Efficient clarification of apple juice using immobilized pectinase on glass surface in a novel support-free plate bioreactor

Abstract

This study presents the development and evaluation of a novel support-free plate bioreactor (SFPB) for immobilization of pectinase and its application in apple juice clarification. The SFPB was developed as an alternative to the conventional packed-bed bioreactor to address operational limitations when comparing at an equal surface area. Pectinase was covalently immobilized on the inner glass surface using 3-aminopropyl triethoxysilane and glutaraldehyde. The immobilization efficiency in the SFPB was 2.38 times higher than in the PBB. The immobilized enzyme retained approximately 70% of its initial activity after 225 min, compared to 47% in the PBB. Kinetic analysis showed increased Km and decreased Vmax for the immobilized enzyme, indicating reduced substrate affinity and catalytic activity. Under optimal conditions (pH 5.5, 50 °C, 0.8 mL min⁻¹, 45 min), the SFPB reduced turbidity from 325 to 4.3 NTU and increased clarity from 12.6 to 89.3%. These results demonstrate that the SFPB is a simple, reusable, cost-effective, and efficient system for industrial juice clarification.

Introduction

Packed-bed bioreactor (PBB) is commonly employed system for enzymatic processes utilizing immobilized enzymes, in which solid support materials such as glass beads are packed and arranged within a fixed chamber¹. The surfaces of these supports are often functionalized with some chemical substances, which enable the covalent binding of enzymes. The desired catalytic reactions take place when the substrate solution flows through the interstitial spaces between the packed beads, enhancing interaction with the immobilized enzymes². Despite the benefits of employing PBBs with solid supports like glass beads, their utilization has encountered specific operational challenges in continuous enzymatic operations. The packed arrangement of supports in the bioreactor may create surface constraints and mass transfer limitations during enzyme immobilization. Furthermore, handling their delicate nature can complicate industrial operations and potentially increase contamination risks^3,4. Moreover, replacing immobilized enzymes after activity decline often requires disassembling the bioreactor and refilling it with freshly prepared supports, increasing operational time, costs, and complexity and necessitating the engagement of several specialists, also raising the risk of contamination through repeated chemical treatments⁵. These limitations may restrict their application on an industrial scale and demonstrate the importance of innovative reactor designs that minimize such drawbacks while enhancing operational efficiency.

To address these restrictions, the current study proposes a novel immobilization strategy by directly binding pectinase enzymes onto the entire glass surface of a fabricated plate bioreactor through a covalent method, eliminating the need for any kind of support. This design simplifies the immobilization and regeneration process, as the internal glass surfaces can be easily treated and recharged without dismantling the entire system. Additionally, by reducing dependence on fragile supports and minimizing associated operational steps, the approach aims to improve enzyme stability and reusability while reducing costs. This novel technique not only deals with the operational issues of PBBs but also opens up new possibilities for developing efficient and sustainable bioreactors on an industrial scale.

The food and beverage sector, especially in fruit juice manufacturing, has increasingly utilized bioreactors for clarification processes. Juice clarification enhances product quality, sensory characteristics, and shelf life through the removal of suspended solids and decreasing turbidity^5–7.

Enzyme-based clarification, mainly using pectinase, has become a favored alternative to conventional procedures, including physical, chemical, and membrane-based processes, owing to its specificity, efficiency at mild conditions, and environmental compatibility^8–10. Pectinase comprises a class of enzymes that catalyze degradation of pectin structure, resulting in a transparent juice with enhanced color and flavor^11,12. The main disadvantage of free enzymes is their poor stability and inability to be reused, which has led to a shift toward using immobilized enzymes for enhanced durability and repeated use. Immobilization enhances catalytic efficiency by preventing enzyme aggregation, auto-proteolysis, and chemical degradation¹³. while also providing a longer half-life and greater resilience to pH and temperature fluctuations^14,15. Also, a key point to consider is that one of the most important advantages of immobilized enzymes is their suitability for use in continuous processes and consequently in bioreactors especially packed-bed types, which is likely impossible for free ones.

Numerous studies have so far focused on the use of immobilized enzyme-containing PBBs to carry out various continuous processes, such as juice clarification, and all of them have reported significantly better outcomes compared to free enzymes^10,16–18. Generally, conventional PBBs are well-regarded for their relatively straightforward design and reliable performance in standard continuous operations. While PBBs are extensively utilized in various industries, the application of solid supports in viscous fluids like fruit juices often encounters hydrodynamic and operational limitations. To enhance enzyme stability and overcome the inherent limitations of conventional PBBs, such as pressure drop, channeling, and support degradation, various strategies have been explored in recent years¹⁹. While traditional macroscopic supports, such as glass beads and alginate, are common, they often suffer from low enzyme loading and mass-transfer limitations. Recently, advanced nanostructured materials, such as magnetic hybrid nanoflowers²⁰, metal–organic frameworks^21–24, silica nanospheres²⁵, and Pickering emulsions²⁶ have been developed to provide high surface areas and robust protection.

Other approaches have focused on modifying the reactor configuration itself, such as employing fluidized bed bioreactors²⁷ or using carrier-free immobilization techniques like cross-linked enzyme aggregates²⁸. Despite these considerable advancements, challenges such as the high cost of support synthesis, complex regeneration protocols, and the physical breakdown of solid carriers under continuous flow of viscous fluids (like fruit juices) still persist, highlighting the need for completely support-free continuous systems. Therefore, acknowledging the importance of support removal in continuous processes in PBBs, this study evaluates the use of a novel support-free plate bioreactor (SFPB) for the continuous clarification of apple juice. The research compares key parameters such as enzyme kinetics, the reusability of the immobilized enzyme, and the amount of immobilized protein content per equivalent surface area between the conventional PBB and the newly fabricated plate bioreactor. The objective of this work is also to identify the potential advantages of this novel system in comparison to traditional packed configurations, providing recommendations for enhancing operational capacity, improving efficiency, and reducing costs in industrial applications.

