.1 . .0. . .. ._ 0... : n 0.: . . I4 .14 I010! ..I. _ . :2 . .0 . ....4. 40.001.70.01 100 40.0.0 00. ..0. 0. .. . .11. .00 .331... ...0 I..... by“... .. -0: ...0._._ .0. 3:04.00 140. )0 . 00.. 0. .’f.....s...0wu:.. .00. 3.03.12.05.90 .02.... . 0”».59J34h. ..u ..0»J\.a r. -0 .. 0.5.0.. . . . .. . . . a . or. .000. . . .... ...A. : . ). . . ... .. I.. . . .9000.“ .1 . . ‘30:“.03593 0. .0. .0 02.1.1.0... ’0. 305.010.03) l..'.n‘..uu£0,00. ’0. 0 0.. 0100; Inn-.03...- a0... ...... 0.10.01... (Jaw. .. . 0 . 1.... . 0..... 0 3&0}; .00. .. .. . 0.. . 0.100 .0. . 5.00. . . s. . '0. . v .0300 . .0 1 Ir. .0 . .. . I 0’ I _ .. . . 00 . .... .. o0 . I.. .0 .. .... ...l 0 . . 5.. .00000... .00. 3.. .. . ..‘040‘. ("“00 0. 0 .00 {.0010 “mg”..- .—Q.. '0 ‘00..“ 1001 v 0.050.. 0.... ..w. v.0 .00 . 0. ..00 n . . :0 0 . . 000.. . . A0... 0 90 . .W.‘ 00!. ..." 00.7 0 0.. .. . y 0 u.“ .....I.0 . . n ‘10 .5. . gm 09.!0 04"... 00.00.2nfi! . .... 0.1.03.0. 0.00... 0.. .7. .. . 90 0.. ..212. u. .01. . K0! . .0 . . . 0 . . . 0 . . . 0 .1 . .... .2. .P ... .. . - u. .0 . . I ... . 2 . .. . 0. ......H 0 ... A»? . r 0:. 0.0.0.234 .050... 00 I... ..s. 300.0%...0. 1... l0 . w. 0..... a 0.9%.? ....0 . . . .090. ..m. ... 0. ... .0 . .0. .. _. 0 .0. . a I 1.0...0 .. 2 .... .. . 0. .. . .00... . 03......71... I..-4.0.0.0005. .. ......Quflaw...‘..0 .2114... ...0. 0,. J. .. . )4... 1 w. : 0. 4. . 0 l .. 9 .. 1:12.... .0 .... 0.. 0. ... :0... . . r . . . . iIVIDP‘I .000 0 . 00:30.00 .00 ..J“."..0\I ”wining. I . . . . .00. .....00’0. .... ".00.. a I . 0 . 0. 0 n 0200.. :10 ...0. .01. 30.0.0.0 .. ... . 0.. .. ..0.00 ‘ {Shiv .Ciu" 00. 3?.’0.0. 3.0: .0 0’00 o u 00 c 0 0,0. . . . . . . . .. . 1.0. . ...... .. 00.. . D 00.0... 0 ' 0 '0 0 . . . o. 0.. . . Or .. .s. 0.0. .9 0000.0 0.. .I 0 .. . u 0: 4. v.0 . 10.0955070...’ 0..-(.98... . Q2 0 4.... . . . . . . .. . 0..0 u... 0 ... . 7 I. 0 ...0 . . .. . £00... . .. .. .. .. . ...I . «0 .. ... 00.. . . ...... _ . .00. .1000. . ..0'0 0 .0. 00.4.0 .0 I... 0.3 .. . 00:. ....0 . f0. 0. 9 'H‘ . a . c. . . . 0 0 v.” . y _ .0! _ 0.0 I .0 0 .\0 . . 0 I. . 0 l ... .. .... ..V . 0. ...l .0 . .900. 0a.. ..0 f2... .... ....0 fl. .. . aflpafirrt 3.1.5.0.... . . ...... ... ...“. h‘w...:.l. .nJfl...“ .. . . . .. . . .0 .3 . .. _ . ..,0 I t 9 01:, .. . .. I... 0. . ... v.u0.1.II0\ ..0! 0.30... .00. . 0..... 0.0 0.0.. :0... . . . . . . . .0 33“....” ... . 00. .0. . . . .. . . _. 00. .o 0. v. . . . .0 I ... . ......0. .00...... .1. ... ...l ... . 0 .. 0.0... 1000.0".Kubfi.’ .... ..0’.§40.,0.£ H‘Pva‘O.’ 0070. 0 . . . _ . . , . ...: 0 .... . ”EH .0 01. ..00W.~.:. 0.0 0.00. r 0. \. 000.. .. 0.0.... 00 1.. v - . 0 .... I . 0. .... 0 ...! v0. 0-0.05.0...(0. .... ...: .. .. .3... .....0.........J 5...... 2w $3.11.... .. ......finram...’ , . . . _ . . .. .. ... ...:.........._: ...... . 1... ,. ....“ 3..-... ...... ... .. .. ...u. ,. .. T .. -.. i . . .. 1... -........ ...; .. . .. #0.. . 0 0. 00 0.0 I .0 . . .l u . . . _ . . . . 0 0.0 0.. 0 0 . . 0 . ... . 0 ... 7 7' 0.. 40 . O 0.0. . . 00.“: . . ... 0 . . 9D,}. . 0 . . .. .\..0 ..0 .....9. .. . .10 . ......00 .... 9.03.30 .. . 0.0.0 .0 0! ... ... . 0 070 . 20.00 0 . . .0. ... .... .. .. ... .0 o ... .... ..- 0... . . . .. . . .. . . . r! T . ‘WHNH...0.0—0 fl‘umuflx 0% 0 0.3.? .05 3.000‘. “0.00.0".0..:0~'0 . . .. . “30.00%”... 00”“. 0- .00. L” 0.... 00.... V304..- . .0. .0 ”I.. 0v.0 00 .0“ . 3 .‘J 000.200 0... ....9 0 .00....0. . 5w 0. 0100 . . .. . ... . 000...... .M010 ...! . .....m.0 Il‘ \UHD. ....- . I... a 0 .n . I. I. . . . . . . . . . 0 0 . . . . ._ . v..l ..00 I. u 0‘. 0a . 0.0. 3.0 .0.0 .. I- .b.“_.. .00.. 0 . . .. . . .A\.h . 0.0 D07? Jtln’0\.:..0l- . . . . . .0‘0. 01 0‘ 3.0.9 .0 II, I . 30000.0. ..0. (.0 O - . 0. . .0. 0 I.. .. .. 0.. .00! . . 00.... _ 0. 0“ I 00.!- , . «bifurv; 0 0 v . 00 ...... £0,010 .001 0.. . . . ..00. . 0... 0. 0 0 0 0 v n. 0 0.‘ . . .0. . I . . \ s 0 0 I. IV 1 00 011.. 0‘ .0. .00 .20. . . .. . . . .. 010 ... . . n. x . . . O . ......0... ... .. .0 . . \. .. 1...... .. . . _ . . .....Dluo; 0..:0 .8!:.....1..:,f0. . .. . . . . . .. ... .0! 0. .. .... ... r . . . ... r. 0.. . . . . 0 0..... .. 0. . 1 . ...; . ... . a - . ... . . . .. . P30. . 07.0.5.0 0.. .0... 9.... .. .8113009000... .. VI. .... ..00, . . . . . . .0“?! ... . .. 0.4 I. ... .. o . .0 0.. 00 I: .0 .. 0 ; ... ..0 ., . l 00. _. 0. .0001... ... 0: -.. 0.1.0 0. ”01‘ 00 c . -.....08 .0 .... .d. . In... D 0 .. 10.... . ..0 *0 I.....«X.~0n.... ... . .. 0.80.. 9 .. . 0. 00. . . .0 . .0. .0...0... .l 0 . .. ._ I . .. . . \: ...... 0. 3n .... .... 000'. 0 ' ‘0..00 . . 0 . . . .. ...0‘0 .. . r. 006. . . 07.. 1.0.431". .I...r... .310... 0 I... .R.u....._.....3w a. ..--.. .... . ..-.w... .. ... 0.. . . . ., .... .. .... .0 .0. . . .. 3 n. . ... ...... 3.200 ...: .0 I0. SIOIOV?!‘ l‘? I: 00. ..00 00:00.00. 0 0.... . .... . , .. . 0... 5.1.0 .0 0’ _. 1 u ..Yoa 0 . . _ - ..1.’ ...Pc ......1. _. ... 0.. I..... .. .0... .II . . .10 . . . .000“ . 0. . .09 .... 0 .. .N 00... . 06 . ....0 .....t. 10.0. ...-... ..I 0. ...0. $000.09.- t. 0.2!... 0 :i—f’J‘l . .0 0. 0.00. . . . . . . u I 0. . I"... 0m . n. 0 .0. 0.90 03 O. 0.0., ‘II. .. |l. 0.0 0. .‘ 1, 0 -0 0000. 3-0. ...000.0000~ 0 .000 ... .0..000. 00% r0....:. . . . . .. . . . .. . . . . "V 0.. 0 $0.. .- ... 00. 0 .. . I.. _ . «0. . 0. ..II ...-...... ... .. 5.0:... ..fiiv). 3.}... v . . 80...... .. l I... . . . . .. . . . . .. n. . .0 1.....05. . I. .... . 0.0.. 0.? .. .. .. .|..a......... ! .v. 57...: .LB .00 grill: 1. fl 0. 0 J #0 00500 9' .v.:.10¢00 . . $10.“, , . . . 0. 0 . 0. .. .... 00,020 . ' ... (00 0 0 2'30; 030000-7120 ...0 ..0 ..10. .0 .0... .0". 00.. 0 I A, «pvt-000"“ .031 ”0000'0—0-3-01 3.005.. 03 . . J0.0I‘..u0. 0 . 0.. .00.. ....s . ...0000 0. u u. i ..‘..0 . 0‘50... 00‘.-I.0‘....0v.. . 0.0 . 0. . .. .. . .. . . . . .. . . .. ... . 0 . .. .. .. . . at}... 0..: 00 00470300.}. 6 not! 0 .J. 0.00.. 0.905330 0 . . . . . . . . _. .... . .. . 0 ..0: 0 v _ I ..0 I .0 0. 0. 70.0.. . 0 I .-.V .00. . ... . . .0! 0.6.0. . It .. ...}. A...) ...0??0.2 .. .00 I .. ‘7! .00 . .3. ... . 500.1; . . . .0. . . . . . . . . ...... . 0 ....I ..0. I. ...I... 00 . 0. r0. 0. . 0.300. I 00. . 00.0.00... 00.02.01. 0 . .330. or “w. ...-..- ‘00: .... .0‘0 5. . . . . .. . .... ... .0 ...-27.70.130.100 .0 0. .0 0. .4“. 0:00. . 0 T. . . .33. 3. 00. y 0. T000030 ...-10.000.00.8Jn; .....0: .....f .. . . . . . . . l . 0 0 00.. z .0 0 . .. .. C «.0. ... . 10.5".1'0. ...: 0:00.000 . J ~II.V...0”.0. I..-p.00 ..0...000.0“n..'00=01000 . .0-u.0’ 00‘ . . .. . . .J 0.. ... . .01: . . 0 0.. 9. I, . . ..t00 0.0. . . 0..-- 0’30“.” 9. 30.90.40 . 0 0.00.3... 0 0 . . . .. . .. . . .. 0.... £15.. . ...0 #010... 0. 3V:- . u. .0 00 . 0 0 .. 0.0.. Q .. 0.0!).0‘00 0 00"...» 0.... ‘ 30.00.3"010 0 . . . . .. 1 0 ..0 0 .90. .. ...0 . 0 .1 v0 1 0.50.0 ...9000. 0. 00100.... 0.0I.'0.0..00.00.00 0. 0.0.. .v. 00 2.0, _ . £00.. . .000.10 0 3.03.0 00 . .0... 0 .. .0. . ...: t . . . . . . . 3000.314? .0 0. 0.2.20.0.‘W1 . ll’ylo...0 .0050 3". . 0. .. 0 00 . )00 00.0w ..0. . .. 0 ..I . , .v 0 .0 . . 7.1.“. ...WL... . . . . 0 '0 . . . . .. 9.0 .0 .. I.lII 0.. 00. . u ’0 . . . . 0 0 a .#.$..0|7l...0u1.d0h.00 9 1000!.) 0 .0. .0): 30.00.300.00 «000. 530...... . ... $00.10! I. . I.. 00. 0.0 ..l 1 x00 .0 . I. .. ., .. 3‘20... .00.. 0.1 .0. . .0 00 0 0.0 0 ...I I" t 11.01.00.510. 0... .0707 .103”: 0 . ..000..0..0!.00 ...00 0.9 010000 93.15001 0 03.0 .0..” ..0 I ‘0. f . O ..-0 . . . .I .. .. . ...: 1.303... .. .... . . I). 00 .. .. I .. . .0075! 00-0,.00. .. . 100.0 00.0 00 '0 00.0 .‘0000 .000. 004.00 0.... l :00. .. u . ....~ 0 9 00...... 0 0 n 0&0.» .0. . .w .. . .0 . ,0 0.2. .. . 1 .I .. 0.! 0r . ... 0...". I ...: 1:69.003}... ... . .0 (:00. ...... .. £01.37...me ..h. . 0? 0 . 0.....093. : ...0.w.€... ..0I0 00.. .... 4‘11: .0 £3.00... 0 - .2. ... . ..00. 0. ..0. _ 01000.00. iff: . 0! .0 ‘0 0.0 - .L.-..Nr 99,3070." 00 .0. 000001..) .- .. ..’ .0 0..0... 0 300‘. .. ..00. L0 0 0.70 0P.0I.I. I . 0. . . I. I ...... ...-001?..70791 :33?" 0;“... .70. J00... 0.‘.§ll£.!.\“ 0.007.000 . . . , . . . .0..§qu. 600.0 .. .0 .0 r0 ‘ ...0 .r .0 ..o ..--.10... 0.. .. ..0 .0. J... ...-.0000. 94.0... t. '0 00.10.34."- \.4 ... I“ . IV’QflQf...o......?.. r 71300.5.3: . . . .. ... . 0. . I. .. 0‘. ... .. .30. 0...) . . .....3 0.0.02.3".04 0 0.0.3... I? a). . . . t! 9.002,? 6 J“. 1:00:59. .. . . . . . ...0. 0.. 0 .0 I :5 .. 3*“..31... I . I20. I..)dwl. ...‘2: 9.er 0 J .... .. . .. 00. 3.. .. 0 . .0?! K0. . . . . . . ... . , . .... C9 .0... .0. .. . .. . 1.0.... 0 .. . 0.00.. : I .0 50’: ”3'0 .050.‘ ’fl '00-‘00. .9. .000. i.’ I In (.0 0. not: 00.... a. t ’,,w 0300“!»0 00 I..0 ,0 3.00 0.. .... I \(u . . . . . 0 0 .0. ll. 0 I . .Iu00000f0.. ... ...I... .0. 0 o. , . a. 000’. .- 0.. 0. . .0 . 0 0. . .1 I0 (or... ‘0? 09.0.10. 0. ..(l’!i0..0..000.0... 0... ... «00 5‘} .I ...... 0 . 000 {afluflnwcfhufi “31.03.... ...0... “090.449. H .... . n. .0.. . . . . !. .P.‘ stilt... I. ":30. .322030. 0. «V0 ... .0.» ......flFSV. v r I . . ....f 00".... . .. 0. 000.0 .1 .. . .. . 100.0009. .0 .0.0I0.. 0.. 0.. a 0.0.0 00 .. \0010 .. .0010 ‘v '00... .301... .0 00.. ... 0. 0 .I . ...0.0I .. .. .50.}. . . .. L . .... 0000 00.0 0.V.. 00. . 000 ! 00.00. git-v0.0.0.0... .30. .‘0m’00‘lw'td' ‘0‘ I . . . . . . . 07.0....4 .x... .- 0 00. ..l .0 ....0 .00 . . . . C006 . 0 .0 . ...‘V C... . 00. L .— ,. ..u......1..4..u..a...nuu.1....fi.. ..u. ..fi.§.$.ia... . é... ...... ....tu r . _ . . _ . . . . ... .. 3....-. . .. -... . _ . . . . ......... ...: ......e... .... ...r. 0 00 . . v . 0’ _ . . . . n 0 . . . . . . . . . . . .. 0 . ... . .. . . .1133... 5..., ......n.......... era...“ a: ....» ...“. 4 1. ...... 3...“... . . ... ...- .... -..... - Layaway... 11.35... .. 5.. . . . . _ . . . , .. ...; 1 .. .. 11001500 ,0 .0P.~. . 00..“ ....” .J. 0‘!!!» "001,". —é.f I.- {A} .9000 . “0 . ...I 0 00. .. I50. 0?; .00 I. 0:. :00... .0} x 0.... . an 00:000. ... . . . . . _ .. . . I . . ..0 0.0.... a.) ... 0 ... ... .0 .5000. . .. .0 .. 0.. .... . .. . 0“,. . .2908." A 7‘. . _. $0. .205“; to....00.0..9. ! .... .0. ...»! I.. .00.... .‘w. . . _ . .0. . 0... .. 1—0010...» 2.31.0 020...... .0833... I: 09.3.0.1...) 19...... 3.... 0.1.1.: ... 00.03.33 0.30.... . .. . {.302 3.04.»; 0 4.0....» 00.. {.0300 2110.0 . 0 ...-I.. 7.2.1... I... ..Q 3.. .....- 0 ...: . Lo. . 0 . . . a. . .....0 ... I .19.:30... ....0. t0 .0... ... . .0 .1.» 0..0l.. ..00 3.0.... 4.0.00... .. II ottJfi} Ud..."5? 0.00. .. :00... 01.00.00“ 0200 20.0.... 0.9.0.0 . 0.... 20. 2.1.100.“ )\ 000 $0.\. 0. 00.... ... 0 0 00. 0. . .. ... 0 0. 0 30.0.0. ....0....vfl..v.vz. .. ..U0P'.0 .0 0.0....) 2 .0057...’ 11...“... . 0...“. .. 000...”.9 $00.0 . ””00. .70 ...? -000 . . I..... ...... 7.l.$ T00. . ...-\- ..00.JV .90.. . ..0 I 0 .0 . .20. :05 0 0.0 I .0. . I. ... . . 0 0.... ... ...: . 0.0.5.00. .0: (1.300. ...90.’Io buffalv. .0ufifi0-J0mfll‘.gfl0..t. .Il . ”1.40.00. . Lil.u00..€.0.0.0003 ...-0.0a... . . 0000. a . 0 3.“! 0050.. 0 0.. 200.0. “0 0...\00P..... . 8.0.0 .0: r. 0.0.. . . . , ,0“. .0 . .700. . 0 v 2.03... 0.. I0. 91.40.... 0.0“": u L . ‘ 0 . .. . -‘ . . .. . .0 .1. In I . . . p0. 0000...... . 000 .. .0.0 0". 0.0.0.! 000...}. .0‘0 0...... _ 000. 0 . 1 0... luv 0 00.0 .0 0 - 0 . . .. .0 : 0:00-00. .0 0a- ..I ‘00" 1 0 00.0. v . .’0.o . 00‘..- 30.057. 05“; .00.O0. 0000, u 2 . . 0.00. 0.00.000. l... . .0... .... 00. ' ...0 .. ...-I . 0f. 20‘ . . .. .0009...‘ 0.. . 0 . . 00 00.3. .0000. 0 .11 0. .. 00‘) ......mbdwutu..nra.."r.fiu..a...»w... a.“un...s..a.....vr._ ”...... . . a . ...- 32...... ... . my}.-. . ., 2.3.3... LUST... ......i. .. W ... . . ..u...........q.v.ye..2...“. ......nz... .. . . .- .0..00..II...... .... $2013.! I....sa_..1 . 0 . T u . -.. .. ..- . . l. v . A .0 ...: I .35 ... .030. . .-.. 0‘0 \ 000 00. . ... I! v 0...0 0.. .00 3.0.0. (0. . 0 .0 . ....1.. 0 0 . . . 0 . O 0 0 ‘ . .. . o. .2140: v. I.— ..0.‘ 7..’p(.0.....l .. . 00....08.‘ 00000000" 00000.0‘000. 0409.010. ‘0... 0000.. . ..00 0.0 0 030 I0...>..60 .00“. .00 4....0..000. ...-.30... . 0.0V, 0. 00-0 . 1’.90V.|?." 00.|‘.0t.000 .“ 0....0I.0..U..3L.01.. .0...~...Ju.l.l..d..Juhuuord‘fluui 0..}. . .... . if?! I .92.... 0.0 . 0. 0 .. 3:31.... *0... ..0 ..0 39.! 8...... _ o 0...... ....0 . . . . ,. . . «r. L...........v... ......0 . 