Use of Pectinases in Juice Clarification Processes: A Scoping Review

Pectin is an important constituent of the middle lamella and cell wall of plants. They are characterized as heteropolysaccharides with D-galacturonic acid in the main chain linked by α-1,4 glycosidic bonds (Figure 1). In addition, the structure of pectin presents residues of L-rhamnose, arabinose, galactose and xylose and esterification with methyl or acetyl groups [1,2]. Several microorganisms can produce compounds and enzymes capable of degrading pectin and this variety of producible compounds has several applications at the industrial level, especially pectinases produced by filamentous fungi [3,4]. Pectinase is widely used in the fruit juice industry since fruits naturally have a high concentration of pectin. From a commercial perspective, this pectin concentration can cause a turbid appearance in juices. Pectinase will act by degrading the pectin present, leaving a better visual aspect in the product [5-7]. Pulp treatment, fruit juice extraction and clarification are examples of steps where microbial pectinases can be applied as they contribute to viscosity reduction and juice clarity and increase juice yield [8,9]. Clarification using enzymatic treatment is regarded as an alternative to traditional physical-chemical methods and is proving to be an increasingly promising method [8,10]. As they have desirable and advantageous characteristics, industrial enzymes are increasingly in focus, requiring constant research to optimize their production and the discovery of new sources, aiming at cost reduction and process improvement [11]. Given this, there is a need to make enzymes the target of studies, seeking the emergence of new enzyme systems and the discovery of new sources of pectinases. Pectinases are enzymes widely used in paper, food and textile industries for several commercial applications. Because they are effective and cost-effective enzymes, they are increasingly employed in the food industry for clarification in beverages such as fruit juices [12-14]. This study aimed to gather evidence of using pectinases exclusively from microbial sources for the enzymatic clarification of different fruit juices, given the need to discover new sources of pectinases and aim at the emergence of new enzyme systems.

Figure 1:Pectin structure and its functional groups (adapted from Oumer 1 and Cosgrave2).

This is a scoping review that followed the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and met the guidelines of the Cochrane Handbook for Systematic Reviews of Interventions, The Cochrane Collaboration.

Strategic searches, study selection and data extraction

The systematic review was last updated in March 2022. The manual search was performed by searching for eligible articles in the list of references of the studies included. Articles were searched in ScienceDirect, PubMed and PMC (PubMed Central) databases on the NCBI website using the descriptors “pectinase,” “clarification,” “beverages,” “juice” and “pectin” with the Boolean operator “AND.” There was no time limitation; however, there was a language limitation and only articles published in English were included. Only original research articles that used pectinase of microbial origin for juice clarification were included. The excluded articles were as follows: those that used pectinase from sources other than microbial ones; papers that, despite using pectinase, did not use clarification; publications that did not use pectinase in clarification and also studies not published in English and those that were duplicates.

After a systematic search of the databases, 525 publications were identified as potentially eligible. However, duplicates were removed in the pre-selection, resulting in 522 studies (Figure 2). Of these, 395 were excluded for not meeting the inclusion criteria (Table 1). A total of 127 studies remained. After reading the titles and abstracts, 77 articles were selected to be read in full and finally, 33 studies were selected that fit the main focus of the proposal for this study. Appendix 1 shows the excluded studies because they did not meet the current criteria for this systematic review [10,15- 45]. Table 1 shows a summary of the results found in the articles selected after full-text reading and their main findings. It is observed in the studies included in the review that the microorganisms that are most prominent in the production of pectinases are from the genus Aspergillus sp., more specifically Aspergillus niger [23,27,33,34,39,44,45]. Other species of Aspergillus sp. Can also be observed in other studies [10,19,26,28,32,35]. India (28%) and China (27%) have the most research papers published in the English language on pectinases in juice clarification, as shown in (Figure 3). The pectinases constitute a complex and heterogeneous enzyme group. Although some of the research included in the review deals with proteases and tannases, the focus of this review was on Pectin Methyl Esterase (PME), Pectin Lyase (PL) and Polygalacturonases (PGs), including their divisions into endo polygalacturonase (endo-G) and Exo-polygalacturonase (Exo-PG). In 24 of the 33 studies included in the review, polygalacturonases were the most commonly used enzymes. Of these 24, four reported using Exo-PGs, seven reported using Endo-PGs and 13 reported using only Pgs. According to Table 1, PLs were used in four studies and SMCs in two. In addition, eight studies reported using only pectinases without specifying their type. As for the types of juices, apple juice was notably the most studied by researchers.

