Dietary Role of Phytase to Improve Minerals Bioavailability for Bone Conformation

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1 World Journl of Medicl Sciences 11 (4): , 2014 ISSN IDOSI Pulictions, 2014 DOI: /idosi.wjms Dietry Role of Phytse to Improve Minerls Biovilility for Bone Conformtion Sehm S. kssem, Mdih M. Adel kder, Ghd E.A. Awd, Ens N. Dnil, Moetzz M. Alshfei, A.M. Kholif nd Emtenn M. Hnfi 1 Deprtment of Food Science nd Nutrition, 2 Deprtment of Chemistry of Nturl nd Microil Products 3 Deprtment of Diry Science 4 Deprtment of Animl Reproduction nd A.I., Ntionl Reserch Center, Giz, Egypt Astrct: Phytte ccumultes in the seeds (whet, mize, rice, soy en, etc) during the ripening period. The unique structure of phytic cid offers it the ility to strongly chelte with ctions such s clcium, mgnesium, zinc, copper, iron nd potssium to form insolule slts. It therefore dversely ffects the sorption nd digestion of these minerls nd consequently ffect one frmtion in mongstric nimls ecuse they lck the intestinl digestive enzyme phytse. It lso negtively impcts protein nd lipid utilistion. It is of mjor concern for individuls who depend minly on plnt derivtive foods. Supplementtion of phytse in diets results in increse in minerl sorption. This study ims to throw light on the possile use of kind of lctocillus species, Lctocillus reutrei, to get rid of phytte onds entrpping minerls nd trces in cerels nd mke it ville for our odies nd one conformtion. The Experiment ws designed on Forty cup chicks of oth sexes (1 dy old) were divided into 4 groups (10 ech) Control group fed on sl diet (G1), irds fed on Bsl diet eside phytse Enzyme derived from lctocillus reuteri (G2), irds fed on sl Diet eside cteril suspension of lctocillus reuteri (G3) irds fed on sl Diet eside commercil phytse derived from A.niger funugs (G4). All chicks hd free ccess to feed nd wter. Enzymes were given to G2 nd G4 nd justified ccording to feed intke to e 600 FTU/ Kg. At the end of the experiment (fter 6 weeks) Birds were slughtered, clssified to mles (M) nd femles(f). The collected lood smples were centrifuged nd the hrvested plsm ws nlyzed for the levels of Clcium, phosphorus, Iron, zinc. Trnsverse section of the one of femur of oth mles nd femles were tken from the different groups, dried nd sent for evlution of porosity using Automted Helium Pycnometer. The sme one smples were fired nd ground to e nlyzed for the minerls C, pi, Zn nd Fe concentrtion using tomic sorption. Results reveled tht the serum showed elevted level of clcium, phosphorus, iron nd zinc in groups of irds supplemented with phytse enzyme specilly G3 if compred to those not supplied y the enzyme. This reflected on one where the sme groups showed less one porosity nd higher level of minerliztion. The study concluded tht use of phytse enzymes or the producing cteri in monogstric niml my improve the iovilility of minerls nd controls one porosity. Key words: Osteoporosis Bone Density Minerls Mize Phytse Enzyme INTRODUCTION nd susceptiility to frcture. Osteoporosis is silent disese until it is completed y frctures tht cn occur Osteoporosis literlly trnsltes s porous ones following miniml trum [1]. or when the holes etween one ecome igger, mking Frctures re climed to ffect 50% of women nd it frgile nd lile to rek esily. Osteoporosis is 30% of men ged over 50 yers [2]. Bone is mde up of progressive systemtic skeletl disese chrcterized y one tissue, one mrrow, periosteum, endosteum, low one mss nd micro-rchitecturl deteriortion of nerves nd lood vessels. Bone tissue contins n one tissue, with consequent increse in one frgility extrcellulr mtrix with n orgnic nd inorgnic mtrix. Corresponding Author: Emtenn M. Hnfi, Ntionl Reserch Center, Dokki, Giz, Egypt. Cell:

