Neosartorya glabra polygalacturonase produced from fruit peels as inducers has the potential for application in passion fruit and apple juices

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Neosartorya glabra polygalacturonase produced from fruit peels as inducers has the potential for application in passion fruit and apple juices Poligalacturonase de Neosartorya glabra produzida a partir de cascas de frutas como indutores tem potencial para aplicação em sucos de maracujá e maçã


Introduction
Pectinases are enzymes with important industrial applications (IRSHAD et al., 2014), representing approximately 25% of the commercial enzyme production (MAKKY;YUSOFF, 2015).These enzymes can be divided into protopectinase, de-esterifying and depolymerizing pectinases according to their mode of attack of the pectin backbone.Polygalacturonases (PG) are the depolymerizing pectinase most used in industries and also the most studied (NEVADITA et al., 2013).PG hydrolyses the α-1,4-glycosidic linkages between the galacturonic acid residues in pectin, an acidic polysaccharide complex that occurs mainly in the middle lamella of higher plants (RIDLEY et al., 2001;ZENI et al., 2011;TU et al., 2013).
Pectinases are secreted in high amounts by some fungal species and are used in biotechnological applications, for example, in the liquefaction and extraction of plant tissues, the degumming of natural fibres, the reduction of viscosity, increase in yield and clarification of fruit juices (UENOJO; PASTORE, 2007).In addition, the study of fruit peels as a cheap carbon source for pectinase production is a growing segment in the biotechnological industry (BATOOL et al., 2013).
There are a great variety of fruit juices on the market, such as apple and passion fruit juices, which are commonly consumed and well-established products.
The culture of passion fruit has grown over the last 30 years on the tropical fruit market, on account of the fast economic return and distribution throughout the year (MELETTI, 2011).The most cultivated species in the world is the yellow passion fruit species (Passiflora edulis f. flavicarpa).Brazil is the world's largest producer and consumer of this fruit, accounting for 50-60% of the total world production (OLIVEIRA et al., 2016).In 2013, the world apple production was approximately 80 million tons according to FAOSTAT (2016), and the productivity of the apple in Brazil is around 15-30 tons/ha (SEBRAE, 2016).Apples and passion fruits are rich sources of pectin.Apple pulp contains from 0.6 to 1.5% pectin while passion fruit pulp contains 0.5% pectin (CANTERI, 2011).Thus the application of polygalacturonases to viscous and jellied fruit juices is an alternative to make them more transparent and homogeneous (SIN et al., 2006;ONGARATTO;VIOTTO, 2016), since these enzymes can break down the pectin present in the cell wall, resulting in a reduction in the solids content, facilitating filtration and increasing the overall juice production.The aim of this work was to describe the production of PG by Neosartorya glabra with fruit peel as the carbon source, and analyse the effect of crude PG on the apple and passion fruit juices from the viscosity and light absorbance.

Microorganism
Neosartorya glabra was isolated from the State Forest of Bebedouro in Bebedouro city, São Paulo State, Brazil.This is a conservation area, managed by the Instituto Florestal do Estado de São Paulo (IFESP), and the forest has 99.41 hectares, an altitude of 570 meters, warm weather (17.2-22.7°C)and a vegetation consisting of pines and eucalyptus (IF, 2016).
Neosartorya glabra was identified and preserved by the URM Culture Collection at the Federal University of Pernambuco (Brazil).Stock cultures are being preserved under the register number of 7294 and the fungus was maintained in the laboratory on PDA medium slants (Himedia) at 4ºC.

