**3. Enzyme Production and Potential Applications**

The use of pomace for the production of enzymes obtained from the agro-industrial processing of foods is an interesting strategy for producing high-added value products (Table 3). One of the main pomaces explored in the production of enzymes is obtained from apple processing. Recent studies point out that apple pomace can be used to obtain different enzymes without an additional carbohydrate source [44–48]. For instance, the production of lignin peroxidase and manganese peroxidase were reported from the fermentation of apple pomace with *Phanerochaete chrysosporium* BKM-F-1767 [48]. In this study, apple pomace was indicated as the most versatile residue to produce these enzymes in comparison to brewery residue, pulp and paper residue, and fishery waste.

The production of amylase, cellulose, pectinase, and xylanase was reported for fermentation with *Rhizopus delemar* F<sup>2</sup> [44]. Similarly, the production of pectinase was reported in another study carried out with *Aspergillus parvisclerotigenus* KX928754 where the fermentation was optimized in terms of pH, temperature, and the period of fermentation [45]. Similarly, the combination of two *Bacillus* strains, *Bacillus subtilis* and *Bacillus pumilus*, was indicated as a relevant strategy to produce pectinase from apple pomace [46]. In this study, the authors optimized the fermentation by exploring the effect of solid content and the ratio between *B. subtilis* and *B. pumilus* in the production of this enzyme.

*Fermentation* **2021**, *7*, 299


**Table 3.** Production of enzymes from the fermentation of apple, grape, olive, tomato, orange, pea, and carrot pomaces.

*Fermentation* **2021**, *7*, 299



CMCase: carboxymethyl cellulase, SmF: submerged fermentation, and SSF: solid-state fermentation.

Combining apple pomace with other sources of nutrients can improve enzyme production yields. This factor was considered in the experiment carried out by Singh et al. [49], who used dahlia tuber powder (source of inulin) to produce inulinase with apple pomace. These authors optimized the fermentation in terms of moisture, fermentation period, and pH. Another interesting strategy to obtain extracts rich in enzymes from apple pomace consist in generating mutant strains such as those indicated by Guleria et al. [47]. In this case, the new mutant of *Cellulosimicrobium* sp. CKMX1 E<sup>5</sup> increased the production of xylanase in relation to its parent strain.

Grape is another relevant substrate for the production of enzymes. In this case, the production of tannase was obtained from the fermentation with *Aspergillus niger* NRRL3 [50]. Similarly, the production of cellulose using *Bacillus subtilis* was also obtained from the fermentation of grape pomace [51]. Another recent experiment indicated that the production of laccase from grape pomace was dependent on the starter culture [52]. In this case, *Pleurotus pulmonarius* was more efficient for producing this enzyme than *Pleurotus ostreatus*. Moreover, the authors also indicated that solid-state fermentation was more appropriate than semiliquid and submerged fermentations.

The effect of adding wheat bran in grape pomace for the production of different enzymes was studied in a recent experiment [53]. The fermentation with *Aspergillus niger* successfully produced more tannase by combining wheat bran with grape pomace than using only wheat bran. However, the presence of grape pomace limited the production of xylanase and β-glucosidase and slowed the production of polygalacturonase. Additionally, the authors also reported a dependency on time for the production of polygalacturonase and carboxymethyl cellulase (higher enzymatic yields were obtained after 96 h of fermentation). Additionally, Papadaki et al. [54] reported the production of amylase, carboxymethyl cellulase, polygalacturonase, protease, and xylanase from a substrate composed of white grape pomace, olive mill wastewater, red grape pomace and wheat bran. *Aspergillus niger* was used to obtain these enzymes.

Olive processing for oil extraction also generates a valuable substrate for microbial enzyme production. For instance, a recent experiment with olive pomace indicated that tannase could be obtained from the fermentation with *Kluyveromyces marxianus* [55]. Another relevant example that supports the use of this pomace in the production of enzymes is the study carried out by Leite et al. [56]. In this case, the authors fermented the exhausted olive pomace with *Aspergillus niger* and reported the production of xylanase and cellulose. In the case of lipase production from grape pomace, the effect of *Aspergillus* species was evaluated in a recent study [57]. *Aspergillus ibericus* was a more efficient species in relation to *Aspergillus niger* and *Aspergillus tubingensis*. Interestingly, a related experiment with exhausted olive pomace reported the production of pectinase from the growth of the microalgae *Crypthecodinium cohnii* [58]. Additionally, no significant differences in terms of substrate concentration (5 vs. 8 g/L) in the production of this enzyme were reported.

Tomato is another relevant source of pomace that can be utilized in the production of enzymes. Proteases could be obtained from tomato pomace using *Aspergillus oryzae* according to recent studies [59,60]. Moreover, the study carried out by Belmessikh et al. [59] indicated that the production of protease from tomato pomace was more efficient in solidstate rather than submerged fermentation. The optimization also indicated that casein and NaCl levels are significant factors in improving the production of protease.

