Mobilization of Phenolics through Enzymatic Treatment

Application of enzymes is widespread in many industrial processes. These are mainly hydrolytic enzymes that are utilized in the textile, pulp and paper and food and feed industries in large quantities. Among them, the carbohydrases have gained relevance in the liberation and enhancement of bioactive phenolic compounds trapped by gly- cosidic bonds inside the plant cell-wall structures (Madeira et al. 2015). Cellulases, hemicellulases and pectinases are frequently used for enzyme-assisted extraction purposes. The importance of (3-glucosidases in the extractable phenolic aglycone production has been described in several studies so far (see Table 12.2). Other enzymes can also be associated with the liberation of phenolic aglycone moieties. For instance, the tannases (tannin acyl hydrolases) hydrolyze ester and depside bonds in hydrolyzable tannins (mainly tannic acid) to liberate gallic acid. Tannins are present in a variety of plants processed in the food industry (Sagar et al. 2018); therefore, their amount in by-products is considerable. The released gallic acid then can be utilized as intermediate compounds in various organic synthesis processes and food industrial applications (Zhang et al. 2014).

The carbohydrase-assisted extraction of bioactive phenolics from food processing by-products has several advantages over the physical and solvent-based methods, such as the high efficiency, large-scale application and eco-friendly reaction conditions (Shahidi and Yeo 2016). In contrast to the fermentation systems, enzymatic approaches might not cause phenolic loss due to the stable pH conditions and the absence of phenolic degrading and/or polymerizing enzymes of the fermenting microorganism (Zambrano et al. 2018). Moreover, low-cost enzyme preparations with high carbohydrase activity can be obtained via fermentation of various agro-industrial wastes (Tako et al. 2015; Behera and Ray 2016). The produced cocktails then can be used for efficient phenolic extraction; some examples are shown in Table 12.4. Note that wheat bran is a preferred substrate for carbohydrase production.

In most studies, however, commercially available enzyme preparations are applied as extraction biocatalysts. From pectinase preparations, for instance, various types of Pectinex, Grindamyl, Ultrazym, Rohapect and Macer cocktails were used in previous applications (Laroze et al. 2010; Oszmiariski et al. 2011; Cerda et al. 2013). Frequent cellulolytic additives are the Viscozyme and Celluclast multi-enzyme cocktails. The Olivex, Rohavin and Lallzyme (3 are mixed cellulase and pectinase preparations (Laroze et al. 2010; Dal Magro et al. 2016), while the Rohalase OS contain cellulase, xylanase and (3-glucanase activities (Szydlowska-Czerniak et al. 2010). Their usage in a mixed form is also frequent. For instance, the cocktail of Pectinex Ultra Clear and Lallzyme (3 (0.75 U/g, in a ratio of 0.52) proved to be the best treatment condition for high-yield bioactive phenolic extraction from Concord grape juice (Dal Magro et al. 2016). After 52 min incubation at 51°C, the total phenolic acid concentration increased from 23.9 to 28.8 mg/L. In another study, an enzyme cocktail (a mixture of cellulase, pectinase and protease; 50:25:25) at 3.8% concentration at 49°C and pH 6.7 for 85 min offered the highest recovery of phenolic compounds (218 or 301 mg GAE/g of extracts depending on the extraction solvent) from pomegranate peel residues (Mushtaq et al. 2015).

De Camargo et al. (2016) evaluated the use of enzymes as a pretreatment to recover phenolic compounds from wine residues. It was observed that two commercial enzymes, the Pronase (mixture of nonspecific endo- and exoproteases) and Viscozyme, increased the extraction yield of the soluble phenolic compounds from 265 to 302 mg GAE/g DM and from 322 to 371 mg GAE/g DM. respectively. Moreover, this increase correlated with the antioxidant capacity measured by the DPPH radical scavenging method. A recent experiment series tested different commercial enzymes (i.e. Celluclast, Termamyl, Pectinex Ultra SPL, Pentopan 500BG,

TABLE 12.4

Crude Enzyme Cocktails, Their Production and Application for Phenolic Compound Extraction

Condition for

Enzyme

Production

Microorganism

Enzyme Cocktail Produced

Treated Material

Reaction Condition

Phenolic Extraction Yield

References

Wheat bran based SSF

Rhizopus oryzae

Cellulolytic

Finger millet (Ragi)

Enzyme to substrate ratio 1:0.6 (v/w), 30°C, 4 h

2.3 mg/mL

Yadav etal. (2013)

Wheat bran based SSF

Rhizomucor miehei

Cellulolytic

Black grape, apple, pita hay a

Enzyme to substrate ratio 1:0.1 (v/w), 50°C, 5 h. pH 5.0

5393, 1732, 2207 mg GAE/100 g DM

Zambrano et al. (2018)

Oats based SSF

Monascus anka

Cellulolytic

Oat powder

102 U/g enzyme amount, 30°C, 6h

0.23 mg GAE/g DM

Bei et al. (2018)

