Patent Application: US-22169594-A

Abstract:
an enzyme composition having a synergetic phytate hydrolyzing activity comprising a phytase having phytate hydrolyzing activity at a ph of from 2 . 5 to 5 . 0 and an acid phosphatase having phytate hydrolyzing activity at a ph of 2 . 5 , in a low ratio corresponding to a ph 2 . 5 / 5 . 0 activity profile of from 0 . 8 / 1 . 0 to 3 / 1 . said enzyme composition preferably displays a higher synergetic phytate hydrolyzing efficiency through thermal treatment . fungal enzymes , especially those from aspergillus , are preferred . use of said enzyme composition in food , feed and fodder products to improve phytate hydrolysis .

Description:
the present invention describes an improved enzymic degradation of plant phytin by an appropriate acid phosphatase / phytase mixture due to a synergetic interaction between both enzymes . in nature , phytin acts as the primary storage form of both phosphorus and inositol in many plant seeds and cereals . during sprouting , phosphorus is liberated from phytin by action of the phytase , present in those seeds and cereals . as those plant raw materials are used in the food and feed industry , phytin is a potential phosphorus source for man and animal . however , bioavailability of this phytin is limited for monogastrics : plant phytases are inactivated in the gastro - intestinal tract by stomach acids , by which their in vivo efficiency is low . therefore , processes can be developed to improve bioavailability of phytin phosphorus and hence bioavailability and absorption of essential dietary minerals . a . enzymic pretreatment of plant raw materials to increase digestible phosphorus ; whereas plant phytases can play an important role in the pretreatment of plant raw materials , feeds and food ( neutral ph ), they are of lower importance to the in vivo act ion ( stomach ph ( 2 )). in vivo phytin hydrolysis can be achieved better by microbial , more specific fungal phytases , which can develop a high activity at the acid stomach ph , and have a high ph - stability . phytin hydrolysing enzymes are produced by fungi belonging to the aspergillus , mucor , rhizopus , botrytis ,. . . genus . these fungi produce a mixture of acid phosphatases ( e . c . 3 . 1 . 3 . 2 ) and phytases ( e . c . 3 . 1 . 3 . 8 ), both hydrolysing phytin , but with a different specificity to phytate and phosphate - monoesters . however , most of these organisms have a low enzyme production level . as the production level of these enzymes is of utmost importance to render the process economic , aspergillus ficuum nrrl 3135 was selected as a highly productive strain . alternative fungi are aspergillus niger and aspergillus terreus , with a lower production level . although both fungal acid phosphatases and fungal phytases can hydrolyse dissolved purified phytin , acid phosphatases show little activity on the phytin in plant raw material , feed or food , both in vitro and in vivo . they were therefore , until recently , neglected as phytin hydrolysing enzymes . however , despite the low phytin hydrolysing activity of acid phosphatases when used as sole enzyme source , phytase action on phytin can be improved by supplementation with acid phosphatase , due to a synergetic interaction between both enzymes , dosed at fixed ratios of acid phosphatase / phytase activity . the synergetic action is present at an appropriate composition and low ratio of both enzymes . besides ph - stability , thermostability of the phytin hydrolysing enzymes is an important factor as to food and feed production processes . as the current compound feed production process often relies on pelletted feed , enzymes that are added to the feed prior to pelleting should withstand the high temperatures achieved in the feed mill ( 75 °- 80 ° c .) to assure a predictable phytin hydrolysis . a . ficuum phytase is partly denaturated at these temperatures , by which an overdosis of 30 % is necessary . on the contrary , a . ficuum acid phosphatase is more stable , by which denaturation is less , and an acid phosphatase / phytase mixture is more stable to the feed pelleting process than phytase itself . thus by using a mixture of acid phosphatase and phytase instead of phytase as sole enzyme , plant phytin hydrolysis is improved , not solely as a result of a higher thermostability of this enzyme mixture , but mainly as a result of an improved synergetic interaction between both enzymes as the ratio ph 2 . 5 / 5 . 0 phytate hydrolyzing activity will increase by the different thermal degradation of both enzymes . fungal a . ficuum or a . niger acid phosphatase ( e . c . 3 . 1 . 3 . 2 ) and a . ficuum phytase ( e . c . 3 . 1 . 3 . 8 ) are both able to hydrolyse dodecasodium phytate in liquid , but their main activity is different : phytase has a main activity on phytate ( 1 ) and a side activity on mono - phosphate esters ( 2 ); acid phosphatase has a main activity on mono - phosphate esters and a side activity on phytate ( example i ). the ratio ( 1 )/( 2 ) of aspergillus ficuum phytase activity on phytate and disodium - β - glycerophosphate amounts to 6 . 6 , while a . ficuum acid phosphatase ratio ( 1 )/( 2 ) amounts to 0 . 13 , indicating the high specificity of phytase to phytate . for aspergillus niger acid phosphatase the ratio ( 1 )/( 2 ) amounts to 0 . 17 . as employed furtheron , acid phosphatase activity always refers to phytate hydrolysis . the acid phosphatase has one optimum activity at ph 2 . 3 , while phytase has an optimum activity at ph 5 . 0 , with a second activity peak at 2 . 