FACULTADES DE CIENCIAS QUÍMICAS, INGENIERÍA Y
CARACTERIZACIÓN FISICOQUÍMICA Y ESTUDIO
CINÉTICO DE LA HIDRÓLISIS ENZIMÁTICA DE LOS
FRUCTANOS DE MAGUEY MEZCALERO POTOSINO
TESIS QUE PARA OBTENER EL GRADO DE
DOCTOR EN CIENCIAS AMBIENTALES
M.C. CHRISTIAN MICHEL CUELLO
ÁNGEL RUIZ CABRERA
DRA. BERTHA IRENE JUÁREZ FLORES
DR. JORGE FERNANDO TORO VÁZQUEZ
FACULTADES DE CIENCIAS QUÍMICAS, INGENIERÍA Y
CARACTERIZACIÓN FISICOQUÍMICA Y ESTUDIO
CINÉTICO DE LA HIDRÓLISIS ENZIMÁTICA DE LOS
FRUCTANOS DE MAGUEY MEZCALERO POTOSINO
TESIS QUE PARA OBTENER EL GRADO DE
DOCTOR EN CIENCIAS AMBIENTALES
M.C. CHRISTIAN MICHEL CUELLO
DR. MIGUEL ÁNGEL RUIZ CABRERA
DRA. BERTHA IRENE JUÁREZ FLORES
DR. JORGE FERNANDO TORO VÁZQUEZ
DRA. ANA PAULINA BARBA DE LA ROSA
DR. GREGORIO ÁLVAREZ FUENTES
PROYECTO REALIZADO EN:
Laboratorio de Ingeniería en Alimentos del Centro de Investigación y Posgrado
(CIEP) de la Facultad de Ciencias Químicas de la Universidad Autónoma de San
CON FINANCIAMIENTO DE:
La Secretaría de Desarrollo Agropecuario y Recursos Hidráulicos (SEDARH) así como de la Fundación Produce A.C. San Luis Potosí
A TRAVÉS DEL PROYECTO DENOMINADO:
Métodos de extracción, caracterización y usos de fructanos de Agave salmiana en San Luis Potosí
BECA-TESIS DEL CONSEJO NACIONAL DE CIENCIA Y TECNOLOGÍA (CONACyT)
A Dios por todo.
A los miembros de mi comité tutelar: Dr. Miguel Ángel Ruiz Cabrera, Dra. Bertha
Irene Juárez Flores y Dr. Jorge Fernando Toro Vázquez por sus conocimientos,
valores, ejemplo y amistad.
A los Profesores de la Facultad de Ciencias Químicas Dr. Marco Martín González
Chávez, Dr. Mario Moscosa Santillán, Dr. Raúl González García, Dra. Alicia
Grajales Lagunes y a la Ing. Cecilia Rivera Bautista por su apoyo en la realización
de este trabajo de investigación.
A los miembros de la empresa “Productores de Mieles y Jarabes de maguey de Zaragoza de Solís” de Villa de Guadalupe, San Luis Potosí; especialmente a: Ing.
Victor López Flores, Sr. José Luis Rojas Galván y Sr. Bernardo Leija Padrón por
su apoyo y colaboración y amistad.
Al Dr. Juan Rogelio Aguirre Rivera por sus consejos y amistad.
A mis compañeros de la Facultad de Ciencias Químicas: Lore, Rodo, Gabys,
Chino, Juanito, Manuelito, Alex, Pedro, Yanoula, Dulce, Sandra, Oziel y Sonia. Y a
mi compañero y amigo Noé por su apoyo, experiencia y conocimientos
A mi esposa Lucía.
A mis padres María de Lourdes y Fernando.
i General Index
1. Abstract ... 1
2. Literature Review ... 3
2.1. Fructans ... 3
2.1.1. Classification and structure ... 4
2.1.2. Properties and applications of fructans ... 6
2.1.3. Fructose syrups from fructans ... 9
2.1.4. Thermal hydrolysis of fructans ... 10
2.1.5. Enzymatic hydrolysis of fructans ... 10
2.1.6. Fructans in powder form ... 11
2.1.7. Fructans in concentrated form ... 13
2.2. Utilization of mezcal agave fructans ... 14
2.2.1. Molecular structure and degree of polymerization of fructans from maguey mezcalero ... 15
2.3. Literature cited ... 18
3. JUSTIFICATION ... 26
4. OBJETIVES ... 27
4.1. General objetive ... 27
4.2. Specific objetives ... 27
5. OBTENTION OF A POWDER WITH HIGH FRUCTAN CONTENT FROM AGAVE SALMIANA ... 28
ABSTRACT ... 28
5.1. INTRODUCTION ... 28
5.2. MATERIALS AND METHODS ... 30
5.2.1. Raw Material and Extraction of Juice ... 30
5.2.2. Preparation of Agave Juice and Spray-Drying ... 31
5.2.4. Powder Moisture Content (MC) ... 32
5.2.5. Powder Yield (PY) ... 32
5.2.6. Equilibrium Moisture Content of Powder ... 32
5.2.7. Statistical Analysis ... 33
5.3. RESULTS AND DISCUSSION... 34
5.3.1. Carbohydrates profiles in various parts of Agave salmiana ... 34
5.3.2. Physical Properties and Carbohydrates Content of Agave Pine Juice ... 36
5.3.3. Moisture Content and Powder Yield from Spray-Drying Experiments. ... 38
5.3.4. Equilibrium moisture content of the spray-dried product (powders) ... 45
5.4. EXPERT COMMENTARY AND 5 YEAR VIEW ... 46
5.5. CONCLUSIONS ... 47
5.6. ACKNOWLEDGEMENTS ... 47
5.7. REFERENCES ... 48
6. STUDY OF ENZYMATIC HYDROLYSIS OF FRUCTANS FROM AGAVE SALMIANA: CHARACTERIZATION AND KINETIC ASSESSMENT ... 52
ABSTRACT ... 52
6.1. INTRODUCTION ... 53
6.2. MATERIALS AND METHODS ... 55
6.2.1. Obtention of the Agave Fructan Powder ... 55
6.2.2. DP Characterization of Agave Fructan ... 56
6.2.3. Carbohydrate Characterization of Agave Fructan and Chicory Inulin ... 57
6.2.4. Solid phase microextraction (SPME) of volatiles compounds ... 58