Figure 2:Flowchart of the article selection process (PRISMA).

Figure 3:Distribution of studies included in the review by country.

Table 1:Articles included in the scoping review.

(**) No information.

It was cited in 19 out of 33 articles. Fruit juices such as orange, papaya, mango, banana, citrus and peach were also highlighted and the results were promising in all the included studies. The pectinases employed were effective in clarification, removing turbidity and improving the aspect of the studied juices. Most studies find a range of 40 °C to 60 °C as the optimal temperature for enzyme activity; however, there is promising research with temperatures below 40 °C, such as 27 °C, 30 °C and 35 °C as the optimal temperature, [20,23,40] and thermal stability in a range from 25 °C to 50 °C [34]. There are also studies where temperatures above 60 °C are described as the optimum temperatures [2,18]. where the enzyme presents stability ranging from 60 °C to 70 °C and works where 65 °C is the optimum temperature for the enzyme [24]. Most studies report acidic pH as the best for enzyme activity, more specifically in the range of 3.5 to 7.0. This result is probably because most enzymes come from filamentous fungi that commonly produce more acidic enzymes, while bacteria tend to produce more alkaline enzymes. Good pH stability was found in some research: a PG from the bacteria Streptomyces halstedii with pH stability of 7 to 11 [21] a PG from the fungus Neosartorya fischeri with pH stability in a pH range of 3.5 to 6.5 [24] and an Endo-PG, also from the fungus Penicillium oxalicum, with pH stability of 2.2 to 7.0 [22].

Pectin, also called pectic substances, is the compound hydrolyzed by pectinases. It is a heteropolysaccharide rich in sugars such as galacturonic acid and methanol. They have an insoluble part, protopectin, which is hydrolyzed by the protopectin’s, which is capable of turning this protopectin into soluble pectin, the pectinaceans [46,47]. Enzymes started to be discovered in the middle of the XIX century. However, only at the beginning of the twentieth century did they start to have an industrial application. The first enzymes applied industrially were the pectinases of fungal origin, with applications focused on wine and fruit juice production. Only years later, enzymes of bacterial origin began to be employed in this sector [12, 48].

Pectinases, or pectinolytic enzymes, are widely distributed in higher plants, modifying pectin during the fruits’ natural ripening process. They efficiently hydrolyze pectin polymers, having a more specific action because they are a heterogeneous group composed of Pectin Methyl Esterase (PME), Pectin Lyase (PL) and polygalacturonases (PGs) (Figure 4) [14,22,49,50]. PME is responsible for removing the ester grouping from the pectin structure, producing pectic acid and methanol in the process [51]. The PGs act in the depolymerization process, catalyzing the hydrolytic cleavage of the Poly Galacturonic Acid (PGA) chain in water, splitting the α1-4 glycosidic bond into galacturonic monomers, the most studied group within the pectinases family. PGs are divided into two subgroups: endo-polygalacturonases (Endo- PG), which can hydrolyze PGA randomly, producing trigalacturonic and digalacturonic acids and Exo-polygalacturonases (Exo-PG), which can hydrolyze PGA into Mon galacturonic acid [52,53,54]. PL also acts in the depolymerization of pectin and can break the glycosidic bonds of pectic acid by catalyzing the β-elimination between two esterified galacturonic acid residues [55]. Enzymes capable of modifying or degrading polysaccharides are called active carbohydrate enzymes (CAZymes).

Figure 4:Different types of pectinases and their attack points within the pectin molecule [26].

According to their structural similarity and an amino acid sequence, they are grouped into five families in the Carbohydrate Active Enzyme (CAZymes) database (http://www.cazy. org): enzymes able to hydrolyze glycosidic bonds between carbohydrates; (II) Glycosyl Transferases (GTs): enzymes in charge of the biosynthesis of glycosidic bonds; (III) Polysaccharide Lyases (PLs): enzymes that fragment polysaccharide chains by a β-elimination mechanism; (IV) Carbohydrate Esterases (CEs): enzymes that catalyze the de-esterification of methyl or acetyl esterified polysaccharides and (V) Auxiliary Activities (AAs): Enzymes that degrade lignin and cleave the monooxygen bond of the polysaccharide [56]. Pectinases are part of a complex enzymatic group with the catalytic ability to hydrolyze the pectin glycosidic bonds by reactions of de-esterification (esterases) or depolymerization (hydrolases and lyases) [57]. According to the CAZy database, the deesterifiers belong to family IV, the class of carbohydrate Esterases (CEs), while the depolymerizers belong to families I and III, the class of glycoside Hydrolases (GHs) and polysaccharide Lyases (PLs), respectively. Next, the main classes of pectinases discussed in this review and their respective families are presented.
• GH28 - Polygalacturonase (PG):
I. Endo – polygalacturonase (Endo-PG) (EC 3.2.1.15);
II. Exo - polygalacturonase (Exo-PG) (EC 3.2.1.67);
• CE8 - Pectin methylesterase (PME) (3.1.1.11);
• PL1, -3, -9 - Pectin lyase (PL) (EC 4.2.2.10);

