Statistical tools application on dextranase production from Pochonia chlamydosporia ( VC 4 ) and its application on dextran removal from sugarcane juice

The aim of this study was to optimize the dextranase production by fungus Pochonia chlamydosporia (VC4) and evaluate its activity in dextran reduction in sugarcane juice. The effects, over the P. chlamydosporia dextranase production, of different components from the culture medium were analyzed by PlackettBurman design and central composite design. The response surface was utilized to determine the levels that, among the variables that influence dextranase production, provide higher production of these enzymes. The enzymatic effect on the removal of dextran present in sugarcane juice was also evaluated. It was observed that only NaNO3 and pH showed significant effect (p<0.05) over dextranase production and was determined that the levels which provided higher enzyme production were, respectively, 5 g/L and 5.5. The dextranases produced by fungus P. chlamydosporia reduced by 75% the dextran content of the sugarcane juice once treated for 12 hours, when compared to the control treatment.

Such substance is responsible for many problems in the sugar industry.Among them we highlight the reduction in the concentration and lower rate of sucrose recovery, increased sugarcane juice and syrups viscosity (Khalikova et al. 2005), and a quality deterioration of the finished product, sugar (Batista 2014, Morel du Boil andWienese BRUNA L. SUFIATE et al. 2002), besides the formation of opaque, irregular, elongated and caramel tone crystals (Jiménez 2009).Apart from the loss of sucrose and the final product quality deterioration, its presence in the sugarcane juice also results in increased equipment wear and compromise of the sucrose content quantification, which is used as the base for sugarcane producer's payment (Singleton et al. 2002).
Dextranases are enzymes that specifically break down dextran molecules of high molecular weight into smaller dextrans, thereby reducing the viscosity of both, massecuite and molasses.With dextranase use, the elongation of the grain ceases, the boiling time is shortened, and the product flows more smoothly due to reduced viscosity (Cuddihy et al. 2000, Jiao et al. 2014).
The fungus P. chlamydosporia, used in this present study, is a facultative parasite of eggs and female nematodes found in the soil in various regions of the world, having been isolated directly from soil samples or eggs and nematode cysts (Lelis 2014, Podestá 2015).Although there are numerous studies in the literature using the fungus P. chlamydosporia toward their ovicidal and nematicidal activity, and proteases and chitinases production (Araújo et al. 2008, 2009, Braga et al. 2008, 2010, 2011, Frassy et al. 2010, Silva et al. 2010, Soares et al. 2015, Tobin et al. 2008) there are no reports as to their use for dextranases production.
Therefore, the objective of this study was to optimize dextranases production by fungus P. chlamydosporia, and its applicability in removing the dextran compound present in the sugarcane juice.

FUNGUS CULTIVATION
The isolated fungus P. chlamydosporia VC4 originated from Brazilian soil, provided by the Parasitology Lab of the Veterinary Medicine Department, Universidade Federal de Viçosa, was maintained on a 2% (w/v) solid Potato Dextrose Agar medium (PDA 2% (w/v)) under refrigeration, at 4°C.Before inoculation in the liquid culture medium, the fungus was activated, and peaked to a new petri dish with PDA medium.The colonies were transferred in the form of discs and incubated for 10 days at 28°C.

OBTAINMENT OF THE ENZYMATIC EXTRACT
For the inoculation in liquid culture medium, were removed from edges of the petri dishes four equally sized disks.These disks were inoculated into 250 ml Erlenmeyer flasks containing 100 ml of liquid medium previously autoclaved at 121°C for 15 minutes.The liquid medium contained dextran 10 g/L, NaNO 3 10 g/L, KH 2 PO 4 4 g/L, MgSO 4 .H 2 O 0.5 g/L, KCl 0.5 g/L, ZnSO 4 .7H 2 O 0.178 g/L, FeSO 4 .7H 2 O 0.18 g/L.After inoculation, the flasks were kept at 28°C and 180 rpm for 7 days.After 7 days, the Erlenmeyer flasks content was filtered and centrifuged at 10,000 X g for 20 minutes, yielding the crude extract.

ENZYME ACTIVITY MEASUREMENTS
The dextranase activity was measured by assessing the amount of reducing sugar produced from dextran hydrolysis by the 3,5-Dinitrosalicylic acid The dextranase activity was measured using reactions with different incubation temperatures: 30, 40, 50 and 60°C, keeping all the previously described conditions, except for the buffer pH, where citrate-phosphate buffer solution 100 mM utilized had a pH value of 6.0.

Reaction time effect on enzyme activity
The dextranase activity was measured using different reaction incubation times: 5, 10, 15, 20, 25 and 30 minutes.In this assay, and all other following, were used the temperature and the pH which resulted in higher enzyme activity.

Ingel activity assay
In order to confirm the dextranase activity presented on the crude extract of the fungus P. chlamydosporia, an in-gel activity assay was warried out.The crude extract was applied to 10% native PAGE gel containing 1% blue dextran, and a voltage of 80 V was used.The gel was incubated in citrate phosphate buffer 100 mM pH 6.0 for 1 hour at 50°C.The dextranase activity was detected as a clear band on blue background.

