EFFICIENT EXTRACTION AND CLARIFICATION OF FRUIT JUICES USING CONCURRENTLY PRODUCED XYLANO-PECTINOLYTIC ENZYMES

their physical (clarity, viscosity and soluble solid), functional (total polyphenolic content) and sensory characteristics (acceptability). In this study, various conditions like enzyme dose, treatment time and stirring speed have been optimized. The extraction and clarification efficiency were found to be optimum at enzyme dose between 2:8 and 4:16 IU/g pulp, treatment time between 30 to 60 min and stirring speed of 50 and 60 rpm for different fruit juices. Enzymatic treatment enhanced the physicochemical, organoleptic and nutritional properties and generated juice with improved yield and clarity. After xylano-pectinolytic enzymatic treatment of various fruit juices, maximum increase in yield (95%), filterability (40%) and maximum decrease in viscosity (58%) was found in case of Tamarindus indica. Maximum increase in clarity (39%) and polyphenolic content (37%) was observed in case of Aegle marmelos . Maximum increase in reducing sugars was found in case of Fragaria ananassa along with other properties. All these attributes of xylano-pectinolytic enzymes indicate an adequate prospect for bio-industrial research.


INTRODUCTION
As awareness about the human health is taking primary seat in today's lives, the health benefits of fruit juices are being recognized and the demand of fresh, crystal clear, less viscous and less turbid juice is increasing day by day.Fruit juice is a rich source of vitamins, dietary fibers and minerals (Mark et al., 2018).They contain antioxidants, which aid in the elimination of free radicals and the reduction of oxidative stress in the body.Fruit juice keeps our body hydrated, activate the metabolism and helps in the detoxification of body.Fruits are more sensitive to microbial activity, which make them highly putrescible with a short shelf life (Hmar et al., 2018).Preservation of these fruits for a longer duration is not only energy consuming but also costly.In addition to this, Food and Agriculture Organization (FAO) revealed that approximately 45% of fruits are wasted every year because of post-harvesting spoilage or are repudiated due to lower quality.To overcome this, contemporary techniques are implemented to convert them into superior quality products such as juices, which is simple and easy while maintaining their functional, physical, sensory and organoleptic properties.Fruit juices obtained by conventional methods such as diffusion, extraction and decanter centrifuges are cloudy, viscous and turbid due to the presence of polysaccharides (Starch, pectin, cellulose and hemicellulose), thus do not meet the consumer expectations and also there is high risk of cross-contamination (Sharma et al., 2017).These conventional methods have several disadvantages such as high initial and ongoing cost and low yield (Vaillant et al., 2001;Sulaiman et al., 1998).As crystal clear and less viscous juice is important to consumers, researchers working in this area are looking for alternative methods to improve this feature (Nachal et al., 2023).In many studies, the use of pectinase and xylanase enzyme for clarification of fruit juices has been presented as an efficient alternative to improve yield and clarity The cost of producing enzymes is a critical factor in determining whether or not they can be applied in industries.The co-production of enzymes by a single bacterial isolate, as well as use of agricultural waste as a production substrate, can reduce production costs (Kaur et al., 2019).Concurrent production of enzymes can save energy and sustaining expenses.Thus, this co-production of xylanopectinolytic enzymes by a bacterial isolate appears to be acceptable for use in fruit juice industry, lowering the cost of enzyme production and increasing the feasibility of using these enzymes for extraction and clarification.Both xylanase and pectinase enzymes used in this study are produced extracellularly by B. pumilus AJK, and no expensive materials were used in their production.This pectinase enzyme preparation also contains a blend of seven enzymes (Pectin esterase, polymethylgalacturonate lyase, polygalacturonate lyase, exopolymethylgalacturonase, exo-polygalacturonase, endo-polymethylgalacturonase and endo-polygalacturonase) that have been reported to give significant results (Sharma et al., 2019).Due to simultaneous extracellular enzyme synthesis using low-cost substrates, the approach is commercially viable for fruit juice industry.This study looks into the treatment of various fruit pulps with crude xylanopectinolytic enzymes under optimal conditions to evaluate if these enzymes are suitable for extraction and clarification of fruit juices.
The purpose to effectuate this research work is to investigate the efficiency of crude xylano-pectinolytic enzymes in extraction and clarification of fruit juices, so as to improve their physical (clarity, viscosity and soluble solid), functional (total polyphenolic content) and sensory characteristics (acceptability).In this study, various conditions like enzyme dose, treatment time and stirring speed have been optimized.The extraction and clarification efficiency were found to be optimum at enzyme dose between 2:8 and 4:16 IU/g pulp, treatment time between 30 to 60 min and stirring speed of 50 and 60 rpm for different fruit juices.Enzymatic treatment enhanced the physicochemical, organoleptic and nutritional properties and generated juice with improved yield and clarity.After xylano-pectinolytic enzymatic treatment of various fruit juices, maximum increase in yield (95%), filterability (40%) and maximum decrease in viscosity (58%) was found in case of Tamarindus indica.Maximum increase in clarity (39%) and polyphenolic content (37%) was observed in case of Aegle marmelos.Maximum increase in reducing sugars was found in case of Fragaria ananassa along with other properties.All these attributes of xylano-pectinolytic enzymes indicate an adequate prospect for bio-industrial research.