6.2.5. Analysis of volatiles compounds by GC/MS ... 58
6.2.6. Enzyme ... 58
6.2.7. Hydrolysis of Agave Fructan and Chicory Inulin ... 59
6.2.8. Mathematical Modeling of the Hydrolysis Kinetics ... 59
6.3. RESULTS AND DISCUSSION... 62
6.3.1. DP Profile of Agave Fructan ... 62
6.3.2. Carbohydrates Profiles of Substrates ... 64
6.3.3. HPLC Analysis of the Hydrolysis Kinetics ... 67
6.3.4. Hydrolysis Kinetics... 68
6.4. ACKNOWLEDGEMENTS ... 73
iv Tables Index
Table 2.1. Fructan content in dry basis of different vegetables. ... 3
Table 2.2: Functional properties of inulin and derived ... 8
Table 5.1. Physical properties and carbohydrates content of agave pine juice. ... 37
Table 5.2. Experimental drying conditions for spray-drying of agave pine juice and powder properties evaluated. ... 39
Table 5.3. Regression coefficients (coded variables) and variance analysis of the linear model (Eq. 3) for evaluating the effect of the spray-drying conditions on the properties of the powder (p<0.10). ... 41
Table 6.1. Values of the rate constants (k) and their respective R2 determined through regression method for each experimental condition. ... 60
v Figures Index
Figure 2.1. Fructan structures present in nature. a) Inulin-type fructan, b) Inulin-type fructan neoserie, c) levan-type fructan d) graminan-type fructan. ... 4
Figure 2.2. Fructans presents in Agave tequilana Weber var. Azul, a) graminan-type fructan, b) inulin agavine-type fructan, synthesized only by plants of Agave genus. ... 5
Figure 5.1: HPLC separation of sugars from agave pine juice. S: Sucrose, G: Glucose, F: Fructose, U: Uknown ... 35
Figure 5.2: Carbohydrate profiles in various parts of Agave salmiana quantified by HPLC-method... 35
Figure 5.3. Variation of moisture content of the powder with inlet air temperature (a) and carrier agent concentration (b) during spray-drying of agave pine juice. ● Gum arabic, ▲ Maltodextrin (10 DE). ... 43
Figure 5.4. Powder yield as a function of carrier agent concentration (a) and as function of inlet air temperature (b). ● Gum arabic, ▲ Maltodextrin (10 DE). ... 44
Figure 5.5. Moisture sorption isotherms of the spray-dried product and reference powders (inulin and freeze dried juice powder) fitted to the GAB model. ... 46
Figure 6.1. MALDI-TOF-MS spectra, in positive-ion mode of Agave salmiana fructan. (a) Low mass spectra (DP from 1to 6), (b) High mass spectra (DP from 4 to 21). ... 63
Figure 6.2. HPLC separation of sugars from agave fructan and chicory inulin used as substrates. S:Sucrose, G: Glucose, F:Fructose, LA: Lactic acid. ... 64
Figure 6.3. Gas chromatogram of polar volatile compounds of powder fructan obtained with PEG fiber and Stabilwax capillary column. ... 65
Figure 6.4. Chromatographic representation of substrates degradation and release of sugars during the enzymatic hydrolysis. (a) Chicory inulin, (b) Agave fructan. S:Sucrose, G: Glucose, F:Fructose, LA: Lactic acid. ... 68
Figure 6.6. Variation of the rate constant k with temperature (a,b). Chicory inulin; ∆
Fructose is one of six carbon monosaccharide which is widely distributed in
plant foods in a variety of forms, such as free monosaccharide or as glucose
complex joined to form the disaccharide sucrose or polymerized to form fructans.
By their structure, fructans can be oligo-or polysaccharides, depending on the
fructose units and have a D-glucose molecule.
Fructans are widely used as ingredients in functional foods for their
technological properties and their health benefits. Due to its nature of
non-digestible polysaccharides, they have applications as prebiotics, stimulating the
growth and activity of beneficial bacteria in the colon such as Bifidobacteria and
Lactobacilli, also have observed positive effects in reducing the glucose level blood
lipid homeostasis, mineral availability and effects of immunomodulation. For its
functional properties, also have the capability of modifying the texture, form gels,
retaining moisture, and stabilize the food, so they are used as substitutes for fats
and sugars principally. Fructans from chicory (Helianthus tuberosus), artichoke
(Cynara scolymus) and tubers of dahlia (Dahlia coccinea) have been the most
studied and widely used in a variety of products, particularly as dietary fibers, or
proposed for the production of syrups and fructo oligo saccharides fructosados
commonly called FOS (polymers formed by from 2 to 10 molecules of fructose).
