Abstract
A total of five pectin-rich banana peels, including those of two Musa paradisiaca cultivars (nendran and kadali) and three Musa acuminata varieties (matti, palayankodan, and robusta), were used for pectinase production by Aspergillus flavus via solid-state fermentation (SSF). Comparative analysis revealed highest pectin content in the palayankodan peel (18.09 ± 0.29%). The palayankodan peel variety presented 135 ± 2.2 U/gds pectinase activity in SSF using A. flavus, whereas robusta peels presented 108 ± 1.9 U/gds pectinase activity in SSF. A one-factor-at-a-time experiment was performed, and the variables moisture, pH, and fermentation period affected enzyme production. A central composite design was used to optimize pectinase production in SSF via three prominent variables (moisture content, pH, and fermentation period). The experimental result was statistically significant (p<0.01), and twofold enzyme production was achieved. The crude pectinase was extracted from the fermented medium and used as a clarifying agent. The pectinase-treated orange juice presented a decreased turbidity compared to the untreated control. The amount of total sugar and total suspended solids was reduced, whereas the total pH increased. Therefore, the A. flavus strain can be utilized for large-scale production of pectinase, which could meet the growing industry demands.
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Pectin-rich Banana Peel Varieties: A Low-cost Biomass for Pectinase Production by Aspergillus flavus in Solid-state Fermentation and its Effect on the Clarification of Orange Juice
Thankappan Sarasam Rejiniemon,a Najla A. Alshaikh,b Ashraf Atef Hatamleh,b,* and Selvaraj Arokiyaraj c,*
A total of five pectin-rich banana peels, including those of two Musa paradisiaca cultivars (nendran and kadali) and three Musa acuminata varieties (matti, palayankodan, and robusta), were used for pectinase production by Aspergillus flavus via solid-state fermentation (SSF). Comparative analysis revealed highest pectin content in the palayankodan peel (18.09 ± 0.29%). The palayankodan peel variety presented 135 ± 2.2 U/gds pectinase activity in SSF using A. flavus, whereas robusta peels presented 108 ± 1.9 U/gds pectinase activity in SSF. A one-factor-at-a-time experiment was performed, and the variables moisture, pH, and fermentation period affected enzyme production. A central composite design was used to optimize pectinase production in SSF via three prominent variables (moisture content, pH, and fermentation period). The experimental result was statistically significant (p<0.01), and twofold enzyme production was achieved. The crude pectinase was extracted from the fermented medium and used as a clarifying agent. The pectinase-treated orange juice presented a decreased turbidity compared to the untreated control. The amount of total sugar and total suspended solids was reduced, whereas the total pH increased. Therefore, the A. flavus strain can be utilized for large-scale production of pectinase, which could meet the growing industry demands.
DOI: 10.15376/biores.20.4.11022-11040
Keywords: Banana peels; Pectinase; Aspergillus flavus; Orange juice; Clarifying agent
Contact information: a: Department of Biotechnology, AJ College of Science and Technology, Thonnakkal,
Thiruvananthapuram, India; b: Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia; c: Department of Food Science & Biotechnology, Sejong University, Seoul 05006, South Korea; *Corresponding authors: ahatamleh@ksu.edu.sa; ashrafatefhatamleh123@gmail.com; arokiyaraj16@gmail.com
Graphical Abstract
INTRODUCTION
Microbial pectinases are widely recognized as a heterogeneous group of enzymes capable of hydrolyzing pectin (Abdollahzadeh et al. 2020). They have wide applications in several industrial sectors, such as the oil and juice extraction, animal feed preparation, fermentation, wastewater treatment, pharmaceutical, pulp, and paper industries (Sharma et al. 2013; Singh et al. 2024). Microbial pectinases are produced commercially by various microorganisms because of their potential to use low-cost substrates, reduce production costs, and increase yields via either solid-state fermentation or submerged fermentation (Kc et al. 2020; Kaul et al. 2024). Recently, pectinase production from fungi has attracted increasing attention. Pectinases from the genus Aspergillus have been widely used for commercial preparations of various agroresidues. For example, Aspergillus niger was used to optimize endogluconases and exogluconate production from mango waste (Santos et al. 2011). In one study, Aspergillus NCFT 4269 was used to produce pectinase by utilizing banana peel as a low-cost substrate (Sethi et al. 2016). In addition, fungi from the genus Aspergillus have the ability to survive, colonize, and produce metabolites on solid substrates (Sandri and daSilveira 2018). Fungi, especially filamentous fungi, prefer solid-state fermentation (SSF) to produce pectinase rather than submerged fermentation because of its potential to improve nutritional properties, reduce contamination, increase enzyme yield, and increase cost-effectiveness (Rojas et al. 2011; Demir et al. 2012; Santos et al. 2015; Alarjani et al. 2024).