06.30 .‘0lu.b0l,a’9‘ ’2' n"!’~0lcou .‘0 ,7 a 05 I I 0 0’0. ’000 . ....0..0 ... L0 ... 000 It. 0300’... 00‘ 0 0 .0... I... 0’. o '0 o 0.0.; . 0 0.0.020. 00.0.00 Ilv .l . .0 ...... ...b. . . . . . . . . . 0 0.0....0.0.0’.0I‘ \‘d.l.’00‘.90 00.5.0 ~ h... ‘IJ! .0 0 Lu! 0 0 . 010 . {0.0.0.700‘0000' 0.31.0010.” 00 ‘ ...-P0‘0DO 000. ..0 00:0...4. . 0 ... ‘0 .0. 000......0... . ’1. ....Q . 0 . . . . . . . . . ‘.0....0I.0..0..0 0.0-15.0...‘0 0..0m1000.«00 .00... 1 0'00 L’3. V. 0 r 000000“.’~ODH0 .0. 0'. 00. 00 . . 0 ..!.'.00.. 0 120-0’0...‘0.0(10 I ... 0 -05.. ..a. . . .00013000 0.01000 . 0.1-1.0 0.0.100... ... 0.... . ..wt‘30.‘ .. ... . .. . ..)..u 0. n . . . .. 3 7.04.0 ..‘V. 0 Cl . 0. .v. .... "unnufisuvfisc... 91.0.33. .... :0.“ .....J. ., :, ..muauéaafljérv. ...... ...u... ......03 ...}... ....v.:..0..: 52.7.”; . . . .... :0... . .. ..., .. . . _ . . . ..fi. . . . . 9 .....u... ...... . ... ..fi..0.n..........\.. .....fh... . . I a . . 3 v .. I. .0140...’ 0. 0.. ......0! 0.00. ...0 0 .0. . . .90200 0.1.0. ...00. Q . 01.. .. . u .. .. . . .. .. . ... . . .. . . ,3... .. . . 0.... . [0.6.4. (.....ntbululaluhun. 0J.LuFUI.-wn0.l...huun0 ...00. . ....0. .. "a rlflvnflmron... us. . 0. 10 .lchu...»0 0.. ...0 II0..H... .0. 0.710.. vliflw..0010w20h0..l\.u..aw.. ..fi. (#03433. .Io. .. a... 04.. 70“»! . 0.0m..0~.1...n L0. . 0v . . .. 0 ? 1-H “10.13.".4 ...... . . . . 0 L . 0 z) ...ruwwzh33HL-fi0u40. .... 10.“.- . . . . . . .. . . .. . .3931... .00. 0.0. . .0. ... .. 000 . 0... 0.. .. 0.02.0800 ....m. . 0.. . . . . . . . . . .. . . . _ .0..- . .. . . I. . 00.0%” 0'0-.WM‘0.0.0.L.:. ”Una” .fl.0uouhu”uuul..nuid..om I ”outflow; 00- .ChrJfiplzufllnuut... '0.- On fl £{mwru.uu.l\.mdv .0 . .N....0.. 0... V I... 0 0'00 000.030.130.000. . . - 0....000; .06.. 0U..J00..67. ...I.. .00.. .adauurnuhwflC 0.0.”..01.... 0... 0 ... 000.“.r\.... nw‘ .0. .0 ."‘03 ll. .0; ....‘I. . . -... 400.00" ...0. .00. I}..I0I.01. 00... . . .. . . 0 . . . . . . 0. .. . . . .. .. . 0 .. ..-. f . :0 0.0.0.0.. 000, . 0,. .0.- .l . . 0 . . D ..0. 1 . . . ......ai ......enfie. .9....u..z.. .. .35., ......t...:..... .. . . .... ..z .. ... ... . . .....ua..n....nu.v;. .. o .............p. ......u than“... in... ... -... -... .. .. ... .. . . _ . .... .. [.0 00.0 00.1 0.“ 01.0..0....0.i,.0.0... 0. . .. {0..-.f‘000.v..0 0000.0)300..000040¢0.I00.000 ... .00.. 0.....0.a000..0......0. 0000.0- .. 8£.0.. .0 00.0000“. .000100: .00.. 090.. 0. 2'0 . .. 0 . . I00 . . .. .. I 030.. 3‘09 . . .I-i..l ...} ...0 ...: 0‘ ..L.....00.I'.00 00 .0 0.: ion‘... ...: 01.9.... 00.900 00...}.00 2... ... 1| I.. 0.0.... o ’0. . . 000.06“! . .. . 0 . ... . 0..l.l.‘n:..0r0 2...... .0 . 0i...000.0..000t.0..0u-..0.. 0.000.“ . 0.0. .....Lviau.» 3005-000. 00- 00,0... ... 0... 0.5.0.0.. . c 0 03” 000.0 0 0.... a . . . . .0000’0..'0 ‘00)! 010..0”0‘IJ.0:0I 0..!000'.,30 Qte0.r..0 all—0.0.80.5}00000‘009'0 .0 . ".0 01.00.03.031‘I.§..00..0.¢00..00.0.00.. 00$..2 .110... . 0 .00‘... I .- ‘0. 0. . . . ...-000.20-.. 0 .00 00 .00.... 0. . . .0 .00..0. . . .0. 0| .00 . .01. . 0 0.00.0.0 30.0.0. 00 a. . .1010 I..???t... 0 [-031.00 310... . . .... .00. .-..o ......i: . . .. 0. .0 .' _ 9. ..000. . $3.01.. 0... DUI-{904.0 . .. 0.0. 1-0.0 . . In . . ".0 0x00 .' .0 0.0 1". ‘0 0.- 10.0000..OI¥.00 l 000 0‘w'300 00000100.}.0'000. .. . ...l...Q00l .30.. 40.0! W" .0 0.1. 0...?! . ‘f..!...... 7.00. 1.3.. .011. ....t )t..0.:..0....0.....00>.. .0110. ...}...29 . .010 0 .o1lwl.f.‘o.l.0.0’bl 0. 3.1.0011 ..0.-£’Jf0.|.0..05 :- 070090” 03.0 I .0 D . . . . ... 0 i .‘a ...),iio. .. ”0. .00... ., 1 I 0.0...00 ..0; . .0 5.0.0 ..0 ..- .. .0‘fi0’..0II-V00fln00003 00$“. .0 .’|.l»¥ln lw‘0 ..o . 0 “0.0.1 0000‘." . 0 . I. I .0 0.0 o 0 000.0. 0 Q... . . 0 Ir 00 I. . l .PIJ n». 9 00.2.0... .\0.r.Ila .110. .. $0.0... - 01 I I . .. 0. . 0.00005..- 0.00 0. .... 0Q . .00 o .0 J... .. 01.8.. . . .....0‘ 9"! 0 I’0\0I .00 0 ‘0‘- 0‘. 00 0!. €30" .00.:70.‘ 0‘0“”...kr0.,.$-.u0.00.0.v 0 . ...0r00000n...000.,0 $00.0! 00600100.. 08.00. ’4 . .i’a.’ :0 00.0 I .‘0‘c 0|.“ .0... .fL sir-0!. 0 1 1:0 000‘ 000000 00 . :0 I. 0‘00. .0090".'0‘0‘0 V .0000 00.0.00... '0 0.”.0 I1— .. .0... .... 4.0.0 0.800330. .0. \20300. .10... 0 1.0.4...- 00 Alli . .00....0. ......00... ...0. J. . . . .0 00.... .0. . . t. .0 0. . .3. .-. .... .....n. .....a "n. ....nwun ... ufi..§uuuu....z.......and?! 5.2.2.303... .. i .... ... I . . . ... . . ._ . .... ....» s ... 000. .0 X 0'. . . 000 . 00 .0 0.0.0.00 .... 0..-00,7 0 . . D .0 . . . .. ..0 . 0... . .. . . . . . . .J) .." .. .000 L . 00.. .nvo 1a. 0.0 . It.‘.l.03.f.u"0 FUJI . roughiifluww. 009”“; 00".0NJ..P’.0..00...V...000000.. a... 000‘..." .0”. ..0.. 0 . 7| 0. . .50. . . ,0 .02.- . . _ . . .‘w .l 00. ..SO .0 .00 4.00.0 . .0 0.! ‘000'. .0 00 0*. ... .. .‘0‘0 .0 a; 0 .0 .0 b ..0 00‘!I‘0f..).0. 0.: 0.0.000 0’10... , .. .. . .. -0. ’ .O 0“. . . .. . . . . . . .. . . ..I00; .I 00 V7 -000. . ..0 01.0.50... 0.0’ a) 0.64.}. .10 390103). 00 03.00.03. . 00. 310200000”. .0003. 01.000000 .0301.0.!000«..0.2 . . . . . . . . . .99 .: w 0. 0 I . . .I‘U0 ... ....0; 0... . . . . .031... . . 0‘. I L0. .0700 0.!!0 ‘T...9.lr00 .lli30.uoi7-|.2.IO0...0.000..1l.. .00 . 6.0.1.900. . .0... .l. 0.00.... 0.007;}..‘0‘O-0 . . . . . 01.. 0.01... .0... . .. I 0.00.. 0 . .0. 7 .0000. . .. . . . 5.0 060‘ 03.0 3.00.0 3v.0.0..0...§0;0.0..0. .30. ....0!..DO I03... «0.15.3I..l.002.00.. 0 .. . . _ . . . .. .0... ..V. .0..K.0.0. 90. Y0 . 2.0 .00 . .Q , 0% I.. . l0. 0 I. \.00\.00'9’0 0 0 .. . 0 .... \ 000,0001‘70‘0 ~0390 00.40.000.09ra‘ 0 00.0 ’ , a 0000 .010 0,. 0 . 01.30.. 0 0 I 10.010.000.90 .00 . 0‘....1...0 13.1.503 .0055... ..t. ’00... 8.....1..0.0..O....0. . .. 103...; 0.... .... 0 . . 32.00:...0 03.0 . . 0 o {0.0 . . . ...I .. . .....0 :85. .... 0... . . 00. 0.700.00‘0-.. .0 I , . 0.00.00.0L .0 .. .. 0. 00 ... 0.0-1.2...- 0...- .. . . . .II 3.. ,n\ 0 . ..t .- . . 00.0 '0 0.00 0 .§;I~P0).0.0.,033 . 51%-. 00‘U000000‘ 000000. ..010.".- ’00900 00 o. I. ‘40.,5‘001 m 0 . . .. . .00.. 1.0 A .-.“.I .0. .....01 .. . o 0 009.9 . 0. . 1.00.01 0.00 .dwU’Q’02I .00 00.00. 00 3 .0!.I.00"0.0 ’0. 1.0.0.0700. 0 000.‘ .‘I 0.0 0.0. 00L. . . . . . ..I... .00.. A, \H. 0.. . 0.... 1 .0 1.0.0 .. . . 2)) 0000.0 .00.“. 0......‘0..(.000....00T . 200'00010 300$... ..Iwi... I..... 0030.. .. . . . .00.... .00 0... 0.. .30. {VI-.0 ’5; .. 0.0.20. 0.090 ..- .. . 10. ).0.0 ...;t‘lfiy’ 30.7 . 59.10/01}... ......Ioi .....1’. I. . ) _. . . 00...... ...-0. ... .0 l 3.16. I. 2 0. o . .1U0.\§..d 9 5.5.0:...00000 0... ”010000.010. ...-0000. 01...:- ..0._ ...-0. . .. . .....00...0. .. . .. .3. £00.... 3.9.31.0“...- . .0..’0 00 I'll .0’1'05 0000.. .039 .0. 0.. 0.! . . . ._ . , . . I . .00 ...“.w‘IDI ‘30.. .I. 01 0.0.r0 3 9 . . .fi330 .0I..vu....0..l.)3~ 0..); 50.0....- -.0.I7.. 90.00 0.35.013..- 2.0.0 . . . . . 0. 0. 5.0.10.0“3 ..u . .40.! 0.0.. 0 .....00. .0... . {904 ... . v... . ".ou ....h.0..!000.u.)-0000I....0fuo as“..u<....«,d.0.0\.lv0¢..u._.n ...“. “nub-1.00 . .000... ...... .... 0..-.0... . , 10.. 0.0 . 0.. .... .. 00.00.25.130... . ...00...‘ 0.000.300.0233.“ 000.04... .0. 1.3.0.0414 ,. .0.0000..0¥.0.. L304..0000!O-00¢000l.. 1.. 00A 001 10.0 . . .. 3.00 .. 0907...: 0.0.0.... . . . 2'0 004001.100” 0”: ...u 0.! ... 0. 0.0” 30.10,..70.’ .. 0 0000. . 0.. .n .190 . x .03 03020.0.- k.v 3”...“ an" ..ti...“. 1.x. . 0:31... 00 .1...09.r0.\0... ...... . 0.., .0 0.. .. .. 30...... : 0.. .. 0 .0 $90 . . . .0‘I‘....00IO‘M. . ’00. 50’ p . .-....w’ 3000 .0. 00.0 I.0..)0 0.0.000 \ ‘05.... 01.0 000:3. . . . .00.- . 000 00. I s . ...... .. ...s-fifiJ..0... ...-0.0.. - .....0.» 3... 3......” 0.0.3.0.. .....r .0. 3.2.0... .0. 3.0.. 13.30... 0 . 0.003‘500 000. {-0.1 0‘ fig]. n00£F0.Da-0000:0 .....i::.‘:: .0000. '.0.000000’l‘:0..ol0a.0 . . .. 0. . 0 o)...\.000{ «I. 0 20.0.0! 0.250400... 00 L... 03000.. 0.. 00 a 00.0 0.0.0.0.. 9.0 .0...- 0... 5. . . . 0 v . .0 . )0 ... . . 6.00....N0m: . 8.500 a 00.0(I.’.v 6? ... 0(0’03’00 0.15.0.9. aid-...: ...)?!000LI000E.‘ 0000...: . _ 0.30 .$ .“deto . .... .0: 01.0 . 0... L 44 . .0 . 0.00.00 .0 \ 0 0. .....w‘)...’ .90 ... 0.. 000.00 .07.- .0030...0!f.0000'0.1. . . . .0520 I . . .03... . 0.16.000 3.11.000... 0 35.31, .0.- ..0. .0... IQ .00. J .I“’.... ‘4’ ...-.0 [AU-(.0. .0. . . . 001.3300: . 00 . 0.0 ...‘0600000.}!3.:.’.0!.00‘..2.0...v‘aafil 00.... 0’00! -.- .30 .07 l .0.QI 0.000 00.000. 0.010.}! .... . ....0.1I. .0009?! / 0 00. .0. 000." C. 0 ....0 .0.I.. 0.90: 0.0.0.0.. I 09.. 9‘00 0 . . A. 0 ... .0. . 00v.l’0.0 9!0..$10.0.00-00. 000 .0}. \r .0 . ‘0. 00 . .0 . . . . .. .0... .. . .iv. a 0i. “.11 005’..00.0 . . .0.000-l.vn‘ I..,Qo00u...0~...0uullu‘0.00Jw0.N.f\“ .00 ".0......0NJN01UEI....»“deiflrulwHMfi”N’|.0.-0.,0WMWM0 0 0.00-0.00?! . 0 .0 . . 0 0 00.. 9:00 . . . .. .. . . . . . . . . . . . .. 3.00.0 : ..mfié......”.....u........fi-Lani... a... . ... ......i .. «.....ur.......n....fi.u ......x...: “.55.... .r ......sfu .. .. .. . . .. . . . . _. . . . . .01. 00‘ 0..-’0.» .0 fl. .1 0020.000. . 20.000. .10 . 000 :3. 0 I. 00.011000. ..0 010.0 .0 ...00'0. . ,0: u 00 .m. 00. II 0 .00 .0... X . . ..JJVLNJwAJtpYJWKHNNmflna _. . . . . 0-00).!9’.0_. 01.0 0‘00. ...0. >13? 00. . .0. s .00 '00.... 00 0'00 0 .0 . .I 0.0.9.100 10.3.! v 0.0 . 03.0 .10...‘ 0.00000 .... 0..0 . . .. . 3.0.0030." .. .330 .1 .0. 5.30.0000"... ..‘0.00 0|..- . 0 0M1. 0. .00.. . 0.0 . ..00..\ 0..-.00.). b. a . . .. .0; ......0 .0..TI.... ..00 ..0. II.’0..’000.A0 l 000- .09 0.0.. 0|...l. ... . .‘0 3.0.0.000! 0". 0. . ..0 . 0 v a. . Y . .0 0 .l!..il0000 .000‘ 0 0.0 ... ..&.0 00 - ll 0 . . . 0 I . .. : i ...... .... .........a.... .....3. 3...... .. .....n .1. ....u..?......r.n..........5 .12... ...... _ , ... . _ ..00 I.. . .03....0‘0. .0 3.01.0.0}... .0. ...-.0239“... 03000.00 0'33..0:....0..0“. (\30..0..0..0 0. .. .0000. .0 I.. ..0 .u . . .. J0. 20.... 01.0 . .. .vrb’.‘ .051 ‘93300. .00.. 003‘li3’I 0.0.. .F 0...... . ..00 0.., In. .‘000‘; .9. 010‘ . 0.000174. 0 0.I I). 0 3. 0.. . 300000;! 0 0.0. ... .0... .9 .. ... 03.1.0 . 3. 3.00.0... .0. ..IOZ 00)... ......5‘0 00 0.30.04.00.30: . ...-I .00... {Q .0 3.0.0.0... 0...... ...0‘.l\...I0.0... . . r . .. .l.....0 ."rug .... 0 . 4-. .0 . 0. ..9 .0300. '0 0.I‘!.I’O.. 00 . 0300.0 00.0.. 00.0.. .... .0 3.1:). 0.0.00 L. II. .00 100:0 0... a ..0 D‘ .‘0... .I 1-00.0. . . u 0. .\ .... 14.... .0 0.". “0a.... .1 0.. .. .r .0310 -\.}ia....3. . 0 . 10.00094 In ...??nullluzl.“ .. . . . ._ . ...O. .. 0a,. .l...0 1 a0 00’- .0... . . . .. .I...V. ...-0.00 0. . ' 00 . 0.003, 0| "Oai 301.0 I . . . . . . . . . 00 ...? "5.... . . . _. . .3 .2. .0. .3...” . . . . . . . (vase... ...”...uuu . .0000. .. . ...}... 0.0.013“? . 2 I. . 1.056.‘ .30 300A... {3000 0.1 .. 0.3.00. 0:00 0 o 0 0.0 0000. 0 0. J00. 70/0 2000.0 0 ....0... ‘1 _'.v..-.VO $0.00 .0 «I . .. I 0 . .. .v).0 00 0a 010310.“...0000 H0 . 00 . 9.0.000\ 0.00.! ..00 9L0. 0 I .9. 0.. 0000070"- . . . ‘0}. . 0 u . . I 00 p . .0 . “2.x“: .Jir‘. 3‘0 .‘0000 I . 3000020. 0.0...0. 0.00 . ”000 ..0-0-0 0 903.900.1000; Dunk-0 000 0nI00.. 000.1000...» .-0.‘ o. .. .. . . M ’ .fiuyuvr thWI‘ 0.... A. 0 .00. 0| 0. . 0 ".0000 0 . 0 .0 .0. .. . . . .. . .. . .. . . .0... I u . . . . 00500....10 .1 .....0 r. 3!. 0.1 .00 .5 .. ...-)0llr00..01 Oou‘.0ll’... . .. €00.00 . 0.\0! V 000 . 0 0000.00.30- .s '00 ... 0.0.0.7.! 0.. ‘... 0.0. .I ... . 0.0.109"... (00.0.0 1.. . . 0 0 . ..0. .9.»0m.0.0..'... 0.000.‘001.0\ 0.00 000‘!VI . . . . . . . . . .. 0000 I. .00, 00 00.0.0..0000.I.'. . .00 .... .0 I... 0'00! . 0.0 ’0. 00‘ 0 ... (0'0 .3004flow0‘.’ 0.00. 0 ..l .100.‘ 03-010....0 ... . . .. . . .. .. .. . \ .30 v.0..'lht\vuu'w.fl0n.lo.u 00..0.»'(“M00....0."~u0.0l000 0......7'00. .0 .0. ... .N.0”JID 000 ..0 4.00 .0 1 .10 0 0 0'0 0 . . . . .000. ... . . . . . 40.000090} . 0 I .. 0. . . . . . ! u . I . I . _ . . C. . .0 . 00'” .... .. 0 .10.! .0 000.! . 0 . . .. 0.9.! . . _ .4 00 .$..¢..I.J...00 ... rumba.“ 33.0.... Won‘t”... . .. .. 509."...1003 51130.30 .. 1 . u. .. ”...; 00.0“. . 3..-... 0.00 -0“ 00... 0|".- .1w..l. I..: .0 1... 0, OIL-I..; 0 20. .... ......it \t...l‘.n..:..”fl. “6”..qu .0.0.l.00-0..l(l$00l-l . .0) ....V ....0.‘ ....0... . . .. . . 10.00 0,0 .01... 0. 4 i. 0 . .0.... 0... 0 ..00 0 ... u . 0 00 0 . .0 .. . 0 . £9.00 ... . .0. 0 I .0. .. 0. 00. . ...0 0. .. . . . . . . . . . . Q. I...“ 0 . 99... 0‘00- .0. .01 0 000. . .r .0. . _ . . . I 0 ....)