5 . enzyme mixtures of phytase and phosphatase are furtheron characterized by the ratio ( a / p ) of their activity at ph 2 . 5 ( a ) and ph 5 ( p ) on phytate . pure phytase a / p ratio is found to be approximately 0 . 6 / 1 and pure acid phosphatase a / p ratio is found to be 1 / 0 . a . ficuum nrrl 3135 and a . niger phytase and acid phosphatase solutions were obtained by methods known in the art . an in vitro phytase test was set up on standard pig feed , low humidity level ( 70 %), ph 2 5 and 40 ° c ., 3 hours incubation , to determine the active component in phytase preparations ( figures ). a . ficuum nrrl 3135 phytase preparations with different a / p ratio were dosed to pig feed , either based on ph 2 . 5 activity ( ua ) or based on ph 5 activity ( up ) on phytate . there existed no direct correlation between phytase ph 2 . 5 activity and phytin hydrolysis , although the degradation took place at ph 2 . 5 , and the acid phosphatase was capable of hydrolysing dissolved sodium phytate in liquid ( fig1 ). on the other hand , phytin hydrolysis could be correlated to phytase ph 5 . 0 activity , especially when phytase preparations were separated in preparations with a / p ratio & lt ; 1 . 5 / 1 and preparations with a / p ratio between 1 . 5 / 1 and 3 / 1 ( fig2 ). from this it could be concluded that the acid phosphatase activity on the feed phytin was not dominant . however , a variation between the different phytase preparations , dosed at the same ph 5 . 0 activity , was still detected . this variation was found to correspond with a different acid phosphatase activity in the phytase preparations ( example ii ): as the a / p ratio increased from 0 . 6 / 1 to 3 / 1 , and consequently the acid phosphatase amount increased , phytin hydrolysis in pig feed was favoured and the efficiency ( amount of phytin phosphorus released by the phytase preparation ( ph 5 . 0 activity )-( g pp / 500 up ) of the phytase mixture increased ( fig3 ) . the importance of a . ficuum acid phosphatase 1 / 0 in a phytase preparation with a / p & gt ; 0 . 6 / 1 was proven by in vitro hydrolysis of pig feed phytin by phytase preparations 0 . 6 / 1 and 1 / 1 , with and without supplementation of acid phosphatase 1 / 0 to an a / p ratio = 2 / 1 ( example iii ). moreover a synergetic effect between the phytase and acid phosphatase was observed . dosing the same phytase 0 . 6 / 1 activity ( up ) to the pig feed , supplemented with acid phosphatase to an a / p ratio 2 / 1 , phytase efficiency increased from 0 . 55 to 1 . 25 g pp / 500 up , with a synergetic effect of 0 . 49 g pp / 500 up . the synergetic effect between a . ficuum phytase and acid phosphatase on the pig feed was maximal at an a / p ratio of 1 . 5 - 2 / 1 and displayed a decreasing effect when a / p increased to 3 / 1 . above this ratio , no additional synergetic effect can be detected ( fig3 ). feeds differ from each other in feedstuff composition and might , as such , influence phytase action , as well as synergetic interaction between phytase and acid phosphatase preparations . different swine and poultry feeds , such as piglet , sow , pig , broiler and layer feeds were incubated in vitro with phytase preparations a / p 1 . 6 / 1 , 0 . 6 / 1 and a mixture of phytase 0 . 6 / 1 and acid phosphatase 1 / 0 to a / p 2 / 1 . all phytase and acid phosphatase efficiencies differed strongly for the different feeds ( f . e . phytase 0 . 6 / 1 : 0 . 25 -& gt ; 1 . 5 g pp / 500 up ; acid phosphatase : 0 . 025 -& gt ; 0 . 15 g pp / 500 ua ), indicating that the occurrence of phytin in the different plant raw materials is different , and influences its enzymic hydrolysis . the synergetic effect of a . ficuum acid phosphatase , detected in standard pig feed , was transferable to the other feeds , but differed strongly for the different feeds ( 0 . 25 -& gt ; 0 . 94 g pp / 500 up ). the influence of phytin origin on the synergetic effect between phytase and acid phosphatase , as supposed above , was tested by the in vitro phytin degradation of different plant raw materials which are frequently found in animal compound feeds : peas , wheat bran , soya beans and rice bran ( example v ). a . ficuum phytase 0 . 6 / 1 was supplemented with a . ficuum acid phosphatase 1 / 0 to an a / p ratio = 2 / 1 . a . ficuum phytase ( 0 . 6 / 1 and 2 / 1 ), as well as acid phosphatase ( 1 / 0 ) efficiencies were strongly variable . although the acid phosphatase had a very low efficiency on the standard pig feed ( 0 . 15 g pp / 500 ua ), some plant phytin , like rice bran phytin , could easily be hydrolysed by this acid phosphatase ( 0 . 8 g pp / 500 ua ). other plant phytin , like wheat bran and soya bean meal phytin , was hardly hydrolysed by the a . ficuum acid phosphatase ( 0 . 2 g pp / 500 ua ). the synergetic effect between the a . ficuum phytase and acid phosphatase , noticed in feed , was only found in those plant raw materials in which the acid phosphatase itself had a low efficiency ( 0 . 2 g pp / 500 ua ): synergy in soya bean meal : 0 . 57 g pp / 500 up ; synergy in wheat bran : 0 . 47 g pp / 500 up . the synergetic effect between the phytase and acid phosphatase was low , when the phytin was already hydrolysed by the acid phosphatase itself : synergy rice bran : 0 . 13 g pp / 500 up ). pea phytin was hardly hydrolysed by both enzymes , and a synergetic interaction could not be detected . consequently , the interaction between a . ficuum acid phosphatase 1 / 0 and phytase 0 . 