Fructans in powder can generate significant advantages such as ease of
application, mixing, conveying and longer shelf life of the product. Spray drying is
one of the methods used at industrial level for food production on a large scale in
the form of powder, granules or agglomerates and have been successfully applied
in the drying of products such as milk, coffee, tea, egg, whey proteins, enzymes
and microorganisms. An advantage of this method is that the residence time is
very short, allowing properties such as flavor, color, odor and nutrients do not
undergo significant alterations.
In recent years, the maguey mezcalero potosino (Agave salmiana) has
2 grade pure fructans as well as FOS. However, to obtain syrups, in most industries
use kilns or retorts to hydrolyze fructans and release their fructose, unfortunately
the high temperatures used in these processes produce unfavorable phenomena
such as the Maillard reactions and the formation of compounds such as phenols,
furfural and hydroxymethyl furfural. The importance of fructose in the food
technology lies in important characteristics such as insulin-independent
metabolism is not enhance the formation of dental caries and is sweeter than
sucrose. The industrial use of enzymes has a wide range of applications such as
controlled depolymerization, transglycosylation, isomerization, oxidation and
reduction of oligo-and polysaccharides, led to a wide variety of products with high
added value by improving their functional properties. Fructans can be hydrolyzed to fructose and fructan β-inulinases Fructosidasas the synergistic action of these
enzymes may be an alternative for the hydrolysis of fructans, because your
application has many advantages: specific action, inhibition of unexpected side
reactions does not generate undesirable by-products, greater efficiency and
For identification and quantification of carbohydrates such as sugars and
fructans chromatography-based techniques combined with a refractive index
detector are the most used. Mathematical models are very useful for understanding
the basic mechanisms responsible for the behavior of natural systems, contributing
to the accurate calculation of time evolution of a system. Thus, the comparison of
measured data with those calculated from models, is an indirect proof of the
assumptions made about such mechanisms.
The aim of this study was develop an efficient process at the laboratory for
extraction, purification and chemical characterization of fructans present in maguey
mezcalero potosino (Agave salmiana). Similarly, establish the experimental basis
for the production of high fructose syrups (food grade and industrial grade) and a
1. Literature Review
The term fructan is a generic name assigned to the polymers of fructose
linked by glycosidic bonds fructose-fructose. When having 2 to 10 molecules of
fructose in the polymer, they are known as fructooligosaccharides (FOS) while a
fructan proper consists of a polysaccharide having a degree of polymerization (DP)
greater than 10 molecules of fructose in the chain (Watlz et al., 2005, Olvera et al.,
2007). Although chains of microbial fructans can reach 100,000 units by weight in
plants hardly exceed 150 units. These polymers are part of the energy reservoir of
a wide variety of plants such as onions, garlic, chicory, artichoke, dahlia and agave
plants (Table 2.1).
Table 2.1. Fructan content in dry basis of different vegetables.
Source Content (g/100g Dry basis)
Pataca (Helianthus tuberosus) 89
Chicory (Chichorium intybus) 79
Dahlia tubers (Dahlia spp.) 59
Onion (Allium cepa) 48
Garlic (Allium sativum) 29
Yacón (Smallanthus sonchifolius) 27 Maguey tequilero (Agave tequilana) 73 Maguey mezcalero (Agave salmiana) 69
(Bautista et al., 2001; Franck y De Leenheer, 2005).
Inulin-type fructans from chicory, artichoke and dahlia tubers have been the
most studied and widely used in a variety of products, particularly as dietary fiber
(Roberfroid, 2000; Roberfroid, 2005; Wack and Blaschek, 2006) or proposed for
production of fructose syrups or fructooligosaccharides (Nakamura et al., 1995,
Wenling et al., 1999, Cho et al., 2001; Zhengyu et al., 2005, Gonzalez-Diaz et al.,
1.1.1. Classification and structure
Following the starch, fructans are the most abundant structural
polysaccharides in nature, are present in many species of plants, fungi like
Aspergillus sp type and bacteria. There are five different groups of fructans, and
are classified according to the present type of bond between the fructose
molecules themselves and the position of the glucose molecule present in the
structure. These groups are: inulin, inulin neoserie, levans, levans neoserie and
graminanos (Figure 2.1) (Lopez et al., 2003, Gonzalez-Diaz et al., 2006,
Mancilla-Margalli and Lopez, 2006; Olvera et al., 2007).
Figure 2.1. Fructan structures present in nature. a) Inulin-type fructan, b) Inulin-type fructan neoserie, c) levan-type fructan d) graminan-type fructan.
Inulin-type fructans consist of linear chains containing fructosyl units linked
together through β link (2-1), and also have a terminal glucose molecule.
5 of glucose. Inulin smaller the known 1-kestose trisaccharide, which for the case of
neoserie inulins, is called neokestose.
Figure 2.2. Fructans presents in Agave tequilana Weber var. Azul, a) graminan-type fructan, b) inulin agavine-type fructan, synthesized only by plants of Agave genus.