The microbial source is preferable for obtaining pectinases, although these enzymes can be obtained from other sources. Microbial enzymes from filamentous fungi have an enzymatic pH close to many fruit juices, around 3.0 to 6.0, while bacteria produce more alkaline pectinases. Among the advantages of microbial sources, we can mention the wide biodiversity and rapid growth, in addition to the ease of genetic manipulation [12,13,58,59,60,61].

In the food industry, pectinases are applied in several processes, including reducing the viscosity of fruit pulp, causing better extraction and filtration and can also be used for juice clarification because of their action in the degradation of the pectin present in fruits. Filamentous fungi and yeasts are the main producers of acid pectinases, widely employed to clarify fruit juices and wine production [14,62,63]. Fruit juice is naturally cloudy since fruits have a high concentration of pectin that forms colloids in the juice, causing turbidity, which is not attractive to consumers. Moreover, the traditional juice extraction processes are not very attractive because they consume much energy. Because of this, enzymatic treatment with pectinases is seen as an effective alternative [5,64]. Pectinases are necessary for juice manufacturing because fruits rich in pectin tend to generate juices with higher viscosity and turbidity. Thus, these enzymes are employed in the process, aiming to hydrolyze the polysaccharides into simpler sugars, reducing the viscosity of the juice and giving it a clearer appearance, thus attracting consumers. In addition, these macromolecules contribute to increasing juice redemption, reducing processing time and stimulating the release of phenolic compounds from fruit peels. Treating fruits with pectinases also aids extraction and further reduces the viscosity and turbidity of juices [6,7,64]. Originally, the suspended particles in the juices would be removed by traditional methods through physical processes such as centrifugation or chemical methods such as the addition of tannic acid. However, these methods can interfere with the final product, which is not advantageous considering that the visual aspect of the product directly impacts consumer choice. In this scenario, enzymatic clarification proves to be increasingly promising for its ability to improve product appearance and its ability to improve product quality and product coloration [65-68].

The disadvantages of traditional methods include the juice’s low recovery and being a long and time-consuming process, with some inefficiency. Moreover, the addition of chemicals to aid these processes can cause changes in color, aroma and even flavor of the juice. Therefore, enzymatic clarification becomes preferable to traditional methods because of its high catalytic efficiency, high degree of specificity and low energy consumption [69,30]. Pectinases are responsible for the breakdown of structural polysaccharides in fruit pulps and can reduce the pulp’s viscosity, turbidity and consistency. If the fruit has too much pectin, it can cause a low juice yield and pectinases increase the pulp’s binding capacity, improving this characteristic and improving the visual aspect [70-72,30]. The enzymatic process seems to be one of the best choices for juice production. In this sense, enzymes, being biocatalysts, have activity, substrate specificity and the ability to work in mild environmental conditions; thus, they are environmentally friendly [73,74].

In addition to improving the pulp yield, the enzymatic treatment can eliminate free radicals and increase the amount of reducing sugars, resulting in a clear juice without opacity [75]. Pectinases are increasingly receiving more attention in the world scenario, impacting several areas because of their actions and applications, as an effective biological catalyst and having an environmentally friendly character. Studies with apple juice are the most abundant [62]. The successful application of enzymes for the depectinization of various fruit juices, including apple, banana, orange, lemon, pineapple, grape, pomegranate, mosambi, mango, papaya and guava, has been reported by some researchers [ 76,77,15]. conducted a very promising study with Exo-polygalacturonase from Sporothrix schenckii, achieving a turbidity reduction of approximately 80% in this type of juice. Similar to Diano et al. [26] with pectinase from Aspergillus sp., Saxena et al. [27] and Deng et al. [26] with Aspergillus niger and Ázar et al. [36] with Calonectria pteridis using polygalacturonases also achieved promising results in this type of juice. As in these studies, the enzymes used were of fungal origin, the pH was around 4.0 in all works and the temperature ranged from 40 °C to 60 °C, not exceeding this limit. As mentioned before, enzymes of bacterial origin tend to be more alkaline than those of fungal origin. A clear example is demonstrated in the study by Tapias et al. [21], using polygalacturonase from Streptomyces halstedii to clarify plum and grape juices.