Fruits and chemicals
All the fresh and ripened fruits were purchased locally.Pectin and birchwood xylan were purchased from Sigma, USA.Other chemicals used were of analytical grade and purchased from Himedia, India.

Microorganism and enzymes production
Bacillus pumilus AJK (MTCC Accession No. 10414) was used to co-produce xylano-pectinolytic enzymes.The crude enzymes concoction was produced under submerged fermentation (Kaur et al., 2010).Erlenmeyer flasks (250 mL) containing 50 mL of medium (peptone, 0.5 %; MgSO4, 10 mM, pH 7.0), was supplemented with 2% of washed and dried wheat bran and 2% citrus peel.The media was inoculated with 2% of 21-hour-old bacterial culture and the flasks were incubated at 37°C for 60 hours under shaking conditions at 150 rpm.After incubation, crude enzymes were obtained as clear supernatant by centrifugation at 10,000 rpm for 15 minutes, and stored at 4°C until further use.The bacterium produced 270 IU/mL of xylanase and 70 IU/mL of pectinase.

Pulp preparation
All the fruits were washed with tap water, deseeded manually and blended using food processor with appropriate amount of distilled water.No dilution was done for Citrullus lanatus because it already contains 92% of water.

Evaluation of extraction and clarification conditions
To determine the best conditions for extraction and clarification of fruit juices, pulp of various fruits was treated with crude xylano-pectinolytic enzymatic concoction.The pH of fruit pulps was adjusted to 6.0 and all the experiments were performed at 50°C temperature.For efficient removal of xylan and pectin, conditions such as enzyme dose (1:4 -5:20), treatment time (40-80 min), and stirring speed (40-80 rpm) were optimized for maximum yield, clarity, and enhanced qualities.Following each enzymatic treatment, the suspension was immediately cooled in an ice water bath kept at 4 o C to stop the enzymatic reactions before being centrifuged at 2000 g for 5 minutes.The obtained supernatant (juice) was tested for yield, clarity and other quality characteristics.Simultaneously control experiments were also carried out.

Analysis of physicochemical parameters
Various analyses were carried out for each juice sample in order to evaluate the parameters like yield, clarity, pH, reducing sugars, TSS, TDS and polyphenolic content.Other parameters like viscosity, acidity, and filterability were also analyzed.

Determination of yield, pH, TSS and TDS
The volume of supernatant obtained following pulp treatment with enzymes was measured to calculate the yield of the juice, which is stated in terms of mL per 100 g of pulp and pH was measured using digital pH meter (Eutech).On the other hand, total soluble solids (TSS), expressed in o Brix and TDS were measured using a hand-held refractometer and TDS tester (Eutech), respectively.

Determination of clarity
The clarity was determined by measuring percentage transmittance of juices at a wavelength of 660 nm using UV-vis spectrophotometer taking distilled water as blank.

