mean value, \* black-hull oilseed, \*\*striped-hull oilseed

was increased from 4.6% d.b. to 17.7% d.b., representing a variation of 6.3%.

Table 6. Sphericity () of different seeds

equipment for the seed.

**Table 5.** Regression equations as a function of moisture content (x, %d.b.) with their respective coefficient of

Equivalent diameter (mm)

1,0

(a) (b)

Sphericity

(c) (d)

**Figure 5.** (a) volume (*V*), (b) equivalent diameter (*De*), (c) thousand seed mass (*W1000*), and (d) sphericity ( ) of dark chia seeds with different moisture content (1) 4.6%, (2) 6.5%, (3) 8.7%, (4) 10.0%, (5) 12.5%, (6) 15.3%, (7) 17.7%).

1,1

1,2

1,3

1,4

Figure 5. Volume (*V*), equivalent diameter (*De*), thousand seed mass (*W1000*), and sphericity () of dark chia seeds with different moisture content

Moisture content (%d.b.) 1234567

Moisture content (%d.b.) 1234567

The sphericity varied between 65.8% and 67.6%, values higher than the data reported for sunflower and safflower seed, but lower

**Seed [Reference] Sphericity (%)**  Amaranth [24] 82 # Fenugreek[16] 60.79 – 64.06 Quinoa [22] 77 – 80 Rapeseed [28] 93 – 92 Safflower [17] 58 – 62 Sunflower\* [18] 49 – 52 Sunflower\*\* [18] 47 – 50 Soybean [23] 80.6 – 81.6

The high value thus suggests that the seeds tend towards a spherical shape. Thus, the value of the generally indicates a likely difficulty in getting the seed to roll. This tendency to either roll or slide should be necessary in the design of hoppers and dehulling

As can be seen in Figure 6, the equivalent diameter (*De*, mm) increased linearly from 1.28 to 1.36 mm when the moisture content

The high *ϕ* value thus suggests that the seeds tend towards a spherical shape. Thus, the val‐ ue of the *ϕ* generally indicates a likely difficulty in getting the seed to roll. This tendency to either roll or slide should be necessary in the design of hoppers and dehulling equipment for the seed.

As can be seen in Figure 6, the equivalent diameter (*De*, mm) increased linearly from 1.28 to 1.36 mm when the moisture content was increased from 4.6% d.b. to 17.7% d.b., represent‐ ing a variation of 6.3%.

efficient of static friction increased linearly with an increase in moisture content. Similar trends

Moisure content (%db) 5 10 15 20 25

> Cumin-galvanized iron Cumin-aluminum Flaxseed-galvanized iron Flaxseed-aluminum

Sunflower-galvanized irom

**Figure 7.** Effect of moisture content (x, % d.b.) on coefficient of static friction. Dates correspond to the adjusted func‐

The engineering properties of dark chia seeds were evaluated as a function of the moisture content, in the range of 4.6% to 17.7% d.b. and their behavior was compared with amaranth, cumin, flaxseed, fenugreek, quinoa, rapeseed, safflower, soybean and sunflower. The princi‐ pal dimensions of dark chia seed (length, width and thickness), geometric diameter, specific surface area, volume, equivalent diameter, and thousand seeds mass and static coefficient of friction on galvanized sheet and aluminium increased linearly as increasing the seed mois‐ ture content. Chia seed is one of the smallest (similar to amaranth and quinoa), and very

The sphericity did not present significant differences in the range of moisture content stud‐ ied for dark chia seed. The most spherical seeds which were compared with chia seed ones were rapeseed, amaranth, soybean and quinoa. An increase in moisture content yields a de‐

Quinoa-galvanized iron

Chía-galvanized iron Chia-aluminum.

Moisture content (%d.b.) 2 4 6 8 10 12 14 16 18 20

fenugreek [16], safflower [17] sunflower [18] soybean [23] and rapeseed [28] seeds.

Volume (V, mm<sup>2</sup>

) Equivalent diameter ( De mm) Thousand seed mass (W1000, g)

Moisture-Dependent Engineering Properties of Chia (Salvia hispanica L.) Seeds

Figure 6. Effect of moisture content (x, % d.b.) on volume seed (V), equivalent diameter (De) and thousand seed mass (W1000). Date corresponds to

The *V* and *W1000* of dark chia seeds linearly increased with moisture content (Figure 6). The similar trend was reported for

The frictional characteristics are important for the proper design of agricultural product handling equipment. Friction between a seed and a surface has an influence on the movement of particles on oscillating conveyors, separation on oscillating sieves and unloading and loading operations. The static coefficient of friction of dark chia seeds was determined on two structural surfaces: galvanized iron and aluminum. The values obtained were higher for aluminum (mean: 0.30, minimum: 0.26, maximum: 0.37) than for galvanized iron (mean: 0.28, minimum: 0.25, maximum: 0.34). Increments of 28.4% and 29.5% were recorded for the galvanized iron and aluminum surfaces, respectively, as the moisture content increased from 4.6% to 17.7% d.b. The reason for the increased friction coefficient at higher moisture content may be that the water present in the seed offered a cohesive force on the contact surface and the seed became rougher and sliding characteristics are diminished [22, 26]. For both structural surfaces, the coefficient of static friction increased linearly with an increase in moisture content. Similar trends were reported for cumin, flaxseed, quinoa

http://dx.doi.org/10.5772/53173

393

Figure 7. Effect of moisture content (x, % d.b.) on coefficient of static friction. Dates correspond to the adjusted function reported by ( ) [25], (- . - )

were reported for cumin, flaxseed, quinoa and sunflower (Figure 7).

and sunflower (Figure 7).

Coefficient static of friction

0,0

tion reported by () [25], () [13], () [27], () [22] and () [15].

**4. Conclusions**

light.

0,2

0,4

0,6

0,8

1,0

[13], (---) [27], (- .. -) [22] and (**–**) [15].

the adjusted function reported by [25].

Properties (V, De, W1000)

1,10

1,15

1,20

1,25

1,30

1,35

1,40

**Figure 6.** Effect of moisture content (x, % d.b.) on volume seed (*V*), equivalent diameter (*D*e) and thousand seed mass (*W*1000). Date corresponds to the adjusted function reported by [25].

The *V* and *W1000* of dark chia seeds linearly increased with moisture content (Figure 6). The similar trend was reported for fenugreek [16], safflower [17] sunflower [18] soybean [23] and rapeseed [28] seeds.

The frictional characteristics are important for the proper design of agricultural product han‐ dling equipment. Friction between a seed and a surface has an influence on the movement of particles on oscillating conveyors, separation on oscillating sieves and unloading and loading operations. The static coefficient of friction of dark chia seeds was determined on two structur‐ al surfaces: galvanized iron and aluminum. The values obtained were higher for aluminum (mean: 0.30, minimum: 0.26, maximum: 0.37) than for galvanized iron (mean: 0.28, minimum: 0.25, maximum: 0.34). Increments of 28.4% and 29.5% were recorded for the galvanized iron and aluminum surfaces, respectively, as the moisture content increased from 4.6% to 17.7% d.b. The reason for the increased friction coefficient at higher moisture content may be that the water present in the seed offered a cohesive force on the contact surface and the seed became rougher and sliding characteristics are diminished [22, 26]. For both structural surfaces, the co‐ efficient of static friction increased linearly with an increase in moisture content. Similar trends were reported for cumin, flaxseed, quinoa and sunflower (Figure 7). friction coefficient at higher moisture content may be that the water present in the seed offered a cohesive force on the contact surface and the seed became rougher and sliding characteristics are diminished [22, 26]. For both structural surfaces, the coefficient of static friction increased linearly with an increase in moisture content. Similar trends were reported for cumin, flaxseed, quinoa

Moisture content (%d.b.) 2 4 6 8 10 12 14 16 18 20

fenugreek [16], safflower [17] sunflower [18] soybean [23] and rapeseed [28] seeds.

Volume (V, mm<sup>2</sup>

) Equivalent diameter ( De mm) Thousand seed mass (W1000, g)

Figure 6. Effect of moisture content (x, % d.b.) on volume seed (V), equivalent diameter (De) and thousand seed mass (W1000). Date corresponds to

The *V* and *W1000* of dark chia seeds linearly increased with moisture content (Figure 6). The similar trend was reported for

The frictional characteristics are important for the proper design of agricultural product handling equipment. Friction between a seed and a surface has an influence on the movement of particles on oscillating conveyors, separation on oscillating sieves and unloading and loading operations. The static coefficient of friction of dark chia seeds was determined on two structural surfaces:

iron and aluminum surfaces, respectively, as the moisture content increased from 4.6% to 17.7% d.b. The reason for the increased

Properties (V, De, W1000)

1,10

the adjusted function reported by [25].

and sunflower (Figure 7).

1,15

1,20

1,25

1,30

1,35

1,40

Figure 7. Effect of moisture content (x, % d.b.) on coefficient of static friction. Dates correspond to the adjusted function reported by ( ) [25], (- . - ) [13], (---) [27], (- .. -) [22] and (**–**) [15]. **Figure 7.** Effect of moisture content (x, % d.b.) on coefficient of static friction. Dates correspond to the adjusted func‐ tion reported by () [25], () [13], () [27], () [22] and () [15].

### **4. Conclusions**

As can be seen in Figure 6, the equivalent diameter (*De*, mm) increased linearly from 1.28 to 1.36 mm when the moisture content was increased from 4.6% d.b. to 17.7% d.b., represent‐

> Moisture content (%d.b.) 2 4 6 8 10 12 14 16 18 20

**Figure 6.** Effect of moisture content (x, % d.b.) on volume seed (*V*), equivalent diameter (*D*e) and thousand seed mass

The *V* and *W1000* of dark chia seeds linearly increased with moisture content (Figure 6). The similar trend was reported for fenugreek [16], safflower [17] sunflower [18] soybean [23] and

The frictional characteristics are important for the proper design of agricultural product han‐ dling equipment. Friction between a seed and a surface has an influence on the movement of particles on oscillating conveyors, separation on oscillating sieves and unloading and loading operations. The static coefficient of friction of dark chia seeds was determined on two structur‐ al surfaces: galvanized iron and aluminum. The values obtained were higher for aluminum (mean: 0.30, minimum: 0.26, maximum: 0.37) than for galvanized iron (mean: 0.28, minimum: 0.25, maximum: 0.34). Increments of 28.4% and 29.5% were recorded for the galvanized iron and aluminum surfaces, respectively, as the moisture content increased from 4.6% to 17.7% d.b. The reason for the increased friction coefficient at higher moisture content may be that the water present in the seed offered a cohesive force on the contact surface and the seed became rougher and sliding characteristics are diminished [22, 26]. For both structural surfaces, the co‐

Volume (V, mm<sup>2</sup>

) Equivalent diameter ( De mm) Thousand seed mass (W1000, g)

ing a variation of 6.3%.

392 Food Industry

Properties (V, De, W1000)

1,10

rapeseed [28] seeds.

(*W*1000). Date corresponds to the adjusted function reported by [25].

1,15

1,20

1,25

1,30

1,35

1,40

The engineering properties of dark chia seeds were evaluated as a function of the moisture content, in the range of 4.6% to 17.7% d.b. and their behavior was compared with amaranth, cumin, flaxseed, fenugreek, quinoa, rapeseed, safflower, soybean and sunflower. The princi‐ pal dimensions of dark chia seed (length, width and thickness), geometric diameter, specific surface area, volume, equivalent diameter, and thousand seeds mass and static coefficient of friction on galvanized sheet and aluminium increased linearly as increasing the seed mois‐ ture content. Chia seed is one of the smallest (similar to amaranth and quinoa), and very light.

The sphericity did not present significant differences in the range of moisture content stud‐ ied for dark chia seed. The most spherical seeds which were compared with chia seed ones were rapeseed, amaranth, soybean and quinoa. An increase in moisture content yields a de‐ crease in bulk and true density. The bulk density and porosity varied nonlinearly for chia seeds, showing a quadratic concave behavior as a function of moisture content.

x Moisture content (% d.b.)

*μ* Static coefficient of friction, dimensionless

Estefanía N. Guiotto1,2, Vanesa Y. Ixtaina1,2, Mabel C. Tomás2

\*Address all correspondence to: snolasco@fio.unicen.edu.ar

1 Grupo de Investigaciones TECSE. Departamento de Ingeniería Química. Facultad de In‐

2 Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), (CCT La Plata –CONICET) Facultad de Ciencias Exactas, UNLP, La Plata, Buenos Aires, Argentina

[1] Álvarez-Chávez, L. M., Valdivia-López, M. A., Alberto-Juárez, M. L., & Tecante, A. (2008). Chemical characterization of the lipid fraction of mexican chia seed (*Salvia his‐*

[2] Cahill, J. 2003. Ethnobotany of chia, *Salvia hispanica* L. (Lamiaceae). Econ. Bot. 57(4):

[3] Palma, F., Donde, M., Lloyd, W.R., 1947. Fixed oils of Mexico. Part 1. Oil of chia- *Sal‐*

[4] Ayerza Jr., R., 1995. Oil Content and fatty acid composition of Chia (*Salvia hispanica* L.) from five northwestern locations in Argentina. J. Am. Oil Chem. Soc. 72, 1079–

[5] Bushway, A.A., Belyea, P.R., Bushway, R.J., 1981. Chia seed as asource of oil, poly‐

\*Address all correspondence to: mabtom@hotmail.com

geniería, UNCPBA, Olavarría, Buenos Aires, Argentina

*panica* L.). Int. J. Food Prop., 11, 687-697.

*via hispanica*. J. Am. Oil Chem. Soc. 24, 27.

saccharide, and protein. J. Food Sci. 46, 1349–1350.

and Susana M. Nolasco<sup>1</sup>

http://dx.doi.org/10.5772/53173

395

Moisture-Dependent Engineering Properties of Chia (Salvia hispanica L.) Seeds

α Angle of tilt, degree *ε* Porosity of seed (%)

*ϕ* Sphericity of seed

*ρb* Bulk Density (g cm-3) *ρr* True Density (g cm-3)

**Author details**

**References**

604-618.

1081.

The friction caused by the aluminum surface was slightly higher than that presented by the galvanized iron surface.

In general, the variation of the engineering properties of chia seed with the moisture content showed a similar trend to that reported for other seeds, with some exceptions. Nevertheless, they presented different variation ranges. It could be attributed to the seeds morphological and physiological characteristics.

The comparison of the data of the different seeds can be important for the design and adap‐ tation of equipment for transporting, storage and processing.

## **Acknowledgements**

This work was supported by a grant from Agencia Nacional de Promoción Científica y Tec‐ nológica (ANPCyT); Argentina (PICT 2007-1085), Universidad Nacional de La Plata (UNLP) (11/X502), and Universidad Nacional del Centro de la Provincia de Buenos Aires (UN‐ CPBA). E.N. Guiotto is recipient of a doctoral fellowship from the Consejo Nacional de In‐ vestigaciones Científicas y Técnicas (CONICET) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT); V.Y. Ixtaina and M.C. Tomás are members of the career of Scientific and Technological Researcher of Consejo Nacional de Investigaciones Científi‐ cas y Técnicas (CONICET), Argentina; and S.M. Nolasco is a research scientist of Universi‐ dad Nacional del Centro de la Provincia de Buenos Aires (UNCPBA), Argentina.