‘20 .. .0. 0‘14 uifi.”uh.mnflfln."wh.fl . . . . . 00.. 00 .00L’fl' .0“.-. O . 000.0 I.. 0000..., 0. o 0 10013 .W.‘ .00 ..‘00H’0L000l 0.0 Q P...0U\0... 00H“. m0 .00... 30 O: . . 0 0" .0 .0 .00 .. 0.0 L 00.00» ,..0I.00.’..000...1Q . .9000 . 0.)”.0“. ...“..- u-W. .. IMQQIIW; . 0.....60000 . . .. .. ~ .. 0 80.. . . 0 0 0...“..4001Is ... l H .0... . ..... ....0 ..Mb_\:0.0u..n 0.00.0...an ‘\ .n . . _ . . . u . . f‘VI.0.0\ . .01. A .s 00 .00.! . .0 0. 00...? .vvl.b.o.. .. .I. .-.-00.. .IILN00JHH .v. .00.. 00 . . a 0. ... . . ...-0 . . .. 9 )‘0 0 0 ..q.l.0.0094 00 Q0 T 0. 0'0. .0 I. . 0. . '0 0 . 1 . .0 . D ‘ .0 0 . . O... .0 0. I 00. .1.0 00 00. . ...0... 00‘00.. 0.0. 0‘20... .0 ..I..0.00'.. . . $0 a 0'. 10.3000030)’: . 00 . 0 0 0: . 00 0 00 .0. '90., 0 90 0. .300. . 0...I0 .0 ’0 00:0 0 . 0 I1 _ . .r m. (II... 0.0! ~ ...0.0.%...0\0 {In «(L 0 . . . . . . . : 0. . 00.. Q . . 0 0.0000...0;00.. 0!. .0500 0.. u .. 00 .0’0.. .04 .. r0 0 0 . . n . . . I I . .0 i .04 .0.. 0’51.- ..0 . . . l0§'0.d.0.0\.000.040u07ng0.w: ”IV-$0.10- .. . . . . .30 0.! 0.000. It 0 . 0.0.0 .0 '0 .. I \ .0 0 00: Q. .0 ... ... 20.00 .. ...! He'd” .000. 9..- I! .0 00. 01.5“. 0., .0‘.’ . .DrQH ...0. ...r 0 . . 0 0.... . .>! 0, . .\.I)0. 0.».5? . a 0.0.0 I..: I I . . . . .. . . .(00. ...1... . 00. 0..}... 0 .0 0:00 I 00 Ol. . ...09. :0 . . .0‘. . I. .. 0! .‘n . lo . .. .. .- .0. . . 0‘0.....0\‘. ..0.. 0...“on 0 0 . . 0 .... . .t0000001.70..0r.r0 . . . . . . . . . is: . 0 . 0 . 9.0... 00 ..0. 9. 10...... 0. . o I .1 . n .q. . . 0 .. .0 0 u. .0 0 .0 . I! . . . “up”... .u. .u . ...0 v .00.. “$0 0032.00.10.00”: .0II. . . . . .. ... 0. . v _. . . . . . . . . . . . . .. . .. .00..- . . ...0 .500 I.. 0. 0 . . . . Q I.. . .. :82... .. ...“udénu ....i . _ ..ufiflunba....:nn.§ . . , . .. .....3: .....- . . ... .u ......“ ...... .... .. 42... c. . ......2; .. - .. : i... .. . 2 J 0.!0...|. .. 5.0.x . . . , 1 1.0.0.0.. 30.3... 0.310.)..0. . . . . . . : 0 ... ..hNWflI...0-.!0I. . .0 9 r). C)... .. .. . 1‘. . . . . 0.00 . . 20.0.. a. 0. 90 0.. HQ". . .0 . . I. . "‘1". ... I... (DA. (0.0 . 1.0 0 . 1000...... an 00!.{03.!§.000 00 .04... ...Q...0..‘00.0.1.}0..I.00.. . . U .0. .000. '0 0..-‘0‘... 0.. .... . 0‘ .b‘sb’tol .107. .00. 0.. or x-.. w .. . 00m... '0‘ .00 .... 0.1. .a. 0. ..‘r.‘0.0(0... . .(s 0000 r1 (0,0 0000 10.00.00.000: 00....‘. II..0.\U.0I‘. . I 0000 I’M... 0‘0 . .00).! ‘00.... . . ‘4‘: ..0. 0 . 0.0 0 0'0 . I I.. u \00. O 0 . . 00 .v . . . 0.0-. ... . 9 ...) 2’. )- ..ICI 00 0.0.0. 0 fit... J... . l 0000‘. 00.0.0.1..00 0 ..0.7.01..30000‘. .0 0 10’... 004... . ,0.’0.00... 06. 00‘. . ’ .04.!0 “0.0 00001.00... 000 .. 0. 0. . '0 .3 . O . ..Q 0 l0! 0 . 0 O0 . 0. . . . n . ...~.\ ..00 ..I. 0’. . . x .00 .0...- t 7 00 0'.0%_I¢v....1 00.. .00 O 0 . ...; 100.33 .300 1.00.5.0 0. . .0. 3.0 . . 0 ..0.J\ . .0 0 . .0 0. . . . . .. I ... . .0 . ..0..1....0. . 0 .n .0. . ... 0.0. .0. .. . 0.. 00.. v). . -. . .... ..0. . .2170 ....0.’ .. ..“I 0"! ... ‘70....0 .00.0.00.10004’.I.0«\.’0£.i .00. 0.].f .300l 00L 0 ... .i 01 0. 00.5 I... 2 90 D ..00 03“.. 0000 0.. .0. .0)... . .0...I£0000.0. .F00070‘01000 ....n .0. .0..0.0.D. 0.0- 0.. .. 0.0.» 0' 0:0 00 .. \l. 20.). , .P....J. .- . _ v {.00} O. r .‘0 0.. 00.0... I. ...00. .1‘0). .00 0.5 0 y 00' . 1.... y 0.0.». ..0 00000.0. 2000.0 . ..‘20. .. 0‘0... .0 IQ! .. . 0 . a . b. .0 I'.‘ .0. 040.... ...b I ...I 00 0 pIO‘. 0 \00 0.00109 10.0.0 00 .00 11.0... p. 0. 100.01... . .000’.0 00; .. . . 00 .\01"0 . 0.0 ‘320 .H 0200 . ...... OI. .. ’00! 0 A 0 0 .0 0 .I..0l_. 0 . . . .0 0. 0u0l I.. 00‘) .00 :0 0 .0. T 00-. . J’s-.0 .0. 00 DI.-0..a 000'... .04 .....JJ. 0.. . . . 5).. It. . . 00.0.10 . 0.0. 0. . . 0..-0M." ... ......0. ..0! I . . . 0000.00.94.13 . 0 .0. u 0. .00\ I. ‘ .Q .00? .00- .. ‘00. 0 \‘. ‘00.. 000.0.I.\0._'30‘00 000v? v... ... .0 .0.V..00..0.-.C.0000.. . . ,. O. . .0 0 .. .. . . . I. ..0’1.0\00I3 . 00’. 0.0000....~ .0 0 . n .. .30 0 .....4 00.0 0 000 0 ”I .00 0 00-. allzldc.‘ 0 00.00. 00.0 . .00 ...0 00$... .0... 33.. . . . .4 v .0. a. 0 C 000.90 0 0 t. I ...: .00.. 0.. . 0.0.00 0 . :0. . 6.1 .O 00- I.‘ .. . .. . 0 0 3.00.51... .00. 0 . £330.... .1. 0:0"); 0. ...-2...: 0.. 0.300. .0 . £00.. I:.L\... . ..50r‘01....0.0. . . . 300.0000.00 .0 . .. .0... . 0 .00 0 . u .... . .\ . . .. 00100. .. ... ..II..0..‘. ...... .. ....0. «1.0.7.2033... ‘03.: .0 o... ...0. 0 . 0..-0.0 0 . . . 00.....0.....1.m 0000:..000.RO.I ., 0‘... .0. . .3003! .. . . . . . .0 ....000. 0... . . N ..9 . 0.. .0. c 7? . . .0 . 1V5..- ... .lhz0.... ...: .ihn.u.l.0t:l..t 3......91. .... . 0.... 1 5.30.0.0- . .0... .. .00.... 0! .... .0 . .AV (00.4.0 \01 a . . .... . .L . I 0. ... ..Q... . . . . .. . .09.. ...-0. 0 . 0 . . II. 0. £0 7. .lf.‘33~.n. 00‘. . 0. 0. 0....) .L.0000..J..f000—9 . . 0‘ s \f..'..003 4.9". u. . 0 .00 .0000 .. . . o. 00. 0). I.-I.’0..O0.val 00.0 0"0.‘. «000000 01.00. l” .0004 .. . 00’.00 . .. 0.00 00m ..000 . 0 I. o. . . P. 0 ...0 00.0.. 0.0.0 0009‘ _. 0 0.039.002 .... .... ... 42130.0 0 1 .. (1.342.... 031). .0. 0.3.0.“: .00.. I0. 01.3.1.7”? I? . 20.70.. 0,40 ...0. . 0.001... 0.51.0013... 0 ..uJI 00. "as . 1-..... .00.. . . .... 0 .... 0.. . 0.. . . . r . 0. . 00 '0... .. 00. —I ....- 0. \u. 0 .. . 0.1.0.0. .3 V1.00. 0.0.00.1...0 I 01.3.. '0 00:3 2.. ...-.0. 0:0.u000 00:0 ...0.00.0.r. . . . 0.300. .10.. 0 . .. . . 0w: . u 0 0. .0. 0.. . 000 02...: 0.. 0.0.9... .‘I. . I . 000 0 . 0 P I. ...00 .N. .000... fol-.00.. . r... 2.00 0...}..l..(¢..10.00..00.0.7000. .... ... . ..~U30. $101!.) «$00 00 ... . ...: . 0).0.~ .01.... 0 0.» v I... . I M.” a . .....t. 0 .. . 0 . .. .... .. r. . . .. .. . 00 I I. .0 r. 0. . . 00... 90". ...0. .3100; . I. 0.‘.$0.¢....‘.9...l .01I.I’\...\..O.-. 020.00.00.34" .... O00..00000 ubu‘s ...C..0....0. . .0 .0.00 .0 00. . . 0 Q. . 000 u 0 0.000.. I. .\ n10 0.9040 .0. ’0 000. 000.0 . .0 . 0A....00.0*3r.§..01. ’0 0.0..r100. I0. 0. ..pI .0; 0.. . I0 . v.0 . 31”....00 00- v. ”s. s 00 “I... . 0.0.: .. 1.0000" 090. 00.( I0. .0 .0. 0 . 0. . 0. 0 0.. 00 N 0 0 .00 00.. 00... 0.0 0000 0.. .30-; ..I.\0000. ..IAQ'0000.*.. ..0 .. . ‘..‘.10-00 .... I... ,Q‘Iuyrrfl. 0.....0. 70‘ 0.0.009”) v..vm 0:, 00. 0. .00 .... .DO0000000. O. ...I. I ...).0 .00 0000’ 1.00 . I 0 .0. . . 000. 0 v .00.... 01.000000460‘ ll 0”..§.-. 0'... O 0.10 v 40.0.0.0. . 0 it'dfi... . 0 0 . . 10.. ...... 0.. :0 l . . 0!: 00? 00.00 0.. .. J‘I0‘ I 0 9 'l .0'. 00 .0 0 .‘0.‘0.§In pI. 0 It 00 ‘ 0 . .00. .$\ .‘\ ' 0. . . 00:“ . ...J.'.0.|\0o....0 l.0 . m0} . 1 .00 C. ..n 0.00..- . o I o .0. ..VC . ‘0 I. I, 0. .. \‘0000 .. 030- 0..-...... ...! . .0r..n.\..§..0'n..00.n 0 .00”! 100. ....0 Nut; . . 8.0.000 . ..0 . . 09 c‘ 0.. .I . . . . _ . . ... . . . ‘0 ... 0 . .0. 0......f00. 0 .. . . 0 .0 005.009.00.041 ...0 1.0, .. $0 00000) 0 .00.! I . I . . 0 0:0 .000 0 . ... 0. - .... . .. .. 0. ..0. 0. 0‘. . 50.07;." 0‘ 050.0 ‘:.,:0000‘ . 0 .0.’0J\0 .0 £0.00 000.0 010.‘0.I0.. v0 .0 . .09'. 0. ‘. 0"0090. 0.010..- . .0 .u . .. . 00 0...... 09.00 .0.._I. . u. . . 00 I ....Q. ... .101“: .. .0. . ..I 00 «I '00,.000.10 . \0..,.0.U00....00. . . 35.00.. I. .. 0: 0... ”.0 ..00000‘ 0 0.0 . II . .. . tr. 00..‘9 .0 E. . 001050 0 0 .040. . ..70 00 0 . 0‘ ,0 .. .0000. . 0.0.0? I 9 .. 0 0 0. .0 . . 0 . .. . . ... .. . 0. 0 ...”... q. 0. . 1.0.1}: . If. .‘0N3 .0..0n.\...... £1000: .... 02?. 30...... .0. .9; . . . 100.000 . ..0 0:101... . ..0 I. . .. . . o. 90...! 0... 0.0.20 . ... 03.0.. 2 ‘..’.\.0 0.0 . .. ... 0 \s . 0 - .. .00. VI...) 0 V . . .0. 0.000 . L.f..0 . 6.... ..0..:\00..nv..l.. . O: .01“. . 0.04 071.104“... .. 0.... .40. ......l.‘....0......0 .00.. 4 v 0 .... 1.3.1.3.... . ....n 1 .0... .. .... 0.0...“100”. . . . . . . . ..I 0.0.0J00 00.. ....(. .. .. .. .... . . . .r. . .0. 0 .. . . . 3 00 ...-0. ....O 0... . .. . I: . 5. f0.l..$....c.0..... ..l! 30 L ... 30‘ 3:1. 0 . .00 .01]... 0.00.3.2.- ..00 I~0.II.1.. .00. I0 0......0...&.h. . 00 .. 0. . . . . - .0 ... .. .. 30110 .0120. . 0.01.... .. . .. . . .0 .30... 0 .... .V. . . ... .. .....I.. . .110. . -0..\ . . . I.. r. . . 0.0 . 0 3.00000. .0.J0" ..0 50000.0 .‘00 0..” 0V ‘...00I00. ..0....0 0.... ...00‘01 . 0.):0 .00‘?. 0‘ 0.0 0.0.0:.0 a (0.4.09.0 O ..90 .0 0..\..A.‘. .10 \. 0000'l0 00.0. 0 ’i . 0.0.0.00 I0..0 .. .00..0.. 0.0.: 0 .0 0K .0 0 ‘ V- 0 00 ‘00. 0.}! .33000‘... . .. 0%.. .0 0.20.... 0”0§h.¢..\0 3.0.251: .0“....0!.009 ..0.~0.0.0 . . .x|.90..0.v. (4...? ...-.0. 3.0.0110 2:909. 01, . 00.... 0.00 ..n 00. ... 03.9-0.0 0 002.0. . 0 0 00.. 0.0 0. 0... .. ..00 .0 .. f P ... .0.. . 0. 0 .i .02... 0 . L . 0 v .0 . . .1; 0... . C 1.00 .0 .0 0. ..‘I‘-.Q(. .r . 000.04....0 .00206.‘n.0\\‘0. 004... .0400. . . . ..‘..0'.00 ’. .0 s 0 . 0. 0 .40.. 0’0 00 .00 I0.' 00.0.0 W0 S . 00 0.00 . .000 0... 0‘0. ...00 . 0!.‘0020 00.. I0 0. .\0....0l I. \4 .0. a t 0 .. 0‘ .-0010300I' . 0 0 o . 0” 0.0.... .101 . 00 0. 000 2.0.0.. .000... 00 . . 000 . .0 0.r0\ I.. I 000.0 0 ..90 n . . 50.0 .... 0 ’ 00.0 ... 0 0 . 0 .‘I...’ ’.... ...00 .00. If... .0 ... J'OI.’ 01000.. 03,0 .....0 . I0 001 0.0. . . 0.50.. . ii... 0.!\..O 0..... u. 0.. .....- . 0.0330310... . .30.... 9.0.0... 00. c 00. ..I 0.....0 «0.03% .0. .0 ... . ..\. . . 0.....0... 0. \0.\00.'.. ... 74:0... .3000....‘... ’ -.?l.. 3.0.0.30 .00....r 9.7.. ..0 .00 .. 0... ..Q. ..1...0.00‘..... . .. .. .. V Cult-.... .0. _ W . . 0200 . -0 . ... 0 03.00.00.007 IQ§~Y1 000 . . t 0 (090.03“ ”L 0 .0 .0 0 0.. . «1.0 ... y ...-0.. O. . . . . . 0.0 . .. . 70.0 . 0.5L.) .0 ...... . . . .00 0.. .. 0. a ..0 :0. . .1. . .0 . .. . . . 0 9.... . . I0 0 . .0. 0.0.! .I s 0 :IIHHPQ...u". n.... . )0. .20..“ Q.“ . 0 .9010“. .. .0 . ..0 0. . ... o ..0 . . .. 0.0.0.0 i 0- 0 0.0 ' . ...00 .~.,00.00 .. 0 ..-... 0‘ '000‘.... 0 O . 0.0 .. 5 \ 0 00.. 040 v .1.- .0 ... 0 . ... .0... o J. .. . .. 00) 00 0.0 ...t n. . v.70 .. .00 .‘0’ 0 300.. 003.90,. 0001 000.’R.0 0‘00! . 005.. V .00 0 0r 0 0 - IQ0I.0 .00I0‘: 0 . . . . a 0 . 0.1 0. . . V. . . . 0000 00. . . . v . v. . D 010.. .b .. . . . . . . . 5.. 0 {0. 0..; . . . . . . 0 0..0n\ I}..- .0 .. 00 . . . .. . . . 0. pt. I: ’0‘ 0.. 0 0 .0.| 0.1,... . 1,000... $ . .I . .-.: . .. . . J. I I . . . .. .. I 07.0 ..0 . . . 0 . 0000. .0. l.r.0..'38«900.0.0.03.9.m00.fo 00.... .0 C..r‘.21.0.u..xnofit. ”00.0.00...1\ 0.010 20 .200... 0. .0 . I .000. 0 0), v 0.0!... ... . .0.. .. .0 . 0 ... v. . . .. . 0 . . .00.. x. . .Wo. .0...LI.|1000... . ... ... . . . . .1’.‘ 07.9.01. .n...}0000. {2.400000‘00330. 0.900.}. ......10.0.0... ......kfioo..l0 0’0. (9‘? 0000”... .I ”.30.“! . . . 3%... ..00... J0~.x000.0.0.00. on, 000.. 2. 0 0 . ..V. D .0..I0 70... 0 .0 ..0. $33.00 (2059. 0 . .0031: “0 will-v.00 .002.3._0v0...ib.0._b 0000.10.00. ...002. 00.00:! . 7.0 . ..0 4t ..00‘ .. 0000 -0. 3.0 00.0....“ 0 . . v 0‘. “a 0... . . 0. . J .00. 0.0.00.0 0 .0099. . . O0 .... 0 . 100.. s . . Q. . {2001...}.0 .......0..\b .. 0:40. .0 0‘..- .. 50.3.1.2 0,0,.00.‘ - 0 ... I. ..P0 .A‘.0...‘0.00|.C . > .. . . . .0! t 0 $3.0 . 0. 0.00 0.0 .. I ' 00'. 0 . o. . . 0! 0. .... . 0! 90 .0 . 0.. 0.. s . . .0; 000'" 5.7059150 00:10:11,} 3 .0. t. 00.... 0.10.1... I.. 0. . 30.0...) 30.07 0“! {.0 .4050. . . 0. 000. W... .0. 0.0 . 0. ..0. . (0.0, 00 0 . i 0' I I\ 0 0 0 0.0...5. 0‘. ..0 .I0 )0! in 0‘... .0’gg00....01000.0.0 .0.~.0.000090.0 .2300. 0. 05 '0‘ .0000040 .000. ....Itz0.’00u . . .00'000. 000 0.0000000... .70.... 0 00 . . ’0 .. 0 00.0 0.. . ‘1 0“. 000“. 0.0 ......11300 .3000. 0000411....00ut . 0.300: ...I 0.0 ..00.0.( 0.3.0.00. I0 .0 . .0 .0.) 00.0...«0 C... . .. £531.03... . .I. .0. . 0 I .... .0 0 A I . . 0 .0 0 I 0 50 0 .0 0 . 0.. 0 . 20.00 .0.00..I.(r 0. .10 ..0003003‘. 0 .....0Ia.. . 0.0 ......10003...‘ . 000.,“ it '00.0.00\«‘..I~00 . 00in» . ..0 ..00 0. 00 0 000... 000 40..., .. 0.000 . . .. .1.“ 00. .0. 90.. .v 0.. ..Y... C ...0.00.-20(ol.' 0. I... ...: 0. 1.0131100: 5.1.1... 00000...’.Qr0.....0.0\'000.. .10.. 0:0. .....0.0'.’IL. .0‘0..0T!.0&00’.0;'0. 0 I . .0. .0. 0 00 .0 .0. .... 0.30 .0.0 0.9 .0 . 0... 0. . . 00.1.... 0. .0... ...... : .. 00.0.- ..0... 0...” ‘4‘..."0 0 0. .00.. 0.0.....‘- .00-|00 ..0 .000 .‘Jib 00.00 '0 000 1300‘ 0...: \0 0.50 00 0 1.01.0.3} 0.0 0. .4.0 0 00.0 0 0. .0 . . .. , .s O ... U 00 0 ' 0 .0.0. . 00 0., . 0'0 .0001 0. 00 . a 0: .. . .0 0.0 . 0 a . ".0.f0.. 0d 0000.. . 2.000.0000 .‘ ..t 710.0901 0.01.! '00X000’100000: . .00 . .0070 ...-‘1. 