PG production
PG production was evaluated in solid-state fermentation (SSF) and submerged fermentation (SbmF).SbmF was carried out by inoculating 1 mL of spore solution (8 x 10 7 spores) into 125 mL Erlenmeyer flasks previously sterilized at 127°C and 1.5 atm for 15 minutes, containing 25 mL of Vogel's minimal medium (VOGEL, 1964).For the preparation of the Vogel minimum culture medium, the stock solution was diluted 50-fold with distilled water, and a 0.01% biotin solution and 1% carbon source added as described below.The production of PG in SbmF was also evaluated from mixtures of tangerine and orange peels (Pear and Bahia varieties); and from mixtures of lemon peels (Tahiti and Sicilian varieties).All fruit peels used in SbmF were obtained after drying at 50°C for 12 h and grinding into fine particles (1 mm).The cultures were incubated at 30°C (static condition) for 96 h, vacuum filtered using a Büchner funnel and Whatman nº 1 filter paper, and the cell-free filtrates obtained used to determine the extracellular PG activity.SSF was carried out by inoculating 1 mL of Neosartorya glabra polygalacturonase produced from fruit peels as inducers has the potential for application in passion fruit and apple juices Pinheiro, V. E. et al.
spore solution (6.8 x 10 7 spores) into 125 mL Erlenmeyer flasks previously sterilized at 127°C and 1.5 atm for 15 minutes, containing 2 g of dried fruit peels (apple, passion fruit, banana, pear, guava, orange (var.Bahia) and lemon (var.Tahiti) as the carbon source and 3 mL of distilled water.The flasks were incubated at 30°C in an incubator (static conditions) with 70% of humidity for 96 h.The fruit peels used in SSF were obtained by drying at 50°C and retaining on a 0.5 mm screen.After cultivation, the culture media were suspended in 30 mL of distilled water with agitation (100 rpm) for 30 min at 4°C.Subsequently, the cultures were vacuum-filtered using a Büchner funnel and Whatman nº 1 filter paper.The cell-free filtrates obtained were used to determine the extracellular PG activity.

Effect of cultivation temperature and time on PG production
The experiment to determine the effect of cultivation temperature was carried out using SbmF after inoculating the fungus into Vogel's medium containing orange peel (var.Bahia) as the carbon source (1%).The cultures were incubated (static conditions) at 25, 30, 35 and 40°C for 96 h and the PG activity then determined.
The time-course of PG production was also carried out in Vogel's medium (using orange peel from the Bahia variety as the carbon source).The cultures were incubated under static conditions at 35ºC for 7 days.Samples were withdrawn every 24 h, vacuum-filtered, and the extracellular PG activity determined.The mycelium obtained after filtration was triturated with glass beads in a porcelain mortar and suspended in 30 mL of 100 mM sodium acetate buffer, pH 5.0.It was then centrifuged at 13,000 x g for 15 min at 4°C to remove cell debris.The supernatant obtained was used to determine intracellular PG activity.

Effect of the carbon source concentration on PG production
The experiment was carried out using SbmF by inoculating the fungus into Vogel's medium containing orange peel (var.Bahia) as the carbon source.The culture media were supplemented with different concentrations (0.25-4.0%(w/v)) of dried orange peel and incubated under static conditions at 35ºC for 72 h.

Extraction and quantification of pectin in the fruit peels
The pectin contents of the apple, passion fruit, banana, guava, orange and lemon peels were analysed according to Carvalho et al. (2006) with modifications.One gram of dry and crushed peels was boiled with 200 mL of distilled water for one hour.The material was filtered and the volume completed to 500 mL.An aliquot of 100 mL was withdrawn, and 300 mL of distilled water and 10 mL of 1M sodium hydroxide added with continuous stirring, after which it was held overnight without stirring.A volume of 50 mL of 1M acetic acid was then added, and after 5 minutes, 50 mL of 2M calcium chloride was added.The solution was boiled for 1 minute, maintained without stirring for 90 minutes and then filtered through Whatman filter paper (3-micron porosity) and dried.
The % pectin was estimated as follows in Equation 1: Filter paper with the pectin -100 5 dilution previously weighed filter paper Sample weight (1)

PG activity
The PG activities were determined by the method of Miller (1959), using 3,5 dinitrosalicylic acid (DNS).The assay was carried out with 50 µL of the enzyme and 50 µL of 1% (w/v) substrate: sodium polypectate from Sigma-Aldrich in 100 mM sodium acetate buffer, pH 5.0.The samples were incubated at 60°C for 10 min and 100 µL of DNS then added to the assay.The absorbance was measured at 540 nm.The results were obtained using a curve of monogalacturonic acid (0-1 mg/mL) as the standard.One unit of enzymatic activity was defined as the amount of enzyme that releases 1 µmol of reducing sugar per minute under the assay conditions.