6 / 1 in feed can be explained as a result of the normal additive effect supplemented by different synergetic effects on the different plant raw materials which compose the feed . besides a . ficuum nrrl 3135 , phytase and acid phosphatase are also produced by aspergillus niger strains . as a . ficuum , a . niger produces a mixture of extracellular phytase and acid phosphatase . a . niger acid phosphatase 1 / 0 has an identical ph activity profile as a . ficuum acid phosphatase with a ph - optimum of 2 . 3 . following experiments proved the synergetic effect between phytases and acid phosphatases of different fungal origin . in vitro phytin hydrolysis in standard pig feed by liquid a . niger phytase preparations was identical to the hydrolysis with a . ficuum nrrl 3135 phytase preparations ( example vi ). the a . niger phytase efficiency increased from 0 . 75 to 1 . 15 g pp / 500 up as the a / p ratio of the phytase preparation increased from 0 . 9 / 1 to 1 . 6 / 1 . addition of acid phosphatase , either from a . niger or a . ficuum nrrl 3135 , to an a . niger phytase preparation a / p 1 . 15 / 1 to increase a / p ratio to respectively 1 . 9 / 1 and 1 . 6 / 1 , proved the synergetic interaction of these enzymes ( example vii ). the efficiency of both acid phosphatases was similar . when acid phosphatase was added to the a . niger phytase 1 . 15 / 1 , residual phytin phosphorus in the feed was zero , by which enzyme efficiency and synergy could not be calculated correctly , because of depletion of the phytate substrate . a synergetic interaction was also observed between a . niger acid phosphatase and a . ficuum nrrl 3135 phytase : efficiency was doubled ( 0 . 85 -& gt ; 1 . 8 g pp / 500 up ) when a . ficuum phytase 0 . 6 / 1 was supplemented with a . niger acid phosphatase 1 / 0 to a / p 1 . 9 / 1 , with a synergetic effect of 0 . 62 g pp / 500 up ( example viii ). the a . ficuum nrrl 3135 acid phosphatase 1 / 0 has the advantage of an enhanced thermostability in liquid conditions , and during feed pelleting , by which the synergetic effect between phytase 0 . 6 / 1 and acid phosphatase 1 / 0 in pelletted feed can be promoted . the denaturation of a liquid phytase 0 . 6 / 1 solution amounted to 60 %, for both ph 2 . 5 and ph 5 activity , after 1 min . at 80 ° c . and ph 5 ( 0 . 5m acetate buffer ), while an acid phosphatase 1 / 0 solution only lost 10 % of its activity under identical incubation conditions . thermostability under feed mill conditions was simulated . vials were filled with standard pig feed supplemented with a liquid phytase 0 . 6 / 1 or acid phosphatase 1 / 0 preparation . humidity of the mixture was 12 . 2 %. the vials were closed and heated at different temperatures ( 60 °- 90 ° c .) by immersion in a water bath ( example ix ). a . ficuum acid phosphatase 1 / 0 activity ( ph 2 . 5 ) still amounted to 100 % after 10 &# 39 ; heating at 70 ° c ., while a . ficuum phytase 0 . 6 / 1 had already lost 40 % of its activity ( ph 2 . 5 ). at 80 ° c ., acid phosphatase activity decreased to 65 % and phytase activity to 35 %. the higher thermostability of a . ficuum acid phosphatase 1 / 0 , already detected in liquid and feed , was furtheron detected in pilote and industrial mill . pig feed , supplemented with a . ficuum phytase 0 . 6 / 1 or acid phosphatase 1 / 0 liquid ( 3000 ua / kg feed ), was pelleted on pilote scale ( example x ) . temperature of the pellets was controlled by steam addition in the meal conditioner . phytase 0 . 6 / 1 lost on the average 55 % of its activity at a pellet temperature range of 68 . 6 °- 72 . 1 ° c ., while acid phosphatase 1 / 0 only lost on the average 25 % of its activity at 71 . 6 °- 73 . 1 ° c . ( ph 2 . 5 activity ) . an industrial pelleting experiment was set up with dried phytase preparations . phytase 0 . 6 / 1 and phytase mixture 1 . 25 / 1 were mixed in standard pig feed ( 900 up / kg feed ) and passed through an industrial feed mill ( example xi ). the efficiency of the phytase mixture 1 . 25 / 1 in the meal , before pelleting was 160 % of the phytase 0 . 6 / 1 efficiency : 0 . 95 & lt ;-& gt ; 0 . 6 g pp / 500 up . after pelleting , at approximately 75 ° c . ( pellet temperature ), the efficiency of the phytase 1 . 25 / 1 amounted to 190 % of the phytase 0 . 6 / 1 : 0 . 75 & lt ;-& gt ; 0 . 4 g pp / 500 up . this increase can be ascribed to a combination of a higher thermostability of the acid phosphatase 1 / 0 present in the phytase 1 . 25 / 1 preparation and the synergetic effect between both enzymes : the activity loss ( 33 %) of the phytase 0 . 6 / 1 component in the phytase 1 . 25 / 1 preparation , was compensated by the increase of the a / p ratio to 1 . 25 / 0 . 66 = 1 . 9 / 1 in the pelletted feed . the synergetic effect between a . ficuum nrrl 3135 phytase 0 . 6 / 1 and acid phosphatase 1 / 0 , noticed during in 5 vitro phytin hydrolysis and the higher thermostability of the acid phosphatase , might favour in vivo phytin hydrolysis in pelletted feed . in vivo digestion trials with pigs ( example xii and xiii ) were set up to determine the effect of a . ficuum acid phosphatase 1 / 0 , supplemented to a . ficuum phytase 0 . 6 / 1 . phytase or acid phosphatase action in vivo was measured through the faecal digestible phosphorus ( dp ), due to enzymic phytin hydrolysis . in vivo faecal dp is calculated as follows : total phosphorus intake -- total phosphorus excretion . an increase in dp is calculated by the difference of the in vivo dp and the dp calculated during feed formulation . in pig trial i ( example xii ) the dp increased with 0 . 78 g / kg feed by adding phytase 0 . 