001.00 0 ' IL 0|....0I1. n; 0M§60 . . . . . . . “0 0 0 '0 00II0 0 0 ‘0 . 3. .0 9.. 0. 0 0.0 000 0 .0 . .0 . l . 900 . 0 .. . 0 0 0 0.0. I.. I'll. . .I.. 9000 . 0.03.0. .. 0.0.. ....003. . $0.60... 0000“,: .0 I. ..1 .0040: 400.30.. 3.3.0903"? .05.“.00.‘ .0 -... ...0 1.14.0 0... .- 0 0 0 .0 ..0 0 0r . . 0..- 0.0I 00.0.. .00. .0. . 0 ~00 ..s u . 0 0). . 0 . . ~ 00. f 0 0 . 00 0 . . 0.. .\ Q .. . 0. 03.00 3.0.0.? .0 .. .\ . v... .. O. 000 . .J. 0.019.... #00.s.00 39:00 0.000090 00!. I 1| 0.0... .‘ 0.00.0.0. 00.)»). 0..0 0. .00 03.003.040.000.‘ - .0.s 0 V - o . is 0.30”. 0’ '0 0.0. 0 0 . IO .30, p. u (0 .. 0 .l.\: . 0.... O .0. . 0.0. 0.. 0 2 0. .. .u v 00 J 0 0..... ..CI 0 . 5‘...) ..0 ....(30. 0.: .... ...... .....0 0.0:... . .0.P. .03.). 4.00. 10...... 00.4 . 2.0.0. .0 0.. I... .. 0.. 3v0.b:..\..’0. .. I.....» 00 .5 .. - 00.0.1. 90..0.0......0.. .0; ..0. l . 0 .. I 0 0.6. .30.... ... . r ,. . «00. :0 . .. .I. 0.020 t ”a .-. . . ..0 0 . 00 . 00’: ...... .05...’ ....0 V0 71.35.030.10: 0.0.000. . .0191: . .I £f..:l. ...I. . ..Il . .0 . ..0 ..--.. 0.00.! I. 00 0.0 00.3%.... .2700...- Y‘ .000 .10 0 I. 0 i I 000.. 0.0 "00 0. 0 0 t Bu.“ . . .00. .I t .. 0.0 . 20500.0 00.. .- . . . . 0 )0...\ a... . 0... . . {10.0 09.. ..0 a . 9.910. .060 .0 .qn‘ \ . 3.00.. a. . 3.0. 00.... 0 .0 00"), ...... 9:00.. .\}l .00 001 0...0.. 10.0.0.0‘... II}! .00. ..I!000 0.10.. 0.0. 00 v-.. 0.0.. ... ..r” .r. 0.0« . . 00. 0 ..O . 0 00.0.0000 0 c . 0 . . . . I .. ... I . “1.0.- ..00 00-00. 00 UI. $0? .0003! 0: 0 r. ‘. 07.0.0..- 0..000.1_\00\ .. ..(0 0 0 ‘0. 00.01.5010. 60.0310...) 0.030.150.000. :93? ...J. h 0.0.. 0! . .... l0.v0_00'.uvo.0 .0“ ”acid... . .0. .0“. 1/00 .. l 000%. . 00.00 ... .. ‘0.)‘01. 0 .0. 0.. .o . . .0 ' .‘00‘0 ....O'. 40' ... .0! 0.0. . ‘I..‘$‘0 . .. 0 3|. .0.~0|h00..0..01\000. C. ..- 0 0... ....S. I. ..la.\0\00.00...._h00 .20.... .u -300070 ) 0.. 0 .000 .0“:!. 51.0000 00 . 2.0.0.0 .....r 00.0 0. no .. . c . Q. . . .0.. 0 . . 000... . 0.0“). 0. {0500.00 07.01. . r. -0 ..0. .00.).‘300Y’i ,0 0.0. ... 0.0.. .10.:2: 0. .001000 03.2.... .~0 330.. V0.2. .. . . ..0 . . . . . O o. 0 0 _§.M0.0 .0\ - so. 0! .‘l. ...0‘1 000 01.) .0 0.00; 0.00 (...... 003’”! $-0.. 0.00.01. 0100....Q... .0 00.30... 0000.. o I 0 00.. . 0|! . n . . . . V ..0 . u . c «0.4 .. ... . 0. 00G. 0 0 . 0.14010 00.0100‘330 0 '0 0.?000'. 0.00. 000;... .-.01 300:.1. . 0).. l... .. . . 0 . ..0 . 0 v 0 o 0 . .I 110'. .. r. a kldufirgr “nagorrxlagu ..\.D a.'\0 0 .. I. 0 I .0 . 0. . . o a . . .100 o . I O . T.” .00.! 000 L 0 0000... 1.0 0.“ O0 0 . .. . . . . ‘0 I. . 0 I x .. . 9.00... . 30.1020 0 .. . O. 3.000).. .I.. .. 0.5! .0. .0 "'3‘... . r00... )~’.O.0...0 -02 . . .0 I... . 0 . . . .0 0 . o . . . ... . .0. 0.. .. 0 . ‘0‘!. 0 P. . y 0 . ... 0 . ... 0 ..0 L00 ..0. .3‘00.‘ 00‘». 0. .0 \ 0. 010.. 00 I 0 0 . .. - . . . I? 0 . v .0 . r . u w . . 0. 0 \ ... \. . 09 .. . . . .. .... 0... .0 .0... . =r'bs..OI-I’ ...00100’5... .0.» 1 ....IO .0 .0. I0. 0 ..0 .. . . . . 1‘. \ . . , on)”. H ...... .. 0 L... “.00.¢0.\.. . 0.0.. d.-fi;-u0’0c $.3lu‘ 0.00.5“.300... ... 00.00.20.000. I 0.00 0.. .0. .0... .... . .0 ... . . .. . . . . ‘ . u. . .. . . . .l . . ‘1 0 .. . V. 0‘. 00 00.0 0. . ... . 4 0A. a 0 0 -.01 I v . .. I l 0 I. ... - t . 1. .. “I <0. .0 0:000 0..-(000‘. 00 r 0.! . n . . 1 ..0 u 4 . . 0 00. I L . . ... r - '0. 90000 ..t03\«9{.0;\30 300-‘é00gh0 sinurlo. .0.” A. .0 . 0.0 3 0.. .0 . . In! or. n 000 0 . . . . . .I 0.0. . 0IL0. .- 35703.... I352... 04.. O .1501). a . u .I . 40 .00. 0.0 0.0 . ..0 . .0! 00 0 0 l: o. . . QI lllll“III!”IIIHIIHHHIII“IIIIIHHIHIIIIUIHI 1293 01019 1827 This is to certify that the dissertation entitled FUNCTIONALITY OF OAT-WHEAT COMPOSITE FLOURS IN SUGAR-SNAP COOKIES: EFFECT OF METHOD OF MILLING, PROCESSING, OAT CULTIVAR AND WHEAT CULTIVAR presented by Ethel Miriam Nettles has been accepted towards fulfillment of the requirements for Ph.D. Foods degree in , 1 - I . jor pr or Date October 18, 1993 MSU i: an Affirmative Action/Equal Opportunity Institution 0- 12771 LIBRARY Michigan State University PLACE IN RETURN BOX to roman thh chockout from your record. To AVOID FINES return on or him duo duo. DATE DUE DATE DUE DATE DUE ’i usu IoAnAfflnnuuvo Mon/Equal opponunny Immulon Wanna-9.1 <____,.._ _ . , FUNCTIONALITY OF OAT-WHEAT COMPOSITE FLOURS IN SUGAR-SNAP COOKIES: EFFECT OF METHOD OF MILIJNG, PROCESSING, OAT CULTIVAR AND WHEAT CULTIVAR Volume I By Ethel Miriam Nettles A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1993 ABSTRACT FUNCTIONALITY OF OAT-WHEAT COMPOSITE FLOURS IN SUGAR-SNAP COOKIES: EFFECT OF METHOD OF MILLING, PROCESSING, OAT CULTIVAR AND WHEAT CULTIVAR By Ethel Miriam Nettles The functionality of oat-wheat composite flours in sugar-snap cookies was studied. The effect of processing was determined by production of whole grain oat flour from groats and rolled oat flakes. The effect of milling was determined by comparing hammer milled flours to roller milled flours. Three oat cultivars were milled into separate oat flours. Straight grade flours of three different soft wheat cultivars were combined with cat flours to make composite flours containing 15 and 30 percent oat flour. Chemical analyses of the oat flours indicated the method of milling and processing groats into flakes influenced protein content. Roller milling produced oat flour with a finer particle size than hammer milling. Flour particle size influenced lipid analysis, viscoamylograph properties and alkaline water retention capacity. Oat cultivars differed in resistance to particle reduction forces during milling and processing. Oat cultivars differed in protein, ash, lipid, total dietary fiber and b-glucan content. Scanning electron microscopy of aleurone cell walls did not find a relationship between aleurone and subaleurone cell wall width and dietary fiber content. The functional properties of oat-wheat composite flours in sugar-snap cookies were affected by the method of milling oats, processing groats into flakes, level of substitution and wheat cultivar used to produce the composite flour. Cookies made with hammer milled groat flour composites had larger diameters and better top grain scores than cookies made with roller milled groat flours. Cookies made with hammer milled flake flours had restricted cookie spread and poorer top grain scores than cookies made with hammer milled groat flours. Wheat cultivars related effects included diameter, protein content, and textural properties. Incorporation of hammer milled groat flours into sugar snap cookies increased cookie spread and improved top grain scores. Cookies made with 30 percent oat flour had larger diameters, better top grain scores and lower Hunter Color Difference L-values (lightness) than cookies containing 15 percent oat flour. Alkaline water retention capacity was positively correlated to cookie diameter when hammer milled groat flours were used in sugar-snap cookies. To Robert, Martha and Helen Nettles who collectively helped me to become the turtle that got to the top of the fence post. ACKNOWLEDGMENTS Sincere thanks to my major professor, Dr. M.E. Zabik for her encouragement and support. I wish to express appreciation to the members of my guidance committee, Dr. D. Harpstead, Dr. J. Gill, Ms. J. McFadden and Dr. M.A. Uebersax. I wish to thank the following individuals and organizations for technical assistance; Marvin Lenz and the Quaker Oats Company, Patrick Finney, John Donelson and the technicians of the U.S.D.A. Soft Wheat Quality Laboratory at Wooster, Ohio. Invaluable time, discussions and smiles were given by Janice Harte, Sharon Hart, Lillian Oceana, Virginia Vega, Sandy Daubenmire, Micheal Gonzales, Richard Jeeter and Samir Rabie. None of this would have been possible without the love and support of my parents, Robert and Martha along with the special encouragement of my sister, Helen. The unconditional love of Kadoda and Hokey Smokes filled in the empty spaces and always made me smile. iv TABLE OF CONTENTS Page LIST OF TABLES ......................................................................................................... viii LIST OF FIGURES ........................................................................................................ xx INTRODUCTION ............................................................................................................ 1 REVIEW OF LITERATURE ......................................................................................... 3 Composite Flours .......................................................................................... 3 Economic, Nutritional and Consumer Considerations ................................................................................... 3 Composite Flour Research in Cookie Systems ..................... 5 Oat Cultivar Related Properties ............................................................ 11 Oat Protein .......................................................................................... 12 Oat Fiber ............................................................................................... 14 Oat Lipid ............................................................................................... 14 Oat Starch ............................................................................................ 16 Oat Structure and Processing ................................................................. 17 Oat structures and related chemical components ............. 17 Commercial processing ................................................................. 18 Effect of Milling on Cereal Grains ....................................................... 21 Hammer milling ................................................................................. 21 Roller milling ..................................................................................... 22 Particle Size Related Properties ......................................................... 23 Cookie Flour Quality .................................................................................... 27 Physiochemical requirements of Cookie Flour .................... 28 Bleaching Agents ................................................................. 29 Particle size ........................................................................... 29 Starch and Starch damage ................................................ 30 Protein ...................................................................................... 31 Role of other flour components in cookie quality ............. 32 Lipids ......................................................................................... 32 Non-starchy polysaccharides .......................................... 35 Flour moisture ....................................................................... 36 Commercial requirements for quality .................................... 37 MATERIALS AND METHODS ..................................................................................... 38 Oat Flours ........................................................................................................ 38 Mills ....................................................................................................... 39 Wheat flour ......................................................................................... 39 Oat-wheat Flour Composites ...................................................... 39 Experimental design for sugar-snap cookie preparation .......................................................................................... 41 Physical properties of oat groats and flours ...................... 44 Chemical Analyses of Oat Flours .............................................. 46 Lipid extraction procedure ............................................... 46 Total dietary fiber assay .................................................. 47 B-glucan Assay ...................................................................... 48 Oat starch isolation ............................................................ 50 Functional characteristics .......................................................... 52 Sugar-snap cookie preparation and evaluation ................... 