Materials and methods

Chemicals and raw materials

Pectin from apple (poly-D-galacturonic acid methyl ester, degree of esterification 70–75%), D-( +) galacturonic acid monohydrate, 3-aminopropyl triethoxysilane (3-APTES), 3,5-dinitrosalicylic acid (DNS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Gallic acid, hydrochloric acid, acetic acid, glutaraldehyde, glass beads with a 6 mm diameter, sodium–potassium tartrate, copper (II) sulfate pentahydrate, sodium carbonate, Folin-Ciocalteu reagent, bovine serum albumin (BSA), sodium acetate buffer, potassium phosphate buffer, potassium chloride buffer, nitric acid, and other analytical-grade chemicals were also obtained from Sigma-Aldrich (St. Louis, MO, USA). Fructozym® P6-L (Erbslöh Geisenheim GmbH, Germany) with protein content of ~ 40 mg mL⁻¹, was used as the pectolytic enzyme which is a high-activity industrial pectinase derived from Aspergillus niger, specifically formulated for application in the fruit juice industries. All other reagents and chemicals were of analytical grade.

Support-free plate bioreactor

In previous studies, a PBB was used for juice clarification, employing enzymes immobilized on the outer surfaces of glass beads that functioned as carriers and were fully packed within the reactor^16–18. In the present research, building upon the limitations observed in the conventional PBB system, a new design approach was considered to address such challenges (Fig. 1).

Fig. 1.

Schematic structure of the novel support-free plate bioreactor.

Accordingly, an SFPB was designed in which the enzyme is directly immobilized on the functional interior surfaces, eliminating the necessity for any carrier material. This bioreactor was designed to address the operational constraints and mass transfer limitations of the PBB system while facilitating continuous enzymatic reactions post-immobilization. Our approach applies immobilization directly to the entire internal continuous surface of the reactor, specifically developed for continuous enzymatic processes. The SFPB was designed as a single parallel-plate channel with dimensions of 280 × 20 mm (L × W) and an internal gap height of 2 mm. This specific 2 mm gap was selected to maximize the direct contact between the flowing substrate and the enzymes immobilized on the internal surfaces of the bioreactor, leading to a more efficient process. Furthermore, this design ensures a uniform flow distribution across the biocatalytic surface without the hydrodynamic disturbances or channeling effects typically associated with internal packing materials in PBBs. A hot water bath sustained the required process temperature, while a pump regulated substrate flow at both the entrance and outlet. It should be noted that the functional surface area of this plate bioreactor was developed to correspond to that of glass beads in the PBB, thus ensuring comparable results at an equal surface area.

Pectinase immobilization

Pectinase was immobilized on 110 glass beads with a 6 mm diameter for the PBB. Beads were cleaned by washing with 5% HNO₃ at 85 °C for 120 min, followed by drying at 110 °C for 24 h. They were then functionalized using 10% (v/v) 3-APTES at pH 4.0 (adjusted by 0.1 N and 6 N HCl) and 60 °C for 3 h, rinsed, and subsequently dried at 80 °C for 24 h. Glutaraldehyde (2.5% v/v) was applied at room temperature (23 °C for a duration of 2.5 h. These functionalization steps, along with the subsequent immobilization in a pectinase solution (diluted with 0.1 M sodium acetate buffer, pH 4.0, for 5 h), were performed in a separate vessel under gentle stirring. Finally, after thorough washing, the prepared beads were packed into the PBB column. Fresh beads were used for each experimental procedure to ensure enzyme activity was preserved^10,29,30. Enzyme immobilization in the SFPB involved similar sequential functionalization and crosslinking stages, performed under the continuous circulation of reagents throughout the reactor. Particularly, 5% nitric acid, 10% APTES, 2.5% glutaraldehyde, and the enzyme solution were circulated continuously for 1, 2, 1.5, and 3.5 h, respectively, at a flow rate of 0.8 mL min⁻¹ (Fig. 2). Preliminary evaluations confirmed that optimal immobilization could be achieved within these shorter durations due to enhanced mass transfer under continuous flow. The continuous circulation of the enzyme solution was conducted at room temperature (23 °C). Following the final washing step, the bioreactor was dried in an oven at the specified temperature.

Fig. 2.

Schematic illustration of enzyme immobilization on the inside surface of the support-free plate bioreactor.

This continuous flow strategy facilitated more uniform activation of the interior glass surface, improved enzyme binding, and reduced the overall preparation time in comparison to immobilization in PBB. After each reaction, the bioreactor was recharged. To achieve the best approach for recharging the bioreactor, three preliminary treatments were evaluated: (1) using enzymatic flow alone, (2) using glutaraldehyde and enzymatic flow, and (3) using APTES flow, glutaraldehyde, and enzymatic flow. Based on this evaluation, the third approach proved to be the most effective and was therefore selected for bioreactor recharging throughout the entire duration of this study. The protein content in the SFPB and the PBB was determined using the Lowry method and compared. To ensure an accurate comparison, rather than relying on partial sampling, the entire functionalized internal surface of the SFPB and all functionalized beads from the PBB were used. A calibration curve was developed using bovine serum albumin, and the results were reported as the amount of protein (mg) per equal surface area (cm²)³¹.