## **Nomenclature**


crease in bulk and true density. The bulk density and porosity varied nonlinearly for chia

The friction caused by the aluminum surface was slightly higher than that presented by the

In general, the variation of the engineering properties of chia seed with the moisture content showed a similar trend to that reported for other seeds, with some exceptions. Nevertheless, they presented different variation ranges. It could be attributed to the seeds morphological

The comparison of the data of the different seeds can be important for the design and adap‐

This work was supported by a grant from Agencia Nacional de Promoción Científica y Tec‐ nológica (ANPCyT); Argentina (PICT 2007-1085), Universidad Nacional de La Plata (UNLP) (11/X502), and Universidad Nacional del Centro de la Provincia de Buenos Aires (UN‐ CPBA). E.N. Guiotto is recipient of a doctoral fellowship from the Consejo Nacional de In‐ vestigaciones Científicas y Técnicas (CONICET) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT); V.Y. Ixtaina and M.C. Tomás are members of the career of Scientific and Technological Researcher of Consejo Nacional de Investigaciones Científi‐ cas y Técnicas (CONICET), Argentina; and S.M. Nolasco is a research scientist of Universi‐

dad Nacional del Centro de la Provincia de Buenos Aires (UNCPBA), Argentina.

seeds, showing a quadratic concave behavior as a function of moisture content.

tation of equipment for transporting, storage and processing.

galvanized iron surface.

394 Food Industry

**Acknowledgements**

**Nomenclature**

d.b. dry basis

*L* Length of seed (mm)

*T* Thickness (mm)

*V* Seed Volume (mm3

*W* Width of seed (mm)

*W1000* Thousand seed weight (g)

*m* Unit mass of the seed (g) *S* Specific Surface area (mm2

)

)

*De* Equivalent Diameter (mm) *Dg* Geometric Diameter (mm)

and physiological characteristics.


## **Author details**

Estefanía N. Guiotto1,2, Vanesa Y. Ixtaina1,2, Mabel C. Tomás2 and Susana M. Nolasco<sup>1</sup>

\*Address all correspondence to: mabtom@hotmail.com

\*Address all correspondence to: snolasco@fio.unicen.edu.ar

1 Grupo de Investigaciones TECSE. Departamento de Ingeniería Química. Facultad de In‐ geniería, UNCPBA, Olavarría, Buenos Aires, Argentina

2 Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), (CCT La Plata –CONICET) Facultad de Ciencias Exactas, UNLP, La Plata, Buenos Aires, Argentina

## **References**


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[21] Özarslan, C. 2002. Physical properties of cotton seed. Biosyst. Eng. 83(2): 169-174.

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of amaranth seeds. Biosyst. Eng. 89(1): 109-117.

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seed (*Brassica napus oleifera* L.). J. Food Eng. 69(1): 61-66.

[22] Vilche, C., M. Gely, and E. Santalla. 2003. Physical properties of quinoa seeds. Bio‐

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[23] Deshpande, S. D., S. Bal, and T. P. Ojha. 1993. Physical properties of soybean. J. Ag‐

[24] Abalone, R., A. Cassinera, A. Gastón, and M. A. Lara. 2004. Some physical properties

[25] Guiotto, E., Ixtaina, V.Y., Tomás, M. C. and Nolasco, S.M. 2011. Influence of moisture content on physical properties of chia (*Salvia hispanica* L.) seeds, Trans.ASABE 54 (2):

[26] Omobuwajo, T.O., Sanni, L.A., Balami, Y.A. 2000. Physical properties of sorrel (*Hibis‐*

[27] Coşkuner, Y., and E. Karababa. 2007. Some physical properties of flaxseed (Linum

[28] Çalişir, S., T. Marakoğlu, H. Öğüt, and Ö. Öztürk. 2005. Physical properties of rape‐

[29] Baryeh, E. A. 2002. Physical properties of millet. Baryeh, E. A. 2002. Physical proper‐


[21] Özarslan, C. 2002. Physical properties of cotton seed. Biosyst. Eng. 83(2): 169-174.

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[8] Ixtaina, V.Y., Martínez, M.L., Spotorno, V., Mateo, C.M., Maestri, D.M., Diehl, B.W., (2011). Characterization of chia seed oils obtained by pressing and solvent extraction.

[9] Ixtaina, V. Y., M. I. Capitani, S. M. Nolasco, and M. C. Tomás. 2010. Caracterización microestructural de la semilla y el mucílago de chia (*Salvia hispanica* L.). In Proc. XXVIII Congreso Argentino de Química. Buenos Aires, Argentina: Asociación Quí‐

[10] Commission of the European Communities. 2009. Commission Regulation (EC)

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[16] Altuntaş, E., E. Özgöz, and Ö. F. Taşer. 2005. Some physical properties of fenugreek

[17] Baümler, E., A. Cuniberti, S. M. Nolasco, and I. C. Riccobene. 2006. Moisture-depend‐ ent physical and compression properties of safflower seed. J. Food Eng. 72(2):

[18] A.K. de Figueiredo, E. Baümler, I.C. Riccobene, S.M. Nolasco Moisture-dependent engineering properties of sunflower seeds with different structural characteristics J.

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134-140.


**Chapter 18**

**Scale Up of Polygalacturonase Production by**

Polygalacturonases are pectinolytic enzymes that catalyze the cleavage of polygalacturon‐ ic acid chain with the introduction of water through hydrogen bonds. These enzymes have technological, functional and organic applications on food processing and plant-fun‐

They are produced by plants, fungi, bacteria and yeasts. However, the fungi are preferred in industrial scale, since about 90% of enzymes produced may be secreted into the medium

Polygalacturonases involved in hydrolysis of pectic substances are endo-polygalacturonase and exo-polygalacturonase. Exo-polygalacturonase act on polygalacturonic acid monomers terminals, producing monogalacturonic acids. Endo-polygalacturonase act randomly on

Pectic enzymes alone are responsible for one quarter of food production enzymes in the world [3]. These are widely used in fruit juices industry to reduce viscosity, improve and increase the efficiency of filtration and clarification [4], preliminary treatment of grape wine

For fruit juices production with pomo (e.g. citrus), and red fruit (e.g. grape) extracts require addition of enzymes to convert viscous macerated or triturated fruit semi gelled (caused by the partial solubility of pectins and the ability high water retention of solids) to maximize the extraction of juice during pressing, subsequent step of process. Pectinases capable to de‐ grade pectins depolymerize with high methylation are more suitable and include endopoly‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Alcântara et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Solid State Fermentation Process**

Siumara R. Alcântara, Nathalya J. Leite and

Additional information is available at the end of the chapter

and produce large amounts of enzymes [1,2].

galacturonase and pectin metilesterease [7].

polygalacturonic acid, producing oligogalacturonic acid [6].

industry, extraction of tomato pulp and among others applications [5].

Flávio L. H. da Silva

**1. Introduction**

gus interactions.

http://dx.doi.org/10.5772/53152

## **Scale Up of Polygalacturonase Production by Solid State Fermentation Process**

Siumara R. Alcântara, Nathalya J. Leite and Flávio L. H. da Silva

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53152

## **1. Introduction**

Polygalacturonases are pectinolytic enzymes that catalyze the cleavage of polygalacturon‐ ic acid chain with the introduction of water through hydrogen bonds. These enzymes have technological, functional and organic applications on food processing and plant-fun‐ gus interactions.

They are produced by plants, fungi, bacteria and yeasts. However, the fungi are preferred in industrial scale, since about 90% of enzymes produced may be secreted into the medium and produce large amounts of enzymes [1,2].

Polygalacturonases involved in hydrolysis of pectic substances are endo-polygalacturonase and exo-polygalacturonase. Exo-polygalacturonase act on polygalacturonic acid monomers terminals, producing monogalacturonic acids. Endo-polygalacturonase act randomly on polygalacturonic acid, producing oligogalacturonic acid [6].

Pectic enzymes alone are responsible for one quarter of food production enzymes in the world [3]. These are widely used in fruit juices industry to reduce viscosity, improve and increase the efficiency of filtration and clarification [4], preliminary treatment of grape wine industry, extraction of tomato pulp and among others applications [5].

For fruit juices production with pomo (e.g. citrus), and red fruit (e.g. grape) extracts require addition of enzymes to convert viscous macerated or triturated fruit semi gelled (caused by the partial solubility of pectins and the ability high water retention of solids) to maximize the extraction of juice during pressing, subsequent step of process. Pectinases capable to de‐ grade pectins depolymerize with high methylation are more suitable and include endopoly‐ galacturonase and pectin metilesterease [7].

© 2013 Alcântara et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Principal application of enzymes that pectins hydrolyze is clarification or extraction of jui‐ ces. The turbidity may be desirable in some juices (e.g. orange), but not for apple and grape juice, in which are translucent over sold. Turbidity is conferred by colloidal particles consist‐ ing of proteins coated with pectin. Pectinases depolymerize pectins, promoting flocculation and facilitating clarification [7].

Polygalacturonases produced by fungi are more active in pH range of 3.5 to 6.0 and temper‐ ature of 40-55°C. The pratical result of activity of these enzymes is that middle lamella is dis‐ rupted and the viscosity of pectin solutions is decreased as the action of enzyme is maintained [7]. In fact, the characteristics of enzyme related with temperature and pH ef‐ fects will depend on factors of production process, e.g. microorganism used, available nu‐ trients, fermentation temperature, among others.

Industrially, pectinases are produced either by submerged fermentation and solid state fer‐ mentation with *Aspergillus niger* strains, however, the solid state fermentation technique is generally considered more susceptible to higher yields of pectin esterase and polygalacturo‐ nase. Some authors state that this preference occurs because the solid state fermentation al‐ lows the production of crude enzymes more concentrated and therefore a lower cost of extraction and purification [5,8].

Solid state fermentation process is defined as a process that occurs over a non-soluble mate‐ rial, acting as support and nutrients source, with small quantity of water, under the action of fermenting agent [9].

**Figure 1.** Type of bioreactors: (A) Column; (B) Rotating drum; (C) Koji-type; (D) Stirred horizontal [12].

ess will vary in temperature that can be of 10°C above the optimum value [16].

centration of nutrients and products, making difficult the automation [17].

ing the area of trays, which can be achieved using larger trays [14].

once daily [13].

al and low cost at Brazil.

Tray reactor consists of a chamber in which temperature and humidity air controlled cir‐ culates around a series of trays. Each tray contains a thin layer of depth. It is notewor‐ thy that intermittent mixing of medium can be performed, but this generally occurs only

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401

Scale-up using tray reactor cannot be simply by increasing the medium thickness, as this leads to problems of overheating. For this reactor type, scaling-up must be done by increas‐

As described above, tray reactors are limited by heat transfer, but also by mass transfers, and may develop high temperatures and internal gas concentration gradients in height above 40 mm of substrate [15]. Still about the medium temperature, it is almost impossible to maintain this variable in the optimum value for production. However, fermentative proc‐

Although there are numerous projects for industrial bioreactors, it is observed that these have limited development to processes using solid state fermentation. This occurs because there are some limitations of this process, such as the difficulty to remove heat generated by microbial metabolism, heterogeneity of mixture during fermentation, which makes the con‐ trol of cell growth and various parameters such as temperature, pH, agitation, aeration, con‐

Despite these difficulties, the use of semi-solid medium may be advantageous, it allows the use of industrial residues (flours, bark and cake) as substrate, which is abundant raw materi‐

One of the important factors in pectinases production by solid state fermentation is the me‐ dium composition. Appropriate balance between sources of nitrogen and carbon is so im‐ portant to the nutritional requirement of microorganism that the effects of environmental conditions which affect mycelial growth [10].

Water in the system is a limiting factor. This amount of water is related to the medium through of moisture, as regards the percentage of water in total mass. Determination of its value in process is closely related to the substrate nature, requirements of microorganism used and type of end product desired. If moisture level is high, it will result in a decrease of substrate porosity and it will result in lower oxygen diffusion within the medium, conse‐ quently, decrease in gas exchange and increases the risk of contamination, especially bacteri‐ al. For lower levels of moisture needed by the microorganism, there will be greater difficulty in diffusion of nutrients, resulting in a lower growth and, consequently, lower production of desired product [11].

Temperature is also considered a critical factor, as well as moisture, due to the accumulation of metabolic heat generated during fermentation, which directly affects the microorganism germination and product formation. In composting process, this effect is desirable, however, for biotechnology processes, such as enzyme production, the heat must be dissipated imme‐ diately, so that temperature increase does not adversely affect the desired fermentation [11].

There are several types of reactors used in solid state fermentation process. Although there are many projects to industrial bioreactors, these have a limited extent for this type of process [12].

**Figure 1.** Type of bioreactors: (A) Column; (B) Rotating drum; (C) Koji-type; (D) Stirred horizontal [12].

Principal application of enzymes that pectins hydrolyze is clarification or extraction of jui‐ ces. The turbidity may be desirable in some juices (e.g. orange), but not for apple and grape juice, in which are translucent over sold. Turbidity is conferred by colloidal particles consist‐ ing of proteins coated with pectin. Pectinases depolymerize pectins, promoting flocculation

Polygalacturonases produced by fungi are more active in pH range of 3.5 to 6.0 and temper‐ ature of 40-55°C. The pratical result of activity of these enzymes is that middle lamella is dis‐ rupted and the viscosity of pectin solutions is decreased as the action of enzyme is maintained [7]. In fact, the characteristics of enzyme related with temperature and pH ef‐ fects will depend on factors of production process, e.g. microorganism used, available nu‐

Industrially, pectinases are produced either by submerged fermentation and solid state fer‐ mentation with *Aspergillus niger* strains, however, the solid state fermentation technique is generally considered more susceptible to higher yields of pectin esterase and polygalacturo‐ nase. Some authors state that this preference occurs because the solid state fermentation al‐ lows the production of crude enzymes more concentrated and therefore a lower cost of

Solid state fermentation process is defined as a process that occurs over a non-soluble mate‐ rial, acting as support and nutrients source, with small quantity of water, under the action of

One of the important factors in pectinases production by solid state fermentation is the me‐ dium composition. Appropriate balance between sources of nitrogen and carbon is so im‐ portant to the nutritional requirement of microorganism that the effects of environmental

Water in the system is a limiting factor. This amount of water is related to the medium through of moisture, as regards the percentage of water in total mass. Determination of its value in process is closely related to the substrate nature, requirements of microorganism used and type of end product desired. If moisture level is high, it will result in a decrease of substrate porosity and it will result in lower oxygen diffusion within the medium, conse‐ quently, decrease in gas exchange and increases the risk of contamination, especially bacteri‐ al. For lower levels of moisture needed by the microorganism, there will be greater difficulty in diffusion of nutrients, resulting in a lower growth and, consequently, lower production of

Temperature is also considered a critical factor, as well as moisture, due to the accumulation of metabolic heat generated during fermentation, which directly affects the microorganism germination and product formation. In composting process, this effect is desirable, however, for biotechnology processes, such as enzyme production, the heat must be dissipated imme‐ diately, so that temperature increase does not adversely affect the desired fermentation [11]. There are several types of reactors used in solid state fermentation process. Although there are many projects to industrial bioreactors, these have a limited extent for this type

and facilitating clarification [7].

400 Food Industry

extraction and purification [5,8].

fermenting agent [9].

desired product [11].

of process [12].

trients, fermentation temperature, among others.

conditions which affect mycelial growth [10].

Tray reactor consists of a chamber in which temperature and humidity air controlled cir‐ culates around a series of trays. Each tray contains a thin layer of depth. It is notewor‐ thy that intermittent mixing of medium can be performed, but this generally occurs only once daily [13].

Scale-up using tray reactor cannot be simply by increasing the medium thickness, as this leads to problems of overheating. For this reactor type, scaling-up must be done by increas‐ ing the area of trays, which can be achieved using larger trays [14].

As described above, tray reactors are limited by heat transfer, but also by mass transfers, and may develop high temperatures and internal gas concentration gradients in height above 40 mm of substrate [15]. Still about the medium temperature, it is almost impossible to maintain this variable in the optimum value for production. However, fermentative proc‐ ess will vary in temperature that can be of 10°C above the optimum value [16].

