Effect of mill type and mechanical kneading conditions on fermentation kinetics of tef dough during injera making and phytate to mineral molar ratio of injera

E ec o Mill Type and Mechanical Kneading Condi ions on Fe men a ion Kine ics o Te dough Du ing Inje a making and Phy a e o Mine al Mola Ra io o Inje a Yoseph Legesse Asse a1,*, Shimelis Admassu Emi e1, Wo kineh Abebe2, Felicidad Ronda3 1Addis Ababa Uni e si y, Addis Ababa Ins i u e o Technology, School o Chemical and Bioenginee ing, Food Enginee ing G adua e P og am, Addis Ababa, E hiopia 2E hiopian Ins i u e o Ag icul u al Resea ch, Addis Ababa, E hiopia 3College o Ag icul u al and Fo es y Enginee ing, Uni e si y o Valladolid, A . Mad id, Palencia, Spain Abs ac Inje a is E hiopian adi ional e men ed la b ead which made mos ly om e lou . Te g ain is milled and he lou kneaded o ha e ba e -like dough which will e men be o e inje a baking. The in luence o mill ype: hamme mill (HM), disc mill (DM) and blade mill (BM) and mechanical kneading speed- ime combina ions (K1, K5, and K9), on e men a ion kine ics was in es iga ed. Phy a e o mine al mola a io (Fe, Zn and Ca) o e inje a was also in es iga ed as a ec ed by mill ype and di e en kneading condi ions. In bo h milling and kneading le els, mal ose was he highes suga concen a ion ini ially, which ollowed by glucose and uc ose. As e men a ion con inued, a simila end in mal ose b eak down was seen among HM, DM, and BM. Howe e , di e en pa e ns o glucose and uc ose b eak down we e seen on HM han DM and BM. Simila ly, HM had a di e en pa e n in inc emen o lac ic acid concen a ion han DM and BM. Simila end in mal ose concen a ion was seen be ween K1, K5, and K9. Again glucose b eakdown and he inc emen o lac ic acid in K9 we e di e en han ha o K1 and K5. Phy a e/mine al mola a io o BM was signi ican ly di e en (p<0.05) om HM and DM. The e was also a signi ican di e ence (p<0.05) in phy a e/mine al mola a io be ween K1, K5, and K9. Dec eased phy a e/mine al mola a io was seen wi h inc easing o kneading speed ( pm) o a longe pe iod o ime. The e ec o mill ype and kneading speed and ime combina ions on e men a ion kine ics and phy a e/mine al mola a io we e signi ican . Keywo ds: Te , milling, kneading, e men a ion kine ics, phy a e/mine al mola a io. 1. In oduc ion Te [E ag os is e ] is an E hiopian indigenous opical ce eal c op and i has been cul i a ed o many yea s in E hiopian highlands (Demissie, 2001; Viswana h, 2012). P oduc s o e g ain a e nu i ionally well packed because hey a e always consumed as whole g ain wi h a high con en o ca bohyd a e and ibe (USDA, 2007), wi h mo e i on, zinc, and calcium han o he ce eal g ains, including so ghum, whea and ba ley (Abebe e al., 2007). Due o he absence o glu en and glu en- like p o eins, e has ecen ly been ecei ing global a en ion, pa icula ly as a “heal hy ood”, making i igh o celiac disease pa ien s (Spaenij-Dekking e al., 2005), and also because o o he die a y bene i s such as slow- elease o ca bohyd a e cons i uen s use ul o diabe ic pa ien s (Abebe and Ronda, 2014). Te is he main s aple in he coun y mos ly used o make inje a, adi ional e men ed la b ead (Bul osa and Taylo , 2004). Inje a is p epa ed om ba e -like dough, which is p e- e men ed o 2-3 days. Fe men a ion is mos o he ime ini ia ed spon aneously by addi ion o wa e o e lou , allowing he na u ally exis ing mic oo ganisms o g ow (Gashe 1985). P ima y o i s s age e men a ion can also be s a ed by he addi ion o he s a e , e sho, which is a small amoun o