Levans have a linear structure in which fructosyl units have links β (2-6),
such as are inulins, have a terminal glucose molecule. Neoserie Levans are
characterized by an internal glucose molecule to which are added fructosyl units linked β (2-6) to carbon 1 and carbon 6 of glucose. Finally, graminano type fructans
present in its structure a terminal glucose molecule, have links type β (2-1) and β
(2-6) between the fructosyl units and the base molecule or smaller fructans such
In the case of Agave species, observed interesting differences in the
structure of fructans (Figure 2.2). Recent studies using sophisticated techniques
6 Matrix-assisted laser desorption / ionization mass spectrometry coupled to a ion
detector by time of flight (MALDI-TOF-MS) have demonstrated that fructans
present in the Agave tequilana Weber, representing over 60% of total soluble
carbohydrates are composed of a complex structure of graminano-type fructans,
but also by another molecule complex and different from those already reported, to
which type fructan called Agavina, both types of fructans links have β (2-1) and β
(2-6) and are highly branched (Figure 2.2) (Lopez et al., 2003, Mancilla-Margalli
and Lopez, 2006; Huazano, 2008). In regard to Agave salmiana, there have been
no scientific studies to characterize depth fructans present in these juices.
1.1.2. Properties and applications of fructans
A structural feature of fructans, the link type β (2-1) is responsible for these
polysaccharides are not digested like any other carbohydrate, resulting in a low
caloric value and function as dietary fiber (Niness, 1999; Tungland and Meyer,
2002). The caloric value of inulin-type fructan tends to be located on average
between 1.6 and 2.71 kcal / g (Coussement, 1999; Ninnes, 1999; Deis, 2001,
Murphy, 2001), but its sweetness relative to sucrose alone is 30-35%. This is
especially due to the metabolism of short chain fatty acids produced during the
fermentation process suffering fructans in the colon. The glycemic index of inulin is
estimated at zero (Deis, 2001).
The consumption of inulin-type fructans helps prevent arteriosclerosis,
hypertriglyceridemia and cardiovascular disease, which are associated with
high-calorie diets (Roberfroid, 2001). Therefore, by reducing caloric intake reduces the
risk of obesity and diabetes (Gallo and O 'Donnell 2003; Marquina and Santos,
2003). Inulin and oligofructose are widely used as sweeteners for diabetic patients,
but it has shown no effect on blood glucose levels or the secretion of insulin or
glucagon, however other improvements were observed in the general diabetic
condition when used doses of about 40-100 g / day (Niness, 1999; Olesten and
Gudmon-Hoyer, 2000). The intake of carbohydrates can also improve lactose
intolerance (Kaplan and Hutkins, 2000). It also prevents steatitis (inflammation of
adipose tissue) in the liver, especially in obese people (Delzenne, et al., 2002).
7 harmful bacteria whose metabolites accelerate the appearance of ulcerative
lesions are inhibited by the action of symbiotic oligofructosaccharides and
bifidobacteria (Loo et al., 1999; Hellwege et al., 2000; Roberfroid, 2001; Taper and
Roberfroid, 2002). Such claims have been demonstrated by epidemiological
studies that have found that in urban populations with higher incidence of colon
cancer is significantly reduced the incidence of this cancer to diets supplemented
with oligofructosaccharides implement. It was also observed that butyrate
produced during fermentation favors the proliferation of normal cells and
suppresses the growth of differentiated cells and potentially carcinogenic
(Marrquina and Santos, 2003).
By eating hydrosoluble fructans usually lead to a reduction in triglycerides,
cholesterol and lipoprotein (Fiordaliso et al., 1995, Loo et al., 1999; Olesten and
Gudmond-Hoyer, 2000). The hipotriglicemia is explained by the decrease in
plasma lipoproteins VLDL (very low density lipoprotein), since fructans inhibit the
ability of palmitate to triacylglycerol interesterification resulted in reduced hepatic
lipogenesis (Garcia, 2000; Marquina and Santos, 2003). This reduction can be up
to a 19-27% (Pereira and Gibson, 2002, Lee et al., 2004).
Inulin-type fructans have solubility in water less than 60 g/l at a temperature
of 10 °C and 330 g/l at 90 °C (Deis, 2001). In its solid state usually has pure
crystalline forms (Suzuki and Chatterton, 1996), which tend to be hygroscopic and
difficult to maintain in lyophilized form to be used unless modified atmospheres
(Deis, 2001; Yun, 2003). These crystals may have melting points around 200 °C
when it comes to fructans of low degree of polymerization (Yun, 2003). The ability
to be water soluble fructans gives moisturizing properties when used as additives
in the food industry and the ability to form gels creamy when heated in aqueous
media. The values of the viscosity of solutions of fructans are generally higher than
those of other carbohydrates at the same concentration and are generally of higher
thermal stability. Fructans are usually very stable pH ranges found in most foods
(pH between four and seven) and stable in cooling process (Yun, 2003). The
8 °C (Suzuki and Chatterton, 1996). Some fractions may have capabilities
oligofructans reducing (Gennaro et al., 2000).