53 Scanning Electron Micros00py ................................................................ 54 Sample preparation ......................................................................... 54 SEM Procedure .................................................................................... 55 Aleurone cell wall study ............................................................... 55 Statistical Analysis of Data ................................................................... 56 Oat Flour Characteristics ............................................................. 56 Cookie Quality Characteristics .................................................. 56 RESULTS AND DISCUSSION .................................................................................... 58 Great and Flake Analyses .......................................................................... 58 Oat Flour Analyses ....................................................................................... 63 Physical properties of oat flours ............................................. 79 Viscoamylograph properties of oat flours ............................ 93 Visco-amylograph properties of oat starches .................... 103 Alkaline water retention capacities of flours ................... 113 Scanning Electron Microscopy of Aleurone Cell Walls ................ 119 Effect of Milling on Cookie Quality ...................................................... 137 Cookie diameter and top grain scores ..................................... 137 Alkaline water retention capacity ........................................... 144 Cookie surface color ....................................................................... 150 Cookie proximate analysis ........................................................... 157 Moisture retention ........................................................................... 164 Shear compression and breaking strength ............................ 167 Effect of Processing ................................................................................... 171 Cookie diameter and top grain scores ..................................... 173 Alkaline water retention capacity ........................................... 175 vi Cookie surface color ....................................................................... 183 Cookie Proximate Analyses ......................................................... 190 Moisture retention ........................................................................... 198 Shear compression and breaking strength ............................ 199 Interaction with Wheat Cultivars ......................................................... 202 Cookie diameter and top grain score ....................................... 205 Alkaline Water Retention Capacity ......................................... 211 Cookie surface color ....................................................................... 218 Proximate analyses of cookies .................................................. 224 Moisture Retention .......................................................................... 237 Shear compression. and breaking strength ............................ 238 SUMMARY AND CONCLUSIONS ............................................................................... 244 APPENDIX ...................................................................................................................... 260 REFERENCES ................................................................................................................. 312 vii Table 10 11 12 LIST OF TABLES Composition of oat forms: Means for moisture Composition of oat forms: Means for protein Composition of oat forms: Means for ash Thousand kernel weight, means and standard deviations of moisture, protein and ash content of oat cultivar groats and flakes Proximate analysis of oat flours: Means for moisture content. Proximate analysis of oat flours: Means for protein content Proximate analysis of oat flours: Means for ash content. Proximate analysis of oat flours: Means for fat content. Proximate analysis of oat flours: Means for TDF content. Distribution of lipid classes in oat flours. Means and standard deviations of total dietary fiber, fi-glucan content and alkaline water retention capacity of oat flours. Proximate analysis of oat flours: Means and standard deviations viii Page 58 6O 61 62 67 71 74 75 77 78 79 80 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Mean color differences and particle size index values of oat flours by type of mill Mean color differences and particle size index values of oat flours by form Mean color differences and particle size index values of oat flours by cultivar Means and standard deviations of particle size index and color difference data for oat flours. Viscoamylograph means for initial paste temperature of oat flour Viscoamylograph means for peak hot viscosity of oat flour . . . Viscoamylograph means for viscosity at 15 minutes of oat flour Viscoamylograph means for peak cold viscosity of oat flour . . . Viscoamylograph means and standard deviations for oat flours. . Viscoamylograph means for initial paste temperature of oat starch Viscoamylograph means for peak hot viscosity of oat starch . . . . Viscoamylograph means for viscosity at 30 minutes of oat starch . Viscoamylograph means for peak cold viscosity of oat flour Means and standard deviations for viscoamylograph data for oat starches. . . . .. . ix 86 87 88 90 96 97 100 101 102 104 105 106 107 109 27 28 29 30 31 32 33 34 35 36 37 38 Means and standard deviations of percent protein in alkali extracted oat starches. Means for alkaline water retention capacities (AWRC) of oat flours Means and standard deviations of alkaline water retention capacities of oat flours. Means and standard deviations of aleurone cell wall measurements at three positions on the oat groat. Means and standard deviations of groat protein content, total dietary fiber content of oat flours and aleurone cell wall widths.. Effect of milling: Means for diameters of cookies made with oat-wheat composite flours Means and standard deviations of cookie diameter and top grain scores of cookies measuring the effect of milling on cookie quality. Effect of milling: Means for alkaline water retention capacity of oat-wheat composite flours . . Means and standard deviations of alkaline water retention capacity measuring the effect of milling on cookie quality.. Effect of milling: Means for Hunter Color Difference L-values of cookies made with oat-wheat composite flours Effect of milling: Means for Hunter Color Difference a-values of cookies made with oat-wheat composite flours . Effect of milling: Means for Hunter Color Difference b-values of cookies made with oat-wheat composite flours 112 117 119 130 132 141 145 147 151 154 156 156 39 40 41 42 43 44 45 46 47 48 49 50 Means and standard deviations of Hunter color difference values of cookies measuring the effect of milling on cookie quality. Effect of milling: Means for protein content of cookies made with oat-wheat composite flours Effect of milling: Means for ash content of cookies made with oat-wheat composite flours Effect of milling: Means for fat content of cookies made with oat-wheat composite flours . . Means and standard deviations of protein, ash and fat content of cookies measuring the effect of milling on cookie quality Effect of milling: Means for moisture retention of cookies made with oat-wheat composite flours. Effect of milling: Means for shear compression of cookies made with oat-wheat composite flours . Effect of milling: Means for breaking strength of cookies made with oat-wheat composite flours. . . Means and standard deviations of moisture retention, shear compression and breaking strength of cookies measuring the effect of milling method on cookie quality. Effect of processing: Means for diameters of cookies made with oat-wheat composite flours Means and standard deviations of cookie diameter, top grain score of cookies measuring the effect of processing on cookie quality. Effect of processing: Means for alkaline water retention capacity of oat-wheat composite flours. . xi 158 160 162 163 165 166 169 170 172 173 176 180 51 52 53 54 55 56 57 58 59 6O 61 62 Means and standard deviations of alkaline water retention capacity measuring the effect of processing on cookie quality.. Effect of processing: Means for Hunter Color Difference L-values of cookies made with oat-wheat composite flours Effect of processing: Means for Hunter Color Difference a-values of cookies made with oat-wheat composite flours Effect of processing: Means for Hunter Color Difference b-values of cookies made with oat-wheat composite flours Means and standard deviations of Hunter color difference values of cookies measuring the effect of processing on cookie quality. . . . . . ....... Effect of processing: Means for protein content of cookies made with oat-wheat composite flours . Effect of processing: Means for ash content of cookies made with oat-wheat composite flours. . Effect of processing: Means for fat content of cookies made with oat-wheat composite flours Means and standard deviations of protein, ash, and fat content of cookies measuring the effect of processing on cookie quality.. Effect of processing: Means for moisture retention of cookies made with oat-wheat composite flours Effect of processing: Means for shear compression of cookies made with oat-wheat composite flours. . . . Effect of processing: Means for breaking strength of cookies made with oat-wheat composite flours xii 182 186 187 188 189 193 195 196 197 199 200 201 63 64 65 66 67 68 69 70 71 72 73 Means and standard deviations of moisture retention, shear compression and breaking strength of cookies measuring the effect of processing on cookie quality.. Chemical analysis and particle size of soft wheat flours as furnished by Soft Wheat Quality Lab. Effect of wheat cultivar: Means for diameters of cookies made with oat-wheat composite flours Means and standard deviations of cookie diameter, top grain scores of cookies measuring the effect of wheat cultivar on cookie quality. Effect of wheat cultivar: Means for alkaline water retention capacity of oat-wheat composite flours . Means and standard deviations of alkaline water retention capacity of cookies measuring the effect of wheat cultivar on cookie quality. Effect of wheat cultivar: Means for Hunter Color Difference L-values of cookies made with oat-wheat composite flours Effect of wheat cultivar: Means for Hunter Color Difference a-values of cookies made with oat-wheat composite flours Effect of wheat cultivar: Means for Hunter Color Difference b-values of cookies made with oat-wheat composite flours Means and standard deviations of Hunter color difference values of cookies measuring the effect of wheat cultivar on cookie quality . Effect of wheat cultivar: Means for protein content of cookies made with oat-wheat composite flours xiii 203 204 205 210 213 219 220 221 222 223 228 74 75 76 77 78 79 80 81 82 83 84 85 86 Effect of wheat cultivar: Means for ash content of cookies made with oat-wheat composite flours Effect of wheat cultivar: Means for fat content of cookies made with oat-wheat composite flours Means and standard deviations of protein, ash, and fat content measuring the effect of wheat cultivar on cookie quality. Effect of wheat cultivar: Means for moisture retention of cookies made with oat-wheat composite flours Effect of wheat cultivar: Means for shear compression of cookies made with oat-wheat composite flours. Effect of wheat cultivar: Means for breaking strength of cookies made with oat-wheat composite flours . . Means and standard deviations of moisture retention, shear compression and breaking strength of cookie measuring the effect of wheat cultivar on cookie quality. Analysis of variance for protein content of oat forms. Analysis of variance for ash content of oat forms. Analysis of variance for moisture content of oat forms . Analysis of variance for protein content of oat flours. Analysis of variance for ash content of oat flours Analysis of variance for fat content of oat flours xiv 234 235 236 238 239 240 242 273 273 273 