Lignocellulose (LC) waste is the major resource utilized for the bioconversion of industrially useful products. Banana is an important crop in Asian countries and is widely consumed because of its palatability, nutritional value, digestibility, and cost effectiveness (Arokiyaraj et al. 2024). Banana peel is an important source of LC substrate and is generated throughout the year in India; hence, it can be utilized for its possible use as a cheap biomass (Sajish et al. 2025). In Asia, banana varieties are cultivated in thousands of hectares and generate tons of industrial waste. Banana peel is one of the major waste products derived from banana fruit. Banana peel comprises cellulose (7 to 10%), hemicelluloses (6 to 9.4%), pectin (10 to 21%), and lignin (6 to 12%) (Emaga et al. 2008). It has been recommended as an effective substrate to produce plant cell wall-degrading enzymes under SSF by Aspergillus terreus and Aspergillus niger (Rehman et al. 2014). It has also been used to produce laccase by Trametes pubescens (Osma et al. 2007) and of alpha-amylase by Penicillium spp. and Bacillus subtilis (Akkarachaneeyakorn et al. 2018). Banana peel is also utilized to produce citric acid from Aspergillus niger via koji fermentation (Karthikeyan and Sivakumar 2010), biohydrogen production (Nathoa et al. 2014), bioethanol production, and fungal carbohydrase production (Janveja et al. 2013). Pectinases are inducible enzymes and microbes utilized pectin rich agricultural residues and improved enzyme production (Shrestha et al. 2022; Alfarhan et al. 2024).
Statistical tools are useful for optimizing culture conditions to reduce production costs. The optimization process initially involves identifying the suitable variables or factors affecting the production of enzymes, followed by the identification of their optimal culture conditions or nutrient requirements. The one-variable-at-a-time approach has been frequently employed to optimize bioprocesses; however, several limitations have been reported in this traditional approach (Managamuri et al. 2016; Marraiki et al. 2020). Hence, statistical-based approaches have been recommended for studying the interactions among process variables. The Plackett–Burman design and the two-level factorial design are important statistical tools for identifying variables that have an impact on enzyme production (Al Farraj et al. 2020; Shoba et al. 2025). These statistical tools allow the elimination of insignificant variables, which reduces the number of total experimental runs. The second step of the optimization process helps to identify the optimum level of variables, predict the optimum response (yield), and validate the response via central composite design (CCD) and Box‒Behnken design (El-Sheikh et al. 2020; Kalaiyarasi et al. 2020). Considering the availability of banana fruit peel in Asian countries and the suitability of the substrate for fermentation bioprocesses, this work was performed to utilize low-cost banana fruit peel waste for pectinase production under solid-state fermentation by Aspergillus flavus.
EXPERIMENTAL
Fungi and Culture Conditions
The fungal strain Aspergillus flavus (MTCC 277) was used to produce pectinase. The culture strain was obtained from the Institute of Microbial Technology, Pune, India. The strain was cultured on Czapek yeast extract agar (Himedia, Mumbai, India) plates and incubated for 3 days in an incubator at 28± 1 °C. It was further stored at 4 °C and subcultured every 30 days.
Substrate
A total of five banana fruit varieties were collected, and the peels were utilized to produce enzymes. Two Musa paradisiaca cultivars, namely, nendran and kadali, and three Musa acuminata cultivars, namely, matti, palayankodan, and robusta, were used in this study. All these types of banana fruits were collected from the market and used for analysis. The ripened banana peels were washed and chopped into small pieces (2 cm × 2 cm) (Fig. 1). The peels were dried under sunlight for five days and powdered mechanically. The material was sieved and either used for pretreatment for enzyme production or used directly for pectin content analysis.
