After 25 days of static incubation at 28°C, laccase from *Pleurotus ostreatus* NRC620 showed the highest activity in the fungal culture medium. The optimal pH and temperature values for this enzyme were 3.0 and 70°C, respectively. After 2 hours of incubation at 40°C and 50°C, the enzyme activity retained 68.33% and 59.61%, respectively. After 2 hours of incubation in citrate-phosphate buffer (pH 7.0), the enzyme activity remained at 100%. The addition of 10 mM MgSO₄ and CuSO₄ increased the enzyme activity by approximately 21% and 35%, respectively, while NaCl, MnCl₂, KCl, and CaCl₂ inhibited the enzyme activity. Using ABTS as a substrate, the kinetic parameters (Km and Vmax) of *Pleurotus ostreatus* NRC 620 laccase were 1.99 mM and 16,217 μmol min−1 L−1, respectively. Enzymatic treatment of apple juice samples significantly reduced both pH and viscosity, and this reduction correlated with an increase in storage time. Laccase treatment resulted in a slight decrease in the total phenolic content of apple juice, but no reduction in antioxidant activity was observed.
In recent years, researchers have focused on the application of green biotechnology in the food industry. Laccase is one of the most useful enzymes in the food industry, finding applications in areas such as juice processing, baking, wine stabilization, and improving the organoleptic qualities of food products. 1 Many higher plants and microorganisms secrete laccase, 2 and fungi such as deuteromycetes, ascomycetes, and basidiomycetes can also produce laccase. 3 Laccase (EC 1.10.3.2) is a blue oxidase that reduces molecular oxygen to water using a system consisting of three different copper atoms, thereby oxidizing various phenolic compounds and aromatic amines. During the production of fruit and vegetable juices, enzymatic and nonenzymatic browning are critical issues. 4 Since these substances negatively affect the color, flavor, and aroma of the juice, they must be removed. 5
Of all fruits, apples are the most consumed worldwide and in the European Union. In 2019, apple production ranked third globally, exceeding 87 million tonnes. 6 Apples contain numerous phenolic compounds, including flavonoids and phenolic acids such as caffeic acid and chlorogenic acid. 7 Because apple juice is typically consumed in its clear form, approximately 50% to 90% of the phenolic components are lost during the filtration process. 8 Today, consumers tend to choose minimally processed products, such as cloudy apple juice with high polyphenol content. However, due to its high phenolic content, this type of apple juice is particularly susceptible to discoloration and darkening. 9 Various technologies, including heat treatment methods such as pasteurization at 60–90°C, are used to reduce or prevent darkening of apple juice. 10 However, according to research by Sauceda-Gálvez 11 , thermal processing can destroy volatile chemicals and affect the organoleptic qualities of apple juice. Alternatives to thermal processing methods include supercritical carbon dioxide, ultraviolet radiation, ultrasound, high hydrostatic pressure, or high-pressure homogenization. 12 The efficiency of these technologies and the yield of suitable fruit juices depend on the parameters used and product characteristics. Their widespread use is limited by high costs, adverse effects on the quality of some food products, or inadequate enzyme inactivation. 13,14
Laccase can be used to stabilize and clarify fruit juice. 15 Gökmen et al. 16 recommend the use of laccase for fruit juice clarification because it effectively removes phenolic compounds by converting them into polymers or oligomers that are easily removed by any ultrafiltration membrane, allowing apple juice to maintain stable color and clarity for up to six weeks at 50°C. Purified *Trichoderma* laccase was immobilized on alumina beads and used to selectively remove off-flavor compounds caused by microbial contamination of apple juice. 17
Approximately 80-90% of the volatile components of apple juice are esters and aldehydes, which impart a unique aroma to the juice. 18 Laccase from *Trametes versicolor* was immobilized on an inexpensive support made from natural fiber from young coconut shells for apple juice clarification. 19 Previous studies have investigated the stabilization of apple juice (color and turbidity) using enzyme-free or immobilization methods, or in combination with ultrafiltration. 5,19 However, the effect of fungal laccases on the physicochemical properties of apple juice during storage remains unclear. Therefore, the aim of this study was to experimentally investigate the changes in the physicochemical properties, phenolic compound content, and antioxidant activity of apple juice after treatment with fungal laccases and two-week refrigerated storage. Laccases have the ability to oxidize phenolic compounds, which makes them promising for use in various industrial processes, including juice clarification. This study examined laccases from *Pleurotus ostreatus* NRC 620, focusing on the ideal conditions for their activity and effectiveness in juice clarification. While research on oyster mushrooms (P. ostreatus NRC 620) is still limited, previous studies have examined enzymes from various fungal sources, such as Trametes versicolor and Ganoderma lucidum. The aim of this study was to evaluate the potential application of this enzyme in the food industry and highlight its unique properties, particularly its ideal pH and temperature.