The combination of tomato pomace with other sources of nutrients for enzymatic production has also been explored [61]. Particularly, for the combination with sorghum stalks, the production in a laccase, protease, and xylanase were dependent on the starter culture [61]. In this case, *Pleurotus ostreatus* was associated with a faster but less intense production of these enzymes. Conversely, *Trametes versicolor* had higher production yields but after longer fermentation periods. Another more recent experiment with tomato pomace, wheat bran, and canola meal indicated that the fermentation with *Bacillus subtilis* was associated with high xylanase content [62].

Another relevant pomace for the production of enzymes is obtained from orange processing. In this case, recent experiments explored the generation of pectinase from the fermentation with *Aspergillus niger* [63,64]. It is also relevant to comment that a recent experiment indicated that the use of sugarcane bagasse is a relevant strategy to reduce moisture loss during fermentation and improve the production yield of pectinase from orange pomace [64]. In a similar way, carrot pomace was indicated as an interesting substrate for fermentation, which can be utilized in the production of inulinase [65]. The production of inulinase was affected by moisture content, fermentation period, and pH.

The production of enzymes from pomaces can also be improved by the use of emerging technologies such as microwave heating and ultrasound. This aspect was reported in the production of carbohydrases from apple pomace by Pathania et al. [44]. According to these authors, the intensity of microwaves (as a pre-treatment) had a significant effect on the production yield. The maximum values for amylase, pectinase, and xylanase were reported for the 450 W treatment. Additional power (up to 600 W) caused a reduction in the production of enzymes. In the same line of thought, the use of ultrasound can improve the production of enzymes. Leite et al. [56] indicated that using 750 W and 20 kHz and optimizing the time and liquid/solid ratio (12.4 min and 7.3) maximized the production yields of xylanase (75 U/g) and cellulase (35 U/g).

It is also important to highlight that some experiments to scale up the production of enzymes from pomaces have been carried out in the last decade. One relevant example that explored this aspect was performed by Boukhalfa-lezzar et al. [60] with tomato pomace fermented with *Aspergillus oryzae*. In this study, similar production yields were reported between lab scale and a bioreactor for protease production (12 U/gds after 42 h with 20 g of substrate vs. 13.6 U/gds after 44 h with 5 kg of substrate). Another relevant experiment supporting the increase in the production scale of enzymes was carried out with orange pomace in a tray reactor and a rotating-drum reactor [64]. In this case, differences in production yield were reported between these two reactors wherein the bioreactor with trays had the highest yield. Moreover, both reactors increased the production of pectinase in relation to a previous experiment from the same research group [63].

The purification of enzymes obtained from fermentation is another relevant aspect considered in recent studies. In order to explore potential solutions to improve the separation of enzymes, an experiment with lignin peroxidase and manganese peroxidase explored the use of centrifugation and filtration after the fermentation of apple pomace [48]. The results revealed that centrifugation was more efficient for separating both enzymes than filtration. A recent study compared the use of fractionation with ammonium sulfate and chromatography filtration in the purification of tannase from fermented olive pomace [55]. Both methods led to extracts with increased enzymatic activity wherein the chromatography filtration was more efficient than fractionation with ammonium sulfate (1026.1 vs. 664 U/mg, respectively). A similar outcome was obtained in another study with pectinase from apple pomace (1081.7 vs. 860.6 U/mg for chromatography filtration and ammonium sulfate fractionation, respectively) [45].

Potential applications can also be considered in the context of enzyme production. Since pomace is a by-product from food processing, the use of these enzymes in food production can be suggested. A relevant example is the experiment carried out by Mahmoodi et al. [63]. In this study, cubic pieces of fresh apple were treated with pectinase to produce apple juice. The main effects were the reduction in juice viscosity and increased juice yield, soluble sugar content, and pectate content. A similar experiment with polygalacturonase obtained from apple pomace was efficient for clarifying apple juice [66].

An interesting application for enzymes obtained from pomace fermentation is the detoxification of food. This approach was evaluated by Cuprys et al. [67] who applied laccase from apple fermentation with *Trametes versicolor* to decompose ciprofloxacin (an antibiotic). However, the presence of a reducing agent (syringaldehyde in this study) was necessary to favor the enzymatic degradation of this antibiotic in water. Although the scientific information about the application of microbial enzymes from pomace fermentation

in food processing is limited, the use of these enzymes could be considered to improve the processing of beer, bread, cheese, syrup, and wine [68] in further experiments. Moreover, potential applications in other research areas are in pharmaceutical, chemical, fuel, and paper production [69].

The use of pomace from different sources can be seen as a relevant strategy to favor the production of enzymes. Current scientific evidence indicates that the production of enzymes can be improved by adding complementary sources of nutrients (such as ingredients rich in carbohydrates for pomaces with reduced levels of this nutrient), applying emerging technologies to favor the exposure of substrates, and increasing the scale of production (minimal effect in production yield, to some extent). The purification with different techniques can also be applied and support the progression towards application in other industrial processes.