Determination of the free and immobilized pectinase activity

The DNS method, as described by de Oliveira et al.⁴⁵, was employed to assess the activity of both free and immobilized Pectinase. One unit of enzyme activity is defined as the quantity of enzyme required to produce 1 μmole of galacturonic acid per min under the experimental conditions of this study. Pectinase activity was reported based on specific activity (U/mg protein). In this study, an apple pectin solution was used as the substrate, with D-galacturonic acid monohydrate functioning as the standard for the calibration curve. 1 mL of free pectinase at a concentration of 1 mg mL⁻¹ was mixed with 2 mL of a 1% w/v apple pectin solution prepared in 0.1 M acetate buffer (pH 5.0) and incubated at 50 ℃ for 15 min. Following incubation, 1 mL of the reaction mixture was transferred to a test tube containing 1 mL of DNS reagent, heated in a boiling water bath for 5 min, and subsequently cooled in an ice-water bath. Afterward, 3 mL of distilled water was added, and the absorbance was measured at 540 nm. The activity of immobilized pectinase was assessed by circulating the substrate in the SFPB and the PBB under optimized conditions of 50 ℃ and pH 5.5 for 15 min. To ensure an accurate comparison of specific activity across all three systems (free enzyme, PBB, and SFPB), an equal total volume of substrate was used, and it was continuously recirculated in the bioreactors for the exact same reaction time (15 min). Enzyme activity was subsequently measured using the same procedure described above. The activity recovery of the immobilized enzymes was calculated using the following equation:

1

Effect of flow rate on the efficacy of immobilized pectinase in SFPB and PBB

The influence of flow rate on the efficacy of the immobilized pectinase in SFPB and PBB was assessed throughout a range from 0.2 to 10 mL min⁻¹. A 1% (w/v) pectin solution was used as the substrate, with the temperature consistently maintained at 50 °C and pH of the buffered pectin solution at 5.5. The quantity of reducing sugar in the samples was determined by the DNS method as stated earlier. The residence time at each flow rate was calculated using the following equation: In this formula, V_v represents the vacant volume of the bioreactor in mL, and F denotes the flow rate in mL per min.

2

Comparative activity and stability of free and immobilized pectinase

The effect of pH on the activity of free and immobilized pectinase in SFPB and PBB was determined by conducting enzymatic hydrolysis on pectin solutions prepared in different buffers with pH values ranging from 2 to 8 at 50 ℃ for 15 min. In addition, the pH stability of both free and immobilized enzymes was assessed by pre-incubating them in various buffers with pH levels ranging from 2 to 8 for a duration of 30 min; subsequently, the residual activity was measured under optimal conditions as described earlier. The influence of temperature on the activity of free and immobilized pectinases was investigated by performing the reaction at various temperatures (30–9 ℃), pH = 5 for free enzymes, pH = 5.5 for immobilized enzymes, and a reaction time of 15 min for both cases as optimal conditions. Additionally, the thermal stability of enzymes was evaluated by pre-incubating free and immobilized pectinase at different temperatures (30–60 ℃) for 0–120 min and then completing the process under optimum condition to measure the enzymes’ residual activity. It is important to note that the flow rates employed in SFPB and PBB were 0.8 and 0.5 mL min⁻¹, respectively.

Assessment of kinetic parameters for free and immobilized enzymes

The Michaelis–Menten constant (Km) and the maximum reaction rate (V_max) were determined in optimum conditions as follows:

3

where [V_max] represents the maximal reaction rate, [S] denotes the pectin concentration in the reaction medium (2–20 mg mL⁻¹), and K_m signifies the Michaelis–Menten constant^5,32.

Reusability of immobilized pectinase

To evaluate the reusability of immobilized enzymes in SFPB and PBB, a fresh 1% w/v pectin solution (pH: 5.5 using a 0.1 M sodium acetate buffer) was circulated in both bioreactors. It should be noted that the immobilized enzymes were exposed to fresh substrate every 15 min, and the hydrolysis reaction was conducted at 50 °C under optimum conditions. The residual enzyme activity in both bioreactors was measured using the DNS method throughout the process.

Surface characterization

Before and after pectinase immobilization on a flat glass made of the same material used for constructing the bioreactor, the surface morphology was investigated by a field emission scanning electron microscopy (TESCAN MIRA, Czech Republic). Images were captured at a magnification of 500 × .

Clarification of apple juice

Preparation of apple juice

The apples were bought at a local shop in Tehran, Iran. The fruits were cleaned, halved, and juiced using a household fruit juicer. The extracted raw juice was subsequently filtered through Whatman No.1 filter paper using a vacuum pump and kept in a refrigerator until needed.

Fruit juice clarification

The results from the previous section indicate that the SFPB demonstrated superior efficiency compared to the PBB. Consequently, the SFPB was utilized to evaluate its effectiveness in more practical conditions during studies on clarifying apple juice. The process time of the apple juice was determined by measuring the turbidity over a period ranging from 0 to 75 min to investigate how it affects the clarification in the SFPB. During this experiment, the optimal conditions identified included a temperature of 50 °C and a flow rate of 0.8 mL min⁻¹. The apple juice was clarified using two methods: (1) continuous clarification using immobilized enzyme in the SFPB and (2) the free enzyme method. For this purpose, apple juice was subjected to immobilized enzymes by circulating in the SFPB considering optimal conditions (50 °C, 45 min, and a flow rate of 0.8 mL min⁻¹). On the other hand, for the free enzyme method, 407 μL of free enzyme solution, equivalent to the protein amount immobilized on the entire surface area of SFPB, which is equal to 124 cm², were added to 11.2 cm³ of apple juice, which corresponds to the internal volume of the bioreactor. The optimal conditions for this stage were a temperature of 50 °C and a duration of 45 min. The obtained results of treatments were then compared to untreated apple juice. for convenience, they were abbreviated to untreated apple juice (UAJ), apple juice clarified with free pectinase (AJF), and apple juice clarified using immobilized pectinase in a continuous process in SFPB (AJIC).

Turbidity and clarity

A TURBO 350 IR portable turbidimeter Weilheim, Germany, was utilized for measuring turbidity (NTU) in various samples at room temperature. A spectrophotometer, calibrated to 540 nm, was employed to evaluate clarity, and the transmittance percentage (%T) was calculated from the results of samples that were diluted four-fold with distilled water³³.

