**1. Introduction**

200 Food Industrial Processes – Methods and Equipment

Young, J.C.; Zhu, H. & Zhou, T. (2006). Degradation of trichothecenemycotoxins by aqueous

Yousef, A.E. & Marth, E.H. (1985). Degradation of aflatoxin M1 in milk by ultraviolet

Yu, J.; Bhatnagar, D. & Ehrlich, K.C. (2002). Aflatoxinbiosynthesis. *Revista Iberoamericana de*

Zain M.E. (2011). Impact of mycotoxins on human and animals. *Journal of Saudi Chemical*

Zinedine, A.; Brera, C.; Elakhdari, S.; Catano, C.; Debegnach, F.; Angelini, S.; De Santis, B.;

Faid, M.; Benlemlih, M.; Minardi V. & Miraglia, M. (2006). Natural occurrence of mycotoxins in cereals and spices commercialized in Morocco. *Food Control*, Vol. 17,

ozone. *Food and Chemical Toxicology*, Vol. 44, pp. 417–424.

energy. Journal of Food Protection, Vol. 48, pp. 697–698.

*Micología*, Vol. 19, pp. 191-200.

*Society,* Vol. 15, pp. 129-144.

pp. 868–874.

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Pectins are polysaccharides ubiquitous in the plant kingdom and constitute the major component of plant cell walls. The pectinases are a group of related enzymes capable of degrading pectin. Therefore, this group of enzymes have been used for decades in the food and winemaking industry for the processing of fruit juices (Mohnen, 2008; Prade et al., 1999; Ribeiro et al., 2010).

The pectinases are synthesized by plants and microorganisms, the latter being used for industrial production. Microorganisms are used to produce many enzymes of industrial interest in processes relatively inexpensive and environmentally friendly. Moreover, enzymatic catalysis is preferred over other chemical methods since it is more specific, less aggressive and generates less toxicity (Hoondal et al., 2002; Lara-Márquez et al., 2011).

Advances in biotechnology, especially in the fields of molecular biology and microbial genetics, have led to major advances in enzyme technology and have allowed, in many cases, the development of new producing strains and microbial enzymes. The production of pectinases may also benefit from these technologies.

This article reviews the characteristics of pectic substances, the types and mode of action of enzymes which degrade them and the main applications of commercial preparations of microbial pectinases in the food and winemaking industry, followed by a review of new microorganisms and pectolytic enzymes, evaluating new approaches to their production, marketing and use.

### **2. Pectic substances**

Pectic substances are polysaccharides of high molecular weight, with a negative charge, appearing mostly in the middle lamella and the primary cell wall of higher plants, found in the form of calcium pectate and magnesium pectate. They are formed by a central chain containing a variable amount although in high proportion of galacturonic acid residues linked through α-(1-4) glycosidic bonds partially esterified with methyl groups (Fig. 1).

This molecule is known as pectin, while the demethylated molecule is known as polygalacturonic acid or pectic acid. Several L-rhamnopyranosyl residues may be attached to the main chain through its C-1 and C-2 atoms. In addition, galacturonate residue may be

**3. Pectolytic enzymes** 

1. Hydrolysis (polygalacturonases)

**Enzyme EC Nº Main** 

via:

**Esterases** 

**Depolymerases**  *Hydrolases* 

*Lyases* 

preference these enzymes are classified into three types:

2. Transelimination (pectin lyases and pectate lyases)

I. Protopectinases, which solubilize protopectin forming soluble pectin

their mode of action and final product are shown in Table 1 and in Fig. 1.

Protopectinases Protopectin Hydrolysis Pectin

Endopectate lyase 4.2.2.2 Pectic acid Transelimination Unsaturated

Exopectate lyase 4.2.2.9 Pectic acid Transelimination Unsaturated

Table 1. Pectolytic enzymes classified according to its mode of action

**4. Pectic enzymes in nature: Microbial pectinases** 

residues (Hoondal et al., 2002; Lang & Dornenburg, 2000).

