Production , characterization and evaluation of in vitro digestion of phytases , xylanases and cellulases for feed industry

1 Federal Rural University of Pernambuco – UFRPE, Rua Dom Manoel de Medeiros, s/n, Dois Irmãos CEP: 52171-900 – Recife – Pernambuco, Brazil. 2 Federal Rural University of Pernambuco – Academic Unit of Garanhuns – UAG/UFRPE, Avenida Bom Pastor, s/n, Boa Vista CEP: 55292-270 – Garanhuns – Pernambuco, Brazil. 3 Federal University of Pernambuco – UFPE, Avenida Professor Moraes Rego, n° 1235, Cidade Universitária – CEP: 506070 – 901Recife – Pernambuco, Brazil.


INTRODUCTION
Monogastric animals have a disadvantage with respect to the use of food as compared to ruminants as they are not capable of producing a series of enzymes needed to promote more effective use of food ingested in their diet.Some enzymes may not be synthesized in sufficient quantity to meet the demand of catalysis of nutrients (Pariza and Cook, 2010).
necessary to improve the digestive performance of monogastric animals, as well as increasing the nutritional value and bioavailability of nutrients, either before or after ingestion (Lange et al., 2010).Moreover, the presence of anti-nutritional factors, such as phytate and non-starch polysaccharides, in foodstuffs of plant origin such as grains, limits the use of nutrients by monogastric animals.Phytate, the principal form of phosphorus storage in plants, affects the availability of cations such as calcium, zinc and magnesium, as well as proteins and carbohydrates, forming insoluble complexes and decreasing the absorption of these nutrients (Brandão et al., 2007).
The presence of non-starch polysaccharides is responsible for the increase of viscosity of food in the gastrointestinal tract, and as a consequence, the reduction in the digestion and absorption of amino acids, carbohydrates and minerals, causing reductions in animals performance (Viana et al., 2011).
The specificity of enzymes in their reactions determines that the incorporation of a single enzyme is insufficient to produce the maximum benefit; this suggests that the use of various enzymes in an additive approach in the form of a multi-enzymatic complex be used to provide better use of nutrients from diets (Tejedor et al., 2001).
The lowering of the total costs of livestock production systems, especially with regard to the greater economy in the use of feed on farms, is one of the main factors that influence the research on the production of multi-enzymatic complexes, in addition to achieving more satisfactory results (Lima et al., 2007).The aim of this study was to evaluate the potential of the filamentous fungi, Aspergillus tamarii URM 4634 and Aspergillus japonicus URM 5633 and select the best microorganism for the production of phytase, xylanase and cellulase from submerged fermentation using the inner bark bran of cassava, palm extract and corn steep liquor as fermentable substrates.

Enzymatic production
Two species for enzyme production, A. tamarii URM 4634 and Aspergillus japonicus URM 5633 were selected; the best producer was used of the two selected.The inoculum was standardized at 10 6 spores/mL, in triplicate, with six fermentation systems being produced (Table 1).The fermentations were carried out in 125 mL Erlenmeyer flasks, containing 25 mL of fermentation medium sterilized at 121°C for 20 min.After inoculation, the systems were kept in a temperature-controlled incubator under agitation for 96 h at 30°C.After this period, they were filtered and centrifuged at 4°C, and the supernatant was termed a crude enzyme extract, later used for analytical determinations.

Analytical determinations
The determination of dosages of total proteins of the enzymatic extracts was based on the methodology proposed by Bradford (1976), using bovine serum albumin as the standard and protein content expressed as mg/mL.Phytase activity was determined according to Heinonen and Lathi (1981).One unit (U) of phytasic activity was defined as the amount of enzyme required to release 1 µmol of inorganic phosphate per minute.Total xylanolytic and cellulolytic activities (FPase) were determined according to the methodology described by Bailey et al. (1992) and Ghose (1987), respectively.The reducing sugars released were determined by the method of 3,5-dinitrosalicylic acid (DNS), proposed by Miller (1959).One unit of these activities was defined as the quantity of enzyme required to release 1 μmol of reducing sugars per minute and expressed in units per mL (U/mL).