2,2′-Azooxybis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was purchased from Sigma-Aldrich (Canada). All other reagents were of analytical grade.
The National Research Center’s Microbial Culture Collection Center obtained the known oyster mushroom strain NRC620. After subculture, this strain was stored on potato dextrose agar slants at 4°C. The inoculum preparation method was as follows: 10-day-old, fully developed mycelium was inoculated onto potato dextrose agar plates and incubated at 28°C. After 10 days, three 12-mm-diameter mycelial blocks were removed from the agar media using a sterile metal punch and placed in 250-mL Erlenmeyer flasks with cotton plugs containing 50 mL of sterilized culture medium (pH 5.0, as described previously by Othman et al. 20 ). The cultures were incubated at 28°C for 18 days. The cultures were then filtered through Whatman No. 1 filter paper, and the resulting supernatant served as the enzyme source.
Laccase activity was determined using ABTS as a substrate. The reaction mixture (2 mL) contained 500 μL of 0.3 mM ABTS (dissolved in 0.1 M sodium citrate buffer, pH 4.5) and the required amount of enzyme sample diluted with distilled water. 21,22 Considering that laccase can oxidize ABTS at room temperature (28 °C ± 2), ABTS oxidation was determined by measuring the increase in absorbance at 420 nm (ε 420 = 36,000 cm -1 M -1 ) using an Agilent Carry-100 UV spectrophotometer. One unit of laccase activity was required to oxidize 1 μmol ABTS per minute. Protein concentration was determined by the Bradford method using bovine serum albumin as an internal control. 23,24
After obtaining the enzyme from the oyster mushroom strain NRC 620, its activity was measured at different cultivation intervals for 25 days under static conditions at 28 °C.
To study the effect of temperature on laccase activity, experiments were conducted in the temperature range from 20 to 90 °C. Before adding the enzyme and starting the reaction, the buffer (0.1 M sodium citrate, pH 4.5) and substrate (ABTS) were mixed and incubated for 5 minutes at various temperatures. Enzyme thermal stability was assessed by incubation in 0.05 M sodium phosphate buffer (pH 7.0) at 40, 50, 60, and 70 °C for 2 hours, respectively. Residual activity was then assessed using the ABTS substrate.
The effect of pH on laccase activity was assessed using ABTS as a substrate in 0.1 M citrate-phosphate buffers with a pH range of 2.5 to 7.0. The enzyme solution was incubated at 40°C for two hours in 0.1 M citrate and Tris buffers (pH 3, 4, 6, and 7) to assess pH stability. Residual activity with ABTS as a substrate was calculated after incubation.
The laccase was incubated for 10 min in sodium phosphate buffer (0.05 M, pH 7.0) containing various metal ions (Mg2+, Cu2+, Co2+, Ca2+, Zn2+, K+, Na+, and Mn2+) at concentrations of 2.5 mM and 10 mM, respectively. The substrate (ABTS) was then added to initiate the reaction, and the relative activity was assessed.
ABTS oxidation by laccase at various concentrations (0.025–3 mM) was measured at pH 4.5 to determine the kinetic parameters (Vmax and Km). The kinetic constants of the Michaelis-Menten equation were calculated using a Lineweaver-Burk plot, which plots the reciprocal of the reaction rate as a function of substrate concentration. The kinetic constants were calculated from the Lineweaver-Burk plot using GraphPad Prism version 6.01 software.
After thoroughly washing the apples with tap water, they were cut in half and juiced using a fully automatic Braun MP80 apple juicer (made in Germany). The juice was filtered through four layers of cheesecloth. No enzymes were added to the control group, while 2.0% laccase (the most effective concentration tested) was added to freshly prepared apple juice, which was then stored at 4°C for two weeks.
Titratable acidity (TA) and pH were determined according to the method of Boulton et al.27 . The pH of each sample was measured using a digital pH meter (JENWAY 3510 pH meter). Titratable acidity (TA) was calculated based on malic acid using the following formula.