Wheat bran and sawdust based SSF

Penicillium oxalicum, Trichoderma reesei

Pectinolytic and cellulolytic

Tomato

10% (enzyme/substrate) pectinase (0.81 U), 5% cellulase (0.027 U), 40°C. pH 4.3, 2 h

164.6 mg GAE/100 g fresh weight (FW) with pectinase: 120.2 GAE/100 g FW with cellulase

Neagu etal. (2014)

Wheat bran based SmF

Aspergillus niger, Trichoderma atroviride Trametes Irogii

Cocktails with high P-glucosidase activity

Olive mill wastewater

50°C, 2 h

1.1.0.5 and 0.2 g/L of free hydroxytyrosol depending on the enzyme preparation

Hamza etal. (2012)

Peptone-dextrose medium, wheat straw based SmF

A. niger, Lentinula edodes, Pleurolus djamor, Pleurolus ostreatus var. florida, R. oryzae

Cocktails with endoglucanase, pectinase, xylanase and laccase activities

Rooibos

40°C, 6 h

4.6 g GAE/100 g tea

Pengilly et al. (2008)

Pectinex 3XL and Fungamyl) to recover phenolic phytochemicals from wet and dried red and white grape residues in high amount (Ferri et al. 2016; Tassoni et al. 2016). In the case of wet red grape pomace, about 300 and 350 mg GAE/L phenolic yields were obtained when Fungamyl (2%, 24°C, 2-hour incubation) and Celluclast (1%, 37°C, 2-hour incubation), respectively, were used as biocatalysts. Higher polyphenol yield was obtained from wet pomace than from dried pomace, and, interestingly, the combined enzyme treatments did not cause any increment in the total phenolic content. Concerning white grape samples, Celluclast (2%) treatments for 2 hours and/ or 6 hours at 37°C increased the concentration of some of the individual phenolic compounds (mainly catechins) in dried pomace (Ferri et al. 2017). In another study, Pectinex BE Colour, Vinozym FCE G and Celluclast 1.5 L FG cocktails enhanced the recovery of phenolics from red wine grape skin (Arnous and Meyer 2010). After an initial decrease in the first 2 hours of hydrolysis, the phenol yield increased during the next 4 to 6 hours. This was notable for the Vinozym treatment exhibiting about 20% increase at the sixth hour of incubation when compared to 2-hour values. In red grape pomace, Chamorro et al. (2012) reported significant changes in the phenolic content and antioxidant activity after enzyme treatments. Authors indicated that Laminex (p-glucanases and xylanases from Penicillium funiculosum), Pektozyme (pectolytic enzyme from A. niger) and tannase (from Aspergillus ficuum) in mixed application produced better extraction yield (from 0.11 to 0.14 mg GAE/100 g DM) and antioxidant activity (from 16.5 to 21.7 ptnol Trolox equivalent/g DM) than the enzyme-free sample. In the study of Gomez-Garcfa et al. (2012), Novoferm was the most effective in phenolic mobilization from grape residues, followed by Pectinex Ultra and Celluclast

1.5 L coctails. Antioxidant activity measured by the DPPH method associated well with the phenolic yield increase presenting maximal scavenging capacity of 87%, 83% and 90% after 12-hour treatment with Celluclast 1.5 L. Pectinex Ultra and Novoferm, respectively. Also, a significant increase in the amount of hydroxybenzoates and hydroxycinnamates was identified when a 2:1 mixture of Novoferm 106 pectinolytic and Cellubrix L cellulolytic enzymes was used for grape pomace treatment (Maier et al. 2008). Optimal conditions for the highest phenolic recovery (92%) were an aqueous substrate extraction followed by an enzymatic treatment for 2 hours at 40°C and pH 4.0. Effect of enzyme type, various solvents and incubation time on the release of phenolics from red and bronze muscadine grape skin and seed residues was compared by Xu et al. (2014). The different enzymes used affected differently the phenolics release from the two varieties: a short-time (1-4 hours) hydrolysis with A. niger pec- tinase and almond (3-glucosidase significantly increased the total phenolic yield in both skin residues, while it was inhibited when Trichoderma reesei cellulase was used as catalyst. Long incubation times should be avoided, since the released phenolics started to degrade over a 4-hour incubation. This was attributed to the reaction condition used (50°C, pH 4.8, presence of cell enzymes in the carbohydrase mixtures).