The following formula was employed to compute Percentage of light transmission:

4

pH and acidity

A GLP22 pH meter (Crison Instruments, Barcelona, Spain) was employed to measure the pH of both unclarified and clarified juice samples. Furthermore, juice samples were titrated with 0.1 N sodium hydroxide to ascertain the total titratable acidity (TTA). The phenolphthalein reagent was used as an indicator in this experiment and the results were quantified in g of citric acid per 100 mL of apple juice³⁴.

Total soluble solids and reducing sugar content

A refractometer (Abbe, Bellingham-Stanley, London, England) was used to record the TSS (°Brix) at approximately 23 °C. As previously mentioned, the DNS method was used to determine the reducing sugar content (mg mL⁻¹).

Viscosity

A programmable viscometer (Brookfield Ametek, DV3TLVTJ0, USA) was used at room temperature and calibrated to a shear rate of 50 rpm to measure the viscosity of apple juice samples. The results were expressed in mPa.s³⁵.

Pectin presence test

To conduct this test, chilled ethanol was mixed with different fruit juice samples in a 2:1 v/v ratio and thereafter refrigerated at about 4 °C for 24 h. Following this period, the presence or lack of supernatant indicates the existence or absence of pectin, respectively⁹.

Total phenol content (TPC)

The Folin-Ciocalteu method was employed to quantify the total phenolic content of fruit juice samples³⁶.To achieve this, 0.5 mL of a 10% (v/v) Folin-Ciocalteu reagent solution was mixed with 0.1 mL of the fruit juice sample, and the resultant combination was kept in the dark for five min. Following the addition of 0.4 mL of a 10% (w/v) sodium carbonate solution, the mixture was maintained in darkness for 45 min. After measuring the absorbance at 765 nm, the sample’s absorbance was compared to the gallic acid standard curve (10–200 μg/mL) to ascertain the total phenolic content (TPC). The TPC was quantified as mg GAE/mL, indicating gallic acid equivalent per mL of apple juice sample^36,37.

Determination of antioxidant activity of apple juice

To analyze the radical scavenging activity of apple juice samples, ABTS and DPPH assays were applied. For this purpose, an ABTS solution (7 mM) was initially mixed with a potassium persulfate solution (2.45 mM) to make the ABTS solution, which was thereafter incubated in darkness for 16–24 h. Subsequent to incubation, it was diluted with distilled water to attain an absorbance of 0.7 ± 0.02 at 734 nm. Afterwards, 1.9 mL of the produced ABTS solution was combined with 0.1 mL of the apple juice sample, which had been diluted 20 times with distilled water. The solutions were vortexed and kept in darkness for 45 min to finalize the antiradical reaction. The absorbance for ABTS radical scavenging activity (ABTS-RSA) was measured at 734 nm³⁰. In the case of the DPPH assay, 1.9 mL of the prepared DPPH ethanolic solution (0.1 mM) was mixed with 0.1 mL of the diluted apple juice sample, which had been diluted 10 times with distilled water. Due to completing the antiradical reaction, the resulting mixtures were vortexed and subsequently maintained in the dark for 45 min. DPPH radical scavenging activity (DPPH-RSA) was assessed by measuring absorbance at 517 nm³⁸.

The following equation was used to determine these antiradical activities:

5

Color parameters

A color reader (CHROMA METER, CR-400, Japan) was employed to determine the color parameters L*, a*, and b* of the apple juice samples³⁹.

Statistical procedure

In this study, one-way ANOVA was used using SPSS software v. 27 to assess significant differences between the samples (p < 0.05). Values are means ± SD (n = 3).

Results and discussion

Evaluation of immobilized protein content in bioreactors

The comparison between the protein content immobilized in the SFPB and the PBB, based on an equal surface area equivalent to 124 cm² is revealed in Table 1. The data demonstrate that the immobilized enzyme loading in the SFPB is approximately 2.38 times higher than that in the PBB system. The notable increase can be due to the distinctive design of the SFPB, characterized by a continuous and smooth surface that facilitates improved surface interactions and the formation of covalent bonds between the enzyme’s amino groups and the functionalized surface. APTES creates a silane coupling agent by introducing primary amino groups to the glass surface, whereas glutaraldehyde functions as a cross-linker between these groups and the enzyme’s amino groups¹⁰.

Table 1.

Protein content immobilized in the SFPB and PBB, based on an equal surface area.

Bioreactor	Protein content (mg/124 cm2)*
SFPB PBB	16.28 ± 0.06 a 6.82 ± 0.03 b

SFPB: Support-Free Plate Bioreactor, PBB: Packed-bed bioreactor. *Values bearing different lowercase letters in the same column are significantly different (p < 0.05).

The uniformity of the surface and the appropriate distribution of these groups are critical for obtaining high immobilization efficiency. The rounded surfaces and spatial constraints of glass beads may lead to non-uniform APTES coverage, a heterogeneous distribution of active groups, and a consequent decrease in the immobilized enzyme loading. The spatial constraints resulting from the curvature of the beads can limit enzyme accessibility to the surfaces. Furthermore, the preparation method contributes to this difference. During the batch preparation of the PBB, the beads were placed in a vessel with gentle magnetic stirring. Even with gentle agitation, the continuous movement and mild friction between the solid beads, the magnetic bar, and the vessel walls can slightly interfere with the uniform formation of the delicate functional layers and limit maximal enzyme immobilization. In contrast, the SFPB utilizes a completely stationary design where solutions flow continuously between the flat plates. This configuration provides optimal conditions for surface functionalization and enzyme binding without any physical disturbance. In conclusion, the SFPB offers a more effective and cost-efficient method for enzyme immobilization, especially in industrial applications. This advantage results from the elimination of physical supports, such as beads, and the establishment of appropriate and consistent conditions for covalent immobilization on surfaces⁴⁰.