Determination of reducing sugars
Following the enzymatic treatment, the amount of reducing sugar in the juices was measured using DNS method (Miller, 1959).The results were represented as the amount of reducing sugar in g per 100 g of pulp using a standard galacturonic acid curve.

Determination of polyphenolic content
Total phenolic content was determined using the Folin-Ciocalteu reagent (Lin and Tang, 2007).Fruit juice (0.1 mL) was diluted appropriately and combined with 2.8 mL of deionized water, 2 mL of sodium carbonate (2%), and 0.1 mL of 50% Folin-Ciocalteau reagent followed by incubation at room temperature for 30 minutes.After that, absorbance of reaction mixture was measured at 750 nm against deionized water used as a blank.Gallic acid was employed as the benchmark.The total phenolic content of fruits was assessed in triplicates using a six-point standard curve (0-200 mg/L), and the results are shown as mg gallic acid equivalents (GAE)/100 g of pulp.

Determination of viscosity and filterability
The viscosity of each juice was measured using a glass capillary viscometer, also known as an Ostwald viscometer and expressed in centipoise (cp).Suction mechanism was applied to draw the juices into the upper bulb, and they were then allowed to flow through the capillary into the lower bulb.Using a stopwatch, the length of time it takes for the juice level to pass between the two markings (one above and one below the upper bulb) is proportional to the kinematic viscosity.On the other hand, the filterability (sec -1 ) was calculated from the reverse of the time taken to filter juice through a whatman filter paper No. 1 under vacuum.

Determination of acidity
Using starch solution (0.5%) as an indicator, 5 mL of diluted juice (1:4, juice: distilled water) was titrated with 0.005 mol/L iodine solution to estimate the overall acidity.Completion of titration process was indicated by appearance of blue color.A comparison was made between the amount of iodine used to titrate each juice and the amount used to titrate ascorbic acid at 0.001% (w/v).The outcomes are given in mg ascorbic acid equivalent/100 g of pulp.

RESULTS
Enzymatic extraction and clarification strategy was tried for increasing the yield, clarity and enhancing various physico-chemical parameters of fruit juices with the concept that xylanase and pectinase enzymes hydrolyse xylan and pectin contents respectively, present in the cell wall of fruits and ultimately would help in better release of crystal clear and less viscous juice with enhanced qualities.So, the commercial preferability and acceptability of fruit juices would increase.The results obtained are discussed under the following heads:

Optimization of extraction and clarification conditions
In order to achieve the best yield and quality of fruit juices, various conditions were examined (Table 1).The enzymatic treatment result showed that the pectinase: xylanase (IU/g) enzyme dose of 3:12 for Actinidia chinensis and Fragaria ananassa, 2:8 for Pyrus communis and Citrullus lanatus and 4:16 for Manilkara zapota, Aegle marmelos and Tamarindus indica was found to be optimum.A treatment time of 50 minutes and 30 minutes was found to be optimum for Actinidia chinensis and citrullus lanatus, respectively, whereas for the rest of the fruits duration of 60 minutes was found to be optimum.A stirring speed of 60 rpm was found to give good results for all the fruits except Citrullus lanatus for which 50 rpm was optimum in our studies.

Effect of enzymatic treatment on various parameters
To appraise the potential of enzymatic extraction and clarification, both control and enzyme treated samples of fruit juices were tested for various parameters (Table 2 and 3).

Effect on TSS
The results showed that an increase of nearly 47.94% TSS was observed in case of Manilkara zapota followed by 35.06% in case of Actinidia chinensis, 31.47% in case of Citrullus lanatus, 29.41% in case of Fragaria ananassa, 15.14% in case of Pyrus communis, 12.08% in case of Aegle marmelos whereas least increase in TSS was observed in case of Tamarindus indica (8.03%).

Effect on filterability
The studies demonstrated that maximum increase in filterability was observed in case of Tamarindus indica (40.36%) followed by Actinidia chinensis (38.21%) while Citrullus lanatus, Fragaria ananassa, Pyrus communis, Manilkara zapota and Aegle marmelos showed a similar level of increase in filterability i.e., in the range of 28.0% to 30.0%.

