucilages obtained from the six species of Opuntia. For this procedure, 0.3 g of dry potassium bromide was mixed with 0.02 g of each dry sample and then compressed with a 3-ton force for 1 min. The samples were analyzed in a Bruker Tensor 27 FTIR spectrophotometer Journal of Food Process Engineering 37 (2014) 285–292 © 2014 Wiley Periodicals, Inc.

Statistical Analysis Statistical analysis of data was performed through analysis of variance (ANOVA) using the statistical software JMP V. 6.0 Statistical Discovery from SAS Institute, Inc. (Cary, NC). Tukey–Kramer (P < 0.05) multiple comparison tests were used to compare species of Opuntia when statistical significance in species was found in general ANOVA (P < 0.05). All experiments were conducted in triplicate.

RESULTS AND DISCUSSION Chemical Composition The chemical composition of mucilage from six species of Opuntia is shown in Table 2. Mucilage content varied widely 287



TABLE 2. CHEMICAL COMPOSITION OF MUCILAGE EXTRACTED FROM SIX SPECIES OF OPUNTIA Chemical component

Opuntia tomentosa

Opuntia atropes

Opuntia hyptiacantha

Opuntia joconostle

Opuntia streptacantha

Opuntia ficus-indica

Moisture Total dietary fiber Soluble fiber Insoluble fiber Ash Calcium (g/100 g) Protein Fat Total carbohydrates

6.32c 68.43ab 64.40a 4.03a 11.26d 3.78b 5.59cd 0.16a 8.24abc

7.62b 57.71b 57.69ab 0.03b 14.01b 7.44a 4.01e 0.06b 16.59a

8.27a 73.01a 67.51a 5.50a 10.82e 3.2b 6.38bc 0.06b 1.46bc

6.32c 65.20ab 65.12a 0.08b 11.72c 1.23c 6.70b 0.20a 9.86abc

3.44e 67.44ab 61.07ab 6.37a 14.19b 6.87a 8.26a 0.19a 6.48c

5.39d 57.23b 51.79b 5.43a 15.13a 4.53b 5.24d 0.09b 16.92ab

a–d Different letters in the same row are significant differences between means of species.

(P < 0.05) in all species. The main chemical component of the six species of Opuntia was the TDF. The highest value of TDF was seen in the species O. hyptiacantha (73.01%) followed by O. tomentosa (68.43%), O. streptacantha (67.44%), O. joconostle (65.2%), O. atropes (57.71%) and O. ficusindica (57.23%). The TDF value obtained in this study was similar (57.83%) to that documented by Rodríguez (2010) for O. ficus-indica. Most of the TDF corresponded to the SF fraction. The O. hyptiacantha had the highest value of SF (67.51%) and did not show a significant difference (P < 0.05) as compared with O. joconostle (65.12%) and O. tomentosa (64.04%), and only presented a partial difference (P < 0.05) with respect to the O. streptacantha (61.07%) and O. atropes (57.69%) species. The O. ficusindica species had the lowest SF value (51.79%). This result was similar to the SF value (56.80%) reported by Rodríguez (2010) for the O. ficus-indica species. The IF values in the mucilage species were as follows: 6.37%, 5.50%, 5.43%, 4.03%, 0.08% and 0.03%, respectively, for O. streptacantha, O. hyptiacantha, O. ficus-indica, O. tomentosa, O. joconostle and O. atropes. The ash content for the mucilages varied from 10.82% (O. hyptiacantha) to 15.13% (O. ficus-indica). In general, the ash content in O. ficus-indica obtained in this study was similar to that documented by Rodríguez (2010), who reported 11.91%; however, our results are different from those found by Sepúlveda et al. (2007), who report an

average value of 37.3%. Calcium content was the predominant mineral in the mucilages. The highest calcium values were for O. atropes (7.44 g/100 g) and O. streptacantha (6.87 g/100 g). The protein content found in the mucilages ranged from 4.01% in O. atropes to 8.26% in O. streptacantha. O. ficus-indica showed a value of 5.24%. Sepúlveda et al. (2007) report an average protein value of 7.3% for O. ficus-indica, meanwhile Rodríguez (2010) reports a value of 6.69%, and both values were slightly lower than our results. The fat content was as much as 0.2% or lower in the mucilage extracted from the six species of Opuntia studied. Total carbohydrates obtained in this study was as follows: O. atropes 18.67%, O. ficus-indica 14.67%, O. joconostle 9.82%, O. tomentosa 8.21%, O. hyptiacantha 6.25% and O. streptacantha 1.62%.

TLC Table 3 shows the monosaccharides, uronic acids and the mucilage detected by TLC. The presence of free monosaccharides (mainly D-xylose) was observed in the methods of Rodríguez (2010) and Zavala (2012). The temperature, extraction time and ratio of cladodes : water used by Rodríguez (2010) and Zavala (2012) could have hidrolyzed the native structure of mucilages during the extraction process releasing some monosaccharides and uronic acids.