Optimal temperature and pH
For the determination of the optimum temperature for enzymatic activities, incubation temperatures that ranged between 40-80°C were used.While for the pH optimum, 50 mM sodium acetate buffer (pH 4.0; 4.5; 5.0; 6.0) and 50 mM Tris-HCl (pH 7.0; 7.5; 8.0) were used, plus phytic acid, xylan or filter paper used as substrates for total phytasic, xylanolytic and cellulolytic (FPase) activities, respectively.Enzyme activities were expressed as relative activity (%).

Thermal stabilities and to pH
For the thermal stability, as well as for the stability of pH, the enzymatic extract was incubated under the same conditions of the optimal temperature and pH studies; this was done with the removal of aliquots every 30 min, for the determination of enzymatic activities, over a total of 180 min.The enzymatic activity was expressed as residual activity (%).

Simulation of in vitro digestion
The simulation of gastrointestinal digestion of monogastric animals was carried out according to the method described by Boyce and Walsh (2007), with a few modifications.Three tests were carried out, using at first, 1% pepsin (w/v) diluted in deionized water, pH 2.5 adjusted with 1 M HCl for the simulation of gastric digestion.In this test, the enzymatic extract was incubated at 39°C for 2 h under agitation (150 rpm).In the second, 1% pancreatin (w/v) was used along with 1% bile extract (w/v) in order to simulate enteric digestion.The enzymatic extract was incubated under the same conditions of the previous test, but for a period of 4 h.The third test was conducted to evaluate the influence of isolated (1%) trypsin on the enzyme complex, being incubated under the same conditions as the simulation of enteric digestion.Each test had their respective controls, constituted only of solutions, without the presence of the enzymatic extract.

Statistical analysis
The data on the specific activity of the enzymes produced by A. tamarii URM 4634 and A. japonicus URM 5633 were analyzed in a 6×2 factorial arrangement, with six systems (1, 2, 3, 4, 5 and 6) and two fungi (A.tamarii URM 4634 and A. japonicus URM 5633).The data were subjected to analysis of variance and averages were compared by Tukey test at 5% level of probability.From the averages of data on optimal temperature and pH and stability of all enzymes to pH and temperature, regression curves were obtained and selected; enzymatic activity was considered the independent variable.Polynomial models were tested and selected based on the coefficient of determination (R 2 ) and residual mean square (RMS).*Data followed by the same lowercase letter in the column and capital letter in the row do not differ from each other, according to the Tukey test at the 5% probability level.For the purpose of calculation the data were transformed into roots (x + 1) and presented as the averages of the observed data and the letter of the transformed data.

Enzymatic production
The fungus, A. tamarii URM 4634 had the greatest production for all enzymes in relation to specific activity (U/mg) in the fermentation system #4 (5.0% palm extract, 2.0% CB and 1.0% corn steep liquor) (Table 2).There was a significant effect (P<0.05) in the interaction among the system factors versus the fungal isolates for all variables: total phytasic, xylanolytic and cellulolytic (FPase) activities.For the specific activity of phytase produced by A. tamarii URM 4634, despite systems #2, 3, 4 and 5 not differing statistically among themselves, system #4 presented the highest enzymatic activity.For the fungus A. japonicus URM 5633, none of the systems differed statistically among themselves; however, system #6 (2.5% palm extract, 2% CB and 1% corn steep liquor) showed the highest phytasic activity (Table 2).Singh and Satyanarayana (2008), working with phytases produced by Sporotrichum thermophile, observed variations between 12.56 and 8250.75U/L in phytase activity through ANOVA statistical analysis, according to the composition of the submerged fermentation medium.This corroborates with data obtained in this study, because different concentrations of waste generated different results of enzymatic activities.
Similarly, xylanolytic activity was greatest when produced by A. tamarii URM 4634 in system #4, differing statistically from the other systems.When the fungus, A. japonicus URM 5633 was used, system #6 presented results of 82.8% better than system #4 which expressed the lowest xylanolytic activity, among all systems.These results show that concentrations of total soluble carbohydrates can interfere directly in enzyme production.
The greatest production of cellulase by A. tamarii URM 4634 also occurred in system #4, showing a specific activity of 50.9% greater than system #6, which had the greatest production when A. japonicus URM 5633 was used.This demonstrated once again the advantage of using A. tamarii URM 4634 as the microorganism for pro-ducing the enzyme complex.