Although there are numerous projects for industrial bioreactors, it is observed that these have limited development to processes using solid state fermentation. This occurs because there are some limitations of this process, such as the difficulty to remove heat generated by microbial metabolism, heterogeneity of mixture during fermentation, which makes the con‐ trol of cell growth and various parameters such as temperature, pH, agitation, aeration, con‐ centration of nutrients and products, making difficult the automation [17].

Despite these difficulties, the use of semi-solid medium may be advantageous, it allows the use of industrial residues (flours, bark and cake) as substrate, which is abundant raw materi‐ al and low cost at Brazil.

**2.2. Substrate characterization**

**2.3. Substrate adsorption isotherms**

values.

weight of sample, g.

**Saturated salt solutions Temperature (°C)**

**Relative humidity (%)** 25 30 35 40

**Table 1.** Equilibrium relative humidity of saturated salt solutions [26]

Measurements of pH, moisture content and mineral waste (MW) followed the standards Brazil [23]. The pectin amount (PC) was determined by gravimetric precipitation method us‐ ing calcium pectate [24]. Reducing sugars (RS) and saccharose were determined by HPLC (High performance liquid chromatography). Concentration of soluble solids (SS) was ob‐ tained by direct reading in refractometer after adding 9 mL of distilled water to 1 g of dry bagasse. It was used 100 g of material to determine the density. This mass was placed in a graduate to determine volume occupied without compression. Size distribution was per‐ formed using 100 g of residue in a Cotengo-Pavitest sieve shaker for 1o minutes in 14, 20, 24, 35, 48 and 60 mesh trays. Result was expressed as weight percentage. Protein was deter‐ mined by semi-micro Kjeldhal method for nitrogen adjusted by spectrophotometry [25]. All characterizations were performed in triplicate. Standard deviation (SD) was based on means

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Triplicate samples were weighed approximately 1 g of product in aluminium crucibles and stored in airtight containers containing saturated salt solutions until reached the equilibrium moisture content for a certain relative humidity range (Table 1). Temperatures of 25, 30, 35 e 40°C were supplied by environmental chamber. Samples were weighed every 24 hours, reached constant weight. After that, samples were transferred to stove at 100°C for determi‐ nation of dry weight. Equilibrium moisture content (xeq) was calculated by Equation 1.

In which: xeq = equilibrium moisture, % dry basis; mi = initial weight of sample, g; ms = dry

*ms* ) ×100 (1)

*xeq* = ( *mi* - *ms*

K(C2H3O2) 23 23 23 23 MgCl2 33 32 32 31 K2CO3 43 42 41 40 NaBr 57 57 57 57 NaCl 75 75 75 75 KCl 86 84 84 83

**Figure 2.** Cashew fruit [22].

Industrial wastes have been used in bioproducts production through fermentation process‐ es. Among these residues may be mentioned the cashew apple (*Anacardium occidentale* L.), which is rich in sugars, organic acids and fiber, that is why it has been used in the produc‐ tion of phenols [18], bioethanol, cashew wine [19], protein enrichment [20], pectinases [21], cellulases, among others.

Therefore, this study aims to characterize the scaling-up of solid state fermentation process for polygalacturonases production, using cashew apple dry bagasse as substrate, *Aspergillus niger* CCT 0916 as microorganism and tray reactor as operating system. For this, it will be performed to characterize of substrate used concerning the physical-chemical properties. It will be constructed and adjusted adsorption isotherms of substrate, showing the relation‐ ship with fermentation process. Initially, there will be characterization of factors that most influence the fermentation process in a laboratory scale. By setting these factors, it will be made to scaling-up of solid state fermentation process using tray reactor. Finally, it will be characterized the crude enzyme extract and its stability over temperature and pH.

## **2. Material and methods**

#### **2.1. Substrate**

Cashew apple bagasse was obtained from fresh cashew fruit acquired at Empresa de Abas‐ tecimento e Serviços Agrícolas (EMPASA) at Campina Grande City, Brazil. First, cashew nut was removed. Next, apple was triturated and pressed to separate the juice. Humid bagasse was dried with air renewal and circulation at 55°C. After drying process, bagasse was ground in TECNAL knife mill.

#### **2.2. Substrate characterization**

Measurements of pH, moisture content and mineral waste (MW) followed the standards Brazil [23]. The pectin amount (PC) was determined by gravimetric precipitation method us‐ ing calcium pectate [24]. Reducing sugars (RS) and saccharose were determined by HPLC (High performance liquid chromatography). Concentration of soluble solids (SS) was ob‐ tained by direct reading in refractometer after adding 9 mL of distilled water to 1 g of dry bagasse. It was used 100 g of material to determine the density. This mass was placed in a graduate to determine volume occupied without compression. Size distribution was per‐ formed using 100 g of residue in a Cotengo-Pavitest sieve shaker for 1o minutes in 14, 20, 24, 35, 48 and 60 mesh trays. Result was expressed as weight percentage. Protein was deter‐ mined by semi-micro Kjeldhal method for nitrogen adjusted by spectrophotometry [25]. All characterizations were performed in triplicate. Standard deviation (SD) was based on means values.

#### **2.3. Substrate adsorption isotherms**

**Figure 2.** Cashew fruit [22].

402 Food Industry

cellulases, among others.

**2. Material and methods**

ground in TECNAL knife mill.

**2.1. Substrate**

Industrial wastes have been used in bioproducts production through fermentation process‐ es. Among these residues may be mentioned the cashew apple (*Anacardium occidentale* L.), which is rich in sugars, organic acids and fiber, that is why it has been used in the produc‐ tion of phenols [18], bioethanol, cashew wine [19], protein enrichment [20], pectinases [21],

Therefore, this study aims to characterize the scaling-up of solid state fermentation process for polygalacturonases production, using cashew apple dry bagasse as substrate, *Aspergillus niger* CCT 0916 as microorganism and tray reactor as operating system. For this, it will be performed to characterize of substrate used concerning the physical-chemical properties. It will be constructed and adjusted adsorption isotherms of substrate, showing the relation‐ ship with fermentation process. Initially, there will be characterization of factors that most influence the fermentation process in a laboratory scale. By setting these factors, it will be made to scaling-up of solid state fermentation process using tray reactor. Finally, it will be

Cashew apple bagasse was obtained from fresh cashew fruit acquired at Empresa de Abas‐ tecimento e Serviços Agrícolas (EMPASA) at Campina Grande City, Brazil. First, cashew nut was removed. Next, apple was triturated and pressed to separate the juice. Humid bagasse was dried with air renewal and circulation at 55°C. After drying process, bagasse was

characterized the crude enzyme extract and its stability over temperature and pH.

Triplicate samples were weighed approximately 1 g of product in aluminium crucibles and stored in airtight containers containing saturated salt solutions until reached the equilibrium moisture content for a certain relative humidity range (Table 1). Temperatures of 25, 30, 35 e 40°C were supplied by environmental chamber. Samples were weighed every 24 hours, reached constant weight. After that, samples were transferred to stove at 100°C for determi‐ nation of dry weight. Equilibrium moisture content (xeq) was calculated by Equation 1.

$$
\times eq = \left(\frac{mi \cdot ms}{ms}\right) \times 100\tag{1}
$$

In which: xeq = equilibrium moisture, % dry basis; mi = initial weight of sample, g; ms = dry weight of sample, g.


**Table 1.** Equilibrium relative humidity of saturated salt solutions [26]

It was used the BET model [27] to fit experimental data (Equation 2):

*xeq xm* <sup>=</sup> *<sup>C</sup>*.*aw* 1 - *aw* 1 - (*n* + 1)(*aw*)*<sup>n</sup>* + *n*(*aw*)*n*+1 <sup>1</sup> - (1 - *<sup>C</sup>*)*aw* - *<sup>C</sup>*(*aw*)*n*+1 (2)

Substrate was hydrated with distilled water to obtain the initial moisture content and it was diluted ammonium sulphate in this water. On polypropylene trays (Figure 3), it was weigh‐ ed 500 g of sterilized medium. Substrate thickness was equal to 40 mm. After spore inocula‐ tion, the medium was incubated at 23°C for 77 hours. Substrate thickness and fermentation temperature had been selected taking into account studies reported in literature [15,16].

 30 (-1) 106 (-1) 0.5 (-1) 25 (-1) 50 (+1) 106 (-1) 0.5 (-1) 25 (-1) 30 (-1) 108 (+1) 0.5 (-1) 25 (-1) 50 (+1) 108 (+1) 0.5 (-1) 25 (-1) 30 (-1) 106 (-1) 1.5 (+1) 25 (-1) 50 (+1) 106 (-1) 1.5 (+1) 25 (-1) 30 (-1) 108 (+1) 1.5 (+1) 25 (-1) 50 (+1) 108 (+1) 1.5 (+1) 25 (-1) 30 (-1) 106 (-1) 0.5 (-1) 35 (+1) 50 (+1) 106 (-1) 0.5 (-1) 35 (+1) 30 (-1) 108 (+1) 0.5 (-1) 35 (+1) 50 (+1) 108 (+1) 0.5 (-1) 35 (+1) 30 (-1) 106 (-1) 1.5 (+1) 35 (+1) 50 (+1) 106 (-1) 1.5 (+1) 35 (+1) 30 (-1) 108 (+1) 1.5 (+1) 35 (+1) 50 (+1) 108 (+1) 1.5 (+1) 35 (+1) 40 (0) 107 (0) 1.0 (0) 30 (0) 40 (0) 107 (0) 1.0 (0) 30 (0) 40 (0) 107 (0) 1.0 (0) 30 (0) 40 (0) 107 (0) 1.0 (0) 30 (0) 40 (0) 107 (0) 1.0 (0) 30 (0) 40 (0) 107 (0) 1.0 (0) 30 (0) 40 (0) 107 (0) 1.0 (0) 30 (0)

**Variables U %(w.b.) E (mL/g) N %(w/w) Tf (°C)**

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**Test**

**Table 2.** Concentrations and tests from factorial design

In which: xeq = equilibrium moisture, % dry basis; aw = water activity, adimensional; C = BET constant; xm = moisture in the molecular monolayer; n = number of molecular layers.

Criteria used to observe the fit were the coefficient of determination (R2 ) between observed responses and predicted by the fitted model and average percentage deviation (P) (Equation 3). The best fits were those with the highest R2 and lowest value of P [28].

$$P = \frac{100}{n} \sum\_{i=1}^{n} \frac{\lfloor \text{Xexp - Xteo} \rfloor}{\text{Xexp}} \tag{3}$$

In which: n = observation numbers; Xexp = humidity of experimental material; Xteo = hu‐ midity calculated by adjusted models.

### **2.4. Fermentation process in laboratory scale**

Microorganism used was *Aspergillus niger* CCT0916, donated by Empresa Brasileira de Pes‐ quisa Agropecuária (EMBRAPA, Fortaleza State – Brazil). Spore concentration was adjusted according to experimental design.

Substrate was hydrated with distilled water to obtain moisture content and ammonium sul‐ fate was added to this volume. In a 250 mL Erlenmeyer flask, it was weighed 10 g of steri‐ lized humidified medium. After spore inoculation, this medium was incubated at fermentation temperature by experimental design for 78 hours.

Enzyme extraction for fermented complex was performed by adding 2.5 mL/g of fermented medium using 200 mM acetate buffer pH 4.5. Samples were then left in water bath for 1 hour at 30°C and filtered through Wattman 1 filter paper.

A 24 factorial experimental design was conducted with 7 experiments at central point to de‐ termine the influence of spore concentration (E), initial moisture (U), ammonium sulfate concentration (N) and fermentation temperature (Tf ) on polygalacturonase activity response (Table 2).

One unit of polygalacturonase activity was defined as the amount of enzyme that releases 1 µmol of galacturonic acid per minute of reaction at 35°C for 30 minutes.

#### **2.5. Fermentation process using tray reactor**

Microorganism used in this stage was also used for optimization in the laboratory scale. Spores concentration, moisture content and ammonium sulphate concentration were deter‐ mined based on experiments performed on laboratory scale.

Substrate was hydrated with distilled water to obtain the initial moisture content and it was diluted ammonium sulphate in this water. On polypropylene trays (Figure 3), it was weigh‐ ed 500 g of sterilized medium. Substrate thickness was equal to 40 mm. After spore inocula‐ tion, the medium was incubated at 23°C for 77 hours. Substrate thickness and fermentation temperature had been selected taking into account studies reported in literature [15,16].

It was used the BET model [27] to fit experimental data (Equation 2):

Criteria used to observe the fit were the coefficient of determination (R2

*<sup>P</sup>* <sup>=</sup> <sup>100</sup> *<sup>n</sup>* ∑ *i*=1

fermentation temperature by experimental design for 78 hours.

µmol of galacturonic acid per minute of reaction at 35°C for 30 minutes.

mined based on experiments performed on laboratory scale.

hour at 30°C and filtered through Wattman 1 filter paper.

concentration (N) and fermentation temperature (Tf

**2.5. Fermentation process using tray reactor**

1 - (*n* + 1)(*aw*)*<sup>n</sup>* + *n*(*aw*)*n*+1

In which: xeq = equilibrium moisture, % dry basis; aw = water activity, adimensional; C = BET constant; xm = moisture in the molecular monolayer; n = number of molecular layers.

responses and predicted by the fitted model and average percentage deviation (P) (Equation

*n* |*Xexp* - *Xteo*|

In which: n = observation numbers; Xexp = humidity of experimental material; Xteo = hu‐

Microorganism used was *Aspergillus niger* CCT0916, donated by Empresa Brasileira de Pes‐ quisa Agropecuária (EMBRAPA, Fortaleza State – Brazil). Spore concentration was adjusted

Substrate was hydrated with distilled water to obtain moisture content and ammonium sul‐ fate was added to this volume. In a 250 mL Erlenmeyer flask, it was weighed 10 g of steri‐ lized humidified medium. After spore inoculation, this medium was incubated at

Enzyme extraction for fermented complex was performed by adding 2.5 mL/g of fermented medium using 200 mM acetate buffer pH 4.5. Samples were then left in water bath for 1

 factorial experimental design was conducted with 7 experiments at central point to de‐ termine the influence of spore concentration (E), initial moisture (U), ammonium sulfate

One unit of polygalacturonase activity was defined as the amount of enzyme that releases 1

Microorganism used in this stage was also used for optimization in the laboratory scale. Spores concentration, moisture content and ammonium sulphate concentration were deter‐

<sup>1</sup> - (1 - *<sup>C</sup>*)*aw* - *<sup>C</sup>*(*aw*)*n*+1 (2)

*Xexp* (3)

) on polygalacturonase activity response

and lowest value of P [28].

) between observed

*xeq xm* <sup>=</sup> *<sup>C</sup>*.*aw* 1 - *aw*

3). The best fits were those with the highest R2

midity calculated by adjusted models.

according to experimental design.

A 24

404 Food Industry

(Table 2).