2 The orgnic mtrix ensures flexiility nd elsticity while, iovilility of minerls nd trces necessry for one one minerls provide strength nd mechnicl rigidity formtion nd control the incidence of osteoporosis to the one [3, 4]. The inorgnic mtrix contins tking poultry s model for monogstric nimls. hydroxyptite crystls nd smll mounts of cronte, citrte, Mg, N, K, Cl nd F. Hydroxyptite crystls re MATERIALS AND METHODS formed y osteolsts, which comine collgen with clcium (C) nd phosphorus (P) to form these crystls. Preprtion of Phytse Enzymes The formtion of hydroxyptite crystls is necessry Microorgnism: Twenty two lctocillus spp. were for overll one size nd one width [3]. so, n indequte tested for their ility to produce phytse (dt is not supply of clcium over the lifetime is thought to ply shown). The screening ws crried out y inoculted significnt role in contriuting to the development of lctocillus spp in fresh MRS medium supplemented osteoporosis [5, 6]. An dequte mount of dietry with 0.1% sodium phytte. Inoculted flsks were protein is essentil to mintin production of hormones incuted in 37 C for 72h under neroic condition. nd growth fctors tht modulte one synthesis [7]. Lctocillus species were su-cultured in MRS roth Low serum phosphte will limit one formtion nd nd preserved in glycerol solution (20%) t working cell minerliztion [8] where it limit osteolst function nd nk t -80 C for further use. This ws n importnt step enhnce osteoclstic one resorption [9]. At ny ge, to ensure tht the strter culture of ech experiment of the the rtio of phosphorus to clcium is proly more sme genertion numer. Among the tested importnt thn the intke of phosphorus lone [7, 8] lctocillus Spp. lctocillus reuteri showed the where the high phosphorus nd low clcium intke my highest production of phytse nd ws selected for led to secondry hyperprthyroidism nd one loss. further investigtion. 0.6 ml of cteril suspension Plnt-sed food products re the min stple ws hving million CFU/ ml tht ws presumly food for humn eings in mny prts of the world. producing 600 FTU They constitute n importnt source of crohydrtes, protein, dietry fire, vitmins nd non nutrients [10]. Phytse Production: Production of phytse derived from Among ll the ntinutritionl components, phytic cid is lctocillus reuteri ws crried out in modified tomto of prime concern for humn nutrition nd helth skimmed milk roth consisted of 200 ml of tomto juice, mngement. The unique structure of phytic cid offers it 5gm skimmed milk nd 1 gm of sodium phytte in one the ility to strongly chelte ctions such s clcium, litter of distilled wter the ph ws djusted to 6.5. mgnesium, zinc, copper, iron nd potssium to form The inoculted flsks were incuted in 37 C for 72h insolule slts. It therefore dversely ffects the under neroic condition. The enzyme ws given to sorption nd digestion of these minerls y nimls irds t 600FTU/Kg the sme dose used in poultry frms [11]. Phytte ccumultes in the seeds (whet, mize, rice, using commercil phytse which hs een derived from soy en, etc) during the ripening period nd is the min A.niger [14]. storge form of oth phosphte nd inositol in plnt seeds nd grins [12]. Phosphorus, in this form, is not Phytse Assy: Phytse ctivity ws determined y utilized y humn eings nd monogstric nimls like mesuring the mount of lierted inorgnic phosphte. dogs nd pigs or irds ecuse they lck the intestinl The rection mixture consisted of 0.9 ml of cette digestive enzyme phytse [13]. Besides, phytte hs lso uffer (0.2 M, ph 5.5) contining 1 mm phytte nd 0.1 ml een reported to form complexes with proteins t oth low of the enzyme solution. After incution for 30 min t nd high ph vlues. These complex formtions lter the 37 C, the rection ws stopped y the ddition of 1 ml of protein structure, which my result in decresed protein 10% trichlorocetic cid. The liquot ws susequently soluility, enzymtic ctivity nd proteolysis nlyzed for inorgnic phosphte s descried erlier [15]. (digestiility). Phytse is n cid phosphohydrolse, This crried out y ddition of 1.5 ml of colour regent which ctlyzes the hydrolysis of phosphte from phytic consists of 1:4 v/v of 2.75 % ferrous sulphte: 2.5% cid to inorgnic phosphte nd myo-inositol phosphte mmonium molidte dissolved in 5.5 % sulphuric cid) derivtives. The ig gol of this study is to investigte One unit of the phytse ctivity ws expressed s the the possile role of different sources of exogenous mount of enzyme required to lierte 1µmol of phosphte dietry phytse (fungl nd cteril) to improve the per min from sodium phytte. 505