Where V and C are the volume (mL) and concentration (0.1 mol/L) of the sodium hydroxide solution used in the titration, respectively. K is the malic acid conversion coefficient, equal to 0.067, and W is the mass (g) of apple juice.
The total soluble solids ( TDS ) content of all juice samples was determined using a PAL-1 pocket refractometer (ATAGO, Tokyo, Japan). After each measurement, the optical lens was rinsed with deionized water, and each apple juice sample was tested three times. The value for each sample was calculated by averaging the three measurements. The mean ± standard deviation for each apple juice sample was also calculated by averaging these results.
The viscoelasticity of the apple juice samples was assessed using a rotational viscometer (RV, Rheotest 2, Germany). The sample was placed inside the “S2″ cylinder of the viscometer. Apparent viscosity was represented by the slope of the shear stress versus shear rate curve, which was calculated from the shear stress and the corresponding curves at various shear rates (from 1.00 to 437.4 s⁻¹). The formula for calculating apparent viscosity is as follows:
Where η is the apparent viscosity (cP), τ is the shear stress (dyn/cm²), γ is the shear rate (sec⁻¹), and (τ) is calculated using the torque (α) and cylinder (Z) values using the following formula: τ = Z . α.
The browning index was determined according to the method of Meidav et al.29 . A 10-ml juice sample was centrifuged at 2750 xg for 10 min. 5 ml of the juice supernatant was mixed with 5 ml of 95% ethanol. The absorbance of the mixture was measured at 420 nm using a Shimadzu UV spectrophotometer (UV-1601 PC).
Total phenolic content (TPC) was determined colorimetrically using the Folin-Ciocalteu reagent as described by Boulton et al. [27 ]. A standard curve of gallic acid was constructed for concentrations from 0 to 500 mg/L ( r² = 0.997). Results are expressed as gallic acid equivalents (mg GAE/mL).
Add 125 μL of distilled water and 2850 μL of FRAP solution to 25 μL of apple juice and leave the mixture in the dark for 30 min. Then measure the absorbance at 593 nm using a Shimadzu UV spectrophotometer (UV-1601 PC). The FRAP reagent was prepared by mixing 300 mM acetate buffer (pH 3.6), 20 mM iron(III) chloride, and 10 mM 2,4,6-tris(2-pyridyl)triazine (TPTZ) (dissolved in 40 mM HCl) in a ratio of 10:1:1. A standard curve was generated using Trolox as the standard ( R² = 0.999), and the results are expressed as μM Trolox/mL.
The antioxidant activity of the treated and untreated juices was determined using the DPPH method to evaluate their ability to scavenge DPPH free radicals. 31 Ten microliters of juice were mixed with 1 ml of a DPPH solution (100 μM) in methanol. After reaction in the dark for 30 min, the absorbance of the mixture was measured at 517 nm using a Shimadzu UV spectrophotometer (UV-1601 PC). The results were expressed as trolox equivalents (μM trolox/ml) based on a calibration curve ( R2 = 0.990).
The data obtained showed that maximum laccase production was observed in NRC 620 oyster mushrooms by the end of the 18th day of fermentation, reaching an activity of 1302 U/L. This served as the basis for determining the optimal cultivation time for laccase production (Figure 1). Although enzyme production increased with increasing cultivation time, the rate of increase was not directly proportional to cultivation time; after 21 days, enzyme activity had increased by only 90 U/L (to 1390 U/L). Therefore, 18 days was ultimately selected as the optimal cultivation time to balance product yield with the economic benefits of increased cultivation time.
Effect of cultivation time on laccase yield in Pleurotus ostreatus NRC 620. Three (12 mm) fungal mycelial blocks were inoculated into 50 ml of sterile medium and then cultured at 28 °C for different times.