Fig. 1. Banana fruit varieties utilized for the preparation of substrates in solid-state fermentation
Pectin Analysis
The pectin content of banana peels was determined as described previously (Begum et al. 2014; Faruque et al. 2016), with slight modifications. Approximately 5 g of powdered banana peel was mixed with 50 mL of double distilled water. The pH of the solution was adjusted to 2.2 ± 0.02 using (0.1 N) hydrochloric acid. The mixture was then heated at 90 ± 1 °C for 1 h and cooled to room temperature, after which the precipitate was observed. The mixture was subsequently centrifuged at 5000 × g for 10 min and filtered through Whatman No. 1 filter paper. To the filtrate, three volumes of 99% ethanol were added, the precipitate was separated, and the filtrate was dried. The dried filtrate was weighed, and the pectin yield (%) was calculated (Eq. 1):
(1)
Fungal Inoculum Preparation
A total of 50 mL of Sabouraud dextrose broth (SDB) (Himedia, India) was added to a 250-mL Erlenmeyer flask. The mixture was sterilized and cooled, and a loopful of a pure culture of A. flavus was inoculated into SDB medium and incubated for 4 days. The mixture was kept on a rotary shaker incubator at 150 rpm until the optimal spore suspension was reached, and it was refrigerated at 4 °C (Sathya et al. 2025).
Solid-state Fermentation
The dried banana peels (nendran, kadali, matti, palayankodan, and robusta) were weighed (5 g) and added to a 100-mL Erlenmeyer flask. The banana peel powder was moistened with a salt solution (0.5% NH4NO3, 0.02% Na2SO4, 0.08% MgSO4⋅7H2O, 0.4%ZnSO4⋅7H2O, 0.2% KCl, and 0.1% Ca(OH)2). The initial moisture content of the solid medium was 60%. The substrates were sterilized for 30 min at 121 °C, and the conical flasks were cooled. The flasks were subsequently inoculated with 2% (v/w) spore suspension and incubated for 4 days (Ahmed et al. 2021). After 4 days of incubation, the amount of pectinase activity was determined.
Extraction
The fermented culture medium was mixed at a ratio of 1:4 (w/v) with phosphate buffer (pH 5.5) at room temperature. Then, the mixture was placed on a rotary shaker at 150 rpm for 20 min. It was subsequently filtered through Whatman No. 1 filter paper, and the clear supernatant was used for the enzyme assay (Shet et al. 2022).
Pectinase Assay
The amount of pectinase (polygalacturonase) activity was evaluated via the DNS (3,5-dinitrosalicylic acid) method with slight modifications. A 0.2-mL aliquot of crude enzyme was mixed with 2.8 mL of 1% pectin solution (phosphate buffer, pH 5.5, 0.1 M). The mixture was incubated at 30± 1 °C for 10 min. Then, 2.5 mL of dinitro salicylic acid (DNS) solution was added to stop the enzymatic reaction. To the blank, 0.2 mL of crude enzyme was added. The test tubes were placed in a boiling water bath and diluted with 5 mL of double distilled water. The optical density (OD) of each sample was assayed spectrophotometrically (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) at 540 nm against a reagent blank (Miller 1959). α-Galacturonic acid was prepared at various concentrations (0.1 to 1.0 mg/mL) and used as the standard. One unit of pectinase activity was defined as the amount of enzyme that released 1 mL of galacturonic acid per minute under standard assay conditions.
Screening of Variables via a One-variable-at-a-time Approach
The SSF was further investigated via a one-variable-at-a-time approach. The pectin-rich palayankodan banana peel, which presented the maximum pectinase production, was selected for optimization studies. To optimize the incubation time, the culture was incubated at 28 ± 1 °C for 2 to 5 days. To optimize the moisture content, the moisture content of the culture medium was adjusted with a mineral salt solution ranging from 45 to 65 °C and incubated at 28 ± 1 °C for 5 days. To optimize the pH for enzyme production, a buffer solution (pH 3.5 to 6.0) (0.1 M) was added to the substrate (moisture, 55%) and incubated for five days. Similarly, the optimum incubation temperature was determined by cultivating the fungal strain in banana peel medium (55% moisture, pH 5.0) for five days at various temperatures (25 to 45 °C) in an incubator.