Effect of temperature on PG activity
The effect of temperature on PG activity was analysed using the extracellular crude extract from N. glabra cultivated on orange peel (var.Bahia).The assays were carried out with the enzyme and 1% (w/v) substrate, and incubation at the temperatures of 30 to 70°C with 10°C intervals.
Thermal stability was determined by a prior incubation of the enzyme at 40, 50 and 60°C for up to 24 h, at pH 5.5.Samples were withdrawn after 10, 20, 40, 60, 120, 150, 180 min and 24 h of incubation, and the residual activities measured as described above.

Effect of pH on PG activity
The effect of pH on the PG activity was also analysed using the extracellular crude extracts from N. glabra cultivated on orange peel (var.Bahia).The assays were carried out using the enzyme and 1% (w/v) substrate, with incubation in 100 mM sodium acetate buffer, from pH 4.0 to 8.5, with 0.5 pH unit intervals.The pH stability was determined by incubating the enzyme in 100 mM McIlvaine buffer (citrate-phosphate) (1:1v/v) from pH 3.0 to 7.5 for 120 minutes at 25ºC.After incubation, the residual activities were measured as described above.The control was that of time 0 (zero), when the residual activity was considered to be 100%.Neosartorya glabra polygalacturonase produced from fruit peels as inducers has the potential for application in passion fruit and apple juices Pinheiro, V. E. et al.

The effect of enzyme application on fruit juice processing
The extracellular crude extract obtained from the cultivation of N. glabra was used as the source of PG.Apple (var.Fuji) and passion fruit (var.Yellow) pulps were used to analyse the action of PG on the fruit juices.The methodology of application was an optimized version of that reported by Kashyap et al. (2001).The selected fruits were washed, cut and triturated.The sliced apples were maintained in contact with oxygen for 15 min to avoid the inhibition of pectinase by polyphenols.Twenty grams of sliced and triturated fruits were placed into 125-mL Erlenmeyer flasks and 4.5 U of crude enzyme per gram of fruit added.The control (untreated pulp) was prepared under the same conditions, but using a boiled enzyme extract.The flasks were incubated at 37°C for 5 h with agitation.The fruit juices were then extracted by filtration and the viscosity and turbidity of the extracts studied.The viscosity was analysed using an Ostwald's viscometer.The percentage of decrease in viscosity (A) was calculated according to Roboz et al. (1952), through the Equation 2: Vo is the flow time (seconds) of fruit pulp treated with inactivated enzyme (control); Vt is the flow time (seconds) of fruit pulp treated with active enzyme; Vs is the flow time (seconds) of the inactivated enzyme plus water The difference in turbidity between the treated and untreated fruit juice samples was analysed from the light absorbance in a spectrophotometer at different wavelengths (250-570 nm, with 10 nm intervals) using a glass cuvette.
The untreated sample (control) was the fruit pulp treated with inactivated enzyme.

The effect of the carbon sources on PG production
The effect of the fruit peels as PG inducers was analysed using SbmF (Figure 1A) and SSF (Figure 1B).Glucose was used as the control (repression conditions).Using SbmF (Figure 1A) orange peel was the best PG inducer (4.3 U/mL), followed by lemon peel, which had a production corresponding to 92% of the activity observed with orange peel.The worst PG inducer was banana peel under the conditions analysed.Using SSF (Figure 1B), guava peel was the best PG inducer (1.2 U/mL), with a PG production 16.5% greater than the second-best residue, apple peel.
Table 1 expresses the enzyme productivity (U/gram of dried peel per hour of fermentation) using SbmF and SSF.SbmF showed better enzyme productivity and has the advantages of easy recovery of the extracellular enzymes,