3 Tle 1: concentrtion of phytin phosphorus nd phytte in feed ingredient Phytin-phosphorus (mg/100g DW) Phytte (mg/100g DW) Mize Soy en concentrtes Mize stuff Tle 2: Anlysis of the nutrients' level (%) of poultry feed [17] Moisture DM OM CP CF EE NEF Ash Mize Soy en Concentrte DM= Dry mtter, NEF = Nitrogen free extrct, EE= Ether extrct, CF = crude fier, CP= crude protein, OM= orgnic mtter. Tle 3: Anlysis of Minerls in poultry feed Phosphorus (mg/g) Clcium (mg/g) Iron (ppm) Zinc (ppm) mize seeds Mize stuff Determintion of Phytte in Feed nd Ilel Digest: the erly morning to e sure of complete consumption. Complete diets nd ilel digest hve een collected to Birds in G3 given cteril suspension estimted to estimte the phytin-phosphorus nd phytte levels [16]. produce the sme units of the enzyme, just to compre Powdered smple (4 g) were soked in 100 cm of 2% HCl etween supplementing cteril flor nd enzyme (v/v) for 3 h nd filtered. To 25 cm of the filtrte in supplementtion. At the end of the experiment (fter 6 conicl flsk, 5 cm of 0.3% NH4SCN(q) nd 53.5 cm of weeks) Birds were slughtered, clssified to mles (M) distilled wter were mixed together nd titrted ginst nd femles (F). The collected lood smples were 3 stndrd FeCl 3(q) solution contining g Fe/cm centrifuged nd the hrvested plsm ws nlyzed for until rownish yellow colour persisted for five minutes. the levels of clcium, phosphorus, iron nd zinc. Blnk ws treted in similr mnner. Phytin Phosphorus Trnsverse section of the one of femur of oth mles (1 cm Fe = 1.19 mg Phytin-Phosphorus) ws determined nd femles were tken from the different groups, dried nd the phytte content clculted y multiplying the nd sent for evlution of porosity ccording to vlue of Phytin-Phosphorus y 3.55 Nwy [19]. The ulk nd skeleton volume (v nd v sk) of the studied smples were mesured using Automted Preprtion of Feed: The formulted feeds were nlyzed Helium Pycnometer (Ultr Pyc 1200e y Quntchrome) t for minerls, crohydrte, protein, ft nd sh (Tle 1) 19 psi nd the mient temperture. The ulk volume (v ) ccording to AOA [17]. The formulted feed were justified ws mesured fter covering nd isolting the one to hve 21% protein for the growing chicks (Mize 55%, smple in Pr-film to void invding the pores y Soy en 33%, concentrtes 5%, vitmin mix 0.3%, helium; where the skeleton volume ws mesured for the sodium chloride0.3%, Clcium phosphte 2.5% nd re smples without coverge to enle injection of cooking oil 4%) [18]. the helium into the pore of one smples. Helium hs the ility to invde the nno pore size, down to 10 m. Experimentl Design: Forty cup chicks of oth sexes Porosity of the dry core-shped smples ws then (1 dy old) were purchsed nd divided into 4 groups clculted precisely y sustituting for oth the (10 ech) Control group fed on sl diet (G1), irds fed ulk nd skeleton volume v, v sk in the following on Bsl diet eside phytse Enzyme derived from eqution:- lctocillus reuteri (G2), irds fed on sl Diet eside cteril suspension of lctocillus reuteri (G3) irds ( v v ) sk = 100 fed on sl Diet eside commercil phytse derived from v A.niger funugs (G4). All chicks hd free ccess to feed nd wter. Enzymes were given to G2 nd G4 nd justified The sme one smples were fired nd ground to e ccording to feed intke to e 600 FTU/ Kg. it ws nlyzed for the minerls C, pi, Zn nd Fe using tomic prepred nd dded in smll mount of drinking wter in sorption. 506

4 RESULTS Estimtion of phytin phosphorus in irds ingest (fig 1) showed tht feeding chicks on diets supplemented with phytse enzyme specilly those given lctocillus cteril suspension (G3) showed high enzyme ctivity expressed s liertion of the ound phosphorus leving the lest mount of phytin-phosphorus in ird s ingest if compred to levels of phytin-phosphorus in control group (G1). Estimtion of the serum level of minerls showed elevted clcium, phosphorus, iron nd zinc levels (Tle 4) in groups of irds supplemented with phytse enzyme specilly G3 if compred to those not supplied y the enzyme. Screening one porosity (Fig 2) in groups of irds supplemented with phytse showed etter condition specilly those in G3. These results were confirmed y the higher level of one minerliztion in the sme groups (Tle 5). DISCUSSION Osteoporosis is grdul loss of the numer of cross ridges in the orgnic one mtrix, comined with reduction in one minerl density nd loss of one mss. The diet is estimted to hve contriuted to s much s 30-50% to pek one mss nd is significntly influenced y the dietry intke of clcium, phosphte nd vitmin [20]. It hs een widely reported tht the presence of phytic cid my compromise minerl iovilility from infnt foods [21, 22]. Phytic cid (myoinositol hex-phosphoric cid, IP6) is the mjor phosphorus storge compound of most seeds nd cerel grins. It hs strong ffinity to chelte