Effect on acidity
The results showed that maximum increase in acidity was obtained in case of Pyrus communis (38.88%) followed by Tamarindus indica (24.59%),Manilkara zapota, Citrullus lanatus and Aegle marmelos (14.0%-18.0%)whereas least increase in acidity was noticed in case of Fragaria ananassa and Actinidia chinensis which was only 5 to 6%.Absorption spectra of all samples were taken from 200-800 nm in order to determine the release of various phenolics or other related components.Several peaks were observed in the region of 320-380, signify the release of several phenolics compounds (Fig 1 -7).4% juice recovery in case of tamarind fruit when treated with pectinase enzyme but this again is lesser as compared to our results in case of tamarind, which is 95%.The enzymatic treatment showed up to 4% decrease in pH for different fruit juices.This may be due to enhancement in galacturonic acid monomer liberation during hydrolysis of pectin (Wang et al., 2007).The reduction in pH may also be due to ascorbic acid liberation during pectin hydrolysis may also be the reason of reduction in pH (Akesovan et al., 2013).The release of carboxylic groups (acid groups) during pectin hydrolysis may potentially be responsible for pH drops after enzyme treatment.
After enzymatic treatment, significant increase in TSS (up to 47%) was observed.This might be explained by the breakdown of pectin, which liberates galacturonic acid monomers that can be absorbed to glucose molecules and boost the TSS.The enhancement in TSS depict the tissue breakdown at higher degree, resulting in release of compounds such as sugars, which contribute to soluble solids (Chang et al., 1995).Yosuf et al. (1994) observed that treatment of soursoup with commercial pectinex ultra-SPL enzyme significantly enhanced TSS from 6.8 ₒ Brix to 7.3 ₒ Brix.Joshi et al. (2011) have reported that treatment of plum, peach, pear and apricot with pectinase enzyme resulted in increase in TSS from 12.20 ₒ Brix to 14.10 ₒ Brix, 9.0 ₒ Brix to 14.27 ₒ Brix, 10.17 ₒ Brix to 12.20 ₒ Brix and 8.60 ₒ Brix to 11.20 ₒ Brix, respectively.Approximately 21% to 33.5% increase in TDS was observed after enzymatic extraction of different fruit juices.This is due to release of Ca 2+ and Mg 2+ ions when xylano-pectinolytic enzymes break down Ca and Mg pectate, and TDS meter primarily count these ions by monitoring the current flowing between the electrodes.A significant increase in clarity (up to 39.6%) was observed after our enzyme treatment, this may be because cloud particles are made up of positively charged protein-carbohydrate cores encased in negatively charged pectin molecules.Pectin delays aggregation by covering the particles and charging them sufficiently to cause strong electrostatic attraction between them.Due to their small size, mutual charge repulsion between them and the shielding effect of pectin, these turbiditycausing particles are thus kept in suspension.Enzyme catalysis results in electrostatic destabilization leading to creation of ions, which then causes electrostatic aggregation increasing the size of the particles.The larger particle size causes a floc to form, which settles out and allows the clarified juice to be retrieved (Yamasaki et al., 1964).Guimaraes et al. (2022) have reported that treatment of different varieties of mango (Tommy and Palmer variety), apple, banana and white guava with polygalacturonase from Aspergillus japonicus, resulted in 41%, 65%, 9.4%, 11% and 40 % increase in transmittance.Enzymatic treatment of various fruit pulps resulted in up to 57.5% enhancement in amount of reducing sugars.The release of more solid components, including sugars, and a larger degree of tissue disintegration may be linked to the higher value of reducing sugars.Azar et al. (2020) have reported that apple juice clarification with purified polygalacturonase from Calonectria pteridis resulting in 40.93% increase in reducing sugars.Results also showed 37.20% enhancement in polyphenolic content, which may be due to their more bioaccessiblity when enzymes are employed.The release of cell wall-bound phytonutrients, particularly polyphenols from the fruit matrix is enhanced by the breakdown of pectic and cellulosic polysaccharides in the cell wall and middle lamella.Viscosity is frequently regarded as being crucial to the quality of liquid foods.Pectin, a substance with a high degree of hydroxylation, is hydrolyzed, releasing oligomers of galacturonic acid, an acid with a lower degree of hydroxylation.The release of water molecules would result in a reduction of viscosity since hydrolysis would be accompanied by a decrease in the system's ability to retain water (Akesowan et al., 2013).A significant decrease in viscosity (up to 58.33%) of different juices was observed due to breakdown of xylan and pectin in the cell wall by the action of our enzymes.Enzymatic hydrolysis of polymeric carbohydrates increases the degree of liquefaction and reduces the viscosity of fruit juices.As a result, the enzyme treated juices require less time to filter via a Whatman filter paper.Up to 40.3% increase in filterability was also obtained after enzymatic treatment.Enzymatic treatment of various fruit pulps resulted in up to 38.8% increase in titratable acidity.This could be due to better release of ascorbic acid during the hydrolysis of cell wall.