**2.4. Fermentation process in laboratory scale**


**Table 2.** Concentrations and tests from factorial design

ces. Other substances also welcome in process are reducing sugars. These sugars (glucose, fructose, etc.) are sources of quick energy, and are also consumed by microorganism during

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407

However, there must be a balance between nutrient sources. The literature indicates that high concentrations of sugars in fermentation medium, supplying the microorganism needs

Cashew has on average 30% of reducing sugars (glucose and fructose) and about 10% of pectin in your composition. This implies the need for addition of inducer in a bagasse for

Some authors observed that for pectinases production, using *Aspergillus niger* T005007-2 and wheat bran as substrate through a solid state fermentation process, the citrus pectin addi‐ tion, up to 16% (w/w) led to increased enzyme production [30]. Based on these data, and in the proximity of same amount with sugar in cashew bagasse, it was decided not to add in‐

In general, pH is an important variable in any biological process, with optimum values for microorganism growth. Generally, fungi prefer low pH between 4.5 and 5.0 [31,32]. Average value observed for cashew dry bagasse is 4.0, confirming proximity to other values found in the literature for cashew cake dry [17,33]. Thus, bagasse pH characterized can promote the pectinases production without requiring adjustment using buffer solution. Moreover, acid

Bagasse was dried to below 15% (w.b), it became necessary to store a reasonable amount, noting that below 15%, the organic materials retain their properties over time and makes it

Water amount in fermentation medium is a limiting factor and it directly affects the micro‐ organism needs, and the final product type. To use this residue, it will be necessary to adjust the moisture content, since some microorganisms that produce pectinases require higher levels. This quantity of water is related with the environment via two variables: moisture

Moisture regards percentage of water in the total mass of medium. And determination of this value in process is closely related to substrate nature, the requirements of microorgan‐ ism used and the type of end product desired [11]. Water activity indicates that the organ‐

Microorganism growth depends on the water activity, due to influence of osmotic pressure by on exchange membranes. And it can be related to moisture in the substrate used in fer‐

Some authors [8,31] cite the use of various substrates for pectinase production by *Aspergillus niger* with water activity above 0.93. Importantly, low levels of water activity means low availability of water molecules near the cell, making exchange of solutes in solid phase, re‐ ducing metabolism and generating lower rates of growth or synthesis of metabolites. In con‐

pH favors the storage at room temperature without contamination problems.

for its growth, and pectin little used, hence there is little enzymes production [29].

fermentation process [21].

use in a pectinases production.

ductors in fermentation process.

difficult to contamination by bacterias.

ism may grow by fermentation, ensuring product quality.

mentation using sorption isotherms for a given temperature [32].

and water activity.

**Figure 3.** Tray reactor

Enzyme extraction for fermented complex was performed by adding 5.0 mL/g of fermented medium, using 200 mM acetate buffer pH 4.5. Samples were left in water bath for 1 hour at 30°C and filtered on Wattman 1 paper filter.

### **2.6. Enzymatic stability**

To check the stability concerning temperature, crude extract samples were taken to a water bath for 20 min at temperatures between 10 and 90 ° C. For pH, crude extract was diluted in the buffers listed below, thus verifying the pH influence on polygalacturonase activity. After reaching the corresponding pH, samples were incubated for 24 hours at 2°C [46]: 0.1 M gly‐ cine-HCl pH 2.5; acetate buffer 200 mM pH 3.5-6.5; 0.1 M tris-HCl pH 7.5-8.5; 0.1 M glycine-NaOH pH 9.5. Results of thermostability and stability as to pH were expressed as relative activity (%).

## **3. Results and discussion**

#### **3.1. Physicochemical characterization of cashew apple dry bagasse**

In a solid state fermentation process for enzymes production, it is important to know about the substrate composition, because the microorganism uses such as nutrient, for growth and reproduction so as to produce.

In case of pectinases, the inducing substance is pectin. Microorganism will adapt to the envi‐ ronment and for its maintenance, it will produce the enzyme needed to break these substan‐ ces. Other substances also welcome in process are reducing sugars. These sugars (glucose, fructose, etc.) are sources of quick energy, and are also consumed by microorganism during fermentation process [21].

However, there must be a balance between nutrient sources. The literature indicates that high concentrations of sugars in fermentation medium, supplying the microorganism needs for its growth, and pectin little used, hence there is little enzymes production [29].

Cashew has on average 30% of reducing sugars (glucose and fructose) and about 10% of pectin in your composition. This implies the need for addition of inducer in a bagasse for use in a pectinases production.

Some authors observed that for pectinases production, using *Aspergillus niger* T005007-2 and wheat bran as substrate through a solid state fermentation process, the citrus pectin addi‐ tion, up to 16% (w/w) led to increased enzyme production [30]. Based on these data, and in the proximity of same amount with sugar in cashew bagasse, it was decided not to add in‐ ductors in fermentation process.

In general, pH is an important variable in any biological process, with optimum values for microorganism growth. Generally, fungi prefer low pH between 4.5 and 5.0 [31,32]. Average value observed for cashew dry bagasse is 4.0, confirming proximity to other values found in the literature for cashew cake dry [17,33]. Thus, bagasse pH characterized can promote the pectinases production without requiring adjustment using buffer solution. Moreover, acid pH favors the storage at room temperature without contamination problems.

**Figure 3.** Tray reactor

406 Food Industry

**2.6. Enzymatic stability**

**3. Results and discussion**

reproduction so as to produce.

activity (%).

30°C and filtered on Wattman 1 paper filter.

Enzyme extraction for fermented complex was performed by adding 5.0 mL/g of fermented medium, using 200 mM acetate buffer pH 4.5. Samples were left in water bath for 1 hour at

To check the stability concerning temperature, crude extract samples were taken to a water bath for 20 min at temperatures between 10 and 90 ° C. For pH, crude extract was diluted in the buffers listed below, thus verifying the pH influence on polygalacturonase activity. After reaching the corresponding pH, samples were incubated for 24 hours at 2°C [46]: 0.1 M gly‐ cine-HCl pH 2.5; acetate buffer 200 mM pH 3.5-6.5; 0.1 M tris-HCl pH 7.5-8.5; 0.1 M glycine-NaOH pH 9.5. Results of thermostability and stability as to pH were expressed as relative

In a solid state fermentation process for enzymes production, it is important to know about the substrate composition, because the microorganism uses such as nutrient, for growth and

In case of pectinases, the inducing substance is pectin. Microorganism will adapt to the envi‐ ronment and for its maintenance, it will produce the enzyme needed to break these substan‐

**3.1. Physicochemical characterization of cashew apple dry bagasse**

Bagasse was dried to below 15% (w.b), it became necessary to store a reasonable amount, noting that below 15%, the organic materials retain their properties over time and makes it difficult to contamination by bacterias.

Water amount in fermentation medium is a limiting factor and it directly affects the micro‐ organism needs, and the final product type. To use this residue, it will be necessary to adjust the moisture content, since some microorganisms that produce pectinases require higher levels. This quantity of water is related with the environment via two variables: moisture and water activity.

Moisture regards percentage of water in the total mass of medium. And determination of this value in process is closely related to substrate nature, the requirements of microorgan‐ ism used and the type of end product desired [11]. Water activity indicates that the organ‐ ism may grow by fermentation, ensuring product quality.

Microorganism growth depends on the water activity, due to influence of osmotic pressure by on exchange membranes. And it can be related to moisture in the substrate used in fer‐ mentation using sorption isotherms for a given temperature [32].

Some authors [8,31] cite the use of various substrates for pectinase production by *Aspergillus niger* with water activity above 0.93. Importantly, low levels of water activity means low availability of water molecules near the cell, making exchange of solutes in solid phase, re‐ ducing metabolism and generating lower rates of growth or synthesis of metabolites. In con‐ trast, high levels of water activity hinder the diffusion of air through solid particles, leading to a reduction in microbial growth [35]

Observed that the curves 25 and 30°C overlap, indicating a similar behaviour as regards the adsorption of water to these their temperatures, in which case this difference (5°C) showed no influence of temperature on these isotherms, unlike curves 35 and 40°C, in which there is small, but clearly influence of this variable. There is also a greater effect of temperature in‐ terval 25-30°C to 35-40°C. An analysis of the equilibrium moisture behaviour in relation to

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Can be found in the literature several studies that relate initial content of water activity with development of various microorganisms responsible for synthesis products obtained in sol‐

In particular, *Aspergillus niger* is described as the organism that best fits to the fermentation process, being around 0.7 the value of minimum water activity for development of their

Thus, minimum value being 0.7 from water activity to grow *Aspergillus niger*, when using cashew apple dry bagasse as substrate in solid state fermentation process and adsorption isotherms obtained (Figure 4), has an indicative that moisture in substrate at intervals of

25-30°C and 35-40°C should not be less than 17.5 and 19.0%(d.b), respectively.

water activity shows similarity to temperature behaviour.

**Figure 4.** BET model to adsorption isotherms of dry cashew apple bagasse

id state fermentation process.

metabolic activities [11].

Crude protein value is approximately 8% and it is close to the value observed by some au‐ thors [36,37]. This value is important for characterization as serve as a nitrogen source for microorganism. And from that data, it can be observed the necessity of supplementation with alternative sources of nitrogen such as urea or ammonium sulphate.

Physical characteristics with respect to substrate morphology are essential, particularly, in size and porosity, as these properties governing the accessible surface area of micro‐ organism [17].

For granulometric distribution, 80% of bagasse was retained in sieves 20, 24 and 35 mesh, corresponding to particle size of 0.85, 0.70 and 0.42 mm, respectively. This particle size can be used in a solid state fermentation process by *Aspergillus niger*, which was already descri‐ bed in literature, particle sizes for pectinases production from 0.5 to 0.7 mm [39,40].

Average particle size of residual fermentation media must be obtained so that there have been no particles large or small. Particles of small size promote greater surface area and con‐ sequently a higher degree of processing. However, the process itself needs to have a particle size allowing the circulation of air through the mass and waste gases and heat produced, which could harm the efficiency of process [11]. Particles larger interparticles promote more space, reducing the efficiency of nutrients absorption for microorganisms. Furthermore, par‐ ticle size analysis is important in enzyme complex extraction, since finely divided solid car‐ riers facilitate access by the solvent [29].

It is important to remember that, in general, the crops characterization crops can vary dra‐ matically depending on time of harvest, agricultural practices and phenomena related to planting. Is then very important to characterize the substrate for solid state fermentation process and therefore the adjustment of certain parameters, which is also an additional chal‐ lenge is to be considered in using this process.

#### **3.2. Adjust of adsorption isotherms from cashew apple dry bagasse**

Data water activity (aw) and average equilibrium moisture (xeq) of material at temperatures studied (25, 30, 35 and 40°C) was adjusted BET model.

Table 3 are the values of equation BET parameters, average deviation percentage (P) and correlation coefficient (R2 ) for each temperature.

From Table 3, there is the BET model appropriately fit to experimental data because P value indicates a good fit when it is less than 10% and R2 must be as close to unity [38].

When comparing the monolayer humidity values of humidity (Xm) by BET equation, it is noted that the range of 25-30°C to 35-40°C, Xm increased by approximately 0.4%. This is not a common behaviour, but can be explained by two mechanisms: (1) increase of temperature may cause changes in product physical structure, providing a larger number of active sites with affinity for water molecules, (2) or may cause an increase in intrinsic solubility of solute to the product, causing a greater number of water molecules is retained on monolayer [39].

Observed that the curves 25 and 30°C overlap, indicating a similar behaviour as regards the adsorption of water to these their temperatures, in which case this difference (5°C) showed no influence of temperature on these isotherms, unlike curves 35 and 40°C, in which there is small, but clearly influence of this variable. There is also a greater effect of temperature in‐ terval 25-30°C to 35-40°C. An analysis of the equilibrium moisture behaviour in relation to water activity shows similarity to temperature behaviour.

trast, high levels of water activity hinder the diffusion of air through solid particles, leading

Crude protein value is approximately 8% and it is close to the value observed by some au‐ thors [36,37]. This value is important for characterization as serve as a nitrogen source for microorganism. And from that data, it can be observed the necessity of supplementation

Physical characteristics with respect to substrate morphology are essential, particularly, in size and porosity, as these properties governing the accessible surface area of micro‐

For granulometric distribution, 80% of bagasse was retained in sieves 20, 24 and 35 mesh, corresponding to particle size of 0.85, 0.70 and 0.42 mm, respectively. This particle size can be used in a solid state fermentation process by *Aspergillus niger*, which was already descri‐

Average particle size of residual fermentation media must be obtained so that there have been no particles large or small. Particles of small size promote greater surface area and con‐ sequently a higher degree of processing. However, the process itself needs to have a particle size allowing the circulation of air through the mass and waste gases and heat produced, which could harm the efficiency of process [11]. Particles larger interparticles promote more space, reducing the efficiency of nutrients absorption for microorganisms. Furthermore, par‐ ticle size analysis is important in enzyme complex extraction, since finely divided solid car‐

It is important to remember that, in general, the crops characterization crops can vary dra‐ matically depending on time of harvest, agricultural practices and phenomena related to planting. Is then very important to characterize the substrate for solid state fermentation process and therefore the adjustment of certain parameters, which is also an additional chal‐

Data water activity (aw) and average equilibrium moisture (xeq) of material at temperatures

Table 3 are the values of equation BET parameters, average deviation percentage (P) and

From Table 3, there is the BET model appropriately fit to experimental data because P value

When comparing the monolayer humidity values of humidity (Xm) by BET equation, it is noted that the range of 25-30°C to 35-40°C, Xm increased by approximately 0.4%. This is not a common behaviour, but can be explained by two mechanisms: (1) increase of temperature may cause changes in product physical structure, providing a larger number of active sites with affinity for water molecules, (2) or may cause an increase in intrinsic solubility of solute to the product, causing a greater number of water molecules is retained on monolayer [39].

must be as close to unity [38].

bed in literature, particle sizes for pectinases production from 0.5 to 0.7 mm [39,40].

with alternative sources of nitrogen such as urea or ammonium sulphate.

to a reduction in microbial growth [35]

riers facilitate access by the solvent [29].

lenge is to be considered in using this process.

correlation coefficient (R2

studied (25, 30, 35 and 40°C) was adjusted BET model.

indicates a good fit when it is less than 10% and R2

**3.2. Adjust of adsorption isotherms from cashew apple dry bagasse**

) for each temperature.

organism [17].

408 Food Industry

Can be found in the literature several studies that relate initial content of water activity with development of various microorganisms responsible for synthesis products obtained in sol‐ id state fermentation process.

In particular, *Aspergillus niger* is described as the organism that best fits to the fermentation process, being around 0.7 the value of minimum water activity for development of their metabolic activities [11].

Thus, minimum value being 0.7 from water activity to grow *Aspergillus niger*, when using cashew apple dry bagasse as substrate in solid state fermentation process and adsorption isotherms obtained (Figure 4), has an indicative that moisture in substrate at intervals of 25-30°C and 35-40°C should not be less than 17.5 and 19.0%(d.b), respectively.

**Figure 4.** BET model to adsorption isotherms of dry cashew apple bagasse


**Test PPG (U/g) t (h)**

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1 1.06 21

2 14.93 54

3 0.42 70

4 15.91 46

5 0.59 5

6 15.38 54

7 5.00 46

8 10.78 21

9 0 ---

10 24.23 46

11 0 ---

12 7.69 29

13 1.66 54

14 33.27 29

15 0 ---

16 11.31 70

17 2.01 70

18 5.07 29

19 10.28 46

20 14.29 70

21 5.53 78

22 11.37 21

23 8.30 46

**Table 4.** Highest polygacturonase activities (PG) observed for which solid state fermentation assay.

**Table 3.** Adsorption isotherms fitting parameters of cashew peduncle dry bagasse to the BET model

For pectinases production by *Aspergillus niger* in solid state fermentation process, several au‐ thors describe fermentation processes in which it can be seen that water activity which best favourable to synthesis of product is above 0.90. This implies that substrate moisture must be greater than 35%(d.b).

It is therefore extremely important to understand the hygroscopic behaviour of semisolid product used as substrate in a fermentation process, since the quantity of available water in through the microorganism to grow and synthesizing reactions during the production proc‐ ess is a limiting factor.

#### **3.3. Most influential factor in solid state fermentation process on laboratory scale**

As previously noted, there are many factors that affect a solid state fermentation process: amount of water available to microorganism can be quantified by moisture of medium (U), amount of inoculum necessary to overcome the adaptation phase and microorganism to produce the desired products (E), addition of nitrogen source such as ammonium sulphate (N) and fermentation temperature (Tf ). These are variables that will be studied as previously described. It was conducted an experimental design 24 , taking an answer the polygalacturo‐ nase activity. Objective was to determine which variable most affects the process and maxi‐ mize the amount of enzyme produced.

