MATERIALS AND METHODS

This work was conducted in dairy department laboratories-National Research Center, Egypt, from 15 March, 2018 to 10 July, 2018 in two stages as follow:

Pectinase production optimization
Fungus, inoculum preparation and cultivation conditions: The submerged cultivation technique was used for pectinase production on static cultures in the current study. Aspergillus terreus (obtained from dairy department, National Research Center, Giza, Egypt) was cultivated and maintained on PDA medium. Beet pulp powder medium (BPPM) was used for growth and pectinase production⁶. Effect of inoculum size ranged from 1-8% (v/v), incubation period from one to seven days, initial pH of growth medium in a range between 3 and 8 and different nitrogen source (ex: ammonium sulphate, ammonium chloride peptone, yeast extract and urea) on pectinase production was studied. The medium of each flask was filtered and collected for pectinase activity assay according to method of Buga et al.¹⁴.

Electron microscope scanning: Orange peel fibers were dried and then treated with laboratory produced pectinase (40 U pectinase/1 g DM of orange peel fibers/100 mL buffer acetate at pH 6.5) for 24 h at 40°C and 100 rpm in a rotary shaker as described by Azzaz et al.⁶. The enzymatically untreated orange peel fibers (control) were kept in flask at the same condition. Finally, scanning electron micrographs of both treated and untreated fibers were taken to observe the effect of enzyme treatment.

In vitro study: Two in vitro experiments were conducted to evaluate impact of the laboratory pectinase (produced from Aspergillus terreus, each kg of it contains 253340 unit) compared with Polyzyme^TM (A commercial pectinase manufactured by Zeus Biotech Limited -India, each kg of it contains 240000 unit) on rumen fermentation characteristics. According to Ismail et al.¹⁵, a 500 mg sample of the formulated dairy animal’s diet (its feed ingredients and chemical composition was shown in Table 1 was weighed into 120 mL serum bottles. The bottles (3 replicates) were separately supplemented with rumen liquor, buffer solution and Polyzyme^TM and the produced pectinase solutions at different levels (0, 200, 400, 600 and 800 IU kg^–1 DM of the dite). Rumen liquor was collected from the rumen of slaughtered rams then moved directly to the laboratory in separate warmed oxygen-free plastic jars. The obtained liquor was mixed with the buffer solution at 39°C under carbon dioxide continuous flushing¹⁶. The bottles were sealed and maintained at 39°C in a shaking water bath (20 oscillations min^–1) for 24 h. After 24 h of incubation the pH value, total gas production volume, NH₃ and total volatile fatty acids concentrations were determined¹⁷.

Table 1:	Chemical composition of the control diet and their ingredients
DM: Dry matter, OM: Organic matter, NDF: Neutral detergent fiber, ADF: Acid detergent fiber, CP: Crude protein, NFC: Non-fiber carbohydrate, EE: Ether extract, Concentrate: Roughage ratio of the control dite was 1:1 on DM basis, concentrate feed mixture (CFM) consisted of 30% corn grains, 30% sugar beet pulp, 22.6% soybean meal, 15% wheat bran,1% limestone, 0.4% minerals and 1% NaCl, roughage part of the diet consisted of 50% berseem hay and 50% wheat straw

The in vitro dry matter (DM) degradability was determined according to the AOAC¹⁸ methods, while neutral detergent fiber (NDF) and acid detergent fiber (ADF) degradability were determined according to Van Soest et al.¹⁹ methods.

Statistical analysis: Data obtained from this study were statistically analyzed by IBM SPSS Statistics for Windows²⁰ using one way ANOVA procedure to compare means. The significance among means were tested through Duncan's multiple range tests with probability level (<%5)²¹.

RESULTS

Effect of initial pH of fungal growth medium on pectinase production: As shown in Fig. 1, pectinase production by Aspergillus terreus grown on different pHs of beet pulp powder medium (BPPM) showed its highest values (20.36 μmole mL^–1 min^–1) at pH 4.0. More over when the pH level increased, the enzyme production decreased. Based on the obtained results, the initial pH of the medium was adjusted to pH 4.0 in subsequent experiments on modified BPPM.