ba e kep om he p e ious dough (Pa ke e al., 1989). Du ing e men a ion s ages, he main e men ing mic oo ganisms a e lac ic acid bac e ia (Lac obacillus species) (Gashe 1985) and yeas (Saccha omyces species) (Gi awesen and Bis a 1982). These mic oo ganisms esul is he all o pH, gas p oduc ion and dough ising and a e esponsible o desi ed inal p oduc la o and acidi y (Ume a and Faulks 1988). Se e al s udies ha e epo ed he bene icial in luence o e men a ion in imp o ing bo h nu i ional and sani a y quali ies o oods (Nou , 2009; S anbe g & Lo i, 1997). Acco ding o Nou & Mo a jemi (1997), P oduc ion o o ganic acids wi h low molecula weigh , such as ace ic and lac ic acid, educes pH and may hus limi con amina ion by oodbo ne pa hogens. G eine &Konie zny (2006) also epo ed ha e men a ion can be a eason o he ac i a ion o se e al endogenous enzymes including phy ases and may hus esul in p oduc s wi h dec eased an i-nu i ional ac o s. Hammes e al., (2005), s a ed ha he le el o which enzymes like phy ases a e ac i a ed depends on he e men a ion kine ics, which in u n, depends on he condi ion o aw ma e ials used. Depending on he mechanical o ces and empe a u e du ing he g inding p ocess, di e en milling o g inding me hods ha e been seen o p oduce lou s wi h di e en pa icle size and damage s a ch le el (Kadan e al., 2008). A epo by Abebe e al (2015), a e also s a ed ha lou pa icle size and deg ee o s a ch damages a e in luenced by mill ypes which a e used o g ind ce eal g ain. On he o he hand, Li e al., (2014) s a ed lou pa icle size and damaged s a ch ex en a ec enzyma ic hyd olysis o lou . De la He a e al., (2014) s a ed he pa icle size o lou also a ec he amoun o ca bon dioxide which e ained du ing e men a ion. Howe e , in o ma ion is lacking on he e ec s o mill ype on e men a ion kine ics o e dough du ing inje a making p ocess and i s e ec on mine al o phy a e mola a io since e possessed bo h signi ican ly. Dough p ocessing is an essen ial ac o which a ec s he quali y o b ead o inje a. Kneading is one o he mos impo an mechanical s eps in indus ial dough p ocessing (Esselink e al., 2003). Di e en s udies we e made on he e ec o dough mechanical kneading, on he mo-mechanical p ope ies o dough’s (Angioloniand Rosa, 2005), dough heological p ope ies (Angioloniand Rosa, 2007) and b ead quali y (Kim & De Rui e , 1968). All s udies showed ha in luences o dough kneading condi ions a e signi ican . Howe e , he e ec o mechanical kneading on e men a ion kine ics and mine al o phy a e mola a io du ing e dough e men a ion is s ill lacking. The e o e, he aim o his s udy was o in es iga e he in luence o mill ype and mechanical kneading condi ions on e men a ion kine ics o e dough du ing inje a making p ocess; and mine al o phy a e mola a io o e inje a. 2. Ma e ials and me hods 2.1 Ma e ials Based on popula i y among E hiopian e a me s and use s, Qouncho e a ie y (DZ-C -387) was selec ed and ob ained om Deb eZei Ag icul u al Resea ch Cen e o he E hiopian Ins i u e o Ag icul u al Resea ch (EIAR). Te sample was he me ically s o ed in cool and d y place using polye hylene bag. Be o e milling, e g ain was cleaned by si ing. 2.2 Milling p ocess Hamme mill (HM) (Pe en 120, Finland) i ed wi h 0.8 mm sie e, s one-disk mill (DM) (co age e g ain-milling, Denma k) and blade mill (BM) (Nu i Bulle NB-101B, China) we e used o ge he whole lou o e sample. The sample was milled o abou 7 min in case o BM. 2.3 Dough p epa a ion, e men a ion and inje a making Te dough was p epa ed acco ding o Pa ke e