The highest activities found for each assay and the fermentation time it was noted, are available in the Table 4. The greatest polygalacturonase activity (33.27 U/g) found, dur‐ ing the execution of experimental design, was obtained under the initial conditions: 50% (w.b) initial moisture, 106 spores/g, 1.5% (w/w) ammonium sulfate and 35°C at 29 hours of fermentation.

Scale Up of Polygalacturonase Production by Solid State Fermentation Process http://dx.doi.org/10.5772/53152 411


**Parameters Temperature (°C)**

410 Food Industry

be greater than 35%(d.b).

ess is a limiting factor.

of fermentation.

(N) and fermentation temperature (Tf

mize the amount of enzyme produced.

described. It was conducted an experimental design 24

25 30 35 40

Xm 5.64 5.61 6.33 6.07

C 12.59 11.28 39.72 49.85

n 14.39 15.02 13.35 13.18

R2 0.9998 0.9986 0.9948 0.9941

P (%) 0.86 2.70 4.79 4.11

**Table 3.** Adsorption isotherms fitting parameters of cashew peduncle dry bagasse to the BET model

For pectinases production by *Aspergillus niger* in solid state fermentation process, several au‐ thors describe fermentation processes in which it can be seen that water activity which best favourable to synthesis of product is above 0.90. This implies that substrate moisture must

It is therefore extremely important to understand the hygroscopic behaviour of semisolid product used as substrate in a fermentation process, since the quantity of available water in through the microorganism to grow and synthesizing reactions during the production proc‐

As previously noted, there are many factors that affect a solid state fermentation process: amount of water available to microorganism can be quantified by moisture of medium (U), amount of inoculum necessary to overcome the adaptation phase and microorganism to produce the desired products (E), addition of nitrogen source such as ammonium sulphate

nase activity. Objective was to determine which variable most affects the process and maxi‐

The highest activities found for each assay and the fermentation time it was noted, are available in the Table 4. The greatest polygalacturonase activity (33.27 U/g) found, dur‐ ing the execution of experimental design, was obtained under the initial conditions: 50% (w.b) initial moisture, 106 spores/g, 1.5% (w/w) ammonium sulfate and 35°C at 29 hours

). These are variables that will be studied as previously

, taking an answer the polygalacturo‐

**3.3. Most influential factor in solid state fermentation process on laboratory scale**

**Table 4.** Highest polygacturonase activities (PG) observed for which solid state fermentation assay.

From regression of polygalacturonase activity data and values of factors studied, it was con‐ structed a first order model with 95% confidence for peaks of enzymatic activities.

```
fff PPG = – + 0.86N + 0.88T – + 0.14UN + 1.56UT – 0.48EN – + 0.93NT f 8.66 + 7.80U 2.50E 2.76UE 2.52ET (4)
```
The highest peak of polygalacturonase activity calculated by the model (30 U/g) was ob‐ tained at higher levels for initial moisture content, for the lower concentration of inoculum and higher for fermentation temperature, observing that ammonium sulfate concentration

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According to Pareto graphic (Figure 6), variable that most influenced fermentation process was initial moisture content of medium (U), and its interaction with variable concentration of inoculums (E), confirming claims reported in literature about the amount of water is thus

Authors [41] examined the effect of temperature on a solid state fermentation process using *Aspergillus niger* 163 and apple pomace as a substrate in a bioreactor rotary drum with 15L of solid medium. Temperature was studied within the range 22-60°C, observing their influence on polygalacturonase activity. Inoculated spore concentration was equal to 5x108 spores/ml. Temperature of 35°C was found to be more susceptible to production of polygalacturonases

Similarly to what was described in this paper, authors [42] studied the influence of ammoni‐ um sulfate concentration (from 0.25 to 0.45%), pH (4.82 to 6.12) and fermentation time (50-90h) on endopectinase enzyme production in a solid state fermentation process, using as substrate apple pomace and *Aspergillus niger* PC5. It was observed that ammonium sulfate concentration have positive effect on enzymatic activity. However, the effect was insignifi‐

It was evaluated the scale-up process using a tray reactor, setting the mass of humid medi‐ um in 500 g. Spores concentration, moisture content and ammonium sulphate concentration were determined based on experiments performed on laboratory scale. Thus the conditions of fermentation process was 50%(w.b) of initial moisture content, 106 spores/g of inoculum

**3.4. Scaling-up of solid state fermentation process using tray reactor**

did not influence significant on the response.

a limiting factor [11,12].

**Figure 6.** Pareto graphic

cant compared to fermentation time.

enzymes.

Model validation was done using Test F. This test shows the ratio between calculated value of F and F tabulated, knowing that the latter was equal to 2.75. When this ratio is greater than 1, regression is significant. Not only to a statistically significant regression, but also pre‐ dictive value of ratio between this two F's must be greater than four [40].

Regarding the determination coefficient (R2 ), it is known that the maximum value is 1, meaning that between the experimental data and curve, there is no waste and any variation about the mean is explained by regression.

In Equation 4, calculated F was equal to 7.99. Thus, the ratio between calculated F and tabu‐ lated is equal to 2.91, meaning that this equation is statistically significant. The R2 was equal to 0.8695, showing a good fit of experimental data to this equation.

Figure 5 indicates the profile of curve representing synergistic effect of studied factors on peaks of polygalacturonase activity response. These figures show the influence of initial moisture content (U), concentration of spores in inoculation medium (E), ammonium sul‐ fate concentration (N) and fermentation temperature (Tf ) on polygalacturonase activity response (PPG).

**Figure 5.** Response surface for polygalacturonase activity.

The highest peak of polygalacturonase activity calculated by the model (30 U/g) was ob‐ tained at higher levels for initial moisture content, for the lower concentration of inoculum and higher for fermentation temperature, observing that ammonium sulfate concentration did not influence significant on the response.

According to Pareto graphic (Figure 6), variable that most influenced fermentation process was initial moisture content of medium (U), and its interaction with variable concentration of inoculums (E), confirming claims reported in literature about the amount of water is thus a limiting factor [11,12].

**Figure 6.** Pareto graphic

From regression of polygalacturonase activity data and values of factors studied, it was con‐

Model validation was done using Test F. This test shows the ratio between calculated value of F and F tabulated, knowing that the latter was equal to 2.75. When this ratio is greater than 1, regression is significant. Not only to a statistically significant regression, but also pre‐

meaning that between the experimental data and curve, there is no waste and any variation

In Equation 4, calculated F was equal to 7.99. Thus, the ratio between calculated F and tabu‐

Figure 5 indicates the profile of curve representing synergistic effect of studied factors on peaks of polygalacturonase activity response. These figures show the influence of initial moisture content (U), concentration of spores in inoculation medium (E), ammonium sul‐

lated is equal to 2.91, meaning that this equation is statistically significant. The R2

fff PPG = – + 0.86N + 0.88T – + 0.14UN + 1.56UT – 0.48EN – + 0.93NT **<sup>f</sup> 8.66 + 7.80U 2.50E 2.76UE 2.52ET** (4)

), it is known that the maximum value is 1,

) on polygalacturonase activity

was equal

structed a first order model with 95% confidence for peaks of enzymatic activities.

dictive value of ratio between this two F's must be greater than four [40].

to 0.8695, showing a good fit of experimental data to this equation.

fate concentration (N) and fermentation temperature (Tf

Regarding the determination coefficient (R2

about the mean is explained by regression.

**Figure 5.** Response surface for polygalacturonase activity.

response (PPG).

412 Food Industry

Authors [41] examined the effect of temperature on a solid state fermentation process using *Aspergillus niger* 163 and apple pomace as a substrate in a bioreactor rotary drum with 15L of solid medium. Temperature was studied within the range 22-60°C, observing their influence on polygalacturonase activity. Inoculated spore concentration was equal to 5x108 spores/ml. Temperature of 35°C was found to be more susceptible to production of polygalacturonases enzymes.

Similarly to what was described in this paper, authors [42] studied the influence of ammoni‐ um sulfate concentration (from 0.25 to 0.45%), pH (4.82 to 6.12) and fermentation time (50-90h) on endopectinase enzyme production in a solid state fermentation process, using as substrate apple pomace and *Aspergillus niger* PC5. It was observed that ammonium sulfate concentration have positive effect on enzymatic activity. However, the effect was insignifi‐ cant compared to fermentation time.

## **3.4. Scaling-up of solid state fermentation process using tray reactor**

It was evaluated the scale-up process using a tray reactor, setting the mass of humid medi‐ um in 500 g. Spores concentration, moisture content and ammonium sulphate concentration were determined based on experiments performed on laboratory scale. Thus the conditions of fermentation process was 50%(w.b) of initial moisture content, 106 spores/g of inoculum concentration, 1.5%(w/w) of ammonium sulphate concentration, 40 mm of substrate thick‐ ness [15] and 23°C of fermentation temperature [16].

**3.5. Enzymatic stability as temperature and pH**

stabilize it completely.

enough to fulfil the expected function [7].

**Figure 8.** Thermostability of polygalacturonase enzyme

tween 70-90°C [7].

In summary, the enzyme is maintained by a delicate balance of noncovalent forces such as hydrogen bonds, ion pairing, hydrophobic interactions and van der Waals force [45]. Thus,

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Temperature has the activation and deactivation effect on enzyme activity. Continued in‐ creases of temperature beyond the maximum or optimum for enzyme activity leads to pro‐ tein denaturation, which involves the deployment of large segments of polypeptide chain [45]. At the other extreme, enzymes inactivation by cooling can occur when nonpolar forces are involved and association of polypeptides. Low temperature reduces the strength of such interactions can promote the dissociation of subunits and compromise enzyme activity [7].

With respect to pH, all of ionisable groups of proteins undergo transitions pH dependent on the basis of intrinsic pKa values of amino acid residues. Many of these transitions will cause impacts on the stability of the enzyme and, in a narrow range of pH, can act together to de‐

Knowing the pH and temperature stability is important for selection of enzymes compatible with the prevailing conditions for application potential so that the enzyme persist long

It can be observed (Figure 8) that temperatures of between 30 and 50°C, polygalacturonase activity remained with up to 80% of its maximum activity, and its optimum temperature at

Considering the greater use of pectic enzymes is in fruit juices industry, the processing of juices is normally done at 30-50°C. Thus, produced enzymes are been active during the process. For inactivation, the binomial time versus temperature must be considered, as well as chemical characteristics of juices. Usually, enzymatic inactivation is made be‐

40°C. After 50°C, relative enzymatic activity falls abruptly, reaching zero at 70°C.

variations in temperature and pH are important in the analysis of enzyme activity.

In Figure 7, there are the behavior of polygalacturonase activity (PG) and process productiv‐ ity (Prod) as function of fermentation time.

**Figure 7.** Polygalacturonase activity and productivity versus fermentation time.

Under conditions described previously, it was observed a peak of polygalacturonase activity of 15.76 U/g at 69 hours of fermentation, corresponding to the highest productivity.

Comparing the maximum activity obtained with reactor tray, and maximum activity ob‐ tained at laboratory scale, it is clear that production was 45 times lower. In this regard, the first fact to note is the internal temperature of medium. When using tray, there is an increase of at least 10°C above of initial value. Thus, there may be denatured enzyme.

Other factors can change during fermentation. One is the medium moisture, because with increase of temperature, there is an increased in evaporation rate of water, reducing the amount available to microorganism, which complicates the production process.

It can be observed that the time for adaptation of microorganism is greatly increased. Re‐ membering, the greatest polygalacturonase activity in laboratorial scale was obtained at 29 h of fermentation. In the fermentation with tray, the highest activity obtained was reached at 69h of fermentation.

When the subject is solid state fermentation process, a large number of studies were con‐ ducted in a laboratory scale. It is relatively easy to control certain parameters for enzyme production. On this scale, many productions show great promise. However, beyond this process to pilot-scale bioreactors, which contain greater amounts of substrate, difficulties in carrying out the fermentation are revealed. Thus, it is characterized one of difficulties in scaling-up solid state fermentation processes [28].

### **3.5. Enzymatic stability as temperature and pH**

concentration, 1.5%(w/w) of ammonium sulphate concentration, 40 mm of substrate thick‐

In Figure 7, there are the behavior of polygalacturonase activity (PG) and process productiv‐

Under conditions described previously, it was observed a peak of polygalacturonase activity

Comparing the maximum activity obtained with reactor tray, and maximum activity ob‐ tained at laboratory scale, it is clear that production was 45 times lower. In this regard, the first fact to note is the internal temperature of medium. When using tray, there is an increase

Other factors can change during fermentation. One is the medium moisture, because with increase of temperature, there is an increased in evaporation rate of water, reducing the

It can be observed that the time for adaptation of microorganism is greatly increased. Re‐ membering, the greatest polygalacturonase activity in laboratorial scale was obtained at 29 h of fermentation. In the fermentation with tray, the highest activity obtained was reached at

When the subject is solid state fermentation process, a large number of studies were con‐ ducted in a laboratory scale. It is relatively easy to control certain parameters for enzyme production. On this scale, many productions show great promise. However, beyond this process to pilot-scale bioreactors, which contain greater amounts of substrate, difficulties in carrying out the fermentation are revealed. Thus, it is characterized one of difficulties in

of 15.76 U/g at 69 hours of fermentation, corresponding to the highest productivity.

of at least 10°C above of initial value. Thus, there may be denatured enzyme.

amount available to microorganism, which complicates the production process.

ness [15] and 23°C of fermentation temperature [16].

**Figure 7.** Polygalacturonase activity and productivity versus fermentation time.

69h of fermentation.

scaling-up solid state fermentation processes [28].

ity (Prod) as function of fermentation time.

414 Food Industry

In summary, the enzyme is maintained by a delicate balance of noncovalent forces such as hydrogen bonds, ion pairing, hydrophobic interactions and van der Waals force [45]. Thus, variations in temperature and pH are important in the analysis of enzyme activity.

Temperature has the activation and deactivation effect on enzyme activity. Continued in‐ creases of temperature beyond the maximum or optimum for enzyme activity leads to pro‐ tein denaturation, which involves the deployment of large segments of polypeptide chain [45]. At the other extreme, enzymes inactivation by cooling can occur when nonpolar forces are involved and association of polypeptides. Low temperature reduces the strength of such interactions can promote the dissociation of subunits and compromise enzyme activity [7].

With respect to pH, all of ionisable groups of proteins undergo transitions pH dependent on the basis of intrinsic pKa values of amino acid residues. Many of these transitions will cause impacts on the stability of the enzyme and, in a narrow range of pH, can act together to de‐ stabilize it completely.

Knowing the pH and temperature stability is important for selection of enzymes compatible with the prevailing conditions for application potential so that the enzyme persist long enough to fulfil the expected function [7].

**Figure 8.** Thermostability of polygalacturonase enzyme

It can be observed (Figure 8) that temperatures of between 30 and 50°C, polygalacturonase activity remained with up to 80% of its maximum activity, and its optimum temperature at 40°C. After 50°C, relative enzymatic activity falls abruptly, reaching zero at 70°C.

Considering the greater use of pectic enzymes is in fruit juices industry, the processing of juices is normally done at 30-50°C. Thus, produced enzymes are been active during the process. For inactivation, the binomial time versus temperature must be considered, as well as chemical characteristics of juices. Usually, enzymatic inactivation is made be‐ tween 70-90°C [7].

**Author details**

**References**

859-868.

Nova 2007; 30 388-394.

Alegre: Artmed; 2010.

France.

Siumara R. Alcântara 1

, Nathalya J. Leite 1

2 Universidade Federal da Paraíba, João Pessoa, Brazil

Process Biochemistry 2001; 37(5) 497–503.

Bioproducts Processing 2011; 89(4) 281-287.

Iranian Journal of Biotechnology 2011; 9 50-55.

zymes: a review. Process Biochemistry 1998; 33(1) 21-28.