Effect of nitrogen sources on pectinase production: Data of Fig. 2 showed that among five nitrogen sources tested for screening their effect on pectinase production, Ammonium sulfate was found to be the best nitrogen source producing the highest level of pectinase activity (21.96 μmole mL^–1 min^–1) by Aspergillus terreus. These data indicating that the source of nitrogen should be inorganic for the best level of pectinase production.

Effect of incubation period on pectinase production: Production of pectinase on modified BPPM was monitored for a period of seven days (Fig. 3). The highest pectinase activity (23.81 μmole mL^–1 min^–1) was recorded after 4 days of incubation with Aspergillus terreus.

Fig. 1:	Effect of initial pH of fungal growth medium on pectinase production

In addition, when the incubation period increased, the enzyme production decreased.

Effect of inoculum size on pectinase production: Aspergillus terreus has exhibited different responses to variations in inoculum size ranged from 1-8% (v/v). The results of Fig. 4 showed that maximum pectinase activity (26.53 μmole mL^–1 min^–1) was obtained at 7% inoculum size and further increase in the inoculum size led to decrease of pectinase activity.

Scanning electron micrographs for orange peel fibers: It was evident from the photographs (Fig. 5a, b) that the orange peel fibers were separated and the peel surface became softer after pectinase treatment as a result of pectin hydrolysis.

In vitro study: All levels of the produced pectinase and Polyzyme^TM addition increased (p<0.05) ruminal DM, NDF and ADF degradability for the treated diets compared with the control one, which gave the lowest values of diet degradability parameters (Table 2). The maximum produced pectinase efficiency (%) for DM degradability was obtained at 600 IU kg^–1 DM reached 11.44% over the control.

Table 2:	Pectinases effects on degradability parameters of experimental diets
DM: Dry matter degradability, NDF: Neutral detergent fiber degradability, ADF: Acid detergent fiber degradability, Enzyme efficiency (%): DM (%) (treated diet)-DM (%) (control diet)/DM (%) (control diet)×100, a-dMeans at the same column with different superscript are significantly (p<0.05) different

Fig. 2:	Effect of nitrogen sources on pectinase production

Fig. 3:	Effect of incubation period on pectinase production

Fig. 4:	Effect of inoculum size on pectinase production

Also, the Polyzyme^TM takes the same trend and its efficiency (%) for dry matter degradability reached 7.82% over the control: It’s obvious that the produced pectinase is superior over Polyzyme^TM for diet degradability improvement. On the other hand, there are no marked changes in the ruminal NH₃ concentration and the total gas production (TGP) after control diet treatment with the produced pectinase or Polyzyme^TM (Table 3). The total volatile fatty acids (VFA) concentration show significant increase after diet treatment with the produced and commercial pectinases (Table 3). In contrary, the ruminal pH decreased significantly after control diet supplementation with the produced and commercial pectinases.

DISCUSSION

Medium initial pH has profound effect on the microbial growth, cell osmotic pressure, nutrient uptake and enzymes production and secretion²². The obtained data were in good agreement with many of previous studies that concluded that optimum pectinase activity has been given from different fungus within the acidic pH range^23-24. While other studies stated that pH ranged from 6.5-7 is optimum for pectinase production by A. niger and P. chrysogenum^6,25. The reduction of pectinase enzyme activity after increasing medium pH level is may be due to occurring partial or irreversible denaturation in the enzyme protein²³.

Impact of nitrogen sources on pectinase activity were extensively studied by Murad and Azzaz¹ and Azzaz et al.⁶. The obtained data of current study was in line with those of El Enshasy et al.³ who found that ammonium sulfate was the optimum source of nitrogen for pectinase production. While other studies revealed that organic nitrogen sources was the best for pectinase production^6,26.

Fig. 5(a-b):	Scanning electron micrographs for orange peel fibers were taken (a) Before and (b) After treatment with crude pectinase

Table 3:	Dietary pectinases supplementation effects on ruminal parameters (in vitro)
TGP: Total gas production, TVFA: Total volatile fatty acids, a-dMeans at the same column with different superscript are significantly (p<0.05) different

The conflict between data of nitrogen sources impact on pectinase activity in the different studies mainly attributed to vary in microbial strain and/or the cultivation conditions.