According Coussement (1999), there is no experimental evidence indicating
that oligofructans have some degree of toxicity regardless of the amount taken as
part of the diet, although some people have found that intakes above 10 g daily
may come to produce a slight discomfort. Usually oligofructans tolerance does not
increase if exposed to prolonged continuous individual intakes (Olesten and
Gudmond-Hoyer, 2000). Pure inulin high intakes can cause diarrhea due to an
osmotic fluid retention, both in the large and small intestines. Some other
symptoms may be flatulence and bloating. The maximum dose of
oligofructosaccharides that causes diarrhea in humans is 0.3 to 0.4 g per kg of
body weight in men and women respectively (Hidaka et al., 1986). In some people,
the rapid fermentation of fructans may cause a high concentration of hydrogen at
the stomach, which can promote peristalsis of the colon, leading to symptoms
similar to lactose intolerance such as irregular bowel movements, abdominal
bloating and irritability (Olesten and Gudmond-Hoyer, 2000)
Table 2.2: Functional properties of inulin and derived
Dairy products Body and palatability, ability to form gel, emulsifiers, fat and
sugar substitute, synergy with sweeteners
Frozen desserts Texture, depression in freezing point, substitute sugars and
fats, synergism with sweeteners
Spread products Emulsion stability, texture, and capable of being poured, fat
Baked goods Decrease in water activity (aw), sugar substitute
Breakfast cereals Crunch, expandability.
Preparation with fruits
Body and palatability, ability to form gel, emulsion stability, fat
and sugar substitute, synergy with sweeteners
Salad dressings Body and mouthfeel, fat substitute
Chocolate Substitute sugar, humectant
Inulin and its derivatives offer many uses as ingredients in the formulation of
products as listed in Table 2.2. Inulin has similar properties to the starch, while
oligofructose has properties more similar to sucrose (Roberfroid, 2002). Inulin
improves the acceptability of yogurt made with skim milk, imparting greater
creaminess; it also acts as a thickening agent, holds and stabilizes the water gels
(Kip et al., 2005). The gels can be formed by mechanical or thermal effect, and that
obtained by the second has better texture and firmness (Kim et al., 2001). The
ability to form gel is critical for use as a substitute for fat in dairy products, spreads,
dressings, sauces and other products in which the functional properties that give
fats are essential to achieve the desired sensory effects by consumers (Franck,
The vegetable fat replacement by inulin in food processing as wheat bread
and pasta, does not modify the rheological characteristics of dough before baking
or affect the sensory quality of the finished product (Wang et al., 2002; O 'O'Brien
et al., 2003; Brennam et al., 2004). The addition of inulin during the preparation of
chocolate, energy bars and extruded cereals, results in improvements in their
organoleptic characteristics such as flavor, color and texture (Franck, 2002;
Moscato et al., 2006, Aragon et al., 2007).
1.1.3. Fructose syrups from fructans
The fructose demand has increased because it is considered as a
sweetener with low glycemic index (GI) (GI = 32) compared with sucrose (GI = 92).
This feature along with its low calorie content (4kcal/g) allows it to be
recommended for consumption by diabetics or some other metabolic disorder
problem (Hernandez-Uribe et al., 2008).
The glycemic index is a classification of the carbohydrates contained in
foods, builds on the response postprandial blood glucose and is a measure of
lower the rate of absorption of carbohydrates and less than the increase in
post-prandial glucose and insulin concentrations (Wolever, 1990). The metabolism of
fructose does not require insulin, because it does not follow the same metabolic
pathway of glucose (Gonzalez-Diaz et al., 2006).
Fructose syrups have a great use in many foods and soft drinks as a
substitute for sucrose, because fructose has a sweetening power two times greater
than sucrose. These syrups have some important functional properties which are
used in food processing such as enhancement of the flavor, color and product
stability, are highly soluble and can be mixed easily with other components,
assuming a crystallization inhibitor (Badui, 1999, Borges da Silva et al., 2006a,
Gonzalez-Diaz et al., 2006).
1.1.4. Thermal hydrolysis of fructans
An alternative to achieve hydrolysis of inulin and release fructose is the
thermal process, which involves the breaking of internal glycosidic linkages of the
polysaccharide. In order to achieve hydrolysis of inulin and fructose generate in
mezcal factories use a traditional process, since the cones are baked in ovens or
autoclaves hydrolyzing fructans at temperatures above 100 ° C for periods of time
between 36 and 48 hours. In this way, is obtained the fermentable sugars such as
fructose, sucrose and glucose. The advantage of this process is easy to apply,
besides having relatively low operating costs. However, during the cooking process
of agave pineapples produced some unfavorable phenomena such as the Maillard
reactions that result in undesirable compounds such as furfural and
hydroxymethylfurfural (HMF), which causes unpleasant flavors and aromas in the
finished product (Mancilla-Margalli and Lopez, 2002; Waleckx et al., 2008).
1.1.5. Enzymatic hydrolysis of fructans
Currently, the fructose syrup is mainly produced by enzymatic hydrolysis of
corn starch. During this process involves several enzymatic steps: liquefaction of
starch by α-amylase enzyme then comes the saccharification, wherein the
11 Shetty, 1999; Ge et al., 1999, Borges da Silva et al., 2006b, Van der Veen et al.,
2006). However, the degree of conversion achieved is relatively low and the
products obtained consisting of oligosaccharides (5%), fructose (45%) and glucose
(50%) while a product called High Fructose Corn Syrup (HFCS) should contain a
standard 55% fructose to have the same sweetness as sucrose from sugarcane
(Crabb and Shetty, 1999; Borges da Silva et al., 2006a, Sharma et al., 2006).