1 Universidade Federal de Campina Grande, Campina Grande, Brazil

and Flávio L. H. da Silva 2

Scale Up of Polygalacturonase Production by Solid State Fermentation Process

http://dx.doi.org/10.5772/53152

417

[1] Blandino A., Dravillas K., Cantero D., Pandiella S S., Webb C. Utilisation of whole wheat flour for the production of extracellular pectinases by some fungal strains.

[2] Lara-Márquez A., Zaval-Pánamo M G., López-Romero E., Camacho H C. Biotechno‐ logical potential of pectinolytic complexes of fungi. Biotechnological Letters 2011; 33

[3] Gomes J., Zeni J., Cence K., Toniazzo G., Treichel H. Evaluation of production and characterization of polygalacturonase by Aspergillus niger ATCC 9642. Food and

[4] Alkorta I., Garbisu C., Llama M J., Serra J L. Industrial applications of pectic en‐

[5] Uenojo M., Pastore G M. Pectinases: aplicações industriais e perspectivas. Química

[6] Abbasi H., Shafighzadeh H., Rahimi A. Continuos production of polygalacturonases (PGases) by Aspergillus awamori using wheat flour in surface culture fermentation.

[7] Damodaran S., Parkin K L., Fennema O R. Química de alimentos de Fennema. Porto

[8] Antier P., Minjares A., Roussos S., Raimbault M., Vinergra-Gonzalez G. Pectinase-hy‐ perproducing mutants of Aspergillus niger C28B25 for solid-state fermentation of

[9] Couto S R., Sanromán M A. Application of solid-state fermentation to food industry

[10] Raimbault M., Deschamps A., Meyer F., Senez J C. Direct protein enrichment of star‐ chy products by fungal solid fermentation. In: Proc. Giam-V, 1977, Marseilles,

coffee pulp. Enzyme Microbial Technology 1993; 15(3) 254-260.

– A review. Journal of Food Engineering 2006; 76(3) 291-302.

**Figure 9.** Stability of polygalacturonase enzyme in relation to pH

It is observed that the pH's of 2.5 and 3.5 (Figure 9), polygalacturonase activity was highest, almost 100%, characterizing the enzyme as acidic. Relative activity is equal to zero only at pH 9.5.

In literature some authors have reported the viability of various types of waste from proc‐ essed fruits (apple, cranberry and strawberry), as substrates for polygalacturonases produc‐ tion, with *Lentinus edodes* as microorganism, using solid state fermentation process. These authors also observed the effects of temperature and pH on the enzymatic extract. Poligalac‐ torunase produced has good thermal stability up to 50°C and high tolerance between pH 3.0 and 6.5 [46].

Other authors have studied the polygalacturonases production, using submerged fermenta‐ tion with orange peel and passion fruit as substrate and *Aspergillus niveus* as microorganism. In terms of stability, polygalacturonase produced showed the highest activity at 40°C and pH between 3.0 and 4.5 [48].

In general, polygalacturonase enzyme activity has maximum pH ranges between 3.5 - 6.0 and temperature between 40 - 55°C [7].

For most industrial uses, fungal polygalacturonase is useful for high activity and optimal ac‐ tivity at low pH range, serving for most applications in the food industry [16]. Thus, enzyme extract studied can be applied in fruit juice processes, such as Barbados cherry (pH 3.3), or‐ ange (pH 3.0), apple (pH 3.6), passion (pH 3.4), peach (pH 3.3) and grapes (pH 3.1) [47].

## **Acknowledgement**

To CNPq (Conselho nacional de desenvolvimento cientifico e tecnológico – Brazil) and CAPES (Coordenação de aperfeiçoamento de pessoal de nível superior – Brazil) by grants of doctoral fellowships and undergraduate research and research financial support.

## **Author details**

Siumara R. Alcântara 1 , Nathalya J. Leite 1 and Flávio L. H. da Silva 2

1 Universidade Federal de Campina Grande, Campina Grande, Brazil

2 Universidade Federal da Paraíba, João Pessoa, Brazil

## **References**

**Figure 9.** Stability of polygalacturonase enzyme in relation to pH

pH 9.5.

416 Food Industry

and 6.5 [46].

pH between 3.0 and 4.5 [48].

**Acknowledgement**

and temperature between 40 - 55°C [7].

It is observed that the pH's of 2.5 and 3.5 (Figure 9), polygalacturonase activity was highest, almost 100%, characterizing the enzyme as acidic. Relative activity is equal to zero only at

In literature some authors have reported the viability of various types of waste from proc‐ essed fruits (apple, cranberry and strawberry), as substrates for polygalacturonases produc‐ tion, with *Lentinus edodes* as microorganism, using solid state fermentation process. These authors also observed the effects of temperature and pH on the enzymatic extract. Poligalac‐ torunase produced has good thermal stability up to 50°C and high tolerance between pH 3.0

Other authors have studied the polygalacturonases production, using submerged fermenta‐ tion with orange peel and passion fruit as substrate and *Aspergillus niveus* as microorganism. In terms of stability, polygalacturonase produced showed the highest activity at 40°C and

In general, polygalacturonase enzyme activity has maximum pH ranges between 3.5 - 6.0

For most industrial uses, fungal polygalacturonase is useful for high activity and optimal ac‐ tivity at low pH range, serving for most applications in the food industry [16]. Thus, enzyme extract studied can be applied in fruit juice processes, such as Barbados cherry (pH 3.3), or‐ ange (pH 3.0), apple (pH 3.6), passion (pH 3.4), peach (pH 3.3) and grapes (pH 3.1) [47].

To CNPq (Conselho nacional de desenvolvimento cientifico e tecnológico – Brazil) and CAPES (Coordenação de aperfeiçoamento de pessoal de nível superior – Brazil) by grants of

doctoral fellowships and undergraduate research and research financial support.


[11] Pinto, G A S., Brito E S., Silva F L H., Santos S F M., Macedo G R. Fermentação em estado sólido: uma alternativa para o aproveitamento e valorização de resíduos agro‐ industriais. Revista de Química Industrial 2006; 74 17-20.

[25] Le Poidevin N., Robinson L A. Métodos ou diagnosticos foliar utilizados nas planta‐ ções do grupo booken na Guiana Inglesa: amostra geral e técnica de analises. Fertilité

Scale Up of Polygalacturonase Production by Solid State Fermentation Process

http://dx.doi.org/10.5772/53152

419

[26] Rockland L B. Satured salt solutions for static control of relative humidity between 5°

[27] Brunauer S., Emmett P., Teller E. Adsorption of gases in multimolecular layers. Jour‐

[28] Santos M M., Rosa A S., Dal'Boit S., Mitchell D A., Krieger N. Thermal denaturation: is solid-state fermentation a really good technology for the production of enzymes?

[29] Souza R L A., Oliveira L S C., Silva F L H., Amorim B C. Caracterização da poligalac‐ turonase produzida por fermentação semi-sólida utilizando-se resíduo do maracujá como substrato. Revista Brasileira de Engenharia Agrícola e Ambiental 2010; 14(9)

[30] Fontana R C., Salvador S., Silveira M M. Efeito das concentrações de pectina e glicose sobre a formação de poligalacturonase por Aspergillus niger em meio sólido. In: SI‐

[31] Taragano V M., Pilosof A M R. Application of Doehlert designs for water activity, pH, and fermentation time optimization for Aspergillus niger pectinolytic activities production in solid-state and submerged fermentation. Enzyme and Microbial Tech‐

[32] Fawole O B., Odunfa S A. Some factors affecting production of pectic enzymes by Aspergillus niger. International Biodeterioration & Biodegradation 2003; 52(4)

[33] Brandão M C C., Maia G A., Lima D P., Parente E J S., Campello C C., Nassu R T., Feitosa T., Sousa P H M. Análises físico-químicas e sensoriais e pedúnculo de caju submetido à desidratação osmótico-solar. Revista de Ciências Agronômica 2003; 34

[34] Santin A P. Estudo da secagem da inativação de leveduras (Sacchatomyces cerevi‐ siae). Masters dissertation. Universidade Federal de Santa Catarina; 1996.

[35] Abud A K S., Silva G F., Narain N. Caracterização de resíduos de indústria de proc‐ essaento de frutas visando à produção de pectinases por fermentação semi-sólida. In:

[36] Matias M F., Oliveira E L., Gertrudes E., Magalhães M M A., Use of fibres obtained from the cashew (Anacardium ocidentale L.) and guava (Psidium guayava) fruits for enrichment of food products. Brazilian Archives of Biology and Technology 2005; 48

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[25] Le Poidevin N., Robinson L A. Métodos ou diagnosticos foliar utilizados nas planta‐ ções do grupo booken na Guiana Inglesa: amostra geral e técnica de analises. Fertilité 1964; 21 3-11.

[11] Pinto, G A S., Brito E S., Silva F L H., Santos S F M., Macedo G R. Fermentação em estado sólido: uma alternativa para o aproveitamento e valorização de resíduos agro‐

[12] Durand A. Bioreactor designs for solid state fermentation. Biochemical Engineering

[13] Mitchell D A., von Meien O F., Krieger N. Recent developments in modeling of solidstate fermentation: heat and mass transfer in bioreactors. Biochemical Engineering

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[16] Dalsenter F D H., Viccini G., Barga M C., Mitchell D A., Krieger N. A mathematical model describing the effect of temperature variations on the kinetics of microbial

[17] Santos S F M., Macedo G R., Silva F L H., Souza R L A., Pinto G A S. Aplicação da metodologia de superfície de resposta no estudo da produção e extração da poliga‐

[18] Micjodjehoun-Mestres L., Souquet J., Fulcrand H., Bouchut C., Reynes M., Brillouet J. Monomeric phenols of cashew apple (Anacardium occidentale L.). Food Chemistry

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[20] Campos A R N., Santana R A C., Dantas J P., Oliveira L S C., Silva F L H. Enriqueci‐ mento proteico do bagaço do pedúnculo de caju por cultivo semi-sólido. Revista de

[21] Alcântara S R., Almeida F A C., Silva F L H. Pectinases production by solid state fer‐ mentation with cashew apple bagasse: water activity and influence of nitrogen souce.

[22] Brasil Escola: Caju. http://www.brasilescola.com/frutas/caju.htm (acessed 12 July

[23] Brasil. Instituto Adolfo Lutz (IAL). Métodos físico-químicos para análise de alimen‐

[24] Rangana S., Manual of analysis of fruit and vegetable products. New Delhi: Tata

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[37] Holanda J S., Oliveira J O., Ferreira A C. Enriquecimento proteico do pedúnculo de caju com emprego de leveduras, para alimentação animal. Pesquisa Agropecuária Brasileira 1998; 33 787-792.

**Chapter 19**

**Effect of Mucilage Extraction on**

Marianela I. Capitani, Susana M. Nolasco and

Additional information is available at the end of the chapter

Mabel C. Tomás

**1. Introduction**

http://dx.doi.org/10.5772/53171

erals and dietary fiber [5, 7, 8].

dietary fiber [13-16].

**the Functional Properties of Chia Meals**

Chia (*Salvia hispanica* L.) is an annual herbaceous plant that belongs to the *Lamiaceae* family, which is native to southern Mexico and northern Guatemala. The *Salvia hispanica* fruit con‐ sists of four nutlets, similar to an indehiscent achene, which contain a single seed. These nut‐ lets are commonly called "seeds" [1]. Chia seed, together with corn, beans, and amaranth were important crops for pre-Columbian civilizations in America, including the Mayan and Aztec populations [2, 3]. With time its use was abandoned, but by at the end of the last cen‐ tury there was a resurgence of interest in chia due to its nutritional value [4]. Chia is consid‐ ered an alternative crop to diversify and stabilize the economy of Northwestern Argentina [5]. The plant produces numerous small white and dark seeds that mature in autumn [6]. These seeds contain about 30% oil, and they mainly consist of unsaturated fatty acids [4, 7]. Chia seeds are a natural source of omega-3 fatty acids, antioxidants, proteins, vitamins, min‐

Chia meal (residue of the seeds after oil extraction) is a good source of proteins (19-23%) [9], dietary fiber (33.9-39.9%) [10], and compounds with antioxidant activity [7]. It also ex‐ hibits some interesting functional properties for its use in the food industry [11]. Function‐ al properties are generally associated with the presence of proteins [12] and also of

Dietary fiber (DF) consists of a heterogeneous mixture of compounds that are classified ac‐ cording to their physical properties and effects of their intake into: soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) [17], referring to the solubility of fibers in water. Plant secretions such as pectins and gums, components such as mucilage, and chelating

and reproduction in any medium, provided the original work is properly cited.

© 2013 I. Capitani et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,


**Chapter 19**

## **Effect of Mucilage Extraction on the Functional Properties of Chia Meals**

Marianela I. Capitani, Susana M. Nolasco and Mabel C. Tomás

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53171

## **1. Introduction**

[37] Holanda J S., Oliveira J O., Ferreira A C. Enriquecimento proteico do pedúnculo de caju com emprego de leveduras, para alimentação animal. Pesquisa Agropecuária

[38] Hennies P T. Produção de pectinases de Penicillium italicum através de fermentação em meio sólido. Masters dissertation. Universidade Estadual de Campinas; 1996.

[39] Martins N., Silva D., Martins E C., Silva R., Gomes E. Seleção de fungos termofílicos produtores de pectinase em fermentação em estado sólido de resíduos agroindus‐

[40] Lomauro C J., Bakshi A S., Labuza T P. Ealuation of food moisture sorption isotherm equations. Part I: fruit, vegetable and meat products. Lebensmittel – Wisseenschaft &

[41] Ferreira C D., Pena R S. Comportamento higroscópio da farinha de pupunha (Bactris

[42] Rodrigues M I., Iemma A F. Planejamento de Experimentos e Otimização de Proces‐ sos: uma estratégia sequencial de planejamentos. Campinas: Casa do Pão Editora;

[43] Berovic M., Ostroversnik H. Production of Aspergillus niger pectolytic enzymes by solid state bioprocessing of apple pomace. Journal of Biotechnology 1997; 53(1) 47-53.

[44] Bari M R., Alizadeh M., Farbeh F. Optimizing endopectinases production from date pomace by Aspergillus niger PC5 response surface methodology. Food and Bioprod‐

[45] Gomes E., Guez M A U., Martin N., Silva R. Enzimas termoestáveis: fonte, produção

[46] Zheng Z., Shetty K. Cranberry processing waste for solid state fungal inoculats pro‐

[47] Venturini Filho W G. Bebidas Não alcoólicas: Ciência e Tecnologia. São Paulo: Bluch‐

[48] Maller A., Damásio A R L., Silva T M., Jorge J A., Terezin H F., Polizeli M L T M. Biotechnological potential of agro-industrial wastes as a carbon source to thermosta‐ ble polygalacturonase production in Aspergillus niveus. Enzyme Research 2011; 2011

gasipaes). Ciência e Tecnologia de Alimentos 2003; 23 251-255.

e aplicação industrial. Química Nova 2007; 30 136-145.

duction. Process Biochemistry 2000; 33(3) 323-329.

Brasileira 1998; 33 787-792.

Technologie 1985; 18 111-117.

ucts Processing 2010; 88(1) 67-72.

2005.

420 Food Industry

er, 2010.

1-6.

triais. In: SINAFERM2005, 2005, Recife, Brazil.