Optimization of Enzyme Production via Response Surface Methodology
A central composite design was used to design the experiment via three independent variables, namely, fermentation period (A), moisture content (B), and pH (C), and their effects on enzyme activity (response Y). The ranges of the independent variables were selected on the basis of the one-variable-at-a-time approach. The fermentation period (low: 3 days; high: 5 days), moisture content (low: 35%, high: 55%), and pH (low: 3.5; high: 5.5) were selected and analyzed at five different levels (Table 1).
Table 1. Variables and Their Actual Low and High Values to Produce Pectinase in the Central Composite Design
For these three independent variables, 20 experimental runs were performed, including six experiments on central points. The experiments were performed in randomized experimental trials, and the experiments were designed via Design-Expert (version 8.1) software. The following second-order polynomial equation was applied to fit the experimental data,
where=independent variable; =independent variable;=response (pectinase activity);=constant; and , , and =regression coefficients. Analysis of variance (ANOVA) was performed, the significance of the designed model was analyzed. A p value<0.05 was considered statistically significant. To evaluate the suitability of this model, factors, such as lack of fit (a significant lack of fit is preferred), adequate precision (>4 is preferable), and R2 values (>0.76 is good), were analyzed. A response surface curve was generated, and an optimum response of the variable was analyzed to study the interactions between the independent variables and pectinase activity.
Juice Clarification Experiment
Orange juice clarification was performed as described previously with slight modifications (Dal Magro et al. 2021). Briefly, 250 g of orange fruit (Citrus × sinensis) was peeled, and the fruit juice was pressed and filtered through cotton gauze. Approximately 100 mL of fruit juice was obtained, and it was equally distributed into five test tubes. Pectinase was added to the fruit juice at various concentrations (2 U/mL to 8 U/mL). Commercial pectinase was added (2 U/mL) (Novozymes, Brazil) to the positive control. Enzymatic treatment was performed for 60 min, and the absorbance of the sample was read at 600 nm via an UV‒Vis spectrophotometer (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan).
Determination of Turbidity, Total Suspended Solids, Reducing Sugars, and pH of the Clarified Fruit Juice
The turbidity of the pectinase-treated juice was determined via spectrophotometric analysis as described previously by Anderson (2005), with slight modifications. After 60 min of treatment, the sample was centrifuged at 5000 × g for 2 min, and absorbance of the supernatant was analyzed at 600 nm via a spectrophotometer (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan). Juice without any enzyme treatment was considered a control. The total sugar content of the fruit juice was estimated as described previously by Kirk and Sawyer (1991), and the result was expressed in g/100 mL. The juice was filtered through Whatman 4 Qualitative filter paper, and the filtrate was used for the analysis. The pH of the juice was measured via a digital pH meter (Hanna, UK). The total soluble solids content was determined via a Bellingham refractometer (Xylem Analytics, 82362 Weilheim, Germany), and the results are expressed as percentages.
Statistical Analysis
The mean results were subjected to analysis of variance and comparison of means was performed with the LSD test at p ≤ 0.05. Different lowercase letters in the same column in the table indicate statistical significance.
RESULTS AND DISCUSSION
Pectin Content
The pectin content in ripe banana peels (nendran, kadali, matti, palayankodan, and robusta) was analyzed, and hydrochloric acid was found to be efficient at extracting pectin from ripe banana peels. The amount of banana peel pectin ranged from 11.31 ± 0.22% to 18.09 ± 0.29% (Table 2). The pectin content was greater in the palayankodan variety (18.09 ± 0.29%) than in the other tested banana varieties. The pectin content of banana peels was consistent with the banana peel contents of other banana peel varieties (Silva et al. 2002).