CONCLUSION
The result obtained in this study showed that pre-treatment of fruit pulp with xylano-pectinolytic enzymes enhanced the juice yield, clarity and various quality characteristics.This study concluded that enzymatic treatment is an effective strategy for the production of crystal clear and less viscous juice along with enhanced qualities.Further, as the enzymes have been derived from the microbial source produced on readily available waste lignocellulosics, the technology becomes more cost-effective and sustainable.This study shows that, xylanopectinolytic enzymatic method for juice extraction and clarification can be used by juice industry for better quality juices.

(
Kashyap et al., 2001; Landbo et al., 2007; Pandey et al., 2022; Panda et al., 2021; Saxena et al., 2015).These enzymes degrade pectin and xylan in the cell wall, reducing viscosity and facilitating separation by centrifugation and filtration.Consequently, the juice is clearer, with more concentrated flavor and color (Abdullah et al.,2007).These enzymes soften plant tissue, thereby causing the release of cell contents, which can be recovered in larger quantities (Sreenath et al., 1984).Xylano-pectinolytic enzymes have emerged as effective concoction for fruit juice extraction and clarification, capable of degrading hemicellulosic polysaccharides, resulting in reduced cloudiness and viscosity, as well as increased nutritional value.A bacterial isolate, Bacillus pumilus, that co-produces xylanase and pectinase enzymes, was employed in this investigation.There have been few reports of a microbial isolate co-producing xylano-pectinolytic enzymes under submerged fermentation like Aspergillus niger F3 (Rodriguez et al., 2018), Aspergillus flavus (Mellon, 2015; Pradal-Velázquez et al., 2018), Aspergillus fumigatus MS16 (Zehra et al., 2020) etc.The co-production of xylano-pectinolytic enzymes using low-cost agro-waste medium will lower the cost of enzyme production and make the process more cost-effective.The purpose of this research was to demonstrate the application of concoction made of crude xylanase and pectinase enzymes in extraction and clarification of fruit juices.The impact of enzyme concoction on yield, clarity, pH, TSS, TDS, reducing sugars, polyphenolic content, acidity, viscosity and filterability of various fruit juices have been studied by optimizing various conditions such as enzyme dose, treatment time and stirring speed.Haile et al. (2022) observed clarification in apple, lemon and mango juice and improvement in other qualities when treated with crude pectinase enzyme from Serratia marcescens.Santana et al. (2022) reported improvement in various physico-chemical properties of guava fruit after treatment with crude multi-enzyme treatment from Rhodotorula mucilaginosa, Rhodotorula orizycola, and Pseudozyma sp.

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7
Figure 1 Absorption spectra of Actinidia sinensis control verses enzymes treated fruit juice Sin et al. (2006) reduced viscosity by exerting a hydraulic action on the cellulose and pectin present in the juices.Singh et al. (2012) have reported 20.7% decrease of viscosity in case of bael fruit.Ninga et al. (2021) have revealed 91% reduction in viscosity after hydrolysis of guava pulp with pectinase enzyme.

Table 1
Optimum enzyme dose, treatment time and stirring speed for extraction and clarification of different fruit juices

Table 2
Comparison of yield, pH, clarity, TSS and reducing sugars before and after enzymatic treatment S.

Table 3
Comparison of polyphenolic content, TDS, viscosity, acidity and filterability before and after enzymatic treatment