Specific activity of the free and immobilized pectinase

Preliminary analyses were performed to assess the specific activity of free and immobilized pectinase enzymes under optimal conditions to evaluate the performance of the immobilized enzyme. The specific activity of free pectinase was 3.16 ± 0.06 U/mg of protein under optimal conditions (50 °C and pH of 5), whereas the specific activities of immobilized pectinase were measured at 2.94 ± 0.07 U/mg (activity recovery of 93%) in the SFPB and 2.75 ± 0.12 U/mg (activity recovery of 87%) in PBB, both at 50 °C and pH of 5.5, using a modified DNS method as described above. The findings clearly indicate differences among the systems, so that, the free enzyme demonstrated the highest specific activity, as anticipated, as it is not influenced by structural or diffusional constraints. The SFPB exhibited a slightly reduced value, suggesting that the enzyme’s catalytic efficiency was well maintained post-immobilization. Conversely, the PBB exhibited the lowest specific activity, attributable to diffusional limitations within the bead matrix and diminished substrate accessibility to the enzyme’s active sites. The findings indicate that the SFPB offers superior preservation of enzyme quality compared to the PBB^41,42.

Effect of flow rate on the efficacy of immobilized pectinase in the SFPB and PBB

The graphs in Fig. 3, demonstrate that an increase in flow rate correlates with a reduction in the duration the substrate interacts with the immobilized enzymes, leading to diminished generation of reducing sugar. Reduced flow rates provide increased contact time between the substrate and immobilized enzymes, resulting in enhanced synthesis of reducing sugars; nevertheless, these rates may elevate costs and affect enzyme stability. In continuous operations like apple juice clarification, it is essential to optimize both reaction conditions (temperature and pH) and the flow rate of substrate to control operational expenses while ensuring adequate reducing sugar production. Given that in SFPB, increasing the flow rate from 0.2 to 0.8 mL min⁻¹ does not substantially influence the yield of reducing sugar, the ideal flow rate of 0.8 mL min⁻¹ has been chosen to minimize time expenditure and cost^17,43. This flow rate provides a suitable residence time of 15 min and produces adequate reducing sugar, without any noticeable change, while decreasing the residence duration from 60 to 15 min. In comparison, the PBB showed a similar pattern, reaching its best performance at a lower flow rate of 0.5 mL min⁻¹. The higher flow rates resulted in a significant decrease due to lower substrate-enzyme interaction and mass transfer restrictions inside the PBB. This optimum flow rate related to PBB was consistent with our previous study¹⁶.

Fig. 3.

Effect of flow rate on efficiency of SFPB and PBB at temperature of 50 °C. SFPB: support-free plate bioreactor, PBB: packed-bed bioreactor. Data are presented as mean ± standard deviation (n = 3). Different lowercase letters indicate significant differences (p < 0.05).

Influence of pH and temperature on the activity and stability of free and immobilized pectinase

As can be seen in Fig. 4a, the free enzyme exhibits greater activity than immobilized systems across most pH ranges, likely due to its structural flexibility, facilitating environmental adaptability and substrate access to the active site⁴⁴. The SFPB demonstrated greater efficiency compared to the PBB. Immobilizing enzymes directly on interior surfaces enhances substrate accessibility and distribution within the plate system. In PBB systems, substrate accumulation and restricted access to bead surfaces reduce efficiency. Preferred flow channels in PBB lead to uneven substrate distribution, which subsequently diminishes the performance of immobilized enzymes. The covalent immobilization of enzymes through APTES and glutaraldehyde can restrict enzyme mobility and decrease activity⁴⁰. The types of chemical cross-linkers and enzymes used can affect the optimal pH following immobilization.

Fig. 4.

Effect of pH on the activity (a) and stability (b) of the free and immobilized pectinase. SFPB: support-free plate bioreactor, PBB: packed-bed bioreactor.

The beneficial effects of immobilization are confirmed by the stability properties of the immobilized enzymes across various pH levels (Fig. 4b). The free enzyme demonstrates a significant reduction in residual activity in acidic and alkaline conditions. The SFPB sustains high residual activity throughout a broad pH range (78.3–100% between pH 3 and 5.5). Furthermore, the PBB demonstrates greater stability compared to the free enzyme but slightly lower stability compared to the SFPB system. Overall, our findings suggest that immobilization, particularly in SFPB, not only preserves enzyme activity but also significantly enhances stability at pH levels under suboptimal conditions. The enzyme’s restricted conformational mobility after covalent attachment likely preserves it from denaturation and environmental stress, accounting for its enhanced stability. Furthermore, these results accorded with prior studies^10,45.

Figure 5a, illustrates that temperature significantly affects the relative activity of the free enzyme, which declines sharply beyond the optimal point due to denaturation and loss of structural integrity. The SFPB and PBB systems demonstrated enhanced relative activity over a broader temperature range, enabling the immobilized enzymes to show significantly higher relative activity compared to the free form. This behavior may be due to the restriction of conformational mobility caused by immobilization, which stabilizes the active conformation and diminishes unfolding. The SFPB showed the most notable influence among the immobilization techniques, suggesting that surface binding to the glass surface provides more favorable conditions compared to PBB.

Fig. 5.

Effect of temperature on the activity of the free and immobilized pectinase (a), Thermal stability of the free (b) and immobilized pectinase in SFPB (c) and PBB (d). SFPB: support-free plate bioreactor, PBB: packed-bed bioreactor.

The thermal stability measurements presented in Fig. 5b–d provide further evidence of the protective benefits of immobilization. At elevated incubation temperatures, the free enzyme exhibited a significant and irreversible reduction in residual activity, while immobilized enzymes demonstrated greater stability, particularly at 50–60 °C, a range where denaturation typically occurs more rapidly. The findings align with our previous research, indicating that immobilized enzymes exhibit greater activity compared to free enzymes, especially at elevated temperatures^5,10.