Optimal temperature and pH
In terms of the optimum temperature for phytase, the greatest value of relative activity was obtained at 40°C; however the variations in activity throughout the analysis were always above 80%, as can be seen in Figure 1.Vats and Banerjee (2005), working with Aspergillus niger, observed that the greatest value of phytasic activity occurred at 55°C, while at 70°C phytase was denatured, ceasing its catalytic activity, which did not occur with the phytase produced by A. tamarii URM 4634.
While for xylanolytic activity, it was observed that the greatest activity occurred at 60°C.The variations in activity throughout the analysis were always above 90%, i.e. xylanase was stable even at high temperatures, thus making xylanase a very promising enzyme for use in foods that will still go through industrialization processes that require high temperatures.These results corroborate the findings of Lemos et al. (2000), who worked with xylanases derived from Aspergillus awamori, which showed maximum results for activities of endoglucanases and β-xylosidases at temperatures of 60 and 55°C.Szendefy et al. (2006), in characterizing the xylanase produced by Aspergillus oryzae, found an optimum temperature between 60 and 65°C.
The optimum temperature for the FPase was verified at 45°C, decreasing by about 35% at 70°C and 80°C, while the activities of cellulase remained always above 60%.Bansal et al. (2012), working with cellulase produced by A. niger NS-2, showed maximum activity values at 30°C.Likewise, Gilna and Khaleel (2011), working with cellulase produced by Aspergillus fumigatus, found the optimum temperature for the activity of FPase to be 32°C, showing that the enzymes produced by the micro-organisms mentioned above feature optimum temperatures less than those produced by A. tamarii URM 4634.
The study of optimal pH showed that the three enzymes produced by A. tamarii URM 4634 showed maximum activity at pH values of 5.0, as shown in Figure 2.These results were similar to those found by Fujita et al. (2003), who worked with phytase produced by A. oryzae, which had the greatest value at pH 5.0.In alkaline pH conditions, the enzyme showed lower catalytic activity.However, in 50 mM sodium acetate buffer, to the extent to which the pH increased, phytase activity also increased, which demonstrates the interference of some ion on the structural conformation of phytase, associated with the pH conditions of the medium.Pal and Khanum (2011) also obtained maximum values for xylanase produced by A. niger DFR-5, under conditions of pH 5.0, showing that the enzyme has better field actuation under acidic pH conditions, since with an increase in pH, the enzyme showed a reduction in its catalytic activity.Singh et al. (2009), evaluating the FPase of Aspergillus heteromorphus, obtained optimal activity at pH 5.0.These results were similar to those found in this study.In both cases, at all other pH values, the enzyme remained active, showing high efficiency of the catalyst in the face of alterations in pH.