Table 2. Pectin Content of Ripe Banana Peels from Various Banana Varieties
Effect of Substrate on Polygalacturonase Production
The amount of polygalacturonase produced varied with the type of banana peel powder (nendran, kadali, matti, palayankodan, and robusta). These pectin-rich peels were used as the solid substrate to produce polygalacturonase in SSF. Table 3 shows the effects of pectin-rich substrates on polygalacturonase production. The palayankodan variety presented 135 ± 2.2 U/gds enzyme activity, whereas robusta presented 108 ± 1.9 U/gds in the SSF. Solid-state fermentation has several advantages over submerged fermentation, as solid substrate‒microbe interactions are highly effective at achieving high product yields (Al Farraj et al. 2020). In the SSF, the water requirement is very low, and product recovery is highly efficient (SavariyarAdimy et al. 2024). Agroresidues, such as cantaloupe and watermelon rinds, are utilized to produce polygalacturonase by Trichoderma species, and improved production has been reported (Mohamed et al. 2013). Zehra et al. (2020) used banana peels to produce pectinase and xylanase via a coproduction strategy. In addition, Arekemase et al. (2020) used banana peel substrates to produce pectinolytic enzymes in submerged fermentation. Doan et al. (2021) used pectin-rich substrates, including wheat bran and banana peel, to produce pectinases by Bacillus amyloliquefaciens.
Table 3. Polygalacturonase Production by Aspergillus flavus in Solid-State Fermentation Using Banana Peels Obtained from Different Banana Varieties
Screening of Variables to Produce Polygalacturonase in SFF
In this study, enzyme production was optimized. The optimum moisture content for polygalacturonase production was 55% (v/w), and the yield was 171 ± 1.9 U/gds (Fig. 2a). Moreover, enzyme production was affected at relatively high and low moisture contents. In the current study, polygalacturonase production reached a maximum (182 ± 2.5 U/gds) after 4 days of fermentation (Fig. 2b). The optimum fermentation period varied based on the type of solid medium, moisture content, and type of microorganism. Ketipally et al. (2019) used orange peel substrate to produce polygalacturonase, and the maximum production of the enzyme was achieved after 7 days of incubation with A. nomius MR103. Barman et al. (2015) used banana peel substrate to produce polygalacturonase and achieved a maximum enzyme yield (6.6 U/mL) within three days of culture with A. niger. In the current study, the maximum enzyme yield was achieved within four days of fermentation, which was consistent with previous reports (Shanmugavel et al. 2018; Nsude et al. 2019).Pectinolytic fungi produced the greatest amount of enzyme activity after 6 days of incubation when orange peels were supplemented with 3.0% ammonium sulfate as a nitrogen supplement (Okonji et al. 2019), which was greater than that observed in the current study.
The pH is an important factor that influences the production of pectinolytic enzymes and regulates the synthesis of fungal enzymes. In the current study, polygalacturonase production was observed at wide pH ranges of 4.5 to 5.5, and the maximum yield was observed at pH 4.5 (213.8 ± 5.4 U/gds) (Fig. 2c). As reported by Adedayo et al. (2021), the initial pH of the culture medium directly influences the fermentation process. The pH of the medium also affects the stability of the culture medium, and the pH of the medium affects membrane permeability and biosynthesis in fungal and bacterial species. The results of the present study were similar to those of Ahmed and Sohail (2020), who reported that pectinolyticenzyme production was high between pH 3 and 5.5. This result is also supported by that of Manan and Webb (2017), who reported that yeast and fungi prefer acidic pH values for optimal growth and enzyme production. The pH value affects fungal cell proliferation, and the active site is altered, which reduces the enzyme activity of the generated enzymes. Although most of the researchers agreed with the optimum pH obtained in the current study, the enzyme yield varied with manufacturing method, microbial species, and substrate. The effect of incubation temperature on enzyme production was studied, and maximum production was achieved at 30 °C (220.5 ± 3.9 U/gds). At lower initial temperatures, the enzyme activity was lower, and it increased at 30 °C (Fig. 2d).