Kinetic parameters

Table 2 presents the K_m and V_max values of the pectinase enzyme evaluated for the free enzyme and two immobilized systems (SFPB and PBB). The data indicate changes in the enzyme’s kinetic properties following the immobilization process in both configurations. The observed reduction in V_max after enzyme immobilization in both systems signifies a decline in the maximum reaction velocity, which directly reflects the rate of substrate conversion and product release from the active site. This decline may be related to limitations in substrate penetration and a reduction in the flexibility of the immobilized enzyme structure, which limits optimal mobility and interaction between the enzyme and the substrate. The SFPB exhibited a lesser reduction in V_max (from 3.11 to 2.98 µmol min⁻¹ mg⁻¹) compared to the PBB (from 3.11 to 2.36 µmol min⁻¹ mg⁻¹).

Table 2.

Kinetic parameters (K_m and V_max) of free and immobilized pectinase (SFPB and PBB) at 50 °C and pH 5.0 and 5.5, respectively.

Enzyme	Km (mg mL−1)*	Vmax (µmol min−1 mg−1)*
Free	9.52 ± 0.06 c	3.11 ± 0.03 a
SFPB	9.81 ± 0.03 b	2.98 ± 0.08 b
PBB	10.63 ± 0.08 a	2.36 ± 0.02 c

SFPB: Support-Free Plate Bioreactor, PBB: Packed-bed bioreactor. *Values bearing different lowercase letters in the same column are significantly different (p < 0.05).

This decrease suggests that immobilizing the enzyme on a uniform glass surface within the plate bioreactor has a minimal impact on catalytic activity, thereby enhancing enzyme function through improved substrate accessibility and reduced mass transfer limitations. Both immobilization methods show an increase in K_m compared to the free state, indicating a reduced affinity of the enzyme for the substrate due to immobilization. This change may stem from limitations in mass transfer and decreased substrate availability at the enzyme’s active site. The K_m values of the two immobilized systems reveal that the PBB exhibited a notable increase in K_m (10.63 mg mL⁻¹) compared to the SFPB (9.81 mg mL⁻¹). This difference may result from the spherical shape of the glass beads and the enzyme’s distribution in this setup, which diminishes the available active surface area and increases the boundary layer surrounding the beads, complicating substrate transfer to the enzyme’s active site. In the PBB, the bead density and the potential for preferential flow (channeling) reduce substrate flow uniformity, consequently decreasing the enzyme’s affinity for the substrate and lowering kinetic efficiency^10,41. Although the SFPB demonstrated superior kinetic properties compared to the PBB, the inherent decrease in Vmax ​and increase in Km upon immobilization remain challenges. To overcome these limitations in future applications, several strategies can be employed. Optimizing the concentration of the cross-linking agent (glutaraldehyde) or incorporating flexible spacer arms⁴⁶ (e.g., polyethylene glycol) could reduce the structural rigidity of the immobilized enzyme, preserving its active conformation and improving substrate accessibility. Additionally, modifying the hydrodynamic conditions, such as optimizing flow rates could further disrupt the boundary layer and minimize mass transfer resistances⁴⁷. Beyond kinetic improvements, future studies should focus on the scale-up of the SFPB to evaluate the commercial feasibility of this support-free system for industrial continuous fruit juice clarification.

Reusability of immobilized pectinase

The activity graph of the immobilized enzyme in the SFPB (Fig. 6) maintained more than 80% of its initial activity even after 150 min. Subsequent to this time, enzyme activity consistently declined and stabilized at around 70%. This discovery highlights the remarkable efficacy of the enzyme immobilization method employed in this system, together with the decreased enzyme consumption during continuous operations. The prolonged elevated activity may result from the principled immobilization technique and the effective use of APTES and glutaraldehyde, facilitating stable enzyme binding to the glass surface and enhancing its resilience against detrimental pressures throughout operations. The gradual decline in activity during subsequent time may result from physical and chemical changes on the bioreactor surface, the degradation of specific glutaraldehyde crosslinks, the displacement of enzyme molecules, or proteolysis. Moreover, a comparison of the enzyme activity in the SFPB and PBB indicates that the enzyme activity in the SFPB was initially superior to that in the PBB. While residual activity diminished in subsequent time, it did so at a more gradual rate in the SFPB⁴¹.

Fig. 6.

Reusability of immobilized pectinase in a SFPB compared with a PBB over 225 min. The hydrolysis reactions were conducted at 50 °C, pH 5.5, with a reaction time of 15 min. SFPB: support-free plate bioreactor, PBB: packed-bed bioreactor.

Immobilizing the enzyme on the plate glass surface enhances substrate accessibility, increases available space, and facilitates a more uniform substrate transfer, leading to consistent enzyme activity. Conversely, the PBB initially exhibited lower enzyme activity and showed a more rapid decline over time. The accelerated decline may be attributed to the limited accessibility of the substrate to the enzymes among the glass beads, together with the potential accumulation of reaction products in the PBB, which obstructs access to new substrates for the active enzyme. The results indicate that the SFPB maintained around 70.2% of its initial activity after 225 min, but the PBB preserved just 47% under the same conditions. Kharazmi et al.¹³ conducted a study investigating the reusability of pectinase enzymes immobilized on magnetic nanoparticles. The findings demonstrated that the residual activity of the immobilized enzyme attained approximately 50% of its initial level following 150 min.