TABLE 3. DETERMINATION OF SUGARS AND URONIC ACIDS IN MUCILAGE EXTRACTED FROM SIX SPECIES OF OPUNTIA (%) Species of Opuntia Sugar

Opuntia tomentosa

Opuntia atropes

Opuntia hyptiacantha

Opuntia joconostle

Opuntia streptacantha

Opuntia ficus-indica

L-arabinose D-galactose D-xylose D-glucose L-rhamnose Uronic acids

33.50ab 21.59d 16.02a 16.21a 2.58c 11.80b

34.36a 26.75c 16.62a 9.05c 1.44e 11.76b

32.82ab 30.83b 17.05a 6.03de 1.41e 11.84b

26.83c 45.48a 12.23c 7.13d 4.09b 5.59d

30.43b 31.83b 14.04b 11.81b 5.40a 8.26c

35.36a 27.26c 16.32a 5.18e 1.93d 13.91a

Different letters in the same row are significant differences between means in species.

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Journal of Food Process Engineering 37 (2014) 285–292 © 2014 Wiley Periodicals, Inc.



FIG. 2. CHROMATOGRAPHIC PROFILE OF THE SUGARS IDENTIFIED IN MUCILAGES EXTRACTED FROM OPUNTIA HYPTIACANTHA AND O. FICUS-INDICA

The differences in the processes employed by Rodríguez (2010) and Zavala (2012) were as follows: Rodríguez (2010) used a ratio of cladodes : water of 1:2 (w/v) and the extraction time was 1 h. Meanwhile, Zavala (2012) used a ratio cladodes : water of 1:8 (w:v) and an extraction time of 2 h. Both methods employed 83C to extract the mucilage. On the other hand, in the modified method, neither the presence of free monosaccharides nor uronic acids was observed only the presence of mucilage. This method uses a ratio cladodes : ethanol at 50% of 1:1 at a temperature of 22C, and no extraction time was used. For this reason, we used the mucilage extracted from modified method to characterize their sugars profile by gas chromatogram.

Gas Chromatogram The chromatograms (Fig. 2) showed the presence of D-galactose, L-arabinose, L-rhamnose, D-xylose, D-glucose and uronic acids, in all samples of mucilages extracted, using the modified method. We select only the chromatogram of two species because the chromatogram peaks of the sugars detected can be better observed. Our results agree with those obtained by Ginestra et al. (2009), Habibi et al. (2004), Majdoub et al. (2010) and Ribeiro et al. (2010). The most abundant sugars contained in the mucilages (in decreasing order, Table 3) were as follows: L-arabinose (26.83–35.36%), D-galactose (21.59–45.48%), D-xylose (12.23–17.05%), uronic acids (5.59–13.91%), D-glucose (5.18–16.21%) and L-rhamnose (1.41–5.40%). This abundance order for all species of Opuntia obtained in our study was similar to the values documented by Nobel et al. (1992) Journal of Food Process Engineering 37 (2014) 285–292 © 2014 Wiley Periodicals, Inc.

and Trachtenberg and Mayer (1981), and is different from those reported by McGarvie and Parolis (1981), Medina-Torres et al. (2000), Habibi et al. (2004), and Majdoub et al. (2010) for mucilage from O. ficus-indica. The differences in sugar composition in mucilages reported by different authors for O. ficus-indica could vary, depending on the source and conditions of extraction, location and other environmental factors such as age of the cladodes, climatic conditions and soil where the Opuntia grow – as was indicated by Cárdenas et al. (1997), Ribeiro et al. (2010), Majdoub et al. (2010) and Guevara-Arauza et al. (2012). Statistical analysis shows a highly significant effect (P < 0.001) on the species depending on sugar content. The D-galactose content in mucilage obtained from O. joconostle (45.48%) was higher (P < 0.05) than the other Opuntia species. The lowest value corresponded to O. tomentosa (21.59%). The highest value of glucose was observed in O. tomentosa (16.21%) (P ≤ 0.05), followed by O. streptacantha (11.81%) and O. atropes (9.05%). The lowest was observed in O. ficus-indica (5.18%). Some authors such as McGarvie and Parolis (1981) and Gibson and Nobel (1990) did not discover the presence of D-glucose in the mucilage of O. ficus-indica. However, several authors did document the presence of glucose (Habibi et al. 2004; Ginestra et al. 2009; Majdoub et al. 2010 and Ribeiro et al. 2010). The values of D-glucose obtained by these authors are lower than our results. The higher values of L-rhamnose (P < 0.05) correspond to the mucilage from O. streptacantha (5.40%). The lowest L-rhamnose values (P < 0.05) were found in O. hyptiacantha (1.41%) and O. atropes (1.44%). 289