Fig. 2. Effects of moisture (a), fermentation period (b), pH (c), and temperature (d) on polygalacturonase activity during solid-state fermentation with A. flavus
The high temperature of the culture medium (>30 °C) was affected enzyme production. The sudden decline in enzyme activity at increasing temperatures may be due to the decreased metabolic function of the organism and the denaturation of the enzyme (Wong et al. 2017). In another study, Abdullah et al. (2018) reported maximum pectinase production at 30 °C in submerged fermentation using the fungal strain Aspergillus niger ABT-5. Moreover, 25 °C was found to be optimal to produce pectinase in Aspergillus fumigatus MS16 under SSF (Zehra et al. 2020). The optimum temperature for pectinase production observed in the current study was similar to that reported in studies in which Aspergillus niger HFD5A-1 (Darah et al. 2013), Aspergillus terreus NCFT 4692 (Sethi et al. 2016), and Aspergillus fumigates R6 (Wong et al. 2017) were used.
Optimization of Pectinase Production by Response Surface Methodology
The optimization of culture conditions for the improved production of pectinase by A. flavus was performed by analyzing the three most significant media components: pH, fermentation period, and moisture content of the medium. The designed CCD matrix and results are presented in Table 4. The results were subjected to regression analysis, and the regression coefficients of the terms were determined. The significance impacts of the selected bioprocess variables on pectinase activity were analyzed via analysis of variance (ANOVA). The PRESS value, R2 value, adequate precision, and lack of fit value were analyzed, and the model suitability was tested. The lack of fit F-value was >0.05 (0.66), which implies that the lack of fit was not significant and that the insignificant lack of fit was good. The “predicted R2” value of 0.9527 was very close to the “adjusted R2” of 0.9776. The adequate precision of the model was >4 (25.456), which indicated an adequate signal of the model. In this model, B, C, AB, BC, A2, B2, and C2 were significant terms (Table 5). Response surface plots were used to study the interactions between the selected independent variables and response (pectinase activity). The influence of the interaction of selected factors (moisture content, incubation time, and pH) on pectinase activity is depicted in response surface plots (Fig. 3). The interaction effects of the initial moisture content of the medium (A) and fermentation period (B) on pectinase activity are presented in Fig. 3a. The 3D graph shows that enzyme activity increased with increasing moisture content up to the optimum level, and decreased enzyme activity was observed at higher moisture levels. In SSF, moisture content is a critical factor that influences the metabolic processes of cells. The low enzyme yield at relatively high moisture levels occurred because the relatively high humidity caused the solid material to form clumps. These clumping particles reduced the interparticle space of the solid medium, which reduced the availability of nutrients to the microorganisms (Bhattacharya et al. 2024). In addition, high moisture content affects the porosity of the solid medium, which prevents oxygen transfer and results in low enzyme activity.
Fig. 3. Response surface plot for the analysis of the interactive effects of variables: (a) interactions between fermentation period and moisture content, (b) interactions between fermentation period and pH, and (c) pH and moisture content
The fermentation period was found to be more significant than that of the moisture content of the medium; as the fermentation period increased, the pectinolytic activity increased. However, as both the fermentation period and moisture content of the medium increased over the optimum period, there was a sharp decline in pectinase activity. This may be due to the viscosity of the particles and the moisture content hindering the active site of the enzymes. In fermentation bioprocesses, pectinase activity generally decreases above the optimum level (Biz et al. 2014), which was similar to the results of the current study. However, in this study, variation in the moisture content of the medium did not significantly affect pectinase activity (p<0.05). The interaction effect between pH and fermentation period on pectinase activity is depicted in Fig. 3b. The 3D response surface plot shows that an increase in pH leads to an improvement in enzyme production. However, an increase in incubation time above the optimum level reduced pectinase activity. This could be because the fungal cells may have reached the decline phase within 4 days and exhibited less enzyme production after 4 days. The sharp decline in enzyme biosynthesis at longer incubation times may be due to the depletion of nutrients (Kaur and Gupta 2017). The interaction effect of pH and moisture on pectinase activity is depicted in Fig. 3c. During enzyme bioprocesses, alterations in environmental parameterscause denaturation of the enzymes decrease pectinase activity. Decreased pectinase activity after the optimum fermentation period has been reported previously by various authors (Kaur and Gupta 2017; Oumer and Abate 2018). The optimized culture conditions for analyzing enzyme activity via Design-Expert software were 58.7% moisture content, 96 h incubation time, and pH 5.09, with a predicted activity of 428 U/gds. Triplicate experiments were performed to validate the predicted response, and the observed result was 438.3 U/gds. The predicted enzyme activity was very close to the experimental value, which validated the designed experimental model (Qi et al. 2009; Roy et al. 2018).