Surface characterization by FESEM

Using Field Emission Scanning Electron Microscopy (FESEM) for evaluating the surface morphology of a flat glass made of the same material used for constructing the bioreactor before and after enzyme immobilization provides direct visual confirmation of effective immobilization. In the micrograph captured prior to the immobilization process (Fig. 7a), the inner glass surface of the bioreactor appeared relatively smooth and uniform, without any noticeable structures characteristic of biological molecules. The surface roughness was consistent with the inherent texture of untreated glass, and no significant coverage or additional structures were observed, as expected in the absence of immobilized enzymes. Conversely, in the image obtained after the immobilization process (Fig. 7b), distinct morphological changes became evident. Irregular formations and denser surface textures were observed compared to the untreated surface⁴³. These modifications suggest the presence of a coating layer, likely composed of immobilized enzyme molecules along with residual glutaraldehyde and APTES. The increased surface roughness and formation of new structures confirm that the enzyme immobilization on the glass surface was successfully achieved. Additionally, the uniform distribution of these structures across the surface indicates that the immobilization process was effectively controlled and consistently applied throughout the bioreactor surface.

Fig. 7.

Field Emission Scanning Electron Microscope (FESEM) images of the SFPB surface before (a) and after (b) pectinase immobilization by glutaraldehyde. SFPB: support-free plate bioreactor.

Effects of treatment time on the efficacy of immobilized pectinase in the SFPB for apple juice clarification

Given that the results of comparing the PBB and the SFPB demonstrated significantly superior performance of the latter in pectin removal, all subsequent experiments, as described in the Materials and Methods section, were conducted using the SFPB. To better understand its performance, the results were also compared with those obtained using the free enzyme. However, before evaluating the performance of the SFPB in juice clarification, it is necessary to determine the time required to achieve an acceptable level of clarity. As can be seen in Fig. 8, the turbidity of apple juice decreased significantly during the first 15 min. This reduction suggests that the pectinase enzyme, immobilized in the SFPB, had a substantial effect on the structure of apple pectin. The breakdown of the intact pectin network reduces the juice viscosity and disrupts its colloidal stability. This process leads to the release and subsequent precipitation of trapped suspended compounds, thereby improving juice clarity. In the following time intervals (30 and 45 min), this effect diminished, which meant that the process reached steady-state. This finding indicates that the primary components responsible for turbidity, especially pectin, were largely degraded in the initial phase, resulting in minimal changes during the later time intervals. The measurements taken at 60 and 75 min showed no apparent differences in turbidity. After this point, only slight improvements in transparency were observed, suggesting that the process may become redundant. Therefore, a total reaction time of 45 min appears to be the most efficient and cost-effective approach for enhancing transparency^16,45.

Fig. 8.

Effect of continuous process duration on apple juice clarification in SFPB at 50 °C and a flow rate of 0.8 (mL min⁻¹), NTU = Nephelometric Turbidity Unit. SFPB: support-free plate bioreactor.

The impact of clarification on the characterization of apple juice

The characteristics of apple juice clarified by both the free enzyme and the continuous SFPB processes under established optimal conditions were evaluated and compared with those of the raw juice sample¹⁸.

Turbidity and clarity

As shown in Table 3 and Fig. 9, both clarification methods significantly improved the optical properties of the apple juice compared to the untreated apple juice (UAJ). The initial turbidity of UAJ was high at 325 NTU, with a corresponding clarity of only 12.58%. Following clarification, the turbidity was drastically reduced to 4.84 NTU in the apple juice clarified with free pectinase (AJF) and 4.34 NTU in the apple juice clarified using immobilized pectinase in a continuous process in SFPB (AJIC). Consequently, the clarity notably increased to 87% for AJF and 89.3% for AJIC. This detailed comparison indicates that the continuous SFPB system performed highly efficiently, achieving clarification levels slightly superior to the free enzyme while offering the operational advantages of a continuous process^18,48.

Table 3.

Physicochemical properties including turbidity (NTU), clarity (%T), pH, total titratable acidity (TTA; g citric acid/100 mL juice), total soluble solids (TSS; °Brix), reducing sugar content (RSC; mg mL⁻¹), viscosity (mPa. s), and pectin presence test of untreated and clarified apple juice samples.

Parameters	Turbidity*	Clarity*	pH*	TTA*	TSS*	RSC*	Viscosity*	Pectin test*
UAJ	325 ± 0.22a	12.58 ± 0.09c	4.2 ± 0.02a	0.24 ± 0.05c	16 ± 0.0a	36.39 ± 0.09c	5.6 ± 0.04a	Positive
AJF	4.8 ± 0.24b	87 ± 0.1b	3.89 ± 0.04b	0.38 ± 0.01b	14.2 ± 0.0b	41.84 ± 0.16b	4.7 ± 0.01b	Negative
AJIC	4.3 ± 0.33b	89.3 ± 0.12a	3.72 ± 0.05c	0.43 ± 0.04a	13 ± 0.0c	45.7 ± 0.12a	4.2 ± 0.08c	Negative

UAJ: untreated apple juice, AJF: apple juice clarified with free pectinase and AJIC: apple juice clarified using immobilized pectinase in a continuous process in SFPB. NTU = Nephelometric Turbidity Unit. * Values bearing different lowercase letters in the same column are significantly different (p < 0.05).

Fig. 9.

The samples clarified by free and immobilized enzyme in SFPB. (a) UAJ: untreated apple juice, (b) AJF: apple juice clarified with free pectinase and (c) AJIC: apple juice clarified using immobilized pectinase in a continuous process in SFPB. SFPB: support-free plate bioreactor.

pH and acidity

As can be seen in Table 3, the declining pH and rising acidity indicate that the pectinase enzyme has promoted the breakdown of pectin, resulting in the release of organic acids. The usage of the immobilized enzyme in the continuous system (AJIC) demonstrated greater efficacy than alternative methods, suggesting that this continuous process in SFPB is appropriate for clarifying apple juice. The pectinase enzyme degrades pectin into galacturonic acid units and smaller fragments. These chemicals have acidic characteristics, leading to a decrease in pH. Galacturonic acid (GalA) derived from pectin is then transformed into other organic acids, such as acetic acid, formic acid, and lactic acid, resulting in a further reduction in pH⁴⁹. Conversely, the release of galacturonic acid and its conversion into alternative acidic molecules result in elevated acidity levels. Moreover, the decrease in viscosity resulting from the breakdown of pectin’s gel structure facilitates the solubility of the generated organic acids in the aqueous phase, enhancing their titratability. These findings align with those reported by Çesko et al.⁵⁰ who observed similar effects of pectinase treatment on the physicochemical properties of apple juice.

