Two-stage in vitro digestibility assay , a tool for formulating non-starch polysaccharide degrading enzyme combinations for commonly used feed ingredients of poultry rations

Aim: An attempt was made to assess the effect of pure enzyme combinations with the objective of formulating customized enzyme mixtures based on sugar release when subjected to two-stage in vitro digestion assay. Materials and Methods: A two-stage in vitro digestibility assay was carried out for commonly used feed ingredients for poultry viz., maize, soy bean meal, sunflower cake, and de-oiled rice bran supplemented with three concentrations of xylanase (5000; 7500 and 10000 IU/kg), cellulase (50; 100 and 400 IU/kg) and â-D-glucanase (100; 200 and 400 IU/kg) were used to formulate various NSP enzymes combinations. In total 27 NSP enzyme combinations (3x3x3) were formulated and the sugar released due to NSP digestion was quantified by phenol sulphuric acid method. Results: The total sugar release was significantly (P<0.05) higher with supplementation of various enzymes combinations for maize, sunflower cake and de-oiled rice bran where as no significant (P<0.05) interaction of various NSP enzymes combinations was observed for soy bean meal. The NSP digestibility was highest in combination (xylanase-5000, cellulase-50 and â-D-glucanase-400 IU/kg), (xylanase-10000, cellulase-50 and â-D-glucanase-200 IU/kg) and (xylanase-7500, cellulase100 and â-D-glucanase-100 IU/kg) for maize, sunflower cake and de-oiled rice bran respectively. In case of sunflower cake, significant (P<0.01) three way interaction was observed among the xylanase, cellulose, and â-D-glucanase enzymes and the two-way interactions between the enzymes were also significant (P<0.01). Conclusion: It is concluded that 'n' number of non-starch Polysaccharide enzymes combinations can be screened for their efficiency to digest non-starch Polysaccharides present in various feed ingredients commonly used in poultry rations by employing two-stage in vitro digestibility assay as a tool.


Introduction
encapsulating nutrients and/or depressing overall nutrient digestibility through gastro-intestinal Polysaccharides are major components of plant modifica-tions.Hence, supplementation of non-starch materials used in rations for poultry.They are polysaccharide degrading enzymes will enhance the macromolecular polymers of monosaccharides linked digestibility of the NSP and utilization of these by glycosidic bonds [1].The Non-starch components to the desired level in the chicken.Polysaccharides are principally non á glucon An attempt was made to assess the effect of pure polysaccharides of the plant cell wall.They are enzyme combinations on commonly used poultry feed heterogenous group of polysaccharides with varying ingredients with the objective of formulating degree of water solubility, size and, structure [2].Noncustomized enzyme mixtures based on sugar release starch polysaccharides (NSP) include celluloses, (The addition of selected carbohydrases will break hemicelluloses, pectines, and oligosaccharides (ádown NSP, releasing nutrients (energy and protein), as galactosides, etc.).They can also be divided into waterwell as reducing the viscosity of the contents) when soluble and water-insoluble fractions; fr which have subjected to two-stage in vitro digestion assay.greater relevance to their nutritional values.Birds do not possess endogenous enzymes capable of cleaving