Effect of different varieties of orange and lemon peels on PG production
Since orange (Bahia variety) and lemon (Tahiti variety) peels were the sources that stood out as inducers of PG production using SbmF, a study using tangerine and other varieties of orange and lemon peels was carried out (Figure 2).Orange peel (Bahia variety) remained as the best residue for PG production by the fungus N. glabra, but orange (var.Pear), tangerine and lemon (var.Tahiti) peels were also shown to be good inducers, presenting 96%, 86% and 90% of the production observed with the Bahia orange peel, respectively.
Tables 2 and 3 illustrate the influence of mixtures of peels of different oranges and the peels of distinct lemons on PG production from N. glabra, respectively.It can be seen that Bahia orange peel remained as the best inducer of PG production even when using mixtures of other orange peels and tangerine (Table 2), whereas lemon peel (var.Tahiti) was the best PG inducer when compared to the mixtures with lemon peel (var.Sicilian) (Table 3).
Analysing Tables 2 and 3 and Figures 1 and 2, it can be seen that orange peel (var.Bahia) was the best PG inducer.Quantitative analyses for the determination of pectin in the dry peels used in this work showed that banana, guava, apple, lemon, passion fruit and orange peels have 19%, 20%, 15.5%, 15%, 16% and 17% of pectin, respectively.The values are close and probably other parameters influenced the PG synthesis, such as: the availability of inducers, structural differences of the pectin contents -branching degree, phenol groups, polygalacturonase-inhibiting protein, and the presence of fungicides, pesticides, ions, detergents, urea, 2-mercaptoethanol, vitamins, phosphorus and others (KAUR et al., 2004;TAI et al., 2013;KANT et al., 2013;SCHWAN-ESTRADA et al., 2000).According to Al-Saadi et al. ( 2009), orange peels contain alkaloids, saponins, terpenes, resins, flavonoids, tannins, phenols and sugars.The orange peels contain 23.8% of sugar, 4%  Neosartorya glabra polygalacturonase produced from fruit peels as inducers has the potential for application in passion fruit and apple juices Pinheiro, V. E. et al.
of protein, 11.86% of water and 5.34% of ash.In addition, according to Canteri et al. (2012), orange peel has a high pectin content (3.5-5.5% of pectin per fresh-fruit weight), which is a good inducer of PG production.Furthermore, this result is very interesting, since orange juice is one of the most widely consumed beverages nowadays and consequently the amount of waste (mostly peel) generated is large.
The cultivation of orange has become an important economic sector in the United States (Florida and California), Brazil, Mexico, Pakistan, China, India, Iran and the Mediterranean countries.Approximately 50-60% of the processed fruit is transformed into citrus waste (peel, seeds and membrane residues), which must be processed in order to avoid the disposal of this residue in the soil resulting in further environmental problems (MARTÍN et al., 2010).Thus orange peel can be used as an alternative and viable pectinase inducer.

Effect of cultivation temperature and time-course on PG production
N. glabra was cultivated at several temperatures for the analysis of the optimal cultivation temperature.
The highest PG activity occurred in the range from 30 to 35°C (Figure 3A) and 35ºC was selected as the optimal temperature.
Using SbmF for the cultivation of N. glabra in Vogel's culture medium supplemented with 1% (w/v) of orange peel (Citrus sinensis var.Bahia), higher extracellular PG activity (3.2 U/mL) was obtained after 72 hours of fermentation under static conditions at 35 o C. The PG activity remained approximately constant after 168 h of fermentation (Figure 3B).The significant secretion of extracellular PG facilitates its recovery from the fermentation medium, being more beneficial for the industry.The cultivation time may be dependent on the amount of nitrogen and carbon source available in the medium, since short periods of incubation may not result in the maximum production of the metabolite of interest.On the other hand, cultures incubated for long periods may lead to depletion of nutrients, cell death of the fungus and degradation of the enzymes (PELCZAR et al., 1996).Maciel et al. (2013) observed that the highest PG production by A. nigri occurred between 48 and 96 h and Patil et al. ( 2012) observed that 72 h was the optimal cultivation time for exo-PG production by Paecilomyces variotii.