EXPERIMENTAL DESIGN
In order to determine which culture medium components significantly influence dextranase production by the fungus P. chlamydosporia (VC4), it was evaluated the following variables by the Plackett-Bruman design (Plackett and Burman , pH and incubation time (days).For this analysis, Minitab 16 software (Philadelphia, Pennsylvania, USA) was used and each factor was analyzed at two levels: -1 to +1 and lower level to the upper level.The variables and their analyzed levels are shown in Table I.The experimental design matrix is illustrated in Table II.method (DNS) (Miller 1959).The enzymatic assay was performed in test tubes, using 750 μL sodium acetate buffer 100 mM pH 5.2, 200 μL of dextran solution at 1% and 50 μL of the enzyme sample.The reactants were incubated at 50°C for 15 minutes.After this time, the reaction was stopped by adding 1 mL of the DNS reagent.Samples were heated for 5 minutes in a water bath.After the boiling bath, 2 mL of water was added to each test tube.The absorbance readings were performed at 540 nm.In order to obtain the amount of reducing sugar from the absorbance values, a standard curve with increasing concentration of glucose was constructed.One dextranase unit (U) was defined as the amount of the enzyme that catalyzes the release of 1 µmol of reducing sugar per minute under the assay conditions.All enzyme activity measurements were performed in triplicate.

INCUBATION TIME EFFECT ON DEXTRANASE PRODUCTION
During a time period of 11 days, every day, an aliquot of 1 mL was removed from each one of the three replicate flasks, from each treatment, transferred to a microtube and centrifuged at 10,000 X g for 20 minutes.These samples were measured for dextranase activity.The liquid culture medium was prepared as described before.Data were analyzed using the software Bioestat 5.0 (Belém, Pará, Brazil), by analysis of variance (ANOVA) with significance level of 5 and 1%.Subsequently, the data were analyzed by Tukey test, with 5% significance.

ENZYMATIC CHARACTERIZATION pH and temperature effect on enzyme activity
In order to access the pH effect over the dextranase activity, an assay was conducted using 100 mM citrate-phosphate buffer with different pH values: 2.2; 3.0; 4.0; 5.0; 6.0; 7.0 and 8.0.Assays were performed under the conditions described above, using the buffer solution in such pH values.BRUNA L. SUFIATE et al.
A central composite design is the most commonly used response surface designed experiment.Central composite designs are a factorial or fractional factorial design with center points, augmented with a group of axial points that let estimate the curvature (Machado 2009).This design was used to apply the response surface methodology, and thus analyze the variables levels obtained from the Plackett-Burman design, as well as their possible interactions.Each variable had its effect evaluated into five experimental levels: -α, -1, 0, +1, + α, where α = 2 n/4 , being n the number of variables and 0 corresponding to the central point.A total of 13 experiments were made, 5 of them being replicates of the center point.Data were analyzed using the software Design Expert 7.0.The variables and their analyzed levels are shown in Table III.The experimental design matrix is shown in Table IV.The other variables not selected in the Plackett-Burman design were maintained as previously described.

DEXTRANASE APPLICATION IN THE DEXTRAN REMOVAL FROM THE SUGARCANE JUICE
For the sugarcane juice extraction, the sugarcane cultivar RB867515 used in this study was obtained from the Department of Animal Science at Universidade Federal de Viçosa.After the harvest, the sugarcane was stored at room temperature for 7 (seven) days.The sugarcane juice was extracted from 5 (five) sugarcanes through a grinder and, subsequently, filtered to remove all residues.The dextran quantification from the sugarcane juice proceeded immediately after extraction.Different amounts of dextranase (0, 5, 10 and 15 U) from the crude extract was added to 100 mL of sugarcane juice, and the residual dextran was quantified

INCUBATION TIME EFFECT ON DEXTRANASE PRODUCTION
The dextranase production was monitored for 11 days (Figure 1).The highest production in absolute values occurred on the eighth day of incubation.
However, the dextranase activities on day 6, 7, 8 and 9 did not differed significantly (p>0.05).Therefore, for the other dextranase production tests, were used 7 days of incubation.reported that Penicillium funiculosum had increased dextranase production on day 7 of incubation, which was also observed in this study.

ENZYMATIC CHARACTERIZATION
The enzyme reaction pH and temperature must be evaluated in order to determine the best conditions for the reaction to occur.It was evaluated the temperature (Figure 2) and pH (Figure 3) effect over dextranase activity.The dextranase activity was higher at 50°C and pH of 6.0, however the enzyme showed high activity between the pH ranging from 4.0 to 7.0.Given these results, the following trials   on dextranase activity were conducted at 50°C and pH of 6.0.
These data corroborate with the results found by Bhatia et al. (2016), who determined that the dextranase produced by P. lilacinus was most active at 50°C and pH of 5. On the other hand, the dextranase produced by Aspergillus subolivaceus showed higher activity at 60°C and pH of 5.5 (El-Tanash et al. 2011), and dextranase produced by Penicillium lilacinum exhibited maximum activity between the temperature and pH ranging from 30 to 35°C and 4.5 to 5.5, respectively (Aslan and Tanriseven 2007).
The in-gel activity assay demonstrates the existence of a band related to the dextranase activity, showing that there is only one form of dextranase present in the crude extract (Figure 4).