Thermal stabilities and pH
In the case of thermal stability (Figure 3), residual phytase activity was greatest at 45°C (78.72%) after 180 minutes.At the other temperatures, phytase activity always remained above 50% even at the highest shows this enzyme to  be thermostable and appropriate for use in different industrial processes.Zhang et al. (2010), when assessing the thermal stability of phytase produced by Aspergillus ficuum NTG-23, found high enzymatic activity at 60°C for 60 min.However, when subjected to the test for stability at 80°C, the phytase completely lost its enzymatic activity, unlike the phytase of this work.
For xylanase, the greatest residual activity was found at 50°C (91.87%) after 180 min.However, the xylanolytic activity always remained above 65% even at the highest temperatures, revealing a thermally stable enzyme.Chidi et al. (2008) noted that the xylanase produced by Aspergillus terreus UL4209 presented activity at 50°C for 5.8 h; however, it was inactivated after 2 h when exposed to a temperature of 70°C, thus showing the enzyme to be sensitive to higher temperatures, unlike the xylanase produced by A. tamarii URM 4634.Liao et al. (2012), working with xylanases produced by Penicillium oxalicum GZ-2, under conditions of submerged fermentation, obtained the best results for stability at 50-55°C.However, at 60°C the xylanolytic activity fell from 78 to 27% after 15 to 60 min of tested incubation, demonstrating that the xylanase produced by A. tamarii URM 4634, as shown in this work, presents greater thermal stability under similar conditions of fermentation.The greatest catalytic activity found for cellulase in terms of thermal stability was observed at 40°C for 180 min, with a maximum residual activity of 60.94%.It was observed that the enzyme showed a significant decrease at the higher temperatures tested, reaching levels of only 25% of residual activity.Delabona et al. (2013) achieved 100% activity of FPase produced by A. fumigatus P40M2 after 40 min at an incubation temperature of 60°C, with a decrease of 25% in residual activity after 60 min.This shows that temperature affects the conformational structure of cellulase over time at higher temperatures, thereby losing its catalysis efficiency.
In terms of pH stability, phytase showed the greatest residual activity in Tris-HCl buffer at pH 7.5 after 180 min (Figure 4).The phytase produced by A. tamarii URM 4634 presented enzymatic activity greater than 60% in all buffers and pH values used.This shows it is stable under diverse pH and ionic situations, which is extremely important for the use of this enzyme in the various food industrialization processes as well as in its use as an additive in feed of monogastric animals.Chantasartrasamee et al. (2004), working with phytases produced by A. oryzae AK9, observed high values of residual activity in the pH range between 2.0 and 7.0.The phytase produced by Rhizopus oryzae, in the study done by Rani and Ghosh (2011), exhibited 75% activity in relation to initial activity at pH values that ranged from 2.5 to 9.5, corroborating the results obtained in this work.
Xylanase had the greatest residual activity in the stability to pH test with Tris-HCl buffer at pH 7.5 for 180 min, showing a high degree of efficiency and stability to pH variations; whether acidic or basic, it is always above 85%.Similar results were obtained by Fang et al. (2008), in which the xylanase produced by Aspergillus carneus M34 presented stability between pH 7.0 and 10 after 60 min and continued with activity above 50% after 12 h of incubation in the pH range between 7.0 and 9.0.
The maximum residual cellulase activity was obtained in sodium acetate buffer at pH 5.0 after 180 min; however, in the other pH ranges, the residual activity of cellulase remained above 55%, showing it to be a stable enzyme despite the pH variations.

Simulation of in vitro digestion
In the simulation study of in vitro gastrointestinal digestion of monogastric animals, it was observed that enzymes produced by A. tamarii URM 4634 demonstrated interference from the simulation conditions and the enzymes present in the stomach and small intestine.This results in variations in their residual activity in the different essays, as can be observed in Table 3.The evaluation of this parameter in the literature is extremely scarce.Boyce and Walsh (2007), working with phytase produced by Mucor hiemalis and evaluating the effects of in vitro digestion on this enzyme, found that phytase had 77% stability under in vitro simulation of gastric digestion, using pepsin, and 85% underin vitro simulation of intestinal digestion using pancreatin and bile extract; in this the isolated influence of trypsin was not evaluated, as was done in the present work.However, it is important to understand what the effects of the enzymatic components of the gastrointestinal tract of monogastric animals are on the action of these enzymes, which justifies this third test.

Conclusion
A. tamarii URM 4634 has a great potential for the production of enzymes for use in the supplementation of feed for monogastric animals in the form of additives, as well as in other industrial processes.The influences generated by digestive enzymes on the enzyme complex produced proved quite promising, since the action of this complex, in some cases, was maximized.

Figure 2 .
Figure 2. Optimum pH of the enzymes phytase, xylanase and cellulase produced by A. tamarii URM 4634 in submerged fermentation with 5.0% palm extract, 2.0% inner bark bran of cassava and 1.0% corn steep liquor.

Figure 3 .
Figure 3. Thermal stability of phytase, xylanase and cellulase enzymes produced by A. tamarii URM 4634 in submerged fermentation with 5.0% palm extract, 2.0% inner bark bran of cassava and 1.0% corn steep liquor.

Table 1 .
Waste concentrations used for the production of submerged fermentation systems.

Table 2 .
Specific activity (U/mg) of the enzymes produced by A. tamarii URM4634 and A. japonicus URM5633.

Table 3 .
Residual activity of enzymes produced by A. tamarii URM 4634 under in vitro simulation of gastrointestinal digestion of monogastric animals.