6 / 1 at 750 up / kg feed ; supplementation of the phytase with acid phosphatase 1 / 0 at 1050 ua / kg feed to increase a / p ratio to 2 / 1 increased dp to 0 . 89 g / kg feed , with a synergetic efficiency of 0 . 055 g dp / 500 up . acid phosphatase , solely added to the feed ( 1050 ua / kg feed ), had no influence at all on the dp level in the feed : dp remained unchanged after the test period . the efficiency of the phytase in this trial was low ( 0 . 52 g dp / 500 up ) because of its high dosage . by addition of acid phosphatase to the phytase , global efficiency increased with 13 % relative from 0 . 52 to 0 . 59 g dp / 500 up . the in vivo synergetic effect in dp for both phytase and phytase / acid phosphatase mixture was also translated in a decreased phosphorus excretion : addition of phytase ( 750 up / kg of feed ) reduced phosphorus excretion with 37 % while the phytase / acid phosphatase mixture reduced the phosphorus excretion with 41 %, compared to the control feed . this supplementary reduction of excretion ( 11 % relative ) can be reflected in a lower cost with respect to the phosphorus excretion legislation . in pig trial ii ( example xiii ), phytase 0 . 6 / 1 was dosed to the pig feed at a recommended level of 400 up / kg . the dp increased with 0 . 58 g / kg feed by addition of phytase , and with 0 . 62 g / kg feed by supplementing the phytase with acid phosphatase 1 / 0 ( 580 ua / kg ) to a / p ratio = 2 / 1 . lowering the phytase level , its efficiency increased to 0 . 725 g dp / 500 up . by addition of acid phosphatase , the overall efficiency increased with 24 % ( relative ) to 0 . 9 g dp / 500 up , with a synergetic effect of 0 . 125 g dp / 500 up . from both trial i and ii , it can be concluded that the in vivo synergetic effect between a . ficuum phytase and acid phosphatase is more pronounced at lower phytase and acid phosphatase level ( example xiv ). as the dosage of the phytase + acid phosphatase mixture decreased from 750 up + 1050 ua / kg to 400 up + 580 ua / kg , the efficiency increased from 0 . 59 to 0 . 9 g dp / 500 up . plant phytin was hydrolysed in vitro in standard pig feed with 0 . 33 % phytin phosphorus by different aspergillus ficuum phytase preparations with a / p ratio , varying between 1 / 0 and 16 / 1 . main components of the pig feed were tapioca , peas , maize gluten feed , wheat gluten feed and soya beans extracted . total phytin phosphorus of the feed was measured according to ellis and morris ( 1983 and 1986 ). a suitably diluted phytase preparation was added to 2 g of the feed , humidity of the mixture was adapted to 70 % by addition of 0 . 2m ph 2 . 5 sorensen - hcl buffer , and the mixture was incubated at 40 ° c . for 3 h . after incubation , the feed was extracted with 40 ml 2 . 4 % hcl ( 3 h ), and residual phytin phosphorus in the extract was measured after ion - exchange chromatography and phytin destruction . one phytase unit on phytate at ph 5 is abbreviated herein as 1 up , while one acid phosphatase or phytase unit on phytate at ph 2 . 5 is abbreviated as 1ua . fig1 gives the percentage residual phytin phosphorus after phytase action in function of the dosed ph 2 . 5 activity : dosing the same phytase ph 2 . 5 activity to the feed ( f . e . 0 . 5 - 1 ua / g ), residual phytin phosphorus ( 95 - 10 %) revealed no correlation with enzyme dosis , indicating that there is no correlation between phytase ph 2 . 5 activity and in vitro phytin hydrolysis in pig feed . fig2 gives the percentage residual phytin phosphorus after phytase action in function of the dosed ph 5 activity : dosing the same phytase ph 5 activity to the feed ( f . e . 0 . 4 - 0 . 5 up / g ), residual phytin phosphorus revealed a correlation with enzyme dosis . moreover , if phytase preparations with a / p between 1 . 5 / 1 and 3 / 1 , and a / p & lt ; 1 . 5 / 1 were separated , the difference in residual phytin phosphorus after phytase action at the same ph 5 dosage became smaller : phytase preparations with a / p & lt ; 1 . 5 hydrolysed phytin phosphorus less efficiently as phytase preparations with a / p between 1 . 5 / 1 and 3 / 1 . fig3 gives the in vitro phytin hydrolysis calculated as the amount of phytin phosphorus liberated by dosing 500 units phytase ( up ) to 1 kg of feed at 40 ° c . for 3 h ( g pp / 500 up ). the efficiency of the different phytase preparations was compared to the efficiency calculated as an additive effect between the two enzymes composing the phytase preparations , phytase 0 . 6 / 1 and acid phosphatase 1 / 0 , as in example iii . the synergetic effect between both enzymes was calculated as the difference between the test result and the calculated additive effect . an increasing synergetic effect can be detected with a maximum around a / p 2 / 1 . above this ratio , no additional synergetic effect can be observed . phytase and acid phosphatase ph 2 . 5 activity was assayed by measuring phoshorus release . 0 . 5 ml of a suitably diluted enzyme preparation was added to 2 ml of a 1 / 1 mixture of 0 . 2m ph 2 . 5 sorensenbuffer and 12 . 5 mm dodecasodium phytate or 25 mm disodium - β - glycerophosphate solution . the reaction mixture was incubated for 10 min at 40 ° c . the reaction was stopped by adding 2 . 5 ml of a 10 % trichloroacetic acid solution . liberated phosphorus was measured spectrofotometrically by adding 5 ml of a vanadate / molybdate reagent according to the official eec method . the ph 5 phytase activity on dodecasodium phytate was measured in a similar way , replacing the ph 2 . 