Therefore, the increase of fructose syrups in may be performed either by the
selective removal of glucose or through chromatographic separation methods
multi-effect, which increases the cost of the process (Toumi and Engell, 2004;
Gonzalez -Diaz et al., 2006). Another alternative for the production of these syrups
is through the direct cleavage of sucrose into its two components: glucose and fructose with the enzyme invertase (α-fructofuranosidase) or with an alkaline
treatment at high temperature and in both cases the products obtained are called
invert sugar (Rubio et al., 2002; Toloti-Carneiro et al., 2005, Yang and
Montgomery, 2007). Remember also that the high demand for corn for biofuel
production and corresponding increase in price has stimulated the search for
alternative sources of starch for the production of fructose or glucose syrups
(Hernandez-Uribe et al., 2008, Morales et al., 2008).
The enzymatic method using the synergistic action of exoinulinasa and
endoinulinasa could be another good alternative for the hydrolysis of these
fructans. One of the main advantages is the use of enzymes which is associated
with its high specificity of action makes no unexpected side reactions occur.
However, this process tends to be more expensive than the traditional method
because of the expense of the enzymes. Several authors suggest that the
enzymatic process more economical, this must be done with an immobilized
enzyme system, to benefit the stability, separation and reuse of the enzyme and
facilitate continued operation of the reactor (Nakamura et al., 1995; Wenling et al.,
1999; Zhengyu et al., 2005, Gonzalez-Diaz et al., 2006; Catana et al., 2007).
12 Fructans in powder form can generate significant advantages such as ease
of application, mixing, conveying and longer shelf life of the product. Spray drying
is one of the methods used at industrial level for food production on a large scale in
the form of powder, granules or agglomerates and have been successfully applied
in the drying of milk, coffee, tea, egg, proteins from serum, enzymes and
microorganisms (Barbosa and Vega, 2000). This process is defined as a unit
operation used to produce powders, where a liquid or a suspension is atomized in
a hot air stream causing the instantaneous dewatering thereof (Geankoplis, 2006).
One advantage of spray drying method is that the residence time is very short,
allowing properties such as flavor, color, odor and nutrients do not undergo
significant changes (Masters, 1991; Mujumdar, 1998).
However, the spray-dried is difficult to apply in the processing of foods rich
in sugars such as fruit juices because of their content of low molecular weight
carbohydrates (fructose, glucose, maltose and sucrose), organic acids (citric acid)
and high water content (Jaya and Das, 2004, Foster et al., 2006). These systems
are characterized by glass transition temperatures (Tg) very low and when in
contact with hot air at a wet bulb temperature above their Tg, these may tend to
structural relaxation and behave as syrups and stick on the walls of the dryer. This
results in low yields, operational problems and difficulty in predicting product quality
(Mani et al., 2002). To reduce such problems it has been used to some practical
methods as cooling and frequent scraping the walls of the dryer and the use of
some additives such as starch, arabic gum and maltodextrins as carriers and
agents to increase the Tg of the mixture.
The amount of these agents depends on the food carriers and the
concentration ranges reported in the literature (Masters, 1991) ranging from 20% to
60% (w/v). However, the amount of additive added is limited by the sensory quality
of final product and are often carried out by trial and error. The combination of
maltodextrins and sugars such as lactose high Tg have allowed the reduction of
solids in the feed (Ruiz-Cabrera et al., 2009). Bhandari and Hartel (2005), have
13 materials using a state diagram (Tg vs aw), helping to establish the best conditions
of temperature, in order to reduce problems bonding during the process.
1.1.7. Fructans in concentrated form
The concentration of liquid foods is a process in which water is removed
carefully in order to obtain a product of appearance and taste similar to the original,
so many benefits are obtained as better stability and presentation, increased
resistance to microbial activity compared with the original food etc. under the same
conditions. Should not be confused with dehydration concentration; a dehydrated
product will always be a solid with a water content of between 2 and 10%, while a
concentrated product is in the form of solution, dispersion or semisolid with a water
content significantly higher (Karel and Daryl, 2003).
The decrease of water content in foods increases its shelf life so that it may
keep in good condition for a longer period of time. During this process reduces the
water activity (aw) 'which is a measure of the availability of water for chemical and
biochemical reactions and the development of microorganisms (Saravacos and
Charm, 1962, Newman et al., 1996). Liquid products such as fruit juices, milk, etc.
Which have a high water content (75-90% wet basis), often need to be
concentrated in order to provide functionality to foods, extend shelf life and reduce
Different methods of concentration and are listed below. The evaporation
consists of removing water from a food by boiling. The water has a boiling point at
100 °C at atmospheric pressure (101, 325 Pa) while the solutes contained in it
have a boiling point above so if subjecting the feed to temperatures above the
boiling point of water and below the boiling point of the solutes, is achieved by
decrease in water content of the feed and concentrate. Using evaporation products
can be obtained with concentrations close to 80 °Brix. However, this method
presents a very significant loss of nutritional and organoleptic properties (taste,
aroma, different from the original colors, etc).
The membrane concentration is a method that has been widely studied due
14 2004). This process uses a selective barrier or membrane, located between two
phases (Tsuru, 2001). The effect of a driving force (such as pressure difference,
difference in concentration etc.) is obtained a retentate flow and a filtrate.
Retentate or concentrate flow is that which fails to pass through the pores of the
membrane and is the phase or product of interest. In contrast filtering is usually
formed by water that gets through the membrane (Avilés, 2007).
Cryoconcentration method or freeze concentration of the food is the partial
freezing and subsequent removal of water formed crystals. The solution resulting
from this process is a concentrate rich in solutes (Habib and Farid, 2006). By this
method can eliminate feed water without damaging the nutritional and organoleptic
properties of food, but otherwise the economic cost of the operation is superior to
other methods and the obtained concentrations do not exceed 60 °Brix.