Effect of the carbon source concentration on PG production
In the study of the effect of the carbon source concentration on the submerged fermentation of N. glabra under static conditions at 35ºC, higher PG activity (4U/mL) was obtained in Vogel's culture medium supplemented  Neosartorya glabra polygalacturonase produced from fruit peels as inducers has the potential for application in passion fruit and apple juices Pinheiro, V. E. et al.
The carbon source concentration is an important parameter to be determined in the optimization of fungal cultivations, because it is a precursor of the carbon chains for the synthesis of all the cell components.Low concentrations may be insufficient for inducing good enzyme levels and very high concentrations may act as inhibitors, since the excess (mainly complex substrates) can contain other sugars (monosaccharides), which have a higher affinity for the membrane transporters and are more easily captured by the fungus than is pectin, thereby decreasing the secretion of pectinase (KUBICEK, 2013).The inhibitory effect was observed with concentrations above 3% (w/v) of orange peel and could be correlated with an excess of substrate, such as fruit processing residues and other compounds that lead to the death of the fungus, such as pesticides, very common in fruit peels (SIDDIQUI et al., 2012).

Effect of temperature on PG activity
The crude PG from Neosartorya glabra presented optimal activity at 50-60ºC (Figure 5A) suggesting good applicability in processes involving high temperatures.Siddiqui et al. ( 2012) reported the optimal temperature as 55°C for the PG from Rhizomucor pusillus.Kashyap et al. (2001) observed maximum PG activity from A. niger at 50°C and Maller et al. (2011) reported the optimal temperature as 55°C for the PG activity from A. niveus.
The PG from N. glabra retained more than 90% of its initial activity after 180 min at 40ºC.At 50ºC, the enzyme presented a t 50 of 150 min and at 60°C it was not stable (Figure 5B).

Effect of pH on PG activity
The crude PG from N. glabra presented optimal activity at pH 5.5 (Figure 6A).This result implies in applications in acidic processes, such as in the citrus juice industry.The PG from N. glabra showed stability at acid pH values when incubated in 100 mM McIlvaine buffer for 120 minutes at 25ºC.The incubation in buffer at pH 3.0 resulted in an increase in residual activity of more than 20% (Figure 6B) when compared to the control (non incubated enzyme).It has been reported that most pectic enzymes show stability under acidic pH conditions (GUMMADI; KUMAR, 2006).An acid solution presents a lot of free H + , which facilitates protonation of the COOH - (glutamic acid and aspartic acid) and NH 2-(lysine, histidine and arginine) groups of the enzymes, directly influencing bonding to the substrate (SILVA; SILVA, 2010).

The application of the enzymatic extract in fruit juice processing
The effect of the crude PG produced by N. glabra in reducing the viscosity of passion fruit and apple pulps was analysed.The treatment of passion fruit pulp with crude PG from N. glabra decreased the viscosity by 80%, whereas treated apple pulps showed a 50% reduction in viscosity in relation to the untreated sample.In the juice industry, the pectin polymer present in the fruit pulp can interact with water molecules in an acidic medium and form a gel, increasing the viscosity.On account of this, it is common practice to apply pectinases to lower the viscosity and the water-binding capacity of the pectin, for easier juice extraction (NAKKEERAN et al., 2011;REHMAN et al., 2013).These results are in agreement with Domingues et al. ( 2014), who analysed the reduction in viscosity of passion fruit juice samples using the enzymatic complex Pectinex 3XL from Novozymes, and verified that the enzymatic treatment was efficient in reducing the viscosity.2013) reported a 17.6% viscosity reduction in papaya juice with the application of an endo-PG.
The turbidity of the treated and untreated pulps was analysed via light absorbance at different wavelengths in a spectrophotometer.The juices treated with crude PG showed different values for absorbance at the different wavelengths as compared to the untreated juice (control).Treated passion fruit juice showed a higher difference in absorbance between 430-500 nm (Figure 7A) and at these wavelengths the light absorbance of the treated samples was shown to decrease by 75%.For apple samples, the  highest difference in absorbance was observed between 320-360 nm (Figure 7B) and at these wavelengths the light absorbance of the treated samples was shown to decrease by 78%.These data suggest the range of wavelengths that could be used to estimate the hydrolysis caused by the enzyme action and the decrease in light absorbance after enzyme treatment, which is an indicator of the decrease in turbidity.