al. (1989) and Zegeye (1997) wi h li le modi ica ion. Amoun o s a e (E sho) equal o 60ml was ini ially added o each kg o lou . The e lou ( om s one-disc mill) was mixed 2:3 (w/w) wi h po able wa e and kneaded by ki chen aid (Moulinex Mas e chi 720, F ance). Dough kneading imes (1, 3 and 7 min) and kneading speed (Speed 1, 6 and 12) we e selec ed based on inje a expo e s p ac ice and kneading machine capaci y. Nine di e en e dough’s we e p epa ed using kneading machine by a ying ime and speed o kneading machine. Based on he senso y esul s o inje a, Kneading # 1 (kneading by speed 1 o 1 min and gi es mode a e o e all inje a accep abili y), #5 (kneading by speed 6 o 3 min and gi es he highe inje a accep abili y) and #9 (kneading by speed 12 o 7 min and gi es he lowe inje a accep abili y) we e selec ed o u he s udies. To s udy he milling e ec s, lou om HM, DM, and BM was mixed simila ly wi h wa e and kneaded by hand in a bowl un il ob aining a homogenous mix u e in he adi ional way. The dough was allowed o e men o 60 hou s a oom empe a u e (30 ± 5 ºC). A e he p ima y e men a ion, 10% o he dough was mixed 1:3 ( / ) wi h boiling wa e , and hea ed o 15 min wi h con inuous s i ing. The ho cooked dough (absi ) was hen mixed back in o he e men ing dough, and su icien po able wa e was added o make a ba e . The ba e was le co e ed o 2 hou s o seconda y e men a ion. Addi ional wa e was added o hin and o m he igh consis ency o he ba e . Finally, hal a li e o ba e was pou ed on o he ho clay g iddle in a ci cula o m. A e 2-3 min o cooking ( adi ional elec ic inje a baking equipmen ) inje a was emo ed and placed in a baske . 2.4 Flou cha ac e iza ion 2.4.1 De e mina ion o phy a es The colo ime ic me hod as desc ibed by Hang and Lan zseh (1983) was used o de e mine he phy ic acid con en o he samples. 2.4.2 Mine al de e mina ion I on, zinc and calcium we e de e mined using me hods 999.11, 968.08 and 985.35 o AOAC (La ime , 2016). 2.5 Fe men a ion kine ics 2.5.1. Change in pH As used by Baye e al., (2013), du ing e men a ion he pH o he slu y was eco ded using a pH me e (Oak on-Eu ech Ins umen s PH6, F ance). The a e o change in pH (_dpH/d ) was calcula ed o each sample as ollows: _(dpH/d ) = pH( +1) _ pH( )/( +1) _ , whe e ‘‘ ’’ s ands o ime (hou s). The maximal alue o _(dpH/ d ) o each sample obse a ion was hen a e aged o gi e he maximal a e o change in pH (_(dpH/d )max). 2.5.2 Ex ac ion p ocedu es The ex ac ion me hod was ha o Be as (1991) in which a 10 g o sample was homogenized wi h 90 mL dis illed wa e using magne ic ba s i e . Fi e millili e s o 1 mol/L HClO4 solu ion was added o a 10 mL aliquo o he homogena e. The mix u e was cen i uged o 15 min a 4000 g a 15 1C, he supe na an was neu alized (pH 7.070.1) wi h 2 mol/L KOH and he olume was adjus ed o 25 mL wi h dis illed wa e . A e 30 min p ecipi a ion on he ice, he solu ion was il e ed on 0.45 mm cellulose il e (Millipo e). 2.5.3 De e mina ion o suga s, o ganic acids, and e hanol HPLC analyses we e pe o med wi h an Agilen 1200 model equipped wi h a 20 mL au oma ic injec ion loop. De ec ion was pe o med wi h a e ac i e index de ec o (RID 156 Beckman) connec ed o a Shima zu STANG-ST3A in eg a o . Ch oma og aphic sepa a ion was pe o med using an ICSep ICE-COREGEL 87H3 column (T ansgenomic)(300 mm 7.8 mm, In e ac ion Ch oma og aphy, F ance) unde he ollowing condi ions: mobile phase 0.001 N H2SO4, low a e 0.6 mL/min, column empe a u e 35 ˚C. 2.6 S a is ical analysis Analysis o a iance was pe o med on he da a o es ablish signi ican (p <0.05) di e ences be ween he samples. The sco es we e hen subjec ed o analysis o a iance using SPSS s a is ical so wa e (Ve sion 16) and means o duplica e esul s we e compa ed by Tukey’s Hones ly Signi ican Di e ence Tes . 