273 274 274 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 Analysis of variance for moisture content of oat flours. Analysis of variance for total dietary fiber content of oat flours. Analysis of variance for L-value of oat flours. Analysis of variance for a-value of oat flours. Analysis of variance for b-value of oat flours.. Analysis of variance for alkaline water retention capacity of oat flours. Analysis of variance for particle size index of oat flours Analysis of variance for initial paste temperature of oat flours Analysis of variance for peak hot viscosity of oat flours. Analysis of variance for 15 minute viscosity of oat flours. Analysis of variance for peak cold viscosity of oat flours. Analysis of variance for initial paste temperature of oat starches. . . . .. . . . Analysis of variance for peak hot viscosity of oat starches . Analysis of variance for 30 minute viscosity of oat starches. Analysis of variance for peak cold viscosity of oat starches . XV 274 275 275 275 276 276 276 277 277 277 278 278 278 279 279 102 103 104 105 106 107 108 109 110 111 112 113 114 115 Analysis of variance for protein content of alkaline extracted oat starches Analysis mill. Analysis capacity Analysis mill Analysis mill Analysis mill. Analysis Effect of Analysis mill Analysis of mill Analysis Effect of Analysis Effect of Analysis Effect of Analysis of variance for cookie diameter: Effect of of variance for alkaline water retention of composite flours: Effect of mill. of variance for cookie L-value: Effect of of variance for cookie a-value: Effect of of variance for cookie b-value: Effect of of variance for cookie protein content: mill. of variance for cookie ash content: Effect of of variance for cookie lipid content: Effect of variance for cookie moisture retention: mill. of variance for cookie shear compression: mill of variance for cookie breaking strength: mill of variance for cookie diameter: Effect of oat processing. Analysis of variance for alkaline water retention capacity of composite flours: Effect of oat processing xvi 279 280 280 280 281 281 281 282 282 282 283 283 283 284 116 117 118 119 120 121 122 123 124 125 126 127 128 129 Analysis of variance for cookie L-value: Effect of oat processing. Analysis of variance for cookie a-value: Effect of oat processing Analysis of variance for cookie b-value: Effect of oat processing Analysis of variance for cookie protein content: Effect of oat processing. Analysis of variance for cookie ash content: Effect of oat processing. Analysis of variance for cookie lipid content: Effect of oat processing. Analysis of variance for cookie moisture retention: Effect of oat processing. Analysis of variance for cookie shear compression: Effect of oat processing. Analysis of variance for cookie breaking strength: Effect of oat processing. Analysis of variance for cookie diameter: Effect of wheat cultivar . Analysis of variance for alkaline water retention capacity of composite flours: Effect of wheat cultivar . Analysis of variance for cookie L-value: Effect of wheat cultivar. Analysis of variance for cookie a-value: Effect of wheat cultivar. Analysis of variance for cookie b-value: Effect of wheat cultivar xvii 284 284 285 285 285 286 286 286 287 287 287 288 288 288 130 131 132 133 134 135 136 137 138 139 140 141 Analysis of variance for protein content: Effect of wheat cultivar Analysis of variance for cookie ash content: Effect of wheat cultivar. ' Analysis of variance for cookie lipid content: Effect of wheat cultivar. Analysis of variance for moisture retention: Effect of wheat cultivar Analysis of variance for cookie shear compression: Effect of wheat cultivar Analysis of variance for breaking strength of cookies: Effect of wheat cultivar. Pearson correlation coefficients and probabilities for cookies made with hammer milled groat composite flours Pearson correlation coefficients and probabilities for cookies made with roller milled groat composite flours Pearson correlation coefficients and probabilities for cookies made with hammer milled flake composite flours Pearson correlation coefficients and probabilities for cookies made with Mariner hammer milled groat composite flours Pearson correlation coefficients and probabilities for cookies made with Ogle hammer milled groat composite flours Pearson correlation coefficients and probabilities for cookies made with Porter hammer milled groat composite flours xviii 289 289 289 290 290 290 291 292 293 294 295 296 142 Pearson correlation coefficients and probabilities for cookies made with Becker composite flours 143 Pearson correlation coefficients and probabilities for cookies made with Caldwell composite flours 144 Pearson correlation coefficients and probabilities for cookies made with Compton composite flours xix 297 298 299 LIST OF FIGURES Figure 10 11 12 Experimental design for oat flour production. Experimental design for effect of processing. Experimental design for effect of milling Experimental design for interaction with wheat cultivars Quantitative extraction procedure for total B-glucan determination in oat flours. Oat starch isolation adapted from Paton (1977). One thousand kernel weights of oat cultivars Scanning electron micrographs of structures and chemical components of the oat groat. Interaction of oat form and mill type on oat flour moisture content. Interaction of mill type, oat form and oat cultivar on oat flour protein content Interaction of mill type and oat form on oat flour ash content. Interaction of oat form and mill type on oat flour fat content. XX Page 40 42 43 45 49 51 59 64 66 69 72 76 13 14 15 16 17 18 19 20 21 22 23 24 25 Interaction of mill type and oat form on particle size index of oat flour. Scanning electron micrographs of coarse and fine oat flour fractions Interaction of mill type, oat form and cat cultivar on Hunter Color Difference values of oat flours. Interaction of mill type, oat form and cat cultivar on initial pasting temperature of oat flour.. Interaction of mill type, oat form and oat cultivar on 15 minute and peak cold viscosities of oat flour.. Scanning electron micrographs of freeze dried alkaline extracted oat starch granules Scanning electron micrographs of freeze dried oat starch gels . . . . . . Interaction of mill type and oat cultivar on alkaline water retention capacity (AWRC) of oat flour. Scanning electron micrograph of Porter groat identifying the three positions on oat groat cross section at which measurements were taken of aleurone and subaleurone cell wall width. Scanning electron micrographs of aleurone and subaleurone cell wall on latitudinal cross section of Mariner groat. Scanning electron micrographs of aleurone and subaleurone cell walls on latitudinal cross section of Ogle groat. Scanning electron micrographs of aleurone and subaleurone cell walls on latitudinal cross section of Porter groat. Aleurone cell wall measurements. xxi 82 83 91 94 98 110 114 116 120 122 125 128 131 26 27 28 29 3O 31 32 33 34 35 Scanning electron micrographs of cross section of Mariner oat groat showing aleurone grains and protein bodies located in the aleurone cells. Interactions for cookie diameter: a) type of mill x level of hammer and roller milled groat flour substitution b) oat cultivar x type of mill. Interaction of oat cultivar x type of mill for alkaline water retention capacity of hammer and roller milled groat composite flours. Interaction of oat cultivar x level of hammer and roller milled groat flour substitution for Hunter Color Difference values of cookie surface color. Interaction of oat cultivar x level of hammer and roller milled groat flour substitution for ash content of sugar-snap cookies Interaction of oat cultivar x level of hammer and roller milled groat flour substitution for cookie shear compression.. Interaction for AWRC of hammer milled groat and flake composite flours. a) oat form x oat cultivar b) oat cultivar x level of hammer milled groat and flake flour substitution . . ' ............. Interaction for Hunter Color Difference values for cookies made with hammer milled groat and flake flours. a) Oat form x Oat cultivar interaction for L value. b) Oat cultivar x Level of hammer milled groat and flake flour substitution for a-value Interaction for protein and ash content of cookies made with hammer milled groat and flake flours. a) Cookie protein content b) cookie ash content. Viscoamylograph properties of hammer milled groat flours. a) Initial paste temperature of oat flour slurries. b) Peak hot viscosity of oat flour slurries.. xxii 134 138 146 152 159 168 177 184 191 207 36 37 38 39 40 41 42 43 44 45 46 Interaction of wheat cultivar and Level of oat flour for alkaline water retention capacity of composite flours of Becker, Caldwell and Compton Correlation by wheat cultivar of cookie diameter and alkaline water retention capacity . Interaction of wheat cultivar, oat cultivar and level of oat flour for cookie protein content. Interaction: of wheat cultivar and level of oat flour for cookie ash content. Correlation between protein content and Hunter Color difference L-values for cookies made with composites of Becker, Caldwell and Compton soft wheat flours. Correlation between protein content and Hunter color difference L-values. for cookies made with composites of Mariner, Ogle and Porter hammer milled groat flours Sugar-snap cookies made with composites of Mariner, Ogle and Porter whole grain hammer milled groat flour and Caldwell soft wheat flour . Sugar-snap cookies made with composites of Mariner, Ogle and Porter whole grain roller milled groat flour and Caldwell soft wheat flour . Sugar-snap cookies made with composites of Mariner, Ogle and Porter whole grain hammer milled flake flour and Caldwell soft wheat flour . Sugar-snap cookies made with composites of Mariner, Ogle and Porter whole grain hammer milled groat flour and Becker soft wheat flour . Sugar-snap cookies made with composites of Mariner, Ogle and Porter whole grain hammer milled groat flour and Compton soft wheat flour . xxiii 212 215 225 227 230 232 300 302 304 306 308 47 Sugar-snap cookie top grain score standards from U.S.D.A. Soft Wheat Quality Laboratory at Wooster, Ohio 310 xxiv INTRODUCTION Oats have been identified as a good source of protein, polyunsaturated fatty acids and soluble dietary fiber. Yet, the use of whole grain oat flour in the United States is mostly limited to baby cereals and ready to eat breakfast cereals. Oat bran usage in the United States has increased significantly since scientific studies linked the intake of dietary fiber with prevention of atherosclerosis, diverticulosis. colonic cancer, appendicitis and reduction of serum cholesterol. Consumers have become more interested in purchasing fiber enhanced foods. Foods targeted by industry for incorporation of fiber are baked goods, breakfast cereals and snack foods. Whole grain oat flour lacks the structural proteins required for most baked products. Sugar-snap cookies are a chemically leavened baked product that is of optimum quality when a minimum of gluten is formed during the mixing and handling of the cookie dough. Soft wheat flours are preferentially used by commercial bakeries for cookies. The sugar-snap cookie formulation is used by the baking industry to evaluate cookie flour quality or to determine if the end product will have desirable characteristics. Cookie doughs made with soft wheat flours must expand and flow during the baking process to enable heat coagulation of flour proteins and loss of moisture. Sugar-snap cookie diameters and tap grain scores are indicators of the rheological properties of cookie doughs during baking. 