CARACTERIZACIÓN FISICOQUÍMICA Y ESTUDIO CINÉTICO DE LA HIDRÓLISIS ENZIMÁTICA DE LOS FRUCTANOS DE MAGUEY MEZCALERO POTOSINO (AGAVE SALMIANA).

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U

NIVERSIDAD

A

UTÓNOMA DE

S

AN

L

UIS

P

OTOSÍ

FACULTADES DE CIENCIAS QUÍMICAS, INGENIERÍA Y

MEDICINA

P

ROGRAMA

M

ULTIDISCIPLINARIO DE

P

OSGRADO EN

C

IENCIAS

A

MBIENTALES

CARACTERIZACIÓN FISICOQUÍMICA Y ESTUDIO

CINÉTICO DE LA HIDRÓLISIS ENZIMÁTICA DE LOS

FRUCTANOS DE MAGUEY MEZCALERO POTOSINO

(

AGAVE SALMIANA

)

T

ESIS QUE PARA OBTENER EL GRADO DE

DOCTOR EN CIENCIAS AMBIENTALES

P

RESENTA

:

M.C. CHRISTIAN MICHEL CUELLO

D

IRECTOR DE

T

ESIS

:

DR. M

IGUEL

ÁNGEL RUIZ CABRERA

C

OMITÉ

T

UTELAR

:

DRA. BERTHA IRENE JUÁREZ FLORES

DR. JORGE FERNANDO TORO VÁZQUEZ

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U

NIVERSIDAD

A

UTÓNOMA DE

S

AN

L

UIS

P

OTOSÍ

FACULTADES DE CIENCIAS QUÍMICAS, INGENIERÍA Y

MEDICINA

P

ROGRAMA

M

ULTIDISCIPLINARIO DE

P

OSGRADO EN

C

IENCIAS

A

MBIENTALES

CARACTERIZACIÓN FISICOQUÍMICA Y ESTUDIO

CINÉTICO DE LA HIDRÓLISIS ENZIMÁTICA DE LOS

FRUCTANOS DE MAGUEY MEZCALERO POTOSINO

(

AGAVE SALMIANA

)

T

ESIS QUE PARA OBTENER EL GRADO DE

DOCTOR EN CIENCIAS AMBIENTALES

P

RESENTA

:

M.C. CHRISTIAN MICHEL CUELLO

SINODALES

PRESIDENTE:

DR. MIGUEL ÁNGEL RUIZ CABRERA

SECRETARIO:

DRA. BERTHA IRENE JUÁREZ FLORES

VOCALES

DR. JORGE FERNANDO TORO VÁZQUEZ

DRA. ANA PAULINA BARBA DE LA ROSA

DR. GREGORIO ÁLVAREZ FUENTES

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i

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

Luis Potosí.

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

y

BECA-TESIS DEL CONSEJO NACIONAL DE CIENCIA Y TECNOLOGÍA (CONACyT)

Convenio No.

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ii

AGRADECIMIENTOS

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

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iii

Dedicatoria

A mi esposa Lucía.

A mis padres María de Lourdes y Fernando.

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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

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ii

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

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iii

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

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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

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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; ∆

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1 Abstract

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

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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

economic viability.

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

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3

1. Literature Review

1.1. Fructans

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.,

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4

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.

(Huazano, 2008).

Inulin-type fructans consist of linear chains containing fructosyl units linked

together through β link (2-1), and also have a terminal glucose molecule.

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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.

(Huazano, 2008).

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

called bifurcosa.

In the case of Agave species, observed interesting differences in the

structure of fructans (Figure 2.2). Recent studies using sophisticated techniques

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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).

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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

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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

Application Functionality

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

substitute

Baked goods Decrease in water activity (aw), sugar substitute

Breakfast cereals Crunch, expandability.

Preparation with fruits

(not sour)

Body and palatability, ability to form gel, emulsion stability, fat

and sugar substitute, synergy with sweeteners

Salad dressings Body and mouthfeel, fat substitute

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9

Chocolate Substitute sugar, humectant

(Franck, 2002).

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,

2002).

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

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10

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

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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).

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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

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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

operating costs.

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

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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

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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

fructooligosaccharides (FOS).

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).

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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

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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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18

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26

2. JUSTIFICATION

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

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3. OBJETIVES

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

base.

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

juice

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

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4. OBTENTION OF A POWDER WITH HIGH FRUCTAN CONTENT FROM

AGAVE SALMIANA

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: mruiz@uaslp.mx

ABSTRACT

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

basis).

Keywords:Agave salmiana, fructans, spray-drying, powder

4.1. INTRODUCTION

The term fructans is a generic name assigned to polymers of fructose linked

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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)

(13-16).

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

Figure

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