3 Resul s and discussion 3.1 Fe men a ion kine ics 3.1.1 Changes in pH The kine ics o dec ease in pH showed he same pa e n in all he h ee ypes o dough’s o bo h milling and kneading a iables (Fig. 1 & 2). Howe e he mean pH a he s a o e men a ion o BM, was highe (6.41) han ha o DM (6.32) and HM (6.23). On he o he hand, he ini ial dough pH was no a ec ed by kneading esul ing in he pH o (6.34) o K1, K5 and K9. Du ing he i s 10h o e men a ion, pH dec eased om 6.23 o 4.22 o HM, and om, 6.32 o 4.13 and 6.41 o 4.36 o DM and BM dough espec i ely. Dec ease in pH was also obse ed due o change in kneading condi ions i.e.; o K1 om 6.34 o 4.16, om 6.34 o 4.1 and om 6.34 o 4.23 o K5 and K9, espec i ely. Acco ding o Nou e al., (1989) he common cha ac e is ic among he di e en dough’s is a apid d op in pH his may be due o back-slopping e ec s. The maximum a e o pH dec ease was simila in bo h milling and kneading le els. Howe e , he e men a ion ime o see highe pH a e o K5 was 2h, which was di e en han ha o K1 and K9 (10h) (Table 1). This di e ence migh be due o he di e ence in kneading speed/ ime combina ion which may a ec he e men a ion kine ics. In all doughs, he a e a which pH was dec eased when he e men a ion ex ended om 10 o 24h. In bo h milling and kneading, he pH was dec eased a slowe a e when e men a ions ex ended om 24 o 48h. Simila esul was epo ed by Baye e al., (2013). The inal pH o HM, DM, and BM we e 3.47, 3.22 and3.32, espec i ely. Final pH o 3.53, 3.51 and 3.80 was no ed o K1, K2 and K3, espec i ely. Le eb e e al., (2002) s a ed ha , when he u iliza ion o e men able ca bohyd a es and he LAB popula ion inc ease, he pH alue o he inocula ed sou dough dec eased. Le eb e e al. (2002) epo ed he ime necessa y o ob ain he minimum pH alue was abou 13 h hough he o al i a able acidi y con inued o inc ease un il 19 h and he pH dec ease was co ela ed wi h he p oduc ion o o ganic acids. Acco ding o Kingamkono e al (1994) and Nou e al, (1989), he pH apidly eached alues below 4.5, p omo ing be e hygienic condi ions. Table 1 Kine ic pa ame e s o pH change du ing inje a sou dough e men a ion HM, DM and BM s and o dough om he hamme , disc, and blade milled lou ; K1, K5, and K9 s and o dough wi h h ee- ime/ pm kneading combina ions. Da a a e exp essed as mean ± s anda d de ia ions Figu e 1 Changes in pH du ing inje a sou dough e men a ion. HM, DM and BM s and o dough om hamme , disc and blade mill. E o ba s ep esen he s anda d de ia ion o means. 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 01210 24 48 pH Fe men a ion ime (h) Sample dough Ini ial ( lou ) pH (_dpH/d ) max Time (h) o each (_dpH/d )max pH a 48h Milling HM 6.23±0.03 0.2±0.00 10 3.47±0.01 DM 6.32±0.00 0.22±0.01 10 3.22±0.00 BM 6.41±0.01 0.21±0.01 10 3.32±0.00 Kneading K1 6.34±0.01 0.22±0.01 10 3.53±0.00 K5 6.34±0.01 0.24±0.01 2 3.51±0.00 K9 6.34±0.01 0.21±0.01 10 3.80±0.01 Figu e 2 Changes in pH du ing inje a sou dough e men a ion. K1, K5, and K9 s and o dough wi h h ee- ime/ pm kneading combina ions.. E o ba s ep esen he s anda d de ia ion o means. 