2 Non-wheat flours substituted into wheat flour to be used for cookie manufacture should not detrimentally affect flavor, appearance or texture of the end product. The flavor of whole grain oat flour has already been made familiar to consumers in the form of breakfast cereals and oatmeal cookies. Cookies made from substituted or composite flours should be of comparable quality to cookies made from 100 percent soft wheat flour. The study had four objectives. The first objective was to determine the effect of two different types of mill on the chemical and functional properties of whole grain oat flour. The second objective was to compare the functionality of oat flour from three different oat cultivars. The third objective was to determine the effect of processing groats into oat flakes prior to milling into oat flour. The fourth objective was to determine the interaction of flours from three different soft wheat cultivars with whole grain oat flours. REVIEW OF LITERATURE Whole grain oat flour has not been reported as being used extensively in chemically leavened baked products. Baby cereals and ready to eat breakfast cereals are the major oat flour products (Weaver et al. 1981). Whole grain oat flour lacks the proteins required to form gluten in baked products. Replacement of wheat flour with a percentage of non-wheat flour reduces the structural organization of gluten within a baked product. However. since sugar snap cookies are not greatly dependent on a gluten structure for the finished product to have the desired quality characteristics. use of a composite flour made of whole oat flour and soft wheat flour could be feasible. Composite Flours Composite flours are flour mixtures in which wheat flour has been replaced by flours from other cereal grains, starches or protein concentrates. Traditionally, many European countries have produced breads prepared from mixtures of wheat and rye flours. Bean flour, potato flour and barley flour were used to prepare breads during World War I and II (de Ruiter, 1978). Composite flours have been developed for economic and nutritional reasons. Economic, Nutritional and Consumer Considerations Economic and nutritional reasons are the basis for use of composite flours In developing countries. Composite flours are used to decrease the amount of wheat imports which contribute to foreign debt (Fellers and Bean, 1988). The Food and Agriculture Organization of the United Nations (FAO) initiated a “Composite 3 4 Flour Program” in 1964 with the objective of using raw materials other than wheat in the production of bread, biscuits, pastas and other similar flour based foods. Nutritionally, composite flours can be formulated to provide extra nutrients such as vitamins, amino acids and trace elements (de Ruiter, 1978). Composite flours in developed countries meet consumer demand for variety and specific nutritional factors in the diet. In the United States, composite flours find their greatest use in providing variety to the diet in such products as multigrain breads, rye and triticale breads, potato bread, oatmeal cookies, corn bread and buckwheat pancakes. Soy breads and breads with increased levels of dietary fiber are examples of nutritionally enhanced breads produced in the United States (Fellers and Bean, 1988). Sievert et al (1990) concluded that a major problem associated with addition of high levels of dietary fiber sources into traditional foods is the detrimental effect these ingredients have on the physical and sensory properties of the foods. Changes in flavor, palatability, appearance and texture are unacceptable to most marketers and consumers. One approach used by research programs has been to establish the percentage of wheat flour that can be replaced by other flours without major changes in quality of the final product and without requiring considerable adjustment in the commercial manufacturing process. An acceptable composite flour should produce baked products and pastas that are of comparable quality to an 100% wheat product. Several researchers have prepared biscuit, cookies and baked goods other than bread from composite flours. Composite 5 flours may be in some ways be more suitable for producing baked goods such as cookies rather than bread. Cookie doughs require less gluten formation than bread doughs. Commercial production requirements for wire cut cookies include dough plasticity, minimum elasticity and a low degree of gluten development (Matz and Matz, 1978). Composite Flour Remrch in Cookie Systems F099 and Tinklin (1972) replaced all purpose wheat flour at levels of 6 and 15 percent by weight in sugar snap cookies with glandless cotton seed flour. Two different particle sized cotton seed flours (CSF) were used in the sugar snap cookies. Fine CSF passed through 100 mesh (150 millimicron) and coarse CSF passed through 80 mesh (100 millimicron) Cookies containing cotton seed flour had significant (p< 0.05) reductions in mean tenderness, height, volume and specific volume (volume/wt). Cookie weight increased as the amount of CSF in the formula increased. Cookies containing coarsely ground CSF were less tender, had less width, volume and specific volume than cookies containing equal amounts of finely ground cotton seed flour. F099 and Tinklin concluded "coarse CSF absorbed moisture more readily or bound moisture more securely than fine CSF”. Cookie spread (width/height) increased with increasing amount of CSF in the formula. Spread of cookies containing CSF appeared to be dependent upon interaction of grind and level of cotton seed flour. Vecchionacce and Setser (1980) substituted liquid cyclone processed cotton seed flour at levels of 12, 24, 36 and 48% by weight for all purpose commercial wheat flour. The cotton seed 6 flour (CSF) on a dry weight basis was composed of 66% protein so that substitution of 48% CSF produced a cookie which contained 15.6% protein. Sodium stearoyl 2 lactylate (SSL) or xanthan gum (XG) were added at the 1% level of combined flour weight to the formula to function as stabilizers. Increasing the level of CSF produced darker cookies with an increasing reddish hue as measured by the Gardener automatic color meter. Sugar-snap cookies containing 24 and 36% CSF had higher spread ratios than the control cookie. Spread ratio was less than the control cookie at the 12% and 40% level of substitution. Addition of SSL produced more tender cookies at the 0, 12 and 24% level of substitution. Xanthan gum significantly increased the shortness value of cookies containing 36 and 48% CSF. Taste panelists rated cookies with increasing amounts of CSF higher in the categories of color, texture and tenderness. Flavor scores were significantly higher when cookies contained cookies with SSL and 24, 36 and 48% CSF compared to cookies with xanthan gum and the same levels of CSF. Tsen et al (1973) prepared sugar-snap cookies using soft wheat flours fortified with three different soy products: full fat soy flour, defatted soy flour or two soy protein isolates. The fortification levels for each soy product in combination with the wheat flour were 8, 12, 16, 20, 24, 30, 40 and 50 percent. The soy products significantly reduced cookie spread and increased cookie thickness as progressively more soy product was blended with the wheat flour. Full fat soy flour (22.2% crude lipid) had less effect on cookie spread than defatted flours at all fortified levels. Tsen attributed this effect to a lower protein level in full fat (40%) soy 7 flour than in defatted (52.6%) soy flour. Sodium-stearoyl 2 lactylate (0.5%) increased the spread ratio of cookies made of any of the types of wheat flours. A sub-study examined the effects of surfactants on cookies fortified with 12% defatted soy flour. The addition of 0.5% sodium-stearoyl 2 lactylate increased the spread ratio of cookies made from wheat flours blended with 12% defatted soy flour. The effect of the surfactants, sodium-stearoyl 2 lactylate and sodium-stearoyl fumarate on cookie spread were compared at five levels of surfactant and two levels of shortening The conclusion was that the individual surfactants increased cookie spread and decreased the levels of shortening required in the cookie doughs prepared with any of the types of soy-wheat composite flours. McWatters (1978) evaluated the cookie baking properties of defatted soybean, peanut, and field pea flours. These flours were substituted at the 10, 20 and 30% level for wheat flour. Soybean flour exhibited high water absorptive properties and cookie doughs required a higher level of water addition. Water absorptive properties at the 20 and 30% replacement levels restricted cookie spread and development of typical top grain. Cookie doughs made with peanut and field pea flours had handling properties similar to the 100% wheat controls. Cookies with all three levels of field pea flour did not differ significantly from the 100% wheat controls in dough and baking characteristics. However, a beany flavor was detected at the 30% level. As the amount of peanut flour increased, cookies had significantly (p < 0.01) decreasing scores for appearance and color. 8 Badi and Hoseney (1976) prepared cookies from grain sorghum and millet flours. The products showed poor spread even with the addition of small quantities of wheat lipids, soy lecithins or soy oils. Sorghum and millet flour cookies lacked the required cohesive properties of a cookie dough. A pretreatment process which consisted of wetting, hydration for 6 hours and air drying to 12% moisture was required to produce sorghum and millet flours that could be combined with wheat flour. Composite flours of wheat flour and treated sorghum or millet flour did produce acceptable cookies. Badi and Hoseney (1978) used corn flour as a partial and total replacement for soft wheat flour in sugar-snap cookies. Cookies made with untreated corn flour had a markedly reduced cookie spread and a poor top grain. A pretreatment process of hydration for 6 hours, air drying at room temperature and addition of 0.6% soybean lecithin was required to increase cookie diameter and improve top grain. Centrifugation and reconstitution of corn flour fractions indicated that enzymatic activity occuring during the hydration process could increase cookie diameters. Corn flour particle size and starch damage were found to be unrelated to cookie spread. Gorczyca and Zabik (1979) substituted 10, 20 and 30% cellulose and coated cellulose particles in sugar snap cookies. The non-coated celluloses differed in particle size, ranging fromaverage particle size 30-35 to 150-225 microns. Soy lecithin was added to the micro Ill method formula at the 2% level total weight of flour and cellulose. Top grain scores and cookie spread decreased as the amount of cellulose added increased. Increasing levels of cellulose 9 substitution increased the moisture content producing cookies with lower levels of breaking strength and shear values. Cookies with 20 to 30% cellulose were softer and thicker while being lighter in color. Cookies containing all five types of cellulose at the 10% level of substitution were judged by taste panelists to be acceptable. Pectin-coated cellulose had been reported to have the greatest hypocholesterolemic effect but produced cookies with the poorest top grain. Oomah (1983) measured baking properties of wheat-oat composite flours. Two types of oat flour were substituted for wheat flour at levels of 5, 10, 15, 20, 25 percent by weight. One of the oat flours was a commercial hammer milled product made from conditioned groats or rolled oats. The second flour was roller milled from groats. Cookies baked with roller milled oat flour had a progressive increase in cookie spread (width/thickness) as oat flour was substituted at higher percentages. There was no significant difference in cookie spread between the 100 percent wheat flour control and the cookies made with commercial hammer milled flour except at the 5 percent substitution level. Hoojjat and Zabik (1984) investigated the effect of navy bean and sesame flours on the baking properties of sugar-snap cookies. Combinations of navy bean - sesame seed flours were substituted for soft wheat flour at the 20 and 30% levels. Navy bean-sesame seed combinations were 20:0, 15:5, 10:10, 5:15, 0:20 for the 20% composite flours. Thirty percent composites consisted of combinations of 30:0, 20:10, 15:15, 10:20, 0:30. navy bean sesame seed flour. Cookies made with navy bean flour had dough handling 10 properties like the 100% wheat flour controls. As the amount of sesame seed flour increased the doughs became more sticky. Cookie diameter decreased and thickness increased with increasing amounts of navy bean and