Chia (*Salvia hispanica* L.) is an annual herbaceous plant that belongs to the *Lamiaceae* family, which is native to southern Mexico and northern Guatemala. The *Salvia hispanica* fruit con‐ sists of four nutlets, similar to an indehiscent achene, which contain a single seed. These nut‐ lets are commonly called "seeds" [1]. Chia seed, together with corn, beans, and amaranth were important crops for pre-Columbian civilizations in America, including the Mayan and Aztec populations [2, 3]. With time its use was abandoned, but by at the end of the last cen‐ tury there was a resurgence of interest in chia due to its nutritional value [4]. Chia is consid‐ ered an alternative crop to diversify and stabilize the economy of Northwestern Argentina [5]. The plant produces numerous small white and dark seeds that mature in autumn [6]. These seeds contain about 30% oil, and they mainly consist of unsaturated fatty acids [4, 7]. Chia seeds are a natural source of omega-3 fatty acids, antioxidants, proteins, vitamins, min‐ erals and dietary fiber [5, 7, 8].

Chia meal (residue of the seeds after oil extraction) is a good source of proteins (19-23%) [9], dietary fiber (33.9-39.9%) [10], and compounds with antioxidant activity [7]. It also ex‐ hibits some interesting functional properties for its use in the food industry [11]. Function‐ al properties are generally associated with the presence of proteins [12] and also of dietary fiber [13-16].

Dietary fiber (DF) consists of a heterogeneous mixture of compounds that are classified ac‐ cording to their physical properties and effects of their intake into: soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) [17], referring to the solubility of fibers in water. Plant secretions such as pectins and gums, components such as mucilage, and chelating

© 2013 I. Capitani et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

agents such as phytates are sources of SDF; whereas cellulose, lignin, some fractions of hem‐ icellulose, phenolic compounds such as tannins and lipid structures such as waxes, suberins and cutins constitute IDF [18].

**2. Materials and methods**

**2.2. Meal without mucilage (Msm)**

*2.2.1. Mucilage extraction*

**2.3. Meal with mucilage (Ms)**

between x136 and x5000.

**2.5. Characterization of meals**

*2.5.1. Proximate composition*

practices Ba 2a

**2.4. Scanning Electron Microscopy (SEM)**


Chia seeds were obtained from commercial sources in Salta, Argentina (25º S and 65.5º W). They were cleaned manually by removing the foreign matter such as stones, dirt and broken seeds. They were packed in hermetic plastic vessels and stored at 5ºC until further use.

Effect of Mucilage Extraction on the Functional Properties of Chia Meals

http://dx.doi.org/10.5772/53171

423

Meal without mucilage refers to the residue obtained after the oil extraction process using a Soxhlet apparatus following the IUPAC Standard Method [33] (*n*-hexane under reflux, 8 h,

The mucilage was extracted of chia seeds previously soaked in water (1:4) for 4 hours at room temperature. This mixture was distributed into plastic trays and covered with alumi‐ num foil and frozen at -80°C, lyophilized, and the mucilage was removed by a sieving proc‐

The data corresponding to the meal with mucilage that was considered in the analysis of the re‐ sults corresponds to that reported by Capitani *et al*. [11]. The meal was obtained after oil solvent extraction (*n*-hexane) in a Soxhlet apparatus (Buenos Aires, Argentina) by thermal cycles at 80ºC for 8 h, following the IUPAC Standard Method [33], of chia seeds previously ground in a

Meals (Msm and Ms) were homogenized and stored in plastic vessels at 5ºC until further use.

The whole seeds and seeds after mucilage removal were adhered to a cover slip, coated with a thin gold film (600 Å) in a sputter coater (Pelco 91000) and observed in a scanning electron microscope (LEO model EVO 40) at 5 kV. Longitudinal sections were sliced with a razor blade, after being plunged into liquid nitrogen to ensure the maintenance of their internal structure, and analyzed by microscopy using the same procedure and magnification ranges

Moisture, crude fiber and ash content were determined according to AOCS recommended


laboratory grinder (Moulinex, horizontal blade grinder, Buenos Aires, Argentina).

90 °C,) of seeds that had previously had the mucilage extracted.

ess (20 sieve mesh ASTM, 840 µm) (3 sections of 15 min each).

**2.1. Seeds**

The functional properties of food components can be defined as any physicochemical property that affects and/or modifies some of its characteristics and that contributes to the quality of the final product. Knowledge about of functional properties such as color, parti‐ cle size, water holding, absorption and adsorption capacity, as well as those linked to the affinity for lipid components is very useful for the food industry, because during the proc‐ essing some modifications can occur that must be taken into account according to the us‐ age of the final product and its marketing conditions [19]. For example, water-holding capacity (WHC) is related to the freshness and softness effect present in bakery products, and the oil-holding capacity (OHC) is related to the un-fatty effect in fried food when it is low and to the juiciness and texture in meat products when it is high [18, 20, 21].

In addition to the characteristics mentioned above, it is important to consider the physio‐ logical effects of the DF intake. Given the capacity of SDF to form gels, it increases the viscosity of the bolus in the gastrointestinal tract, slowing the intestinal transit, making di‐ gestion and the absorption of nutrients more efficient, providing more of a feeling of sati‐ ety. Soluble fiber are fermentable fibers that can be microbiologically decomposed in the colon, producing gases such as carbon dioxide, hydrogen and methane, and short-chain fatty acids (acetic, propionic and butyric) which are absorbed and used as energy sources. Some of the most important beneficial effects of SDF is that it regulates blood sugar and lower cholesterol levels. On the other hand, IDF is responsible for adding bulk to the stool, speeding the passage of stool through the intestine by promoting peristalsis, allevi‐ ating constipation and other gastrointestinal disorders [22, 23]. Both types of fiber may al‐ so reduce the risk of obesity, hypertension, appendicitis, and other disorders [24]. The beneficial effects noted above show the important role that DF play in human intake, and that is why a daily intake of 25-30 g is recommended, with a good SDF/IDF balance (a minimum of 30% SDF and 70% IDF, optimum 50/50 ratio) in order to benefit from both fractions of fiber [25, 26].

Chia mucilage (SDF), a complex carbohydrate of high molecular weight, is an important component of the seed due to its physiological role. The mucilage is secreted when the seed comes into contact with water, generating high-viscosity solutions [27, 28]. Many studies have examined the functional properties of different types of gums (*Linux usitatissimum*, *Opuntia Picus indica, Alyssum homolocarpum, Psyllium plantago)* [29-32]. However, little infor‐ mation has been reported on the functionality of chia seed mucilage as a stabilizing or thick‐ ening agent of food products.

The objective of the present work was to perform a comparative evaluation of the func‐ tional properties of chia meals (*Salvia hispanica* L.) obtained from seeds with and without mucilage.

## **2. Materials and methods**

## **2.1. Seeds**

agents such as phytates are sources of SDF; whereas cellulose, lignin, some fractions of hem‐ icellulose, phenolic compounds such as tannins and lipid structures such as waxes, suberins

The functional properties of food components can be defined as any physicochemical property that affects and/or modifies some of its characteristics and that contributes to the quality of the final product. Knowledge about of functional properties such as color, parti‐ cle size, water holding, absorption and adsorption capacity, as well as those linked to the affinity for lipid components is very useful for the food industry, because during the proc‐ essing some modifications can occur that must be taken into account according to the us‐ age of the final product and its marketing conditions [19]. For example, water-holding capacity (WHC) is related to the freshness and softness effect present in bakery products, and the oil-holding capacity (OHC) is related to the un-fatty effect in fried food when it is

low and to the juiciness and texture in meat products when it is high [18, 20, 21].

In addition to the characteristics mentioned above, it is important to consider the physio‐ logical effects of the DF intake. Given the capacity of SDF to form gels, it increases the viscosity of the bolus in the gastrointestinal tract, slowing the intestinal transit, making di‐ gestion and the absorption of nutrients more efficient, providing more of a feeling of sati‐ ety. Soluble fiber are fermentable fibers that can be microbiologically decomposed in the colon, producing gases such as carbon dioxide, hydrogen and methane, and short-chain fatty acids (acetic, propionic and butyric) which are absorbed and used as energy sources. Some of the most important beneficial effects of SDF is that it regulates blood sugar and lower cholesterol levels. On the other hand, IDF is responsible for adding bulk to the stool, speeding the passage of stool through the intestine by promoting peristalsis, allevi‐ ating constipation and other gastrointestinal disorders [22, 23]. Both types of fiber may al‐ so reduce the risk of obesity, hypertension, appendicitis, and other disorders [24]. The beneficial effects noted above show the important role that DF play in human intake, and that is why a daily intake of 25-30 g is recommended, with a good SDF/IDF balance (a minimum of 30% SDF and 70% IDF, optimum 50/50 ratio) in order to benefit from both

Chia mucilage (SDF), a complex carbohydrate of high molecular weight, is an important component of the seed due to its physiological role. The mucilage is secreted when the seed comes into contact with water, generating high-viscosity solutions [27, 28]. Many studies have examined the functional properties of different types of gums (*Linux usitatissimum*, *Opuntia Picus indica, Alyssum homolocarpum, Psyllium plantago)* [29-32]. However, little infor‐ mation has been reported on the functionality of chia seed mucilage as a stabilizing or thick‐

The objective of the present work was to perform a comparative evaluation of the func‐ tional properties of chia meals (*Salvia hispanica* L.) obtained from seeds with and without

and cutins constitute IDF [18].

422 Food Industry

fractions of fiber [25, 26].

ening agent of food products.

mucilage.

Chia seeds were obtained from commercial sources in Salta, Argentina (25º S and 65.5º W). They were cleaned manually by removing the foreign matter such as stones, dirt and broken seeds. They were packed in hermetic plastic vessels and stored at 5ºC until further use.

## **2.2. Meal without mucilage (Msm)**

Meal without mucilage refers to the residue obtained after the oil extraction process using a Soxhlet apparatus following the IUPAC Standard Method [33] (*n*-hexane under reflux, 8 h, 90 °C,) of seeds that had previously had the mucilage extracted.

#### *2.2.1. Mucilage extraction*

The mucilage was extracted of chia seeds previously soaked in water (1:4) for 4 hours at room temperature. This mixture was distributed into plastic trays and covered with alumi‐ num foil and frozen at -80°C, lyophilized, and the mucilage was removed by a sieving proc‐ ess (20 sieve mesh ASTM, 840 µm) (3 sections of 15 min each).

#### **2.3. Meal with mucilage (Ms)**

The data corresponding to the meal with mucilage that was considered in the analysis of the re‐ sults corresponds to that reported by Capitani *et al*. [11]. The meal was obtained after oil solvent extraction (*n*-hexane) in a Soxhlet apparatus (Buenos Aires, Argentina) by thermal cycles at 80ºC for 8 h, following the IUPAC Standard Method [33], of chia seeds previously ground in a laboratory grinder (Moulinex, horizontal blade grinder, Buenos Aires, Argentina).

Meals (Msm and Ms) were homogenized and stored in plastic vessels at 5ºC until further use.

### **2.4. Scanning Electron Microscopy (SEM)**

The whole seeds and seeds after mucilage removal were adhered to a cover slip, coated with a thin gold film (600 Å) in a sputter coater (Pelco 91000) and observed in a scanning electron microscope (LEO model EVO 40) at 5 kV. Longitudinal sections were sliced with a razor blade, after being plunged into liquid nitrogen to ensure the maintenance of their internal structure, and analyzed by microscopy using the same procedure and magnification ranges between x136 and x5000.

#### **2.5. Characterization of meals**

#### *2.5.1. Proximate composition*

Moisture, crude fiber and ash content were determined according to AOCS recommended practices Ba 2a -38, Ba 6-84 and Ba 5a -49, respectively [34]. Oil and nitrogen content (N) were determined following IUPAC Standard Method [33] and AOAC Method [35], respectively. Protein content was calculated as nitrogen x 6.25. Carbohydrate content was estimated by calculating the nitrogen-free extract (NFE) by difference using Eq. (1).

$$\text{NFE:} 100 - \left( \text{oil} + \text{protein} + \text{crude} \, \text{fiber} + \text{ash} \right) \tag{1}$$

*2.5.5.2. Water Absorption Capacity (WAbC)*

pressed as gram water absorbed per gram sample.

*2.5.5.3. Organic Molecule Absorption Capacity (OMAC)*

*2.5.5.4. Emulsifying Activity (EA) and Emulsion Stability (ES)*

weight gain (g oil/sample g).

tering % 25-30 mm).

This property was determined according to the AACC method 88-04 [41]. Approximate wa‐ ter absorption capacity was first determined by weighing out 2 g (d.b.) sample, adding water until saturation (approx. 35 mL) and centrifuging at 2000 x g for 10 min in a Rolco Model CR-5850, 22-cm radius centrifuge (Buenos Aires, Argentina). Approximate water absorption capacity was calculated by dividing the increase in sample weight (g), by initial weight, quantifying the water needed to complete the original sample weight (2 g d.b.) to 15 g. Wa‐ ter absorption capacity (WAbC) was then determined by placing samples in four tubes, add‐ ing different quantities of water (1.5 and 0.5 mL water above original weight, and 1.5 and 0.5 mL water below; one in each tube), agitating vigorously, and centrifuging the samples at 2000 x g for 10 min in a Rolco Model CR 5850. The supernatant was discarded and the resi‐ due weighed. Average water absorbed was calculated, and WAbC was determined and ex‐

Effect of Mucilage Extraction on the Functional Properties of Chia Meals

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425

This capacity was determined according to the method of Zambrano *et al*. [19]. A three gram (d b.) sample was placed in excess quantity corn oil (approx. 25 mL) for 24 h at room tem‐ perature, and then centrifuged at 2000 x g for 15 min in a Rolco Model CR-5850. OMAC was expressed as the absorbed hydrophobic component and calculated in terms of sample

These properties were evaluated according to the method [36] of Chau *et al*. [40]. Briefly, 100 mL 2 g/100 mL suspension was homogenized using an Ultra-Turrax T25 disperser (Janke & Kunkel, IKA-Labortechnik, Germany) at 7800 rpm for 2 min. Then, 100 mL corn oil (density 0.92 g/mL, Arcor) were added and homogenized at 15,000 rpm for 2 min. Emulsions were centrifuged in a 15 mL graduated centrifuge tube at 455 x g for 10 min, and then emulsion volume was measured. The EA was expressed as the remaining volume of the centrifuged emulsion corresponding to 100 mL of initial emulsion. The emulsion stability was determined by heating the emulsions to 80ºC for 30 min, cooling them to room temperature and then centrifuging the samples at 455 g for 10 min. ES was expressed as the remaining volume of the centrifuged emulsion corresponding to 100 mL of initial emulsion. On the other hand, all emulsions were evaluated by optical characterization using a Vertical Scan Analyzer (QuickSCAN, Beckman Coulter, Fuller‐ ton, USA). The QuickSCAN head scans the entire length of the sample (approximately 65 mm), collecting backscattering (BS) data every 40µm. Thus, it is possible to obtain curves showing the percentage of backscattering light flux, relative to external standards, as a function of the sample height in mm [42]. Coalescence kinetics were determined by measuring the mean values of BS as a function of time in the 25-30 mm zone (Backscat‐

#### *2.5.2. Total, soluble and insoluble dietary* fi*ber*

Total dietary fiber (TDF), soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) were determined according the enzymatic gravimetric method [36].

#### *2.5.3. Neutral Detergent* fi*ber (NDF), Acid Detergent* fi*ber (ADF), lignin, cellulose and hemicellulose*

The vegetable cell was separated into two parts (Van Soest method): cell content (highly di‐ gestible) and cell wall (partially digestible). The cell wall was analyzed and its components (cellulose, hemicellulose and lignin) were determined. The technique makes use of acidic and neutral detergent [35, 37].

#### *2.5.4. Antioxidant activity*

The extraction of phenolic compounds was carried out according to the method of Re *et al*. [38]. Ten mL ethanol were added to 1 g sample, then it was homogenized in Vortex for 2 min, decanted and filtered (0.45 µm nylon paper). The supernatant was transferred into a flask and evaporated using a rotavapor apparatus (BUCHI R124, Germany) to concentrate the sample. It was then redissolved in 1000 µL ethanol.