In a study combined with supercritical fluid and pressurized liquid extractions, Viscozyme L and CelluStar XL (xylanase preparation) enzymes remarkably increased the yield of antioxidative phenolics in blackcurrant pomace samples (Basegmez et al. 2017). In this case, 6% enzyme:substrate ratio, and incubation for 7 hours at 40°C and pH 3.5 was the optimal condition to obtain the highest phenolic yield. Also, Viscozyme L at pH 3.7, 50°C and 12 hours proved to be the most favourable condition for unripe apple samples, resulting in about three times increase in the phenolic yield compared to the control (Zheng et al. 2009). On the other hand, due to the high pectin content of apple, it is easy to explain by the fact that pectinase activities significantly affected the bioactive compound release from apple residues. However, the type of the commercial pectinase is a yield-limiting factor. In the study of Oszmianski et al. (2011), for instance, the antioxidative phenolic content of apple purees obtained after Pectinex AFP L-4, Pectinex Yield Mash and Pectinex XXL pomace treatment was superior to that treated with Pectinex Ultra SPL. In a comparative research, different cellulolytic, hemicellulolytic and pectolytic commercial formulations were screened on raspberry wastes in which Grindamyl CA 150 and Maxoliva cocktails (mostly pectolytic activities) resulted in significant increment in extract phenolic yield (from

11.3 to 15.8 and 15.4 mg GAE/g DM) and antioxidant activity (Laroze et al. 2010). Temperature of 50°C, 1:20 solid/liquid ratio, 1:10 enzyme/substrate ratio, 18-hour incubation and ethanokwater (25/75) solvent were applied as the operating conditions. Interestingly, when the authors increased the Grindamyl CA 150 concentration from 0.5% to 5%, there was no significant change in the phenolics production. In the study of Huynh et al. (2014), enzyme/substrate ratio of 0.2% for Viscozyme L and 0.5% for Rapidase (i.e. pectinase and hemicellulase) were effective to produce phenolics from outer leaves of cauliflower using the reaction condition of 35°C, pH 4.0 and 12-hours incubation time. Treatments with higher enzyme amounts up to 5% did not cause increment in the phenolic yield. In addition, less phenolic yields were obtained at 50°C when Viscozyme cocktail was used as compared to lower operating temperatures, owing to a possible activity loss of the enzyme preparation and thermal degradation of phenolics. For tomatoes, pure cellulase and pectinase (i.e. Carenzyme and Pectinex 3XL) were tested at 40°C and 50°C temperatures, respectively (Neagu et al. 2014). The optimal enzyme/substrate ratio was 5% for Carenzyme and 10% for Pectinex 3XL, resulting in a phenol yield increase from 70.2 to 118 and 123.8 mg GAE/100 g fresh weight, respectively. DPPH inhibition capacity was higher in samples obtained after pectinase treatment compared to cellulase-aided extractions. There were applications for enzyme-assisted extraction of phenolics from oilseed residues as well. For instance, flaxseed meal, the by-product of flaxseed oil extraction process, was subjected to Ultrazym, Viscozyme and different protease treatments by Ribeiro et al. (2013). Though proteolytic preparations are rarely investigated in terms of phenolics release, this study highlighted the importance of pancreatin and alcalase in case of flaxseed meal substrate.

As can be seen from the above discussed studies, the operating conditions of the enzymatic treatment have a determining impact on the release of phenolic compounds. The most important factors are the following: (i) type of the plant material and its particle size and porosity, (ii) type of enzyme/cocktail and its composition and activity, (iii) enzyme to substrate ratio (enzyme concentration), (iv) moisture content of the substrate (solid to water ratio), (v) hydrolysis time, (vi) temperature and pH.

Overall, the enzyme technology is a useful method to extract bioactive compounds from agro-industrial by-products. Using this approach, there are no microbial toxins and phenolic-degrading enzymes potentially formed during the fermentation processes. These products may influence both the extraction yield and the product quality. In addition, the enzyme treatment requires mild reaction conditions in aqueous solutions, which eliminates the environmental risk and toxicological effects caused by organic solvents (Puri et al. 2012). On the other hand, the enzyme-aided extraction can be well-integrated to solvent, ultrasound, microwave and high hydrostatic pressure (HPP) based downstream processing strategies. For example, Mushtaq et al.

(2015) tested and compared aqueous ethanol (80%) and supercritical fluid extraction techniques after carbohydrase treatment of pomegranate peel residues. Depending on the enzymatic treatment prior to the extraction, the supercritical fluid approach resulted in about 1.1 to 1.5 times higher phenolic yield than those obtained with conventional ethanol extraction. In addition, a recent study combined the Celluclast, Pectinex, Rapidase and Viscozyme derived hydrolysis with HPP technology to extract tricin flavonoid and other phenolics from rice hull (Park et al. 2016). More than twice the amount of total phenolics (1.7 vs. 3.5 mg GAE/kg rice hull) could be extracted when Rapidase supported with 500 MPa HPP was used as the extraction technique compared to the traditional ethanol solvent method. The Celluclast and Pectinex were also effective catalysts during the study. In addition, the samples showed improved bioactive properties after combined extraction using Celluclast and HPP. Here, the carbohydrases liberated cell-wall compounds, and then the HPP treatment increased the solvent permeability, improving the extraction yield.

 
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