5 sorensenbuffer by 1m ph 5 acetate buffer . one phytase or acid phosphatase activity unit was defined as the amount of enzyme that liberates 1 μmol of phosphorus / min at 40 ° c . and respectively ph 5 or ph 2 . 5 . the ratio ( 1 )/( 2 ) between the enzyme activity at ph 2 . 5 on phytate ( 1 ) and disodium - β - glycerophosphate ( 2 ) determines the specificity of both enzymes : phytase 0 . 6 / 1 develops highest activity on phytate , while the acid phosphatases develop highest activity on disodium - β - glycerophosphate . table 1______________________________________ ph 2 . 5 ratioenzyme ( 1 ) ( 2 ) ( 1 )/( 2 ) ______________________________________a . ficuum phytase 0 . 6 / 1 77 11 . 7 6 . 6a . ficuum acid phosphatase 1 / 0 490 3685 0 . 13a . niger acid phosphatase 1 / 0 8 . 7 52 0 . 17______________________________________ phytin was hydrolysed in vitro in a standard pig feed with 0 . 33 % phytin phosphorus by different a . ficuum phytase preparations . mean components of the pig feed were peas , tapioca , maize gluten feed , wheat gluten feed and soya beans extracted . total phytin phosphorus of the feed was measured according to ellis and morris ( 1983 , 1986 ). 1 . 4 units ( up ) of a liquid phytase preparation were added to 2 g of the feed , humidity of the mixture was adapted to 70 % by addition of 0 . 2m ph 2 . 5 sorensen - hcl buffer , and the mixture was incubated at 40 ° c . for 3 h . after incubation , the feed was extracted with 40 ml 2 . 4 % hcl ( 3 h ), and phytin phosphorus in the extract was measured after ion - exchange chromatography and phytin destruction . phytin hydrolysis was calculated as the difference in phytin content of the feed before and after treatment with phytase . in vitro phytase efficiency was calculated as the amount of phytin phosphorus ( g pp ) liberated by 500 units phytase ( up ) per kg feed at 40 ° c . for 3 h . as the a / p ratio of the phytase preparations at low a / p ratio ( 0 . 6 / 1 -& gt ; 2 / 1 ) increased , efficiency of the preparations displayed a linear increase : pure phytase 0 . 6 / 1 displayed an efficiency of 0 . 55 g pp / 500 up , while the efficiency of a phytase preparation with a / p ratio = 2 / 1 amounted to 1 . 75 g pp / 500 up . thus , the addition of 500 units ( up ) of phytase activity to 1 kg pig feed results in the liberation of an amount of phytin phosphorus varying between 0 . 55 and 1 . 75 g , depending on the a / p ratio of the phytase . at higher a / p ratio , efficiency increase ( synergy ) is levelled off . table 2______________________________________ a / p g pp / 500 up______________________________________ 0 . 6 0 . 55 0 . 8 0 . 85 1 . 0 1 . 00 1 . 4 1 . 20 1 . 6 1 . 30 1 . 7 1 . 60 2 . 1 1 . 75 3 . 0 1 . 80 16 . 3 3 . 5______________________________________ phytin was hydrolysed in vitro in standard pig feed ( example ii ) by a . ficuum phytase preparations , supplemented with a . ficuum acid phosphatase 1 / 0 to prove the importance of acid phosphatase in an acid phosphatase / phytase mixture . phytase 0 . 6 / 1 was supplemented with acid phosphatase to a / p = 2 / 1 and 3 / 1 by addition of respectively 2 and 3 . 4 ua of acid phosphatase to 1 . 4 up of phytase 0 . 6 / 1 ; phytase 1 / 1 ( 1 . 4 up ) was supplemented with 1 . 4 and 2 . 8 ua of acid phosphatase to a respective a / p ratio 2 / 1 and 3 / 1 . 1 . 4 up of a liquid phytase preparation ( a / p 0 . 6 / 1 , 1 / 1 , 2 / 1 and 3 / 1 ) was added to 2 g of the feed , humidity of the mixture was adapted to 70 % or 80 % by addition of 0 . 2m ph 2 . 5 sorensen - hcl buffer , and the mixture was incubated at 40 ° c . for 3 h . the efficiency of the acid phosphatase itself was determined by dosing 2 ua to 2 g of the pig feed and incubating the mixture as mentioned above . mixing phytase and acid phosphatase preparations to an a / p ratio 2 / 1 and 3 / 1 before addition to the feed , increased the efficiency of the enzyme mixture by both additive and synergetic interactions between both enzymes . for example : addition of 500 up phytase 0 . 6 / 1 to the pig feed liberates 0 . 95 g phyuin phosphorus , while the addition of 500 ua acid phosphatase 1 / 0 liberates 0 . 25 g phytin phosphorus . as 500 up phytase 0 . 6 / 1 have 300 ua ph 2 . 5 activity , 700 ua of acid phosphatase 1 / 0 are necessary to obtain an acid phosphatase / phytase mixture with a / p ratio = 2 / 1 . if both enzymes have a simple additive effect , efficiency can be calculated as follows : ( 500 up phytase * 0 . 95 g pp / 500 up )+( 700 ua acid phosphatase * 0 . 25 g pp / 500 ua )= 1 . 3 g pp (/ 500 up phytase ). the efficiency noticed in the assay is not 1 . 3 , but 2 . 1 g pp / 500 up , implicating a synergetic effect of 0 . 8 g pp / 500 up . table 3______________________________________ g pp / 500 ua / p test add . synergy______________________________________1 / 0 (*) 0 . 15 -- -- 1 / 0 ** (*) 0 . 25 -- -- 0 . 6 / 1 0 . 55 -- -- 0 . 6 / 1 → 2 / 1 1 . 25 0 . 75 0 . 500 . 6 / 1 ** 0 . 95 -- -- 0 . 6 / 1 → 2 / 1 ** 2 . 10 1 . 30 0 . 801 / 1 1 . 15 -- -- 1 / 1 → 2 / 1 1 . 50 1 . 30 0 . 201 / 1 → 3 / 1 1 . 75 1 . 45 0 . 30______________________________________ u : up (*): g pp / 500 ua **: incubation at 80 % humidity add . : calculated additive efficieny syn . : efficiency ( test ) efficiency ( add . ), i . e . the surplus synergetic efficiency phytin was hydrolysed in vitro in different standard feeds ( pig , piglet , sow , broiler and layer ) by different a . ficuum phytase preparations to investigate the synergetic interaction between phytase and acid phosphatase on feeds in general . piglet : barley , wheat , soya beans extracted , wheat bran , whey powder and fish meal sow : tapioca , peas , sunflower seed extracted , rice bran and coconut expeller pig 1 : tapioca , peas , soya beans extracted and wheat gluten feed pig 2 : tapioca , peas , soya beans extracted , wheat gluten feed and maize gluten feed phytase 0 . 