Consistent with other studies, our results indicate that the ideal culture period to achieve peak laccase secretion by fungi is likely to be between 7 and 36 days. 32 According to Ezike et al. 33 , *Trametes polyzona* WRF03 produced the highest amount of laccase by the end of the ninth day of fermentation, with a specific activity of 1637 U/mg protein. Furthermore, Othman et al. 34 found that *Trichoderma harzianum* S7113 secreted a large amount of laccase on the fifth day of culture. The laccase production rate reached a peak activity on the fourteenth day and then gradually decreased. 34 Although enzyme secretion can also occur during the main growth phase, it usually peaks during the intermediate phase and is triggered by the consumption of a carbon or nitrogen source. 34,35
Although laccase from Pleurotus ostreatus NRC 620 exhibited high activity over a wide temperature range from 50°C to 80°C, nearing peak activity (69–98%), its maximum activity was observed at 70°C (Fig. 2a). Outside this temperature range, enzyme activity decreased at approximately 70°C. These results suggest that the enzyme is active at high temperatures, likely because high temperature increases the kinetic energy of the reaction.
Effect of reaction temperature (a) and pH (b) on laccase activity in *Pleurotus ostreatus* NRC 620. Temperatures ranging from 20 to 90 °C were achieved by pre-incubating the mixture at different temperatures for 5 min before adding the enzyme and starting the reaction. The effect of pH on laccase activity was assessed using ABTS as a substrate in solutions containing 0.1 M citrate-phosphate buffer over a pH range of 2.5 to 7.0.
According to Ezike et al.33 , the optimal temperature for *Trametes polyzona* WRF03 laccase is 55 °C, which is the same as that for *Ganoderma lucidum* laccase36 and similar to the optimal temperature (50 °C) for *Trametes polyzona* KU-RNW02737 laccase . Baldrian38 notes that, as for other lignin-degrading enzyme systems, the ideal temperature range for laccase is between 50 and 70 °C.
The results showed that the enzyme exhibited the highest activity at pH 3.0, reaching 94% activity at pH 3.5. However, it remained active over a wide pH range from 2.5 to 7.0 (Figure 2b). Furthermore, it exhibited higher activity in acidic conditions compared to neutral or alkaline conditions. Its activity remained at least 77% over the pH range from 2.5 to 4.5, but only reached approximately 38% at pH 7.0. The optimum pH for laccase from *Trametes polyzona* WRF03 was 4.533, which is the same as the pH for laccases from *Trametes polyzona* KU-RNW02737, *Trichoderma harzanium* 39, *Pleurotus* sp. 40, and *Trametes hirsuta* 41. However, according to the study by Chairin et al. 42 , the optimal pH for laccase from *Polymorpha f. sp.* WR710-1 is 2.2, while the optimal pH for laccase from *Polymorpha f. sp.* IBL-04 is 5.043. The binding of hydroxide anions (laccase inhibitor) to the copper atoms of T2/T3 laccase may be the reason for the decreased laccase activity under neutral or alkaline pH conditions. This may disrupt the internal electron transfer from the T1 center to the T2/T3 center, thereby limiting the enzyme activity23,44
By incubating the enzyme at different temperatures, it was found that both incubation time and temperature affected the enzyme stability. Notably, laccase from *Trametes polyzona* NRC 620 exhibited higher stability at 40℃ and 50℃, retaining 68.33% and 59.61% of its initial activity, respectively, after 120 minutes (Figure 3a). In contrast, under the same conditions (40℃ and 50℃, 120 minutes), laccase from *Trametes polyzona* WRF03 retained 64.38% and 42.92% of its activity, respectively. 33 On the contrary, increasing incubation time and temperature decreased the stability of *Trametes polyzona* NRC 620 laccase; After incubation at 60℃ and 70℃ for 60 minutes, its activity decreased to 39.24% and 1.72%, respectively (Figure 3a). Consistent with the experimental results, laccase from *Trametes polyzona* WRF03 showed higher stability at 40 and 50℃ throughout the thermal treatment process. 33 Similarly, Lueangjaroenkit et al.37 and Chairin et al.42 reported the stability of laccases from Trametes polyzona KURNW027 and Trametes polyzona WR710-1 at 50 °C for 1 hour, respectively. As a useful biocatalyst applicable in various biotechnological fields, laccase should have good stability and performance over a wide temperature range.
Thermostatic stability (a) and pH stability (b) of laccase from *Pleurotus ostreatus* NRC 620. Thermostatic stability was assessed by incubating the enzyme solution in 0.05 M sodium phosphate buffer (pH 7.0) at 40, 50, 60, and 70 °C for 2 h, respectively. pH stability was assessed by incubating the enzyme solution in 0.1 M citrate buffer and Tris buffer (pH 3, 4, 6, and 7) at 40 °C for 2 h. Residual activity was calculated using ABTS as a substrate after incubation.