Total soluble solids (TSS) and reducing sugar content

The application of the clarification method has resulted in a decrease in total soluble solids, as shown in the Table 3. Furthermore, this process resulted in an increase in reducing sugar levels. The immobilized enzymes facilitated the breakdown and hydrolysis of the pectin polymer network, resulting in the precipitation and degradation of various substances, which decreased the soluble solids⁵¹. Additionally, the activity of the pectinase enzyme accelerated the breakdown of pectin into simpler sugars, releasing them into the solution and boosting the sugar concentration. These alterations can affect the flavor and sweetness of the juice, as monosaccharides typically impart a sweeter taste than polysaccharides. The results correspond with those presented by Muller et al.⁵² and Hosseini et al.^16,17, who observed that the utilization of pectinase, especially in its immobilized state, enhances reducing sugar levels and juice clarity by efficiently degrading pectin networks.

Viscosity

Viscosity, a result of pectic substances and other soluble polysaccharides, significantly affects the production of fruit juice. The data in Table 3 demonstrate that the clarifying process using SFPB in AJIC reduced the viscosity of UAJ from 5.6 to 4.2 mPa.s. This change highlights the effect of the immobilized pectinase enzyme on the pectin structure, leading to its breakdown and the release of entrapped water. Additionally, this effect, along with the degradation of the pectin structure into smaller components such as oligosaccharides and monosaccharides, has contributed to the decrease in viscosity. These observations are consistent with the findings of Rozendo et al.⁵³ who demonstrated that pectinase treatment effectively lowers the viscosity of apple juice by breaking down pectin networks and releasing bound water.

Pectin presence test

Table 3 revealed that the pectin presence test was negative in all treatments except for the control sample. The control sample remained unchanged by the pectinase enzyme, confirming the presence of pectin¹⁶.

Total phenol content (TPC)

A decreasing trend in phenolic compounds is observed during the clarification process, as illustrated in Table 3. This reduction is likely attributable to the deposition of some compounds, such as phenolics, which therefore diminishes their content in fruit juice. As well, the existence of supplementary enzymatic activity can alter the profile of phenolic compounds. Phenolic chemicals function as natural antioxidants and may inhibit DPPH and ABTS free radicals. The control sample (UAJ), exhibiting the highest total phenolic content (TPC), revealed the most significant radical scavenging activity. Conversely, the sample subjected to continual clarifying, which revealed a more significant decrease in TPC content, demonstrated the lowest radical scavenging activity⁵⁴.

Antioxidant properties

The DPPH and ABTS scavenging activities are almost equivalent (Table 3); nevertheless, in all samples, the DPPH scavenging value is marginally superior to that of ABTS. These data indicate that the phenolic chemicals in apple juice may possess a greater tendency to scavenge DPPH radicals. These findings are consistent with those reported by Sójka et al.⁵⁵ who observed similar reductions in total phenolic content and antioxidant activity following clarification processes in fruit juices.

Color parameters

The lightness (L), green–red value (a), and blue-yellow value (b) of UAJ, AJF, and AJIC in SFPB were assessed, and the results are presented in Table 3. The clarification method significantly enhanced the brightness index (L), demonstrating its positive effect on the clarity and reduction of turbidity in the raw apple juice sample. Additionally, the data indicate that this clarifying technique led to a decrease in the (a) index and an increase in the (b) index. The reduction in the (a) value, characterized by diminished redness in the juice and a shift toward more neutral colors, is attributed to the deposition of red pigment molecules, such as anthocyanin, as well as some antioxidant. These findings are in agreement with the results reported by Uzuner et al.⁵⁶ who similarly observed improvements in juice clarity and color Parameters following enzymatic clarification using microalgae-derived polygalacturonase.

Conclusion

The SFPB proved to be a successful system for enzyme immobilization without the need for support materials, demonstrating effective pectinase immobilization and activity retention. Kinetic studies revealed that immobilization slightly reduced V_max and increased K_m values, indicating a moderate decline in catalytic efficiency and substrate affinity, with the SFPB performing better than the PBB system. SFPB is capable of reusability in multiple operational times and also allows for easy enzyme recharging, ensuring sustained catalytic performance. FESEM analysis confirmed successful enzyme immobilization by showing increased surface roughness and uniform enzyme coating on the glass surface of the SFPB. Clarification of apple juice using the immobilized enzyme significantly decreased turbidity and viscosity, enhanced clarity, and improved physicochemical properties. The process also caused some changes, like lowering the pH and total soluble solids and raising the acidity. This was because the enzymes broke down pectin and released organic acids and simple sugars. Antioxidant capacity and total phenolic content decreased slightly, reflecting the deposition of phenolic compounds during clarification. Overall, the findings of this study introduce a novel and efficient system that holds promise for enhancing enzymatic processes in both industrial and laboratory settings.

Author contributions

Hamid Mahmood-Fashandi: Investigation, Methodology, Formal analysis, Writing—original draft. Faramarz Khodaiyan (Corresponding Author): Conceptualization, Validation, Funding acquisition, Project administration, Resources, Supervision. Seyed Saeid Hosseini: Formal analysis, Methodology, Validation, Writing—review & editing.

Funding

The authors declare that this research did not receive any specific funding from public, commercial, or not-for-profit organizations.

Data availability

Data will be made available on request.

Declarations

Competing interests

The authors declare no competing interests.