A spectrophotometric method was used to determine the antioxidant activity using a Hita‐ chi U-1900 UVeVIS spectrophotometer (Japan). The antioxidant activity was quantified by a dying assay of the radical cation ABTS+ measuring ABTS+ reduction as the percentage of ab‐ sorption inhibition at 734 nm, just 6 min later. The radical cation ABTS and potassium per‐ sulfate were obtained from Sigma Aldrich. Chlorogenic acid was used as standard antioxidant. Results were expressed as µmol/L Trolox g/sample, considering that chlorogen‐ ic acid diminishes twice the amount of absorption than Trolox [39].

#### *2.5.5. Functional properties*

### *2.5.5.1. Water-Holding (WHC) and Oil-Holding Capacity (OHC)*

Water and oil holding capacities were determined according to the method of Chau *et al*. [40]. Briefly, 1 g (dry base (d.b.)) sample was weighed and then stirred into 10 mL distilled water or corn oil (density 0.92 g/mL, Arcor). These suspensions were centrifuged at 2200 x g for 30 min (Rolco Centrifuge Refrigerate, Model CR-5850, 22 cm radius, Buenos Aires, Ar‐ gentina) and the supernatant volumes were measured. Water-holding capacity was ex‐ pressed as gram water held per gram sample, and oil-holding capacity as gram oil held per gram sample.

## *2.5.5.2. Water Absorption Capacity (WAbC)*

determined following IUPAC Standard Method [33] and AOAC Method [35], respectively. Protein content was calculated as nitrogen x 6.25. Carbohydrate content was estimated by

Total dietary fiber (TDF), soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) were

The vegetable cell was separated into two parts (Van Soest method): cell content (highly di‐ gestible) and cell wall (partially digestible). The cell wall was analyzed and its components (cellulose, hemicellulose and lignin) were determined. The technique makes use of acidic

The extraction of phenolic compounds was carried out according to the method of Re *et al*. [38]. Ten mL ethanol were added to 1 g sample, then it was homogenized in Vortex for 2 min, decanted and filtered (0.45 µm nylon paper). The supernatant was transferred into a flask and evaporated using a rotavapor apparatus (BUCHI R124, Germany) to concentrate

A spectrophotometric method was used to determine the antioxidant activity using a Hita‐ chi U-1900 UVeVIS spectrophotometer (Japan). The antioxidant activity was quantified by a

sorption inhibition at 734 nm, just 6 min later. The radical cation ABTS and potassium per‐ sulfate were obtained from Sigma Aldrich. Chlorogenic acid was used as standard antioxidant. Results were expressed as µmol/L Trolox g/sample, considering that chlorogen‐

Water and oil holding capacities were determined according to the method of Chau *et al*. [40]. Briefly, 1 g (dry base (d.b.)) sample was weighed and then stirred into 10 mL distilled water or corn oil (density 0.92 g/mL, Arcor). These suspensions were centrifuged at 2200 x g for 30 min (Rolco Centrifuge Refrigerate, Model CR-5850, 22 cm radius, Buenos Aires, Ar‐ gentina) and the supernatant volumes were measured. Water-holding capacity was ex‐ pressed as gram water held per gram sample, and oil-holding capacity as gram oil held per

fi

NFE:100 oil protein crudefiber ash -+ + + ( ) (1)

*ber (ADF), lignin, cellulose and hemicellulose*

measuring ABTS+ reduction as the percentage of ab‐

calculating the nitrogen-free extract (NFE) by difference using Eq. (1).

determined according the enzymatic gravimetric method [36].

fi*ber*

*ber (NDF), Acid Detergent* 

*2.5.2. Total, soluble and insoluble dietary* 

fi

the sample. It was then redissolved in 1000 µL ethanol.

ic acid diminishes twice the amount of absorption than Trolox [39].

*2.5.5.1. Water-Holding (WHC) and Oil-Holding Capacity (OHC)*

dying assay of the radical cation ABTS+

*2.5.3. Neutral Detergent* 

424 Food Industry

and neutral detergent [35, 37].

*2.5.4. Antioxidant activity*

*2.5.5. Functional properties*

gram sample.

This property was determined according to the AACC method 88-04 [41]. Approximate wa‐ ter absorption capacity was first determined by weighing out 2 g (d.b.) sample, adding water until saturation (approx. 35 mL) and centrifuging at 2000 x g for 10 min in a Rolco Model CR-5850, 22-cm radius centrifuge (Buenos Aires, Argentina). Approximate water absorption capacity was calculated by dividing the increase in sample weight (g), by initial weight, quantifying the water needed to complete the original sample weight (2 g d.b.) to 15 g. Wa‐ ter absorption capacity (WAbC) was then determined by placing samples in four tubes, add‐ ing different quantities of water (1.5 and 0.5 mL water above original weight, and 1.5 and 0.5 mL water below; one in each tube), agitating vigorously, and centrifuging the samples at 2000 x g for 10 min in a Rolco Model CR 5850. The supernatant was discarded and the resi‐ due weighed. Average water absorbed was calculated, and WAbC was determined and ex‐ pressed as gram water absorbed per gram sample.

#### *2.5.5.3. Organic Molecule Absorption Capacity (OMAC)*

This capacity was determined according to the method of Zambrano *et al*. [19]. A three gram (d b.) sample was placed in excess quantity corn oil (approx. 25 mL) for 24 h at room tem‐ perature, and then centrifuged at 2000 x g for 15 min in a Rolco Model CR-5850. OMAC was expressed as the absorbed hydrophobic component and calculated in terms of sample weight gain (g oil/sample g).

#### *2.5.5.4. Emulsifying Activity (EA) and Emulsion Stability (ES)*

These properties were evaluated according to the method [36] of Chau *et al*. [40]. Briefly, 100 mL 2 g/100 mL suspension was homogenized using an Ultra-Turrax T25 disperser (Janke & Kunkel, IKA-Labortechnik, Germany) at 7800 rpm for 2 min. Then, 100 mL corn oil (density 0.92 g/mL, Arcor) were added and homogenized at 15,000 rpm for 2 min. Emulsions were centrifuged in a 15 mL graduated centrifuge tube at 455 x g for 10 min, and then emulsion volume was measured. The EA was expressed as the remaining volume of the centrifuged emulsion corresponding to 100 mL of initial emulsion. The emulsion stability was determined by heating the emulsions to 80ºC for 30 min, cooling them to room temperature and then centrifuging the samples at 455 g for 10 min. ES was expressed as the remaining volume of the centrifuged emulsion corresponding to 100 mL of initial emulsion. On the other hand, all emulsions were evaluated by optical characterization using a Vertical Scan Analyzer (QuickSCAN, Beckman Coulter, Fuller‐ ton, USA). The QuickSCAN head scans the entire length of the sample (approximately 65 mm), collecting backscattering (BS) data every 40µm. Thus, it is possible to obtain curves showing the percentage of backscattering light flux, relative to external standards, as a function of the sample height in mm [42]. Coalescence kinetics were determined by measuring the mean values of BS as a function of time in the 25-30 mm zone (Backscat‐ tering % 25-30 mm).

## **2.6. Statistical analysis**

The results obtained were analyzed using ANOVA and Tukey's test (p ≤ 0.05), using Infostat software [43].

## **3. Results and discussion**

The nutlet of Salvia hispanica consists of the seed and a pericarp surrounding the seed. The true seed, in turn, consists of a coat (testa), the endosperm and the embryo, consisting main‐ ly of two cotyledons [1]. Basically, the pericarp of the chia seed is similar to that other Nepe‐ toideaes because it shows cuticle, exocarp, mesocarp, layers of sclereids and endocarp. The cells of the mesocarp and exocarp are parenchimatic. Figure 1 shows a scanning electron mi‐ croscopy of a *Salvia hispanica* nutlet. In the exocarp there often are cells that produce muci‐ lage when the nutlets get wet (Figure 1D).

Figure 2 shows SEM microscopy of *Salvia hispanica* L. seeds after mucilage extraction. In these images it can be observed that the mixocarpy phenomenon occurs in the outer layers (cuticle and exocarp). After removing the mucilage, the nutlet surface is characterized by small hill-like eminences, spaced, that cover the entire surface, corresponding to the meso‐ carp cells. Chia seeds presented a similar structure to that of two mucilaginous species (*Car‐ richtera annua* and *Anastatica hierochuntica)*, which could be associated with the presence of concentric aggregates of glucuronic acid [44]. The retention of the mucilage close to the seed can be due to the association of the mucilage with the columella (a secondary wall cell pro‐ duced after mucilage secretion) and portions of the cell wall [45].

**Figure 1.** SEM microscopy of *Salvia hispanica* nutlets. (A) Whole nutlet, lateral view (x150), (B) pericarp surface

Effect of Mucilage Extraction on the Functional Properties of Chia Meals

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427

**Figure 2.** SEM microscopy of *Salvia hispanica* L. nutlets after mucilage extraction. (A) Whole nutlet (x136), (B) nutlet

(x5000), (C) broken nutlet, longitudinal section (x149), (D) broken nutlet (x1500).

surface (x5000), (C) broken nutlet (x150), (D) broken nutlet (x3500).

The proximate composition of chia meals with and without mucilage is presented in Table 1. Both meals were characterized by a high protein content, higher than that reported for sun‐ flower meals of different origin (20.6-23.1%) [46] and canola meals (36.1-40.0%) [47, 48], and within the range of the values reported for linseed meals (38.9-43.3%) [48, 49].

On the other hand, both types of meals presented a high crude fiber content, with values higher than those reported for sesame, soybean, linseed and canola meals, 5.8%, 3.5%, 5.27% and 11.54%, respectively [50, 48].

In Table 2 it is possible to observe that both types of meals presented a high content of TDF, consisting mainly of IDF. Even though the value of SDF was relatively low, this could be attributed to the fact that, during the determination of this fiber, some components were not quantified because they cannot precipitate during the treatment with ethanol, and thus SDF was underestimated [26]. It is worth noting that the meal obtained from seeds that previous‐ ly had their mucilage extracted (Msm) exhibited a statistically higher content of IDF (p<0.05) than that for Ms, at the expense of a significant decrease in its SDF content. These results are consistent with the data obtained from the analysis of NDF, consisting of cellulose, hemicel‐ lulose and lignin (structural polysaccharides that contribute to the IDF fraction), which was statistically higher (p<0.05) in Msm (Table 3). Regarding Ms, it presented a better IDF/SDF balance, with a 89/11 ratio.

**2.6. Statistical analysis**

**3. Results and discussion**

lage when the nutlets get wet (Figure 1D).

and 11.54%, respectively [50, 48].

balance, with a 89/11 ratio.

duced after mucilage secretion) and portions of the cell wall [45].

within the range of the values reported for linseed meals (38.9-43.3%) [48, 49].

software [43].

426 Food Industry

The results obtained were analyzed using ANOVA and Tukey's test (p ≤ 0.05), using Infostat

The nutlet of Salvia hispanica consists of the seed and a pericarp surrounding the seed. The true seed, in turn, consists of a coat (testa), the endosperm and the embryo, consisting main‐ ly of two cotyledons [1]. Basically, the pericarp of the chia seed is similar to that other Nepe‐ toideaes because it shows cuticle, exocarp, mesocarp, layers of sclereids and endocarp. The cells of the mesocarp and exocarp are parenchimatic. Figure 1 shows a scanning electron mi‐ croscopy of a *Salvia hispanica* nutlet. In the exocarp there often are cells that produce muci‐

Figure 2 shows SEM microscopy of *Salvia hispanica* L. seeds after mucilage extraction. In these images it can be observed that the mixocarpy phenomenon occurs in the outer layers (cuticle and exocarp). After removing the mucilage, the nutlet surface is characterized by small hill-like eminences, spaced, that cover the entire surface, corresponding to the meso‐ carp cells. Chia seeds presented a similar structure to that of two mucilaginous species (*Car‐ richtera annua* and *Anastatica hierochuntica)*, which could be associated with the presence of concentric aggregates of glucuronic acid [44]. The retention of the mucilage close to the seed can be due to the association of the mucilage with the columella (a secondary wall cell pro‐

The proximate composition of chia meals with and without mucilage is presented in Table 1. Both meals were characterized by a high protein content, higher than that reported for sun‐ flower meals of different origin (20.6-23.1%) [46] and canola meals (36.1-40.0%) [47, 48], and

On the other hand, both types of meals presented a high crude fiber content, with values higher than those reported for sesame, soybean, linseed and canola meals, 5.8%, 3.5%, 5.27%

In Table 2 it is possible to observe that both types of meals presented a high content of TDF, consisting mainly of IDF. Even though the value of SDF was relatively low, this could be attributed to the fact that, during the determination of this fiber, some components were not quantified because they cannot precipitate during the treatment with ethanol, and thus SDF was underestimated [26]. It is worth noting that the meal obtained from seeds that previous‐ ly had their mucilage extracted (Msm) exhibited a statistically higher content of IDF (p<0.05) than that for Ms, at the expense of a significant decrease in its SDF content. These results are consistent with the data obtained from the analysis of NDF, consisting of cellulose, hemicel‐ lulose and lignin (structural polysaccharides that contribute to the IDF fraction), which was statistically higher (p<0.05) in Msm (Table 3). Regarding Ms, it presented a better IDF/SDF

**Figure 1.** SEM microscopy of *Salvia hispanica* nutlets. (A) Whole nutlet, lateral view (x150), (B) pericarp surface (x5000), (C) broken nutlet, longitudinal section (x149), (D) broken nutlet (x1500).

**Figure 2.** SEM microscopy of *Salvia hispanica* L. nutlets after mucilage extraction. (A) Whole nutlet (x136), (B) nutlet surface (x5000), (C) broken nutlet (x150), (D) broken nutlet (x3500).


The antioxidant activity of the two chia meals compared with other types of meals is shown in Table 4. Both for Ms and Msm, the activity was high, without a significant dif‐ ference between them (p>0.05). These values were higher than those found for wheat bran and sorghum and barley whole grain meals. But they were significantly lower than those found for chia meal obtained as a byproduct of cold-pressing oil extraction. The latter could be attributed to the fact that the meal obtained by pressing shows a higher percent‐ age of residual oil (11.39% d.b.), which contains tocopherols, a class of compound with

Effect of Mucilage Extraction on the Functional Properties of Chia Meals

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429

**Sample Trolox equivalent antioxidant coefficient (TEAC, μmol/g)**

Msm 187.4 ± 33.21 a

Ms**<sup>1</sup>** 226.6 ± 4.13 a

Chia meal from oil pressing extraction**<sup>1</sup>** 557.2 ± 28.18 b

Wheat bran 2 48.5

Sorghum meal **<sup>3</sup>** 51.7

Barley meal **<sup>3</sup>** 14.9

Values followed by different letters differ significantly (p ≤ 0.05), according to Tukey's test.

**Table 4.** Antioxidant activity of chia (*Salvia hispanica* L.) meals compared with other meals

and 3.28 g/g, respectively) and similar to that of linseed meal (6.03 g/g) [48].

Regarding the functional properties, the meal with mucilage (Ms) exhibited a statistically higher absorption and water holding capacity (p<0.05) than that of the meal without muci‐ lage (Msm) (Table 5). This behavior can be associated with the presence of mucilage in Ms, which acts as soluble dietary fiber, capable of holding water inside its matrix [51]. The WAbC of both meals was higher than that observed for canola and soybean meals (3.90 g/g

Both types of chia meals presented a low absorption of organic molecules and oil-holding capacity, being significantly higher in Msm. These differences could be explained in terms of the particle size and the cellulose content of the meal [52, 53]. The determination of OHC is

natural antioxidant activity [11].

1 Capitani *et al*., [11] 2 Iqbal *et al*. [57] 3 Ragaee *et at*. [58] Mean value (n = 3)