6 / 1 was dosed at 0 . 7 up / g and acid phosphatase 1 / 0 at 1ua / g . phytase 0 . 6 / 1 was supplemented with acid phosphatase 1 / 0 to a / p 2 / 1 adding 1 ua acid phosphatase to 0 . 7 up phytase . incubation conditions were similar to example iii ( 80 % humidity ). synergetic effect was calculated as in example iii . both phytase and acid phosphatase efficiency differed strongly for the different feeds . the synergetic effect between both enzymes was found in all feeds , but differed for the different feeds . phytase a / p 1 . 6 / 1 was dosed at 0 . 7 up / g , to different feeds . the efficiency of this phytase confirmed the effect of the acid phosphatase 1 / 0 in an acid phosphatase / phytase mixture . table 4______________________________________g pp / 500 ua / p 0 . 6 / 1 → feed % pp 1 . 6 / 1 0 . 6 / 1 1 / 0 (*) 2 / 1 add . synergy______________________________________piglet 0 . 15 -- 0 . 55 0 . 025 & gt ; 1 . 0 0 . 58 & gt ; 0 . 42sow 0 . 39 1 . 75 1 . 5 0 . 15 2 . 6 1 . 71 0 . 89pig 1 0 . 23 1 . 4 0 . 75 0 . 125 & gt ; 1 . 5 0 . 93 0 . 57pig 2 0 . 32 -- 0 . 90 0 . 15 2 . 05 1 . 11 0 . 94broiler 0 . 15 -- 0 . 55 0 . 075 & gt ; 1 . 2 0 . 65 & gt ; 0 . 55layer 0 . 23 -- 0 . 25 0 . 075 0 . 6 0 . 35 0 . 25______________________________________ u : up (*): g pp / 500 ua & gt ;: phytin phosphorus content after incubation = 0 , by which only minimum phytase synergy can be calculated add . : additive efficiency as enzymic phytin hydrolysis differed strongly between different feeds ( example iv ), it could be expected that phytin hydrolysis depends on the plant raw materials composing the feed . the hydrolysis of plant phytin from different origin , and the efficiency of a . ficuum phytase 0 . 6 / 1 and acid phosphatase 1 / 0 , added solely or in combination , on different plant raw materials , was tested in vitro . phytase 0 . 6 / 1 was dosed at 0 . 35 - 0 . 7 up / g and acid phosphatase 1 / 0 at 1 ua / g feedstuff . phytase 0 . 6 / 1 was supplemented with acid phosphatase 1 / 0 to a / p 2 / 1 adding respectively 0 . 5 and 1 ua acid phosphatase / g . incubation conditions were similar to example ii ( 80 % humidity ). phytin hydrolysis was analysed and phytase efficiency was calculated as in example ii ; the synergetic effect was calculated as in example iii . phytase efficiency on peas was very low ( 0 . 15 g pp / 500 up ), with no synergetic interaction of the acid phosphatase . the greatest synergy was found in those plant raw materials in which phytin was easily hydrolysed by the phytase and hardly hydrolysed by the acid phosphatase , f . e . soya beans and wheat bran . a low synergy was found when the acid phosphatase itself already hydrolysed the plant phytin more efficiently , f . e . rice bran . table 5______________________________________g pp / 500 ua / p % feedstuff pp 0 . 6 / 1 1 / 0 (*) 0 . 6 / 1 → 2 / 1 add . synergy______________________________________peas 0 . 21 0 . 15 0 . 1 0 . 30 0 . 29 0 . 01soya beans 0 . 35 1 . 1 0 . 2 1 . 95 1 . 38 0 . 57wheat bran 0 . 73 1 . 6 0 . 2 2 . 35 1 . 88 0 . 47rice bran 1 . 33 1 . 8 0 . 8 3 . 05 2 . 92 0 . 13______________________________________ u : up (*): g pp / 500 ua add . : additive efficiency in vitro phytin hydrolysis in standard pig feed ( example ii ) by aspergillus niger phytase preparations with different a / p ratio . phytase was dosed at 0 . 7 or 1 up / g feed . incubation conditions were similar to example ii ( 70 % humidity ). phytin hydrolysis was analysed and phytase efficiency was calculated as in example ii . as the a / p ratio of the a . niger phytase preparations increased ( at low ratio ), efficiency of the preparations increased : phytase efficiency varies between 0 . 75 and 1 . 15 g pp / 500 up , depending on the a / p ratio of the phytase . table 6______________________________________ a / p g pp / 500 up______________________________________ 1 . 1 / 1 0 . 75 1 . 5 / 1 1 . 00 1 . 6 / 1 1 . 15______________________________________ in vitro phytin hydrolysis in standard pig feed ( example ii ) by aspergillus niger phytase preparation a / p 1 . 15 / 1 , supplemented with acid phosphatase from aspergillus niger ( an ) or aspergillus ficuum nrrl 3135 ( af ), to increase a / p ratio to respectively 1 . 9 / 1 and 1 . 6 / 1 . a . niger phytase 1 . 15 / 1 was dosed at 0 . 7 up / g feed ; 0 . 5 ua a . niger or 0 . 3 ua a . ficuum acid phosphatase / g were added to the a . niger phytase to increase a / p ratio to resp . 1 . 9 / 1 ( an ) and 1 . 6 / 1 ( af ). acid phosphatases were dosed solely to the feed at 0 . 5 ua a . niger or 0 . 3 ua a . ficuum / g . incubation conditions were similar to example ii ( 80 % humidity ). phytin hydrolysis was analysed and phytase efficiency was calculated as in example ii . the synergetic effect between a . niger phytase a / p 1 . 15 / 1 and acid phosphatases 1 / 0 , either from a . niger or a . ficuum , was calculated as in example iii . table 7______________________________________ g pp / 500 ua / p add . synergy______________________________________a . niger 1 / 0 (*) 0 . 25 -- -- a . ficuum 1 / 0 (*) 0 . 30 -- -- a . niger 1 . 15 / 1 1 . 25 -- -- 1 . 15 / 1 → 1 . 9 / 1 ( an ) & gt ; 1 . 65 1 . 44 & gt ; 0 . 211 . 15 / 1 → 1 . 6 / 1 ( af ) & gt ; 1 . 65 1 . 48 & gt ; 0 . 17______________________________________ u : up (*): g pp / 500 acid phosphatase ph 2 . 