3.1.2 Kine ics o subs a e consump ion and p oduc o ma ion A e e milled using h ee di e en mills, he ini ial suga con en o dough be o e adding s a e cul u e (E sho) was in es iga ed (Fig 3). In all dough’s ob ained om (HM, DM, BM), he dominan suga was mal ose which was ollowed by glucose and uc ose. This ag ees wi h a epo by Baye e al (2013) o ba ley-whea and whea - ed so ghum dough in inje a making. The concen a ion o mal ose a he s a o e men a ion was 535.23 ppm o HM, 615.68 ppm o DM and 501.36 ppm o BM. A e which, i s a s dec easing o he inal alues (ze o) in all dough’s (HM, DM, BM). This ag ees wi h Kulp & Lo enz (2003) epo which s a ed ha mal ose b eaks down in o glucose molecules du ing sou dough e men a ion. Fo he dough om HM, dec ease in glucose (22.64 ppm) and uc ose (56.1 ppm) concen a ion was obse ed du ing 2h o e men a ion. Kulp & Lo enz (2003) explain glucose and uc ose a e he i s suga s o be used du ing e men a ion. These suga s showed inc emen a 10h o e men a ion, a e which, i s a s dec easing o each he inal alues o glucose (18.23 ppm) and uc ose (0 ppm). Kulp & Lo enz (2003) s a ed ha , suc ose is apidly b oken down o glucose and uc ose by yeas enzymes al eady p esen ou side he cell memb ane; his may jus i y an inc emen shown o glucose and 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 01210 24 48 pH Fe men a ion ime (h) HMI and DMI we e no a ied (Table 2). Howe e , BMI had a highe mola a io (Phy a e/i on, phy a e/zinc, and phy a e/calcium) han ha o HMI and DMI. Al hough he samples had simila amoun o mine als, he di e ence in phy a e con en which was as esul o using di e en mill ype jus i ied he eason o ha ing a di e en mola a io. A simila end was seen again be ween K1, K5, and K9 wi h a signi ican di e ence in phy a e con en which caused by mechanical kneading lead o ha e di e en mola a io be ween samples. Phy a e/i on mola a io o samples a ied signi ican ly in he o de o K9 (0.63) < K5 (0.81) < K1 (1.00). The esul showed ha i has less isk o bioa ailabili y. Hallbe g e al., (1989) also s a ed, phy a e/i on mola a io >1 is ega ded as indica i e o poo i on bioa ailabili y. Simila ly phy a e/zinc and phy a e/calcium mola a io o samples we e signi ican ly a ied in he o de o K9 (5.98) < K5 (7.65) < K1 (9.49) and K9 (1.13) < K5 (1.45) < K1 (1.78), espec i ely. The bioa ailabili y o calcium in HMI, DMI, and BMI and K1, K5 and K9 we e less. Acco ding o Mo is and Ellis (1985), phy a e/calcium mola a io >0.24 will impai calcium bioa ailabili y. On he o he hand, Tu nlund e al, (1984) epo ed ha , zinc abso p ion is g ea ly educed and esul s in a nega i e zinc balance when he phy a e/ zinc mola a io is 15. 4. Conclusions The e ec o mill ype du ing inje a making p ocess a ied e men a ion kine ics and dough composi ion signi ican ly. Fe men a ion pa e n and dough composi ions a e also in luenced by using di e en mechanical kneading ime and speed combina ions. Bo h e milling and mechanical kneading me hods play impo an ole in he deg ada ion o phy a e and inc ease mine al bioa ailabili y. BMI which was made om e lou wi h la ge pa icle size had low mine als (Fe, Zn and Ca) bioa ailabili y han DMI and HMI. Inc easing o mechanical kneading speed o a longe pe iod o ime, can dec ease he phy a e con en o inje a and lead o ha e be e mine al bioa ailabili y. Howe e , al hough phy a e/calcium mola a io dec eased wi h inc ease in kneading ime and speed (K9), he calcium bioa ailabili y s ill needs o be imp o ed. ACKNOWLEDGMENTS The au ho s would like o hank Deb eZei Ag icul u al Resea ch Cen e o he E hiopian Ins i u e o Ag icul u al Resea ch (EIAR) o p o iding e a ie y and Addis Ababa Uni e si y o suppo ing wi h acili ies in his esea ch wo k. 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