The fermentation time has a profound effect on enzymes formation in molds²⁷. The current data supported the findings of Ghildyal et al.²⁸ who reported that, maximum production of pectic enzyme from different moulds varies from 1-6 days. In contrast, Rangarajan et al.²⁶ reported that maximum pectinase activity of A. niger has been obtained after 40 h of fermentation. This variance in the optimal incubation period is depending on the growth rate of the microorganism and its enzyme production pattern²⁹. The reduction of pectinase activity with increasing inoculum size above 7% is might be due to clumping of cells which may lead to reduced sugar and oxygen uptake rate and subsequently enzyme release³⁰.

The obvious softness of the orange peel fibers after its treatment with the produced pectinase is supported the previous findings of Azzaz et al.⁶ who observed separation of banana leaves fiber's after their treatment with pectinase. According to Jacob et al.³¹, pectin is the main component of the middle lamella of higher plant tissues. The pectinase treatment may weaken the middle lamella and let for more hydrolysis for orange peel fibers.

The improvement of dietary DM, NDF and ADF degradability after pectinases addition is may be due to separation of middle lamella fibers of feed particles (see electron micrographs). The current data have been supported by results of many in vitro studies that revealed enhancement of dietary degradability parameters after its treatment with fibrolytic enzymes^4,5,11,16-17. Mao et al.³² attributed the improvement in the fermentation pattern for diets treated with fibrolytic enzymes to increase numbers of total bacteria especially Fibrobacter succinogenes in the incubation medium.

Increase TVFA production in the ruminal incubation medium was attributed to increase extent of dietary fiber hydrolysis after pectinases addition which drives the pH toward acidity. It is known that there is an inverse relationship between the production of volatile fatty acids in the rumen and ruminal pH³³. In addition, the concentrate portion of the control diet contains highly fermented carbohydrates combined with low degradable protein (Zein)¹², which may give another reason for increase concentration of TVFA with no change in NH₃ concentration in the incubation medium. The current results suggested that production of propionate was increased while; butyrate and acetate production was decreased. Propionate acts as alternative hydrogen sink in the rumen diverting hydrogen away from the reduction of CO₂ to CH₄, while the production of butyrate and acetate promotes methanogenesis³⁴. This may give an explanation for why the ruminal TVFA production increased with no change in the volume of TGP production.

Finally, it was obvious from this study that the newly produced pectinase show superiority over the commercial pectinase for improving rumen environment and degradability of dairy animal's diets. Higher cost of production is the major constraint for commercialization of new sources of enzymes. In current study, pectinase was produced economically on cheap components, mainly beet pulp as a carbon sources. Concerning with the economics of pectinase production, the fermentation technique (submerged or solid state) may has potent impact on enzyme yield and cost of production. Therefore, it is recommended to apply solid state fermentation for pectinase production by Aspergillus terreus for data comparison.

CONCLUSION

It could be concluded that pectinase production under the optimum cultivation conditions using beet pulp as the substrate (carbon source) will provide animal breeders with highly effective feed additive product with low cost. The laboratory produced pectinase showed superiority over the commercial enzyme for improvement of feeding value of dairy animal’s diets rich in pectin. Utilization of pectin rich agro-residues with locally produced pectinase in dairy animal feeding, may help in overcome of the feed gap beside the good impact on economy and environment.

SIGNIFICANCE STATEMENT

This study discovers one of economic ways for pectinase production by Aspergillus terreus under the optimum fermentation conditions using beet pulp as cheap substrate. The newly produced pectinase may give feed factories highly effective product with low cost. Application of the newly produced pectinase in enrichment of the feeding value of dairy animal’s diets can be beneficial for animal’s breeders who suffering high prices of traditional feed stuff. This study will help the researchers to uncover the critical areas of using of biotechnology for enrichment of the feeding value of the agricultural residues and their impact on environment and public health.