The predominant sugar found in the mucilages was Larabinose. O.ficus-indica (35.35%) and O. atropes (34.36%) had the highest values of L-arabinose and partial differences (P < 0.05) were found between these species with respect to O. tomentosa (33.50%) and O. hyptiacantha (32.82%). According to the hypothetical structure of the mucilage of O. ficus-indica (McGarvie and Parolis 1981; Gibson and Nobel 1990), the branches of the main chain of the mucilage are formed by three D-galactose units that are joined to residues of arabinose and D-xylose. Therefore, L-arabinose is the most abundant sugar in the chemical structure of mucilage. The functional groups of arabinose and D-xylose are more disposed to interact in an intermolecular form. Under these conditions, probably the viscosity generated could be higher when mucilage is exposed to water. This signifies that mucilage with higher L-arabinose contents could possibly generate increased viscosity suspensions as compared with mucilages with lower L-rabinose contents. We found higher acid uronics in mucilage from O. ficusindica (13.91%) which were significantly different (P < 0.05) from the other mucilages. Also, a high content of uronic acids was observed in O. hyptacanthia (11.84%), O. tomentosa (11.80%) and O. atropes (11.76%) where significant differences (P < 0.05) in these mucilages. The uronic acid content is also a molecule of great importance to generate viscous solutions, because the carboxyl groups are able to interact with water molecules or with certain cations, such as calcium. Consequently, we can assume that suspensions of mucilage from all species of Opuntia have a higher level of negative charges capable of interacting with calcium ions causing an increase in the viscosity.

FTIR FTIR spectra provide a chemical fingerprint of materials by correlating their absorption frequencies with known absorption frequencies of bonds. The spectra of mucilages from the six samples of Opuntia, shown in Fig. 3, represent the characteristic frequencies of the functional groups associated with mucilages, such as, carboxylic acid, carboxylate, ether and alcohol groups. The same number and intensity of bands studied by FTIR were found in our mucilages obtained from the six species of Opuntia (Fig. 3). In the mucilage spectrum there are two bands at 1,423 and 1,625 cm−1 associated with the antisymmetric and symmetric COO- stretch typical of carboxylic acid salts present in mucilage, as indicated by Fox et al. (2012). The bands corresponded to the ionized form, no esterified, of carboxylic group (Habibi et al. 2004; Cárdenas et al. 2008). We can assume that the mucilage molecules from the six species of Opuntia had a low DE, as was reported by Sáenz et al. (2004) and Cárdenas et al. 290


FIG. 3. FOURIER TRANSFORM INFRARED SPECTROSCOPY SPECTRUM OF THE MUCILAGES EXTRACTED FROM SIX SPECIES OF OPUNTIA

(2008). Also, in our mucilage samples, there is no evidence of the band at 1,749 cm−1 that is characteristic of mucilage with high DE. The low DE of mucilage is important because the carboxyl groups are free and are able to interact with water molecules, and thus have a high capacity for water absorption. Furthermore, the free carboxyl groups can bind with calcium ions and other mucilage molecules to form viscous structural networks in the presence of water. This is very useful for the food industry using such additives to enhance properties of viscosity and texture in food. Also, a band at 3405.68 cm−1 was found, which corresponds to OH stretching of alcohol and carboxylic acid –OH groups involved in intermolecular hydrogen bonding of mucilage molecules, as mentioned by Habibi et al. (2004). Another band was found at 2933.35 cm−1, which corresponded to the vibrations of –CH and –CH2 present in the mucilage molecule. Furthermore, some bands were observed at 1324.29–1252.34 cm−1 associated with the vibration δC–H, δCH2 and δO–H related to mucilages, according to Habibi et al. (2004), as well as the bands found in the region of 1084.27–1046.47 cm−1 that can be attributed to vibrations of C–C and C–O of the mucilage molecules (Fig. 3).

CONCLUSIONS The optimal conditions for extracting mucilage were proved. Mucilage of the six species of Opuntia had the same type of sugar; however, the amount varies in proportion as to the function of species studied. The sugars found in greatest proportion in all species were L-arabinose, D-galactose, D-xylose, uronic acid, D-glucose and LJournal of Food Process Engineering 37 (2014) 285–292 © 2014 Wiley Periodicals, Inc.


rhamnose. The mucilage studied from wild species contains a higher content of FD and FS than the commercial species O. ficus-indica. All species of Opuntia had low DE evidenced by FTIR studies. For this reason, the mucilage of the five wild species could also be exploited to produce hydrocolloids of importance in the food industry. We recommended studies of viscosity and emulsifying properties of mucilages from wild species to propose specific uses.

ACKNOWLEDGMENTS This work was supported by the Coordinación de la Investigación Cientifica. Universidad Michoacana de San Nicolás de Hidalgo (CIC-UMSNH), project 26.1, 2012. Sarahí Rodríguez González and Eder Zavala Mendoza wish to thank CONACyT for the scholarship received for MSc studies.

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