An alternative for the food processing which does not involve harsh
treatments that damage to sensory and nutritionally product is the concentration at
constant temperature and below the boiling point of water. Recent Investigations
design and build a team capable of generating products with concentrations
between 18 and 80 °Brix, using temperatures not exceeding 50 °C (Avilés, 2007).
The concentration of the syrup is a function of temperature controlled conditions
such as dry bulb and wet bulb temperature.
1.2. Utilization of mezcal agave fructans
The maguey mezcalero Agave salmiana is the agave plant used since prehispanic times as food (“aguamiel”, heads or cooked pineapple, vinegar,
pulque, rum, etc.), building materials and fibers for clothing. The maguey is widely
distributed resource in the highlands of San Luis Potosi-Zacatecas and is probably
the most economically important species in the region, being used primarily as a
source of fermentable sugars for the production of mezcal and as forage for
livestock (Aguirre et al., 2001; Torrentera, 2001). Most of the sugars in the
pineapple are formed by fructans and there are used as energy reserves in the
maguey (Badui, 1999, González-Díaz et al., 2006) and is on the stem where the
15 become a promising raw material for industrial production of fructose syrups, food
grade pure fructans in dried or concentrated form, as well as for production of
The production of "honey" or agave syrup made from “aguamiel” (sap
obtained after castration and scraping of the maguey) is pre-Hispanic and currently
produced commercially in communities throughout the state of Hidalgo, and syrup
from tequila agave juice by producers in the state of Jalisco. However, in the first
case, because they are very small amounts of raw material, has no future in the
industry level and the finished product is handmade with very poor quality. With respect to the syrup produced in the company “Industrializadora Integral del Agave (IIDEA)” is an industrial product of good quality, but with plant installation costs too
high. However, it has been shown that in most industries in Tequila, Jalisco for the
production of fructose syrups is still using the traditional method of production of
tequila, that is, whole or cut pineapple are cooked in brick ovens ( 36 to 48 h) or
autoclave (12 hours) at a temperature above 100 ° C. It is found that under these
conditions of cooking are favored Maillard reactions and induces the formation of
compounds such as phenols, furfural and hydroxymethyl furfural (Mancilla-Margalli
and Lopez, 2002; Waleckx et al., 2008).
1.2.1. Molecular structure and degree of polymerization of fructans
from maguey mezcalero
The molecular structures of fructans vary depending on the species from
which they come, its structural elucidation has been proposed as a taxonomic
marker (Bonnett et al., 1997). In species of the genus Agave have been reported
more than one type of fructan, Sanchez-Marroquin and Hope (1953) and Bathia and Nandra (1979) report the presence of an inulin-type fructan links β (1-2) as the
main reserve carbohydrate in Agave americana and Agave tequilana respectively.
Aspinall and Das Gupta (1959), Satyanarayana (1976) and Dorland et al. (1977)
report that the Agave veracruz is a mixture of fructans with widely branched structure, a glucose molecule and not just internal β link (1-2), but also link β (2-6).
16 presence of both fructans with a DP of 5 and fructans with a DP of 3 with an
intermediate glucose molecule known as neokestose. In 2003, Lopez et al.,
Proposed the molecular structure of fructans present in Agave tequilana Weber,
this molecule has three types of fructans: inulin, levan and Neoinulina, with DP of 3-29 and links between β (2-1) and β (2-6) bonds.
As noted, there are structural differences between species of fructans of the
agave. These findings indicate the need to accurately define the size and
molecular structure of these carbohydrates, for that there are different
physicochemical methods such as Nuclear Magnetic Resonance (NMR), Gas
Chromatography coupled to Mass Spectrometry (GC-MS) and Electrospray
Ionization Mass Spectrometry (ESI-MS) (Ravenscroft et al., 2009).
The analysis by MALDI-TOF-MS is currently the main analytical technique
used for the spectrometric analysis of biomolecules such as peptides, proteins,
oligosaccharides and oligonucleotides. Is also used to analyze larger organic
molecules such as polymers and macromolecules, which are studied with respect
to its space oligomeric distribution terminal group, molecular weight distribution and
polydispersity in space. It has also been used successfully in the investigation of
fullerene dendrimers and their derivatives, non-covalent complexes, kerogen, coal
tar, humic and fulvic acids (Zenobi and Knochenmuss, 1998).
In the analysis by MALDI-TOF-MS, fragments of molecules are mixed with a
solid matrix of organic nature such as trans-3-indoleacrilic, or inorganic salts such
as sodium chloride or silver trifluoroacetate. Said matrix is used to protect the
sample but also to facilitate evaporation and ionisation. The sample is mixed with
the matrix on a metal surface to allow crystallization when the solvent evaporates.
Subsequently these crystals are subjected to short pulses of nitrogen laser at half
high vacuum so that the energy absorbed by the matrix becomes excitation energy
and transferring H+ ions to the sample (ionization), thereby producing species
monocharged. Irradiated surface is heated allowing the desorption of the ions of
solid phase to gas phase.