multivlent metl ions, especilly iron, zinc nd clcium. The cheltes thus formed, give rise to insolule slts of the ove minerls with poor sorption chrcteristics nd hence low iovilility [21]. Low sorption of these nutrients from cerels is considered to e fctor in the etiology of minerl deficiencies in Tle 4: The minerl level ( mens ± SE) in serum of chicks supplemented with mize sed diet nd phytse enzyme Group Phosphorus (mg/dl) Clcium (mg/dl) Iron (ug/dl) Zn (ug/dl) G1F ± ± ± ±4.47 G2F ± ± ± ±2.8 G3F ± ± ± ±6.02 G 4F 18.9 ± ± ± ±6.50 G1M 18.0 ± ± ± ±0.58 G2M 18.8± ± ± ±0.59 G3M 19.83± ± ± ±1.26 G4M 19.27± ± ±6.70 c 90.60±2.15. Different superscript mens significnt difference p 0.05 Tle 5: minerl concentrtion in femur of mle nd femle chicks supplemented with mize sed diet nd phytse of different sources Group (F= femle, M= mle) Phosphorus (mg/g) Clcium ( mg/g) Iron ( ppm) Zinc (ppm) G1F G2F G3F G4F G1M G2M G3M G4M Fig. 1: Estimtion of phytte nd phytin-phosphorus in irds secretion 507

5 Fig. 2: Bone porosity of chicks fed on Mize sed diet nd phytse. infnts. Phytse, which occurs widely throughout hormone [28]. The study concluded tht use of cteril nture, is the requisite enzyme to degrde nd relese or fungl phytse in cerel rich diets improves minerl inorgnic P [23]. iovilility nd consequently the stte of one The prcticl cceptnce of microil phytse in minerliztion nd controls one frgility or osteoporosis. poultry diets chnged the nutritionl requirement for mss Also cteri derived enzyme proved more efficient poultry production. Aspergillus niger-derived phytse ctivity thn fungl derived phytse. feed enzyme, with the cpcity to lierte phytte-ound P, ws commercilly introduced in 1991 [24]. The cteril REFERENCES phytse ws more resistnt to pepsin ctivity thn fungl phytse [25]. Lctocillus reuteri (L-M15) hd the 1. Rizzoli, R., Mngement of the oldest old with highest phytse production nd phytte degrding osteoporosis. Europen Geritric Medicine, 1: ctivity when used s strter in whole whet red 2. Hejzi, J., J. Mohtdini, S. Kolhi nd mking process [26]. The present study declred tht M. Erhimi-Mmghni, Nutritionl sttus lctocillus reuteri, produced higher level of phytse mong postmenopusl osteoporotic women in enzyme when given s cteril emulsion nd its phytse North West of Irn. Asi Pc. J. Clin., Nutr., product proved to e more efficcious thn tht derived 18(1): from Aspergillus niger. This high enzyme ctivity 3. Clrke, B., Norml one ntomy nd expressed s liertion of the ound phosphorus leving physiology. Clin. J. Am. Soc., Nephrol, 3: S131-S139. the lest mount of phytin-phosphorus in ird s ingest 4. Rlston, S.H., Bone structure nd metolism. in G3 if compred to levels of phytin-phosphorus in Medicine, 37(9): control group (G1). Estimtion of minerls level in oth 5. Ntionl Osteoporosis Foundtion. Boning up on serum nd one ( tle 4 nd 5 )showed elevted clcium, osteoporosis: guide to prevention nd tretment. phosphorus, iron nd zinc levels (Tle 4) in groups of Wshington, D.C irds supplemented with phytse enzyme specilly G3 6. Stránský, M. nd L. Rysvá, Nutrition s if compred to those not supplied y the enzyme. prevention nd tretment of osteoporosis. Physiol. The enzyme ctivity showed the lest one porosity in Res., 58(1): groups of irds supplemented with cteril suspension 7. Heney, R.P. nd B.E. Nordin, Clcium effects (Fig.2). Use of phytse enzyme incresed the on phosphorus sorption: implictions for the iovilility of iron which mens protection from Iron prevention nd co-therpy of osteoporosis. J.Am. deficiency complictions such s nemi, significnt Coll. Nutr., 21: decreses in psychomotor nd mentl development nd 8. Heney, R.P., Phosphorus nutrition nd the reduced immune sttus [21]. The use of phytse with tretment of osteoporosis. Myo Clin. Proc., diets rich in cerels will lso increse the sorption of 79: clcium, ffecting the ttinment of pek one mss, 9. Whiting, S.J., J.L. Boyle, A. Thompson, R.L. Mirwld which my e importnt in reducing the risk of frctures nd R.A. Fulkner, Dietry protein, phosphorus nd osteoporosis in lter life [27]. In the sme time the use nd potssium re eneficil to one minerl density of enzyme will increse iovilility of Zinc which is in dult men consuming dequte dietry clcium. J. necessry for one formtion nd stimultion of growth Am. Coll.Nutr., 21:

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