To determine the optimal conditions for enzyme use and storage, we investigated the effect of pH on laccase stability. Exposure to different pH values significantly affected the stability of the protein structure, thereby influencing the stability and activity of the enzyme molecule. The results showed that the enzyme was less stable under acidic conditions, while it demonstrated better stability at higher pH values (neutral and alkaline regions). At pH values of 7.0, 6.0, 4.0, and 3.0, the enzyme retention rates after 120 minutes were approximately 100%, 62.54%, 52.39%, and 11.14%, respectively (Fig. 3b). *Strombus multisus* WRF03 laccase showed higher stability at neutral pH values (5.5–6.5) and lower stability at acidic pH values (below 4.0). After 120 minutes at pH values of 5.5, 6.0, and 6.5, the enzyme retention rates were approximately 82%, 100%, and 93%, respectively. 33 Khairin et al. 42 noted that laccase from Trametes polyzona WR710-1 was stable in the pH range of 6.0 to 7.0, while Sayed et al. 45 showed that laccase was more stable under neutral pH conditions. However, laccase from Cerrena unicolor also exhibited stability under alkaline conditions (pH 9.0) 46 . The laccases studied showed high stability over a wide pH range. This may be an important characteristic for industrial applications.
Since some metal ions have both stimulatory and inhibitory effects on enzyme activity, their effects on enzyme activity must be considered in industrial applications. This is crucial because metal ions are common environmental contaminants that can affect the stability and synthesis of extracellular enzymes. 47 To investigate the effects of multiple metal ions on laccase from *Pleurotus ostreatus* NRC 620, we conducted corresponding experiments. As shown in Figure 4, depending on the type of metal used, increasing the metal ion concentration from 2.5 mM to 10 mM negatively affected the enzyme function. For example, Mg²⁺ , Co²⁺ , Zn²⁺ , and Cu²⁺ could stimulate and activate the enzyme activity, while Na⁺ , Mn²⁺ , Ca²⁺ , and K⁺ could inhibit the enzyme activity. At a concentration of 10 mM, Cu²⁺ and Mg²⁺ ions were the most potent activators of laccase activity from *Pleurotus ostreatus* NRC 620, providing an activation degree of approximately 34% and 20%, respectively. However, at a concentration of 10 mM, Ca²⁺ ions were the most potent inhibitor of laccase, reducing enzyme activity by approximately 60%.
The effect of metal ions on the activity of Pleurotus ostreatus NRC 620 laccase. The laccase was incubated for 10 minutes in sodium phosphate buffer (0.05 M, pH 7.0) containing various metal ions at concentrations of 2.5 mM and 10 mM. The reaction was then initiated by the addition of the substrate (ABTS), after which the relative activity was measured.
Our results are consistent with those of other authors who found that Mg²⁺ and Cu²⁺ enhance the activity of *Trametes polyzona* WRF03³. Castaño et al.⁴⁸ found that laccase from *Xylaria* sp. is stimulated to some extent by copper ions (Cu²⁺). Furthermore, Foroutanfar et al.⁴⁹ and Si et al.⁵⁰ conducted similar studies on laccases from *Paraconiothyrium variabile* and *Trametes pubescens*, respectively. The type II copper-binding site (T2) of this enzyme can be saturated with Cu²⁺ at a given concentration, which may explain the stimulation of laccase activity at higher Cu²⁺³⁹ concentrations. Since white rot fungi laccases are oxidases containing multiple copper atoms, the effects of copper ions on laccase activity are diverse and range from stimulatory and inhibitory to neutral.⁵¹ In contrast, Zhou et al . [52] reported that Cu²⁺ inhibited the laccase activity of Taiwan subterranean termite (Odontotermes formosanus). However, laccases of Cerena sp. HYB07 [53] and Clitocybe maxima [54] were not affected by copper ions.