5 units & gt ;: phytin phosphorus content after incubation = 0 , by which only minimum phytase synergy can be calculated add . : additive efficiency in vitro phytin hydrolysis by a . ficuum phytase ( af ), supplemented with a . niger acid phosphatase ( an ), in standard pig feed . phytase 0 . 6 / 1 was dosed at 0 . 7 up / g ; the phytase was supplemented with 0 . 85 - 0 . 9 ua / g acid phosphatase 1 / 0 to obtain phytase preparations with respectively a / p ratio 1 . 8 / 1 and 1 . 9 / 1 . phytin hydrolysis was analysed and phytase efficiency was calculated as in example ii . the synergetic effect between a . ficuum phytase 0 . 6 / 1 and a . niger acid phosphatase 1 / 0 was calculated as in example iii . table 8______________________________________ g pp / 500 ua / p add . synergy______________________________________0 . 6 / 1 ( af ) 0 . 85 -- -- 1 / 0 ( an )(*) 0 . 25 -- -- 0 . 6 / 1 → 1 . 8 / 1 1 . 5 1 . 15 0 . 350 . 6 / 1 → 1 . 9 / 1 1 . 8 1 . 18 0 . 62______________________________________ u : up (*): g pp / 500 ua add . : additive efficiency in vitro thermostability of a . ficuum phytase 0 . 6 / 1 and acid phosphatase 1 / 0 was tested in standard pig feed ( example ii ) in closed vials , immersed in a water bath , simulating feed mill conditions . 20 g pig feed , containing 1750 ua phytase or acid phosphatase was mixed with 480 g pig feed , resulting in a feed with 3500 ua / kg . vials were filled with 7 g of the enzyme supplemented feed , containing 10 . 5 ua phytase or acid phosphatase . humidity of the feed was 12 . 2 %. the vials were closed and immersed in a waterbath at different temperatures ( 70 °- 90 ° c . ), during 10 minutes . residual acid phosphatase and phytase activity at ph 2 . 5 was measured after enzyme extraction : 3 g of the temperature treated feed was extracted with 50 ml 0 . 1m ph 2 . 5 sorensenbuffer during 30 minutes , and phytase or acid phosphatase activity in the feed extract was determined at ph 2 . 5 on dodecasodium phytate according to example i . the activity , measured in the non - treated feed , was set 100 %, and all activities were calculated as percentage remaining activity . table 9______________________________________ remaining activity (%) t (° c .) phytase 0 . 6 / 1 acid phosphatase 1 / 0______________________________________70 61 10080 33 6790 4 34______________________________________ pig feed was pelletted on pilote scale after addition of liquid a . ficuum phytase 0 . 6 / 1 or acid phosphatase 1 / 0 . both enzymes were dosed at 165000 ua / 550 g premix , which was then added to 55 kg pig feed before pelleting . main components of the pig feed were : peas , rape seed extracted , maize gluten feed and tapioca . temperature of the pellets , leaving the die of the mill , was controlled between 69 ° and 74 ° c . by addition of steam . phytase and acid phosphatase ph 2 . 5 activity of the meal and the pellets was measured after feed extraction : 5 g of the feed was extracted with 50 ml 0 . 1m ph 2 . 5 sorensen buffer during 30 minutes , and phytase or acid phosphatase activity was measured at ph 2 . 5 on dodecasodium phytate according to example i . activity in the meal feed ( 3 ua / g ) was set 100 %. the % humidity of the phytase and acid phosphatase feed decreased respectively from 11 . 6 and 11 . 9 in the meal conditioner to 10 . 6 and 10 . 8 in the cooled pellets . table 10______________________________________phytase acid phosphataset (° c .) remaining activity (%) t (° c .) remaining activity (%) ______________________________________68 . 6 50 71 . 6 7672 . 1 41 73 . 0 7774 . 2 35 73 . 1 67______________________________________ a . ficuum phytase 0 . 6 / 1 and phytase 1 . 25 / 1 preparations were supplemented to pig feed at 900 up / kg feed , followed by feed pelleting on industrial scale . main components of the feed were : peas , maize gluten feed , soya beans extracted and tapioca . phytin phosphorus concentration was 0 . 37 %, measured according to example ii . temperature of the pellets , leaving the die of the mill , was comparable ( 74 . 5 ° c ). phytase survival was measured by in vitro phytin hydrolysis in the pelleted feed : 2 g of the phytase feed was incubated as in example ii ( 80 % humidity ). phytin hydrolysis was analysed and phytase efficiency was calculated as in example ii . the phytase efficiency in the meal feed , before pelleting was set 100 %. phytase 1 . 25 / 1 efficiency in the pelleted feed is 190 % ( 0 . 75 ) of phytase 0 . 6 / 1 efficiency ( 0 . 4 ), whereas its efficiency in the meal feed is only 160 % ( 0 . 95 ) of the phytase 0 . 6 / 1 efficiency ( 0 . 6 ). table 11______________________________________phytase 0 . 6 / 1 phytase 1 . 25 / 1g pp / 500 up % g pp / 500 up % ______________________________________meal 0 . 6 100 0 . 95 100pellet74 . 4 ° c . 0 . 4 66 -- -- 74 . 6 ° c . -- -- 0 . 75 79______________________________________ control pig feed was formulated to 2 g / kg digestible phosphorus ( dp ) ( feed i ), and a total phosphorus content of 5 . 8 g / kg . feed i contained mainly peas , tapioca , maize gluten feed , wheat gluten feed and soya beans extracted . phytase - containing pig feeds were formulated to a total phosphorus content of 4 . 4 g / kg and 1 . 17 g / kg digestible phosphorus , containing the same main components as feed i . they were supplemented with different a . ficuum phytase preparations : 750 up phytase ( 0 . 