Materials and Methods
and digesting the â á linked NSP.The water-insoluble The NSP enzymes investigated in present study NSP can be considered practically undigested by were cellulase, xylanase, and â-D-glucanase.These poultry and only soluble NSP has the potential to be pure enzymes were procured from Advanced Biodigested by birds [3].However, soluble NSP are known Agrotech Limited, Pune, India.The activity of to posses anti-nutritional properties by either cellulase, xylanase, â-D-glucanase and Protease were 1000000, 1200000, 1600000 and 100000 IU/g, respectively.
The in vitro digestibility studies were undertaken for the commonly used feed ingredients for poultry (Table -2).Significant (P<0.05)interaction was viz., maize, soybean meal, sunflower cake and de-oiled observed for xylanase, cellulase and â-D-glucanase.rice bran supplemented with various NSP enzymes At 10000 IU/kg xylanase concentration, the NSP oncentrations (xylanase, cellulase and â-D-glucanase) digestibility was higher at all combinations containing and was assessed by two stage in vitro digestion assay cellulase and â-D-glucanase (19-27 combinations) of [4] and the total sugars released from two stage in compared to control and the sugars release was vitro digestion was estimated.The total sugar released comparable among all combinations containing 10000 from in vitro digestion was assessed as per the IU/kg xylanase (170.01 -207.66 mg/g).At 7500 IU/kg procedure described by, [5].Based on the available xylanase combinations the sugars release was higher in literature various enzyme concentrations were selected all combinations except in combination containing 400 to formulate different NSP enzymes combinations for IU/kg â-D-glucanase.At 5000 IU/kg xylanase commonly used feed ingredients (Table -1).
supplementation the NSP digestibility was highest in combination 3 (xylanase-5000, cellulase-50 and â-D- between xylanase and â-D-glucanase, xylanase and cellulase and â-D-glucanase, twenty seven different cellulase and cellulase and â-D-glucanase were also combinations were formulated.These enzyme not significant (Table -2).The in vitro sugars release in combinations were supplemented to each ingredient control was 116.9 mg/g and increased in various enzyme and subjected to two stage in vitro NSP digestibility supplementation groups which, ranged between 162.9 and total sugars released were measured.
-191.2 mg/g.The in vitro NSP digestion increased Two-stage in vitro digestion assay: About 0.1g of significantly (P<0.05) with xylanase supplementation, ground samples containing different enzyme irrespective of cellulase and â-D-glucanase concencombinations in triplicate were incubated with 3 ml of trations, but dose of xylanase did not show any 0.1 N HCl containing 2000 IU pepsin/ml at 40°C for 45 difference.minutes to simulate the peptic / gastric phase.To the Sunflower cake: The sugars release from sunflower same tubes after 45 minutes, 1 ml of 1 M NaHCO 3 cake with two stage in vitro digestion was 133.20 mg/g containing 2 mg pancreatin/ml were added and in control and was significantly (P<0.01)increased in incubated for 2 hours at 40°C to simulate the enzyme supplemented groups (155.6-175.9mg/g) pancreatic/intestinal phase.At the end, contents were irrespective of the combinations (Table 2).Significant centrifuged and the supernatant was stored in ice for (P<0.01)three way interaction was observed among total sugar estimation.
[ 353 U/g of enzyme) and control.
[9] reported that In vitro studies conducted by [15] revealed higher xylanase and cellulase combination was superior for dose requirement of xylanase (60000 and 240000 sunflower meal than xylanase, cellulase and pectinase IU/kg substrate) along with pectinase and glucanase combination.for barley and rye.The enzyme dose used was higher The sugars release from sunflower cake without than that used for maize, used in the present study and any enzyme supplementation in the present study was as reported by [10] which might be due to higher NSP 133.2 mg/g and was lower than the value reported by content in barley and rye compared to maize.Similarly [10] (Table -2).NSP enzyme supplementation to in-vitro studies with multiple enzyme addition (high, sunflower cake resulted in increased (P<0.01)total medium and low) to extruded and non-extruded corn sugars release compared to non supplemented group.based diets for pigs improved nutritional value in terms The NSP enzymes supplemented to sunflower cake at of coefficient of apparent digestibility (CAD), starch various doses and combination, the in vitro sugars and NDF [16].Further [17] also reported that reconsrelease was significantly (P<0.01)higher than control.titution of maize with added enzymes improved pepsin Among various enzyme combinations comprising of pancreatic digestibility (IVPPD) 20% over control.
The in vitro sugars release from soybean meal in Similarly[23]reported significant increase in the the present study was 116.9 mg/g and ranged between release of glucose, other reducing sugars and in organic 163.43-190.5 mg/g, when supplemented with various phosphorous due to addition of enzyme mixtures to enzyme combinations (1-27) (Table-2).No significant sunflower meal.[10] also reported improved sugars (P<0.05)effect was observed between control and release in enzyme supplemented group.The influence enzyme supplementation for soybean meal (Table-2).ofcellulase was clearly seen in the present study and Though insignificant (39.35-63.55%)higher sugars was in accordance with the findings of the [9,10].release was observed with supplementation of xylanase (5000 to 10000 IU/kg substrate), irrespective De-oiled rice bran (DORB):