The substrate specificity was represented by its kinetic parameters (Km and Vmax); the stronger the binding affinity of the substrate to the enzyme, the lower the Km value and the higher the substrate specificity. 3,21,55 The kinetic parameters (Km and Vmax) of laccase from *Pleurotus ostreatus* NRC 620 were determined using GraphPad Prism 6.0 software by plotting the Lineweaver-Burk plot (Figure 5). When using ABTS as a substrate, the results were 1.99 mM and 16217 μmol min⁻¹ L⁻¹, respectively. Elsayed et al. 21 reported that the Km values for ABTS oxidation were 0.1 mM and 0.064 mM, respectively, indicating a high affinity of Lac A and Lac B isoenzymes for ABTS. Furthermore, the Vmax values were 0.182 μmol min⁻¹ and 0.603 μmol min⁻¹ , respectively. The obtained Km value was lower than that of Trametes polyzona WRF03 (8.66 mM); furthermore, their Vmax value (1429 mmol min⁻¹) was also lower when using ABTS as a substrate.33 Similarly, the Km values of Lentinus squarrosulus MR13 and Trametes sp. AH28-2 laccase concentrations were 0.0714 mM and 0.025 mM, respectively, and the Vmax values were 0.0091 mM min−1 and 0.67 mM min−1 mg−1 (relative to ABTS) , respectively.56,57
The effect of ABTS concentration on the activity of laccase from *Pleurotus ostreatus* NRC 620 was investigated, and a Lineweaver-Burk plot of the reciprocal of the initial reaction velocity versus ABTS concentration was plotted. The oxidation reaction of ABTS with different concentrations (0.025–3.0 mM) of laccase was measured at pH 4.5 to determine the kinetic parameters (Vmax and Km). The Michaelis-Menten kinetic constants were calculated using the Lineweaver-Burk plot of the reciprocal of the reaction velocity versus the substrate concentration. The kinetic constants were calculated from the Lineweaver-Burk plot using GraphPad Prism 6.01 software.
Traditional clarifying enzymes, such as pectinases, hydrolyze pectic substances, reducing viscosity and turbidity. They effectively break down structural polysaccharides and are often used in combination with other enzymes, such as cellulases and hemicellulases, to improve yield and clarity. However, pectinases do not specifically target phenolic compounds, which are the main contributors to turbidity and oxidative browning, particularly in juices such as apple and grape juice. 58 In contrast, laccases catalyze the oxidation of phenolic compounds, polymerizing them into larger, insoluble molecules that can be removed by sedimentation or filtration. This mechanism not only improves clarity but also extends the shelf life of juice by reducing the likelihood of oxidative browning caused by phenolic compounds. Furthermore, laccase-based clarification processes can be carried out under mild processing conditions (pH 3.5–5.5, temperature 25–40 °C), making them suitable for delicate juices without compromising their nutritional or organoleptic properties. 59 Studies have shown that pectinase treatment can clarify juice in 1–2 hours, while laccase treatment typically requires a longer reaction time (3–6 hours) to completely reduce phenolic compounds. However, this process can be optimized by immobilizing the enzyme or by combining laccase with mechanical clarification methods. 60 In this study, enzyme profiling of the crude extract revealed significant laccase and α-amylase activities, while pectinase and xylanase activities were extremely low, and cellulase activity was not detected. Therefore, the reduction in turbidity and phenolic content was mainly due to the action of laccase, while the change in viscosity could be partly due to the action of amylase.
Table 1 shows the physicochemical parameters of freshly squeezed apple juice and laccase-treated samples. The results showed that the yield of freshly squeezed apple juice (71.59%) was lower than that of laccase-treated samples (87.34%). These results are consistent with the findings of Pilnik and Orange 61 , who indicated that the use of enzymes in fruit processing can increase juice yield, improve filtration, and obtain high-quality, clear juice for concentration. The increase in juice yield is mainly due to the increase in the content of soluble sugars in the juice. During enzymatic hydrolysis of fruits, mesoglea and pectin in the cell walls of the product are destroyed and converted into soluble substances such as neutral sugars and acids 62 . The pH value of the enzyme-treated apple juice was significantly lower than that of the control group (P < 0.05), and the pH value of both groups increased significantly during storage (Table 1). These results are consistent with those of Mark et al. 63 , who noted that the pH of cashew fruit juice decreased after storage after heat treatment. Pectin degradation and galacturonic acid formation after enzyme treatment may be responsible for the increase in pH during storage. The pH of enzyme-treated samples remained between 4.05 and 4.31 throughout storage, while the pH of untreated apple juice ranged between 4.12 and 4.33.