**substrate** 

Pectin methyl esterase 3.1.1.11 Pectin Hydrolysis Pectic acid + methanol Pectin acetyl esterase 3.1.1.6 Pectin Hydrolysis Pectic acid + methanol

Endopolygalacturonase 3.2.1.1.5 Pectic acid Hydrolysis Oligogalacturonates Exopolygalacturonase 3.2.1.6.7 Pectic acid Hydrolysis Monogalacturonates

Endopectinlyase 4.2.2.10 Pectin Transelimination Unsaturated methyl-

Pectic enzymes are widely distributed in nature and are produced by bacteria, yeast, fungi (Fig. 2A) and plants. (Lang & Dornenburg, 2000; Whitaker, 1990). In plants, pectic enzymes are very important since they play a role in elongation and cellular growth as well as in fruit ripening (Sakai, 1992; Ward & Moo-Young, 1989; Whitaker, 1990). Pectolytic activity of microorganisms plays a significant role, firstly, in the pathogenesis of plants since these enzymes are the first to attack the tissue (Collmer & Keen, 1986; Whitaker, 1990). In addition, they are also involved in the process of symbiosis and the decay of vegetable

and acetyl residues from pectin giving rise to polygalacturonic acid

The enzymes which hydrolyze pectic substances are known as pectic enzymes, pectinases or pectinolytic enzymes (Blanco et al., 1999). Based on its mode of action and substrate

II. Esterases (pectin methyl esterases and pectin acetyl esterases), which eliminate methoxyl

III. Depolymerases, which break the glycosidic α-(1- 4) bonds between galacturonic residues

Also, the latter enzymes are subdivided into endo- if its pattern of action is random or exoif its pattern of action is at the terminal end (Fogarty & Kelly, 1983; Rexova-Bencova & Markovie, 1976; Sakai, 1992; Whitaker, 1990). The detailed classification of these enzymes,

> **Mode of action**

**Product** 

197

oligogalacturonates

oligogalacturonates

oligogalacturonates

acetylated at the C-2 and C-3 positions, and side chains of residues of neutral sugars may be linked to the galacturonic acid or to the C-4 of the rhamnose residue in the main chain (Caffall & Mohnen, 2009; Mohnen, 2008; Pilnik & Voragen, 1970; Rombouts & Pilnik, 1980).

Fig. 1. Structure (main chain) of low (a) and high (b) methylated pectic substances and site of action of enzymes involved in their degradation

The generic name of pectic substances is used for referring to four types of molecules: protopectin (pectic substance in intact tissue), pectinic acids (polygalacturonan containing >0-75% methylated galacturonate units), pectic acids (polygalacturonan that contains negligible amount of methoxyl groups), and pectins (pectinic acid with at least 75% methylated galacturonate units). Protopectines are insoluble in water, while the rest are wholly or partially soluble in water (Alkorta et al., 1998; Kertesz, 1951).

Pectic substances represent between 0.5-4% of fresh weight plant material (Jayani et al., 2005; Sakai et al., 1993). In addition to their role as cementing and lubricating agents in the cell walls of higher plants, they are responsible for the texture of fruits and vegetables during growth, maturation and their storage (Alkorta et al., 1998; Caffall & Mohnen, 2009). Furthermore, pectic substances are involved in the interaction between plant hosts and their pathogens (Collmer & Keen, 1986; Prade et al., 1999).

Pectins have numerous and important applications in the food and pharmaceutical industries. In the food sector, it is primarily used as a gelling agent, replacing sugars and/or fats in low-calorie food and as nutritional fiber (Panchev et al., 1988; Sakai et al., 1993; Thakur et al., 1997). The pharmaceutical industry offers them as preparations to reduce cholesterol or to act as a lubricant in the intestines thus promoting normal peristaltic movement without causing irritation. In addition, these polysaccharides are used as drug delivery systems, which can also reduce the toxicity of these and make their activity longer lasting without altering their therapeutic effects (Morris et al., 2010; Pilnik & Voragen, 1970; Schols et al., 2009; Thakur et al., 1997).