The authors obtained a good clarification as the enzyme reduced turbidity and viscosity and increased the amount of reducing sugars in the juice. However, the optimal enzyme pH was around 7 to 11. The enzyme was observed to be inactive at acidic pH. Another example of the application of enzymes of bacterial origin is presented in the study by Koshy et al. [16], where pectinase from Bacillus tequilensis is used to clarify papaya juice. The enzyme was promising in clarifying this type of juice; however, its optimal pH was 7.5, higher than those found in enzymes of fungal origin. However, although pectinases in the industry are considered a promising method, some drawbacks, such as instability at adverse temperatures and pHs and difficulty in recovery and recyclability, limit their industrial application [36]. The inherent difficulty in enzymatic recovery and recycling reduces operational efficiency. Consequently, there is a growing interest in immobilization techniques to improve enzyme performance in juice processing. Continuous clarification steps, widely employed in industry, are enabled exclusively by enzyme immobilization [78,79]. The application of enzymes with appropriate physicochemical and kinetic properties is essential to preserve product quality and ensure consumer acceptance. Different fruit matrices require distinct enzymatic treatments, for example, grape juice requires the combined application of pectinases and cellulases for the complete elimination of turbidity and viscosity [80]. The instability of the enzyme leads to a certain delay in its industrial advancement since this characteristic in its commercialization, given the multiple pHs and temperatures, limits its resistance and application in commercial processes [81,82]. Therefore, there is a need for research that can optimize the production of these enzymes, aiming to make them more stable [8,78,79].

Hence, thermophilic enzymes are gaining more and more prominence because of their temperature stability during fermentative processes. In addition, research in enzyme immobilization is increasingly gaining space because of cost reduction since it enables enzyme reuse [9,50]. In a study by Sassi et al. [20] using Penicillium occitanis, temperatures of 35 °C were used to clarify pear, banana and citrus juices with endo-polygalacturonase at pH 7.0. The work showed more promising results in pear and banana juices than in citrus, possibly due to the more alkaline pH of the enzyme. Ajayi et al. [23] achieved very successful results in clarifying tomato juice using polygalacturonase from Aspergillus niger at a temperature of 27 °C, which is lower than the commonly used temperature. In addition to demonstrating the effectiveness of clarification, the authors also point out an increase in juice yield, possibly due to the pectin hydrolysis. Benuci et al. [70] conducted a promising study, immobilizing pectinases and proteases on chitosan spheres by polydialdehyde starch, which is considered an effective method for clarifying pomegranate juice. However, the study reported that it would have been more appropriate to have chosen another support, such as glass, since the enzyme system is stressed during clarification, requiring resistant support for the process.

Pectin is an essential component of the cell wall of plants, conferring its rigidity and is important in the fruit’s development. However, despite suffering degradation by pectinases in the ripening process, it is an aggravating factor in the juice industry for interfering with the visual aspect of the product, often displeasing consumers. Therefore, the clarification process is necessary to improve the quality and visual aspect of the product to please the consumers and the use of enzymes is a viable alternative in this scenario. The enzymatic clarification makes this activity environmentally friendly and ecologically correct and besides its low cost and great effectiveness, it does not interfere negatively with the product. Because of this, we conclude that it is more and more necessary to research new enzyme-producing sources, like filamentous fungi, especially pectinases that are effective for application in the food industry, thus increasing the quality of the products that will be marketed.

The authors thank the National Council for Scientific and Technological Development (CNPq), Coordination for the Improvement of Higher Education Personnel (CAPES), Araucária Foundation and Paraná Western State University (UNIOESTE).

This word was supported by National Council for Scientific and Technological Development (CNPq) grants no458859/2014-1 for Alexandre Maller and Coordination for the Improvement of Higher Education Personnel (CAPES) for Bruna.

B.D: drafting, writing, editing; A.C.S: revising; A.M. revising, writing, editing. Furthermore, all the authors have contributed significantly to the conception, planning e interpretation of this work and approval of the manuscript.

The authors have no conflict of interest to declare.

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