The total acidity (TA) of both untreated and laccase-treated samples showed a decreasing trend with increasing storage time (Table 1). The decrease in acidity was attributed to the conversion of organic acids to carbohydrates or enzymatic reactions, as well as oxidation during juice storage. 64 The total acidity of the control apple juice and enzyme-treated samples was lower than that of other juices (strawberry juice 0.9%, plum juice 2.2%, kumquat juice 1.0%, apricot juice 2.4%, orange juice 0.8%), but similar to that of other juices (e.g., pear juice 0.3%). 62 These differences in untreated freshly squeezed apple juice may be due to various factors such as growing conditions, genetic factors, maturity level, and processing methods. 65 The decrease in total acidity of control and laccase-treated apple juice is consistent with the results presented by Singh et al. 66 regarding the decrease in total acidity of Jin Nuo apple juice after 74 days of storage. On the other hand, Oshmiansky and Wojdylo 67 did not find any significant changes in the acidity of apple juice when studying the effect of traditional clarification methods.
The results presented in Table 1 indicate that the total soluble solids (TSS) value of the laccase-treated apple juice was higher than that of the untreated sample. These results are consistent with the published studies . 68 Furthermore, Table 1 shows that the TSS value of the control apple juice group was 9.58 at the initial time point and reached 11.05 by the end of the storage period. These values are lower than the TSS values of fresh apple juice reported by Hamid et al . 69 (11.2 and 11.80, respectively). The TSS value of the laccase-treated apple juice samples increased significantly, starting from 11.23 and reaching 12.93 after two weeks of storage at 4°C (Table 1). A similar increase in TSS during storage was also observed in citrus fruits, lemons, and sweet oranges. The increase in total soluble solids (TSS) during storage may be due to the hydrolysis of polysaccharides (starch) to monosaccharides (sugars), the increase in concentration due to juice dehydration, and the degradation of pectin in the juice to soluble solids. The increase in total soluble solids (TSS) is likely due to the increase in soluble sugars, which may be formed by the conversion of pectin or cellulose to soluble sugars by pectin or cellulase, respectively, or by the hydrolysis of starch to sugars, as reported by Hamed et al. 69. The effect of laccase on the properties of apple juice can be observed visually, as laccase-treated apple juice exhibits better flowability and lower viscosity than untreated juice. This observation is recorded in Table 1; The viscosity of the enzyme-treated sample was 1.87 cP, while the viscosity of the control sample was 2.95 cP. This significant decrease in viscosity is likely due to the higher water-holding capacity of pectin-like substances and the formation of a cohesive network structure.
In this study, the effect of laccase on the browning index (BI) of apple juice was investigated by measuring the absorbance at 420 nm using a spectrophotometer. The results are shown in Table 1. During storage, the BI of apple juice samples in both the treated and untreated groups showed a gradual increasing trend. BI reflects the degree of browning and can serve as an important indicator of enzymatic and non-enzymatic browning reactions. The absorbance increased significantly during storage (P < 0.05). At the end of storage, the A420 value of apple juice samples in the control and enzyme-treated groups increased by about 217% and 121%, respectively (Table 1). The results indicate that enzyme treatment can effectively reduce the browning degree by about 56%. The results of Bezerra et al. [19 ] are consistent with our results; They used laccase-glutaraldehyde-coconut fiber to clarify apple juice, reducing its original color by 61%.
Although polyphenols in fruit juices have positive nutritional and therapeutic effects on the human body, they can also react with proteins, causing juice cloudiness, sedimentation, or turbidity, thereby altering the flavor and aroma of the product and reducing its shelf life. 71 The aim of this study was to safely reduce the phenolic compound content of apple juice using laccase from Pleurotus ostreatus NRC 620. The results presented in Table 1 show that the total phenolic compound content of laccase-treated apple juice was significantly reduced before storage at 4 °C. Furthermore, the total phenolic compound content also decreased during storage in both samples studied (Table 1). Research by Sandri et al. 72 showed that enzyme-treated apple juice can retain its antioxidant activity and phenolic compound content. However, the results of a study by Lettera et al. 73 show that treatment of orange juice with fungal laccase can reduce the content of phenolic compounds in it by up to 45%.