Conclusion
The use of residues from fruit juice processing as a carbon source, especially orange peel (var.Bahia), was favourable for PG production by N. glabra.This result was very interesting since the use of low-cost substrates is required in industrial processes.Optimal PG production conditions were obtained with 1.5% dried and ground orange peel (Bahia) as the carbon source when using SbmF, cultivating the microorganism in Vogel's minimal medium and incubating at 35°C for 72 h.The PG of the crude extract presented optimal activity at 50-60°C and pH 5.5 The enzyme was highly stable at 40ºC for 180 minutes and retained 50% of the initial activity after 150 minutes at 50°C.The PG was stable in the acidic range after 120 minutes of treatment at 25°C.The enzyme extract obtained under the optimized condition was used in the extraction of fruit juices and in the application tests, where it showed a reduction in viscosity of 80% and a decrease in light absorbance of 75% with the passion fruit pulps, and a reduction in viscosity of 50% and decrease in light absorbance of 78% with the apple pulps.This work contributes to the improvement of knowledge on the influence of physical-chemical factors on the production of polygalacturonase by Neosartorya glabra and its success in the application to passion fruit and apple pulps.

Figure 1 .
Figure 1.The effect of the carbon sources on PG production.(A) shows the PG activities using SbmF and (B) using SSF.The microorganism was cultivated in Vogel's medium and incubated under static conditions at 30ºC for 96 h.Symbols: (*) p < 0.05, (**) p< 0.01 and (***) p < 0.001 versus Glucose according to the Student Newman Kwels test (SNK); ns -not significant.
Neosartorya glabra polygalacturonase produced from fruit peels as inducers has the potential for application in passion fruit and apple juices Pinheiro, V. E. et al. mycelia and spores.SbmF is a process of a homogeneous nature with easy control of the parameters, such as temperature and agitation(FERNANDÉZ, 2009), and more than 75% of industrial enzymes are produced using SbmF (SUBRAMANIYAN; VIMALA, 2012).Camargo et al. (2005) reported the production of 3.6 U/mL of PG by Aspergillus sp. in a medium supplemented with orange (pulp, peel and seeds) as the carbon source.Maller et al. (2011) andMrudula and Anitharaj (2011) observed that orange peel was the best inducer of pectinase production by A. niveus and PG by A. niger, respectively.

Figure 2 .
Figure 2. Effect of tangerine peel and different varieties of orange and lemon peels as carbon sources for PG production using SbmF.The microorganism was cultivated in Vogel's medium in a bacteriological incubator at 30ºC for 96 h.Symbols: (*) p < 0.05, (**) p< 0.01 and (***) p < 0.001 versus Glucose according to the Student Newman Kwels test (SNK); ns -not significant.

Table 3 .
Influence of the mixture of two varieties of lemon peel on PG production.cultivated in Vogel's medium and incubated under static conditions at 30ºC for 96 h.

Figure 3 .
Figure 3.The effect of cultivation temperature (A) and time (B) on PG activity.Symbols: (■) extracellular and (□) intracellular PG activities.The microorganism was cultivated in Vogel's medium (SbmF) using orange peel (var.Bahia) as the carbon source and incubating under static conditions.

Figure 4 .
Figure 4.The effect of the carbon source concentration on PG production.The microorganism was cultivated in Vogel's medium (SbmF) using orange peel (var.Bahia) as the carbon source and incubated under static conditions at 35ºC for 72 h.
Laorko et al. (2010) also used the enzymatic treatment of apple juices before micro-and ultrafiltration tests.Patil et al. (2012) tested an exo-PG produced by Paecilomyces variotii in the treatment of fruit juices, and observed viscosity decreases in orange, apple, grape, banana and guava juices.Tu et al. (

Figure 6 .
Figure 6.The effect of pH on PG activity (A), pH stability of PG at 25ºC for 120 minutes (B).C= control.

Figure 7 .
Figure 7. Estimation of the turbidity of the juices.The values for absorbance of treated and untreated passion fruit juice samples (A), and apple juice samples (B).Symbols: untreated samples -control (•), treated samples (■).

Table 1 .
Comparison between SbmF and SSF in the production of PG using different carbon sources.

Table 2 .
Influence of the mixture of some varieties of orange peel on PG production.