Table 4. Central Composite Design to Produce Pectinase in Solid-state Fermentation
Table 5. Analysis of Variance to Produce Pectinase in the Central Composite Design
Fungal Pectinase Reduced the Amount of Suspended Solids, Reducing Sugars, and pH
The total suspended solids in the raw orange juice were 21.2 ± 0.28%, and the total suspended solids were reduced in the pectinase-treated juice. The total percentage of suspended solids decreased with increasing concentrations of pectinase. The decreased sugar content in pectinase-treated juice is caused by the destruction of sugars by microbial enzymatic activity. The reduction in total suspended solids in the enzyme-treated juice results in the breakdown of suspended particles in the fruit juice. The pH of the raw fruit juice was 6.09 ± 0.16, and it increased slowly in the treated juice (Table 6). In contrast, a significant reduction in suspended solids, reducing sugars, and pH was observed in the juice treated with commercial pectinase. The fact that pectinase activity was produced from banana peels as a substrate in A. flavus cultivation may have significantly contributed to the efficiency of the orange peel clarification produced with banana peels. The present result is corroborated by a previous report by Barman et al. (2015), in which banana peel was used as a medium for pectinase production and used for food processing applications. During fruit juice clarification, nutrient losses are reported in beverages. For example, Haile et al. (2022) used pectinase from Serratia marcescens for the clarification of apple, lemon, and mango juices and reported a reduced total phenolic content. In the clarified juice, the absorbance value at 600 nm was less than that of the control (orange juice without enzymatic treatment). In this study, the pectinase-treated fruit juice presented decreased turbidity, and the absorbance at 600 nm was low (Fig. 4). In commercial enzyme-treated fruit juice, turbidity was reduced significantly, and no turbidity reduction was observed in the negative control (without enzyme treatment). The application of pectinase in juice clarification was reported previously by Ahmed and Sohail (2020), who achieved 61% orange juice clarification within 180 min of incubation at 30 °C with pectinases from Geotrichum candidumAA15. The use of crude enzymes from microbial sources has been recommended for fruit juice clarification, and the application of crude enzymes could reduce the production cost; this type of application does not require any purification process (Begum et al. 2014; Kc et al. 2020; Andrade et al. 2021).
Table 6. Effects of Various Concentrations of Pectinases Extracted from A. flavus on Sugar Content, Total Suspended Solid Content and pH
Fig. 4. Effect of A. flavus pectinase on orange juice clarification. The crude pectinase was applied at various concentrations (2 U/mL to 8 U/mL) in orange juice and treated for 60 min. As a negative control, the enzyme was not supplemented, and commercial pectinase (8 U/mL) was used as a positive control
CONCLUSIONS
- Banana fruit peels are good sources of pectin, and these pectin-rich substrates are natural inducers of the production of pectinase in solid-state fermentation by Aspergillus flavus. Banana fruit peels are considered as waste and can be utilized for the production of pectinase by microbial fermentation to reduce the production cost.
- Central composite design improved pectinase production, and the yield was improved over twofold. A limited number of experiments and resources were used due to the application of a statistical approach.
- The crude pectinase was used as a natural clarifying agent, and it reduced the total suspended solids and sugar level in the orange juice. The efficacy of crude pectinase as a clarifying agent in other fruit juices is another area to explore.
ACKNOWLEDGMENTS
The authors extend their appreciation to the Ongoing Research Funding program (ORF – 2025 – 1358) King Saud University, Riyadh, Saudi Arabia.
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Article submitted: September 7, 2025; Peer review completed: October 5, 2025; Revised version received and accepted: October 21, 2025; Published: October 31, 2025.
DOI: 10.15376/biores.20.4.11022-11040