The Time of Flight ion detector (TOF) determines the mass of the sample in
17 until they strike the detector. An advantage of the TOF detector is its ability to
transmit ions with high kinetic energy, usually up to 20 keV (Medzihradszky et al.,
2000). This technique can determine the main structural characteristics of the
biopolymer which is formed as intermolecular space, distribution of end groups,
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The maguey mezcal is a resource widely distributed in the highlands of San
Luis Potosí-Zacatecas and its exploitation has been a source of employment for
many farmers during times of collection and processing in the mezcal. Its primary
use is as a source of carbohydrates for the production of mezcal. However, the
Agave salmiana is characterized by a large amount of fructans, comparable with
that of chicory, artichoke, why should develop alternative uses for this raw material
for obtaining new products with high added value.
An example may be the manufacture of high fructose syrups and fructans in
powder form and / or concentrated solution; these types of syrups are proving very
attractive as additives in the food industry and are used by people with diabetes,
due to their functional properties that make it a major competitor of sucrose
commercially used. In the case of fructans because of its nutraceutical and
functional properties can have a wide application in the food industry. That is why
this research aims to establish a laboratory at the best operating conditions that will
produce solutions with high concentrations of fructose that can be used in the
manufacture of mezcal or as a basis for obtaining high fructose syrups and
3.1. General objetive
Develop an efficient process at the laboratory for extraction, purification and
chemical characterization of fructans present in maguey mezcalero potosino
(Agave salmiana). Similarly, establish the experimental basis for the production of
high fructose syrups (food grade and industrial grade) and a high fructan powder
3.2. Specific objetives
I) Establish the conditions for extraction, purification and chemical
characterization of fructans and free sugars contained in maguey juice.
II) Evaluate and optimize the spray drying process by characterizing the
glass transition temperature of multicomponent systems
(juice-adjuvants) to obtain a powder with a high content of fructans from agave
III) Perform a kinetic study of enzymatic hydrolysis of fructans present in
Agave salmiana juice, using a commercial inulinase preparation acting in
free form in order to establish the groundwork for producing high fructose
4. OBTENTION OF A POWDER WITH HIGH FRUCTAN CONTENT FROM
L. Moreno-Vilet, C. Michel-Cuello, A. Mota-Santillán, M.M. González-Chávez, A. Grajales-Lagunes, M.A. Ruiz-Cabrera*
Facultad de Ciencias Químicas. Universidad Autónoma de San Luis Potosí. Av. Dr. Manuel Nava No. 6, Zona Universitaria, C.P. 78210, San Luis Potosí S.L.P. México.
Author for correspondence: email@example.com
The aim of this study was to obtain powder products with high fructan
content by spray-drying of the Agave salmiana juice. A laboratory scale spray dryer
(Pulvis GB 22 model) operated at inlet air temperatures of 140-160 °C was
employed. Maltodextrin (10 DE) and gum arabic were used as carrier agents at
levels between 5 and 10 % (w/v). In each experiment, the mass recovered was
recorded and the powder yield was calculated. The sorption isotherm of this
powder in the water activity (aw) range 0.1-0.9 was evaluated. Fructan content of
the powder ranged 48-60 %, with presence of low molecular weight sugars such as
sucrose, fructose and glucose. Statistical analysis (p<0.10) showed that lowest
moisture content and highest powder yield were reached at inlet air temperature of
160 °C using 10 % (w/v) gum arabic. However, the powders were highly
hygroscopic when exposed at environments with aw between 0.5 and 0.9
displaying a high moisture retention capacity, which varied from 10 to 35 % (wet
Keywords:Agave salmiana, fructans, spray-drying, powder
The term fructans is a generic name assigned to polymers of fructose linked
29 molecules, these are known as fructooligosaccharides (FOS), whereas a properly
named fructan is a polysaccharide with a degree of polymerization (DP) greater
than 10 molecules of fructose (1, 2). In the literature, references have been made
to five groups of fructans, which are classified according to their structure and type
of bond such as inulin, levan, graminan, neoseries levan and neoseries graminan
(1-3). The inulin-type fructan, extracted from chicory (Cichorium intybus), artichoke
(Cynara scolymus) and dahlia plant tubercles (Dahlia variabilis) have been the
most commonly used in the food industry due to their functional properties as well
as their health benefits. They are non-digestible polysaccharides, considered as
prebiotics, since they stimulate the growth and activity of beneficial colon bacteria
such as Bifidobacteria and Lactobacillus (4-6). Fructans have been associated to a
decrease on glucose level in blood, homeostasis of lipids, mineral availability and
immunomodulatory effects (7, 8). They possess the ability of modifying texture,
forming gels, retaining moisture, and stabilizing food, for which they are mainly
employed as fat and sugar substitutes (9-12). These fructans have also been
considered for the production of fructose syrups or fructooligosaccharides (FOS)
The Agave plants possess fructans as their main photosynthetic product,
synthesized and stored in the stem, and used by the same plants as a source of
energy and as an osmo-protector during drought and cold stress periods (17, 18).
In the particular case of Agave tequilana, over 60% of the soluble carbohydrates is
represented by a complex mixture, mostly comprised of highly ramified fructans
and neo-fructans (19-21). The main use of fructans from Agaves has been been to
obtain fermentable sugars in the manufacturing of alcoholic drinks such as tequila,
mescal and sotol. The production of powder bases with high fructan content may
pose an alternative toward a better use of the Agave, thus creating a new range of
products. Fructans in powder form can generate important advantages such as
easier application, mixing, shipping and an increase in product shelf life.
Spray-drying is one of the most commonly used methods at industrial level
to produce food at a large scale in powder form, granulated or agglomerated