Phenolic compounds have been shown to have properties such as free radical scavenging, singlet oxygen reduction and quenching, hydrogen atom transfer, and electron donation to free radicals, making them potent antioxidants. 74 Therefore, in this study, DPPH and FRAP-based methods were used to evaluate the effect of laccase on the antioxidant activity of apple juice stored in a refrigerator for 14 days (Table 2). Both methods showed an increase in antioxidant activity during storage, which may be due to the increase in free phenolic compounds or the formation of Maillard reaction products (MRPs), with Maillard reaction products likely being the cause of the increase in antioxidant activity. 75 Non-enzymatic browning reactions (including ascorbic acid degradation, Maillard reactions, and acid-catalyzed degradation of sugars) produce brown pigments (melanoidins). Intermediate ascorbic acid degradation products and sugar degradation products (such as carbonyl compounds) can react with amino acids through Maillard reactions. 76 Although browning of fruits and vegetables during storage has been extensively studied, our understanding of these reactions remains limited. 77 Compared with the FRAP method, laccase-treated apple juice showed significantly lower antioxidant activity by the DPPH method (Table 2), and the antioxidant activity of all samples increased significantly with increasing storage time. Two different methods for determining antioxidant activity were used in this study because their principles differ. The DPPH method measures the ability to neutralize free radicals, while the FRAP method measures the ability to reduce iron ions. Therefore, it is recommended to use multiple methods for determining antioxidant activity to better understand the antioxidant activity of the samples studied. 78
One of the key findings of this study is that *Pleurotus ostreatus* laccase NRC 620 exhibits optimal activity at 70°C and pH 3.0. Compared with other fungal laccases commonly used for juice clarification, such as *Trametes versicolor* and *Ganoderma lucidum* laccases, *P. ostreatus* NRC 620 exhibits higher thermal stability and a more acidic pH. Laccases from *Trametes versicolor* and *Ganoderma lucidum* typically exhibit optimal activity in the range of 50-60°C and at pH values between 3.5 and 5.0. This difference may contribute to improved juice clarification efficiency, especially for acidic juices where stability at lower pH values is critical. The unique characteristic of *P. Compared to other studied fungal laccases, *Pleurotus ostreatus* NRC 620 exhibits the ability to function effectively under more challenging conditions. Its higher optimal activity temperature suggests potential advantages in industrial applications, such as faster reaction rates and reduced microbial contamination. Its low pH, which is well suited to the acidic nature of many juices, may be useful in juice clarification processes. These results justify further exploration for large-scale application, making *Pleurotus ostreatus* NRC 620 a viable alternative to traditional fungal laccase sources. Compared to previous studies, we found that the optimal temperature is 60°C and the optimal pH is 3.0. After reaction at 60°C for 80 minutes, *Ganoderma lucidum* laccase retained 46 % of its activity.79 According to Kurniawati and Nicelle 80 , *Ganoderma lucidum* enzymes exhibit excellent to moderate stability at 25°C and pH values ranging from 5.0 to 8.0, and stability at pH 6.0 and temperatures ranging from 10 to 30°C. In this study, we found that the optimum pH and temperature for enzyme activity for *Pleurotus ostreatus* were 3.0 and 70°C, respectively. After incubation at 40°C and 50°C for two hours, the enzyme retained 68.33% and 59.61% of its activity, respectively. Furthermore, Pleurotus ostreatus NRC 620 laccase exhibited high activity over a wide temperature range from 50°C to 80°C, nearly reaching maximum activity (69%–98%), with maximum activity observed at 70°C.
In conclusion, oyster mushroom laccase NRC620, obtained under static conditions, demonstrated optimal activity and stability across a range of pH and temperature conditions, demonstrating superior stability compared to other enzyme sources. The addition of 10 mM MgSO₄ and CuSO₄ increased enzyme activity by approximately 21% and 35%, respectively. When processed into apple juice, the enzyme reduced pH and viscosity, while the phenolic content decreased only slightly during storage.
The results confirm the potential of laccase in the food industry, particularly in beverage clarification. By specifically breaking down phenolic compounds, laccase not only reduces turbidity and improves clarity but also maintains the quality of fruit juices under mild operating conditions. Unlike traditional clarifying agents such as gelatin, bentonite, and silica gel, laccase does not generate waste or remove pleasant aromas from beverages, making it a more environmentally friendly and sustainable option. Furthermore, compared to other enzymes and filtration methods, laccase offers a targeted and cost-effective solution without compromising product quality.
Kyomuhimbo, HD and Brink, HG. Applications and immobilization strategies of copper-containing laccases; a review. Heliyon 9, e13156 (2023).
Post time: Dec-15-2025