DEXTRANASE PRODUCTION OPTIMIZATION
The Plackett-Burman design allowed us to determine which, of the studied factors, significantly influence dextranase production from P. chlamydosporia fungus.It was evidenced that NaNO 3 significantly affect (p<0.05)dextranase production, and the pH showed a tendency to a significant effect (p=0.054) (Table V).The experimental matrix with dextranase activity values obtained from each run is shown in Table II.Based on the results from the Plackett-Burman experimental design, the central composite design was employed to establish the optimum levels of these two variables (NaNO 3 and pH).A total of 13 trials were conducted with different combinations of the two variables.The central point was repeated five times in order to estimate the error.The experimental matrix with the dextranase activity values obtained are shown in Table IV.
The regression model was statistically tested by the F test, and the analysis of variance (ANOVA) was used for the quadratic model of the response surface (Table VI).The quadratic model was significant with a p-value of 0.0003.The R 2 model was 0.94, indicating this model's reliability.The highest dextranase activity observed was 4.388 (U/mL) at pH of 4.88 and 12.5 (g/L) NaNO 3 concentration.
The results showed that the pH linear term, the pH quadratic term, and the term related to the interaction between pH and NaNO 3 have a significant effect (p<0.05) in the dextranase production from P. chlamydosporia.
The equation below presents the dextranase activity after the terms related to non-significant variables (p>0.05) have been removed: Where Y is the dextranase activity, and X 1 and X 2 correspond to the pH and NaNO3, respectively.As shown in Figure 5, a three dimensional response surface model was generated according to the final dextranase production from P. chlamydosporia fungus.It can be notice that the pH and NaNO 3 reduction results in a higher dextranase production.In other words, the dextranase activity was inversely proportional to the levels of variables pH and NaNO 3 .The 12 hours sugarcane juice storage resulted in increased dextran content from 3.62 to 28.84 mg/100 mL.With the dextranase addition to the sugarcane juice, was observed a lower increase in the dextran content when compared to the control treatment, which demonstrates the dextran removal through the dextranase enzyme activity (Table VII).
With the dextranase addition in a concentration of 15 U/100 mL, the dextran content measured after 12 hours was 7,158 mg/100 mL, which means that a reduction of 75% on the dextran content occurred when compared to the untreated treatment (28.84 mg/100 mL) (Table VII).Tukey's test revealed no significant difference (p>0.05) between the sugarcane juice treated with 10 and 15 U/100 mL of sugar cane juice.The dextran removal from the sugarcane juice through the use of dextranase produced from fungus was reported by Bhatia et al. (2016).In this particular study, when they added 5, 10 and 15 U of partially purified dextranase from P. lilacinus per 100 ml of sugarcane juice observed, after 12 hours, a reduction in the dextran content of 38.89%, 52.74% and 61.11%, respectively.
Among the analyzed factors, pH and NaNO 3 influenced the dextranase production of by fungus P. chlamydosporia.Through the surface response analysis, the NaNO 3 and pH levels that resulted in higher dextranase production were, respectively, 5.5 and 5g/L.
T h e d e x t r a n a s e s p r o d u c e d b y P. chlamydosporia proved to be effective in removing dextran present in sugarcane juice.After 12 hours, when comparing the untreated treatment with the  The values shown are the mean ± standard deviation.The values in parentheses represents the percentage (%) of dextran content removal when compared to the control treatment (without dextranase).Values followed by the same letter were not significantly different (p>0.05) by Tukey test.
treated (15 U) treatment, there was a 75% dextran content reduction.This is the first report on dextranase activity from the nematophagous fungus P. chlamydosporia and it opens new perspectives about the use of this established biological controller in a new biotechnological approach.

Figure 1 -
Figure 1 -Dextranase activity produced by the fungus Pochonia chlamydosporia as a function of the incubation time in submerged fermentation at 28°C and 180 rpm.*Indicates that there was no significant difference in the dextranase activity (p> 0.05) between the marked days by the Tukey test.

Figure 3 -
Figure 3 -pH effect of the dextranase enzyme activity reaction present in the crude extracts produced by the fungus Pochonia chlamydosporia.

Figure 2 -
Figure 2 -Incubation temperature effect of the dextranase enzyme activity reaction present in the crude extract produced by the fungus Pochonia chlamydosporia.

Figure 4 -
Figure 4 -Clear band showing the dextranase activity of crude extract on a 10% native PAGE gel containing 1% (w/v) blue dextran.

Figure 5 -
Figure 5 -Response surface of the dextranase production by the fungus Pochonia chlamydosporia in submerged fermentation.

TABLE VI Analysis of variance for the response equation obtained in the dextranase production by fungus Pochonia chlamydosporia in submerged fermentation.
SS: sum of the squares; DF: degree of freedom; MS: media square.BRUNA L. SUFIATE et al.DEXTRANASE APPLICATION IN THE DEXTRAN REMOVAL FROM THE SUGARCANE JUICE