6 / 1 )/ kg ( feed ii ), 1050 ua acid phosphatase ( 1 / 0 )/ kg ( feed iv ), and a mixture of 750 up phytase + 1050 ua acid phosphatase / kg ( feed iii ) to obtain an a / p ratio = 2 / 1 . concentrated enzyme solutions were added to the meal feed . twelve ( 12 ) pigs were kept in individual pens and were fed the feed for 17 days ( 7 d preliminary and 10 d test ) ( 3 pigs / feed ) with a daily feed intake of 1800 g , and a total intake of 18 kg during the test period . faeces were collected per pig during these 10 days , and total phosphorus excretion was determined on the collected faeces according to the eec method . the apparent phosphorus digestion coefficient ( dc - p (%)) was calculated by the difference between total phosphorus intake and total phosphorus excretion . table 12______________________________________tot - p in tot - p out dc - p dp ( g ) ( g ) (%) g / kg______________________________________feed i : 5 . 8 g p / kg - 2 g dp / kg104 . 4 70 . 9 ( 100 %) 32 . 1 1 . 86feed ii : 4 . 4 g p / kg - 1 . 17 g dp / kg + 750 up phytase ( ph 5 )/ kg79 . 2 44 . 2 (- 37 %) 44 . 3 1 . 95feed iii : feed ii + 1050 ua acid phosphatase ( ph 2 . 5 )/ kg79 . 2 41 . 7 (- 41 %) 47 . 3 2 . 06feed iv : 4 . 4 g p / kg - 1 . 17 g dp / kg + 1050 ua / kg79 . 2 58 . 1 (- 18 %) 26 . 7 1 . 17______________________________________ dc - p : digestion coefficient phosphorus dp : digestible phosphorus totp in : total phosphorus intake totp out : total phosphorus excretion control pig feed was formulated to 2 g / kg digestible phosphorus and 6 . 1 g / kg total phosphorus ( feed i ). feed i contained mainly peas , tapioca , wheat gluten feed and soya beans extracted ; phosphorus was partly supplied by monocalcium phosphate . phytase - containing feeds were formulated to 5 . 2 g / kg total phosphorus and 1 . 4 g / kg digestible phosphorus , containing the same main components as feed i , with omission of mono - calcium phosphate . they were supplemented with either phytase 0 . 6 / 1 , at a recommended level of 400 up / kg ( feed ii ), or a mixture of 580 ua acid phosphatase 1 / 0 + 400 up phytase 0 . 6 / 1 / kg to obtain an a / p ratio = 2 / 1 ( feed iii ). phytase solutions were added to the meal feed . twenty - four ( 24 ) pigs were kept in individual pens and were fed the feed for 17 days ( 7 d preliminary and 10 d test ) ( 8 pigs / feed ) with a daily intake of 1800 g , and a total intake of 18 kg during the test period . faeces were collected per pig during these 10 days , and the total phosphorus excretion was measured on the collected faeces according to the eec method . digestible phosporus ( dc - p % and g dp / kg ) were calculated as in example xii . table 13______________________________________tot - p in tot - p out dc - p dp ( g ) ( g ) (%) g / kg______________________________________feed i : 6 . 1 g p / kg - 2 g dp / kg109 . 8 73 . 4 ( 100 %) 32 . 9 2 . 01feed ii : 5 . 2 g p / kg - 1 . 4 g dp / kg - 400 up phytase / kg93 . 6 58 (- 21 %) 38 . 1 1 . 98feed iii : feed ii + 580 ua acid phosphatase / kg93 . 6 55 . 4 (- 24 . 5 %) 40 . 8 2 . 12______________________________________ dc - p : digestion coefficient phosphorus dp : digestible phosphorus totp in : total phosphorus intake totp out : total phosphorus excretion in vivo phytase efficiency ( g dp / 500 up ) of liquid a . ficuum phytase and acid phosphatase , dosed at different levels to pig feed ( meal ) ( example xii and xiii ). the in vivo phytase efficiency was defined as the amount of phosphorus released during digestion by 500 units ( up ) phytase ( 0 . 6 / 1 or 2 / 1 ) added to the feed ( g dp / 500 up ). the amount of phosphorus , liberated by the enzymes , is calculated as the difference between the formulated digestible phosphorus in the feed , and the digestible phosphorus , calculated from the animal trial as in example xii . as the in vivo efficiency of the acid phosphatase is 0 , the synergetic efficiency is calculated as the difference between the efficiency of the 2 / 1 acid phosphatase / phytase mixture , and the phytase 0 . 6 / 1 . table 14______________________________________ g dp / kgphytase / acid feed g dp / 500 upphosphatase dosis formu - animal enzyme syn -( units / kg ) lation trial effect ergy______________________________________750 up 1 . 17 1 . 95 0 . 78 0 . 52 -- 1050 ua (*) 1 . 17 1 . 17 0 . 00 0 . 00 -- 750 up + 1050 ua 1 . 17 2 . 06 0 . 89 0 . 59 0 . 07400 up 1 . 40 1 . 98 0 . 58 0 . 725 -- 400 up + 580 ua 1 . 40 2 . 12 0 . 72 0 . 9 0 . 175______________________________________ u : phytase ph 5 . 0 units ua : acid phosphatase ph 2 . 5 units (*): g dp / 500 ua e . p . 0 321 004 b1 ( 1988 ). a process for steeping cereals with a new enzyme preparation . e . p . 0 420 358 a1 ( 1990 ). cloning and expression of microbial phytase . borggreve , g . 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( 1972 ). chemical and physicochemical properties of phytase from aspergillus terreus . agr . biol . chem . 36 ( 12 ), 2097 - 2103 . youssef , k ., ghareib , m . & amp ; nour el dein , m . ( 1987 ). purification and general properties of extracellular phytase from aspergillus flavipes . zentralbl . mikrobiol . 142 , 397 - 402 . zyla , k . ( 1993 ). the role of acid phosphatase activity during enzymic dephosphorylation of phytates by aspergillus niger phytase . world j . microbiol . biotechnol . 9 , 117 - 119 . zyla , k . & amp ; koreleski , j . ( 1993 ). in vitro and in vivo dephosphorylation of rapeseed meal by means of phytate - degrading enzymes derived from aspergillus niger . j . sci . food agric . 61 , 1 - 6 . zyla , k ., koreleski , j . & amp ; kujawski , m . ( 1989 ). dephosphorylation of phytate compounds by means of acid phosphatase from aspergillus niger . j . sci . food agric . 49 , 315 - 324 .