sesame seed flour. Composite flours of 20 and 30% sesame flour produced cookies that were tougher than the controls. Increasing the amount of navy bean flour produced cookies that required less force than the controls to break them. Sievert et al (1990) investigated the functional properties of soy polysaccharides and wheat bran in soft wheat products. The soy polysaccharides tested were derived from processing dehulled and defatted soybean flakes. The raw material was primarily cell wall material of the soybean cotelydon. The polysaccharides included mainly cellulose, arabinogalactan, arabinan and an acidic polysaccharide complex. The soft wheat cookie flour was a blend of Pacific Northwest varieties. Cookies were baked by AACC method 10-52. As the percentage of fiber added increased, cookie diameter decreased. Influence on cookie spread varied with the source of fiber. Coarse and fine wheat bran had a significant effect in cookie diameter at the 5% level. Coarse wheat bran had the least effect, soy polysaccharides and fine bran had the greatest effect. At levels greater than 10%, fine bran was worse than coarse bran. Addition of soy polysaccharides produced lighter colored cookies and had a more pronounced effect on top grain characteristics than adding wheat bran. As the amount of soy polysaccharides increased, a desirable top grain was replaced by fine hairline cracks. A similar less pronounced effect was seen for fine wheat bran. Coarse bran 11 affected top grain appearance the least. Adding 15% coarse wheat bran affected top grain characteristics rather than cookie spread. Oat Cultivar Relgfled Properties The origin of oats can be traced back to about 2000 B.C., being grown in the areas surrounding the Mediterranean Sea. (Schrickel, 1986) The two species most frequently cultivated in the world are Avena sativa and A. byzantina. A. sativa has a white or yellow seed coat. A. byzantine has red seed coat. A. sativa is the most popular species grown in the United States and the world. Oat cultivars are grown in agricultural areas based upon environmental conditions and disease situations in the growing area. Webster’s New World Dictionary (1986) defined a cultivar as a variety of a plant species originating and continuing in cultivation and given a name in a modern language. Spring oats grown in the Northern Hemisphere are planted in April or May and harvested in July or August (Schrickel,1986). The planting schedule for oats make the crop more susceptible to disease than other small grains because the regions are warm and humid (Simon and Murphy, 1961). Schrickel (1986) ranked the primary oat diseases in the United States in order of economic importance as being yellow dwarf virus, leaf or crown rust (Puccinia coronata), septoria (Septoria avenae), stem rust (Puccinia graminis), halo blight (Pseudomonas coronafaciens), loose smut (Ustilago avenae) and covered smut (Ustilago kollen'). Cultivars planted in the United States must be resistant to barley yellow dwarf (Red Leaf) virus disease (Freed et al 1986). Barley Yellow Dwarf Virus (BYDV) seriously depresses oat 1 2 yields. Crown rust has been the most widespread and destructive disease of oats, causing reduced crop yield and grain quality in major oat producing areas of the United States (Simon and Murphy, 1961). The best method for avoiding losses due to BYDV and crown rust is to grow disease resistant cultivars or varieties. Oat breeders over the last 50 years have developed several varieties of cats with reasonably good genetic resistance to these diseases. Oat cultivars not only differ in resistance to disease but also in yield, protein content, lipid, fiber and mineral content. There are co-relationships between yield and protein content. A depression in yield due to low levels of rainfall may result in higher levels of protein (Welch et al, 1991). This negative relationship between yield and protein concentration exists for the majority of oat cultivars. One recent exception to the relationship was the Wisconsin grown cultivar, Dal (Burrows, 1986). Miller et al (1980) reported that the environment influenced changes in oat groat phytic acid levels and these influences are similar for different oat cultivars. O_at Protein: Robbins et al (1971) analyzed protein content in 286 different oat cultivars and reported a range of 12.4 - 24.4% protein (db) in oat groats. High protein cats are those that contain more than 17 percent protein on a dry basis (Paton, 1977). According to Kim et al (1979), oat protein Is composed of glutelins (66%), albumins (7.5%), globulins (12.9%) and prolamines (13.9%). Saigo et al (1983) reported that the predominate storage protein in oats is globulins. Pomeranz (1973), Cluskey et al (1979) and Zarkadas et al (1982) 1 3 concluded that oat groats contain good quality protein and that lysine is the limiting amino acid. A unique characteristic of oats is that the nutritional quality of the protein as measured by amino acid profile is only slightly changed as the protein level is increased due to breeding practices (Burrows, 1986). Youngs and Gilchrist (1976) studied protein distribution within oat kernels of a single cultivar. Growing conditions and level of fertilizer application influenced the groat protein concentration. As protein content increased, protein concentration increased in the bran, germ and starchy endosperm fractions of the groat. This deposition pattern was different from that previously found in two wheat cultivars. The bran fraction showed a greater increase in protein (2.5 - 2.8% vs 0.6 - 2.3%) than other oat fractions. Wu and Stringfellow (1973) air classified ground oat flour from one high protein (17.2% db) and one normal protein (12.8% db) variety. The coarse residue or particles > 30p had a higher protein percentage (24.2 and 29.2), fat percentage (3.2 and 2.8) and crude fiber (5.0 and 6.4) than the total groat in both cultivars. For particles less than 30p, protein content decreased with increasing particle size. The crude fiber content increased with increasing particle size. MacArthur and D'Appolonia (1979) studied sugars in oat cultivars with three different protein levels, high (21.1%) ‘ intermediate (19.9%) and low (16.5%) protein levels. The oat flour with the highest protein content also contained the higher percentage (1.3%) of total sugar. The oat brans contained less total sugar (2.6-3.40/0) than the wheat bran (4.9%). The intermediate 14 protein containing oat cultivar contained the most total sugar (3.4%) in its oat bran. Sucrose was the predominate free sugar in eat and wheat flour. The amount of sucrose varied according to the protein level in the cat flour, high protein oats containing the most sucrose and low protein oats containing the least sucrose. Oat Fiber: The major component of soluble fiber found in the kernels of oats (Avena sativa) is (1-3)(1-4)-b-D-glucopyranose or p-glucan (Welch et al, 1991). p-glucan is located throughout the cell walls of the endosperm but is present in higher amounts in the thicker cell walls of the subaleurone layer (Wood, 1989 and Fulcher, 1986). p- glucan is the active serum cholesterol lowering component in oat bran (Anderson and Chen, 1986). The level of p-glucan found in oats can be dependent on cultivar type and levels of nitrogen fertility. Welch and Lloyd (1989) reported that when oat cultivars were grown under similar conditions, kernel levels of p-glucan ranged from 3.2 to 6.3%. The results indicated that plant breeding could influence oat p-glucan levels. Welch et al (1991) reported that increased application of nitrogen based fertilizers led to significantly higher (p <0.05) levels of kernel p-glucan. Cultivar differences in p-glucan levels were more predominate when relatively low levels of nitrogen fertilizer was available. This study indicated a positive relationship between p-glucan levels and protein within a cultivar. Oat Lipid: Oat groats contain a higher concentration of lipids than other cereal grains. Weber (1973) measured the lipid content of seven 1 5 cereal grains and reported values of 7.6% for oat groats as compared to 4.4% for corn, 2.1% for wheat, 2.2% for rice, 1.8% for rye, 3.4% for sorghum and 2.1% for barley. Youngs et al (1977) and Sahasrabudhe (1979) found that digalactosyldiglycerides (DGDG) were the major glycolipid component in the groat lipid (6.9-7.60/0). Phosphatidylcholine was the major phospholipid in groat lipids and in all groat fractions, ranging from 2.8 - 6.1%.. Most of the triglycerides had a high level (ratio of 1 to 2.2) of unsaturation. Lipid concentration and fatty acid concentration in oats is highly heritable (Youngs,1986). Multiple genes are involved in the inheritance of oil content (Frey et al. 1975). Breeding procedures are continually being evaluated to determine if oats can be developed as an oilseed crop (Branson and Frey, 1989). Youngs et al (1977) analyzed two oat cultivars for lipids and fatty acids. The cultivar Dal had high lipid concentration, (8.0%) and Froker, a medium lipid concentration (5.5%). Bran contained more free and bound lipids than the starchy endosperm. Bran contains less palmitic and more stearic acid than the other fractions. Saastamoinen et al (1989) compared the oil content and fatty acid composition of seven of the most commonly cultivated Finnish oat varieties. Average oil content ranged from 6.1 to 7.8%. The environment had an effect on oil content of oats. Low growth temperatures increased the percentage of oil in oats and the synthesis of oleic and linoleic acids while decreasing the concentrations of stearic and palmitic acid. Youngs and Forsberg (1979) and Gullord (1980) concluded that there was no significant correlation between oil and protein content 1 6 in oats. The environment had a greater effect on protein content so variation among oil concentrations was usually less than that among protein concentration. Oat Starch: Starch is the major carbohydrate in oats. The amount varies with cultivars and with the method of extraction. Paton (1977) used an alkaline extraction procedure combined with centrifugation on nine oat cultivars and found starch levels which ranged from 43.7- 61.0%. MacArthur and D’Appolonia (1979) used a centrifugation extraction method for starch from three cultivars containing high, intermediate and low protein levels. The results were starch levels ranging from 67 - 73.5%. The nine oat cultivars analyzed by Paton (1977) were grown at various locations in Canada and the United States. Fertilizer application was at two levels; no fertilizer application or 500 kilograms per hectare (kg/ha). Fertilizer application increased protein content. There was an inverse relationship between protein content and starch content. The cultivars in this study contained higher (57-61%) yields of starch in varieties that had lower (14.3- 15.9%) protein content. MacArthur and D’Appolonia (1979) also reported that in oat groats, starch concentration varied inversely with protein content, the high protein cultivar contained the least starch. The starch granule of the higher level protein oat flour had the highest granule density which indicated a more compact granule structure which might explain differences in pasting properties. The high protein oat flour starch also had a slightly lower water binding capacity. 17 Amylose content and lipid content was slightly higher in high protein oats than in intermediate or low protein oats (27.9 vs 25.5 and 25.9%) with lipid values at 1.11 vs 0.67 and 0.81%. Qaft Structure and Processing Oats are a member of the gramineae or grass family and contain similar structures as those found in wheat, barley, corn and rice (Fulcher, 1986). However, the oat kernel has specific structural and chemical components that are different than other cereal grains. These greatly influence processing characteristics of oats. The oat kernel is divided into four distinct parts: pericarp, seed coat, germ and endosperm (Kent, 1983). Oats are covered by an additional layer outside the pericarp. This layer is the husk and unlike the husk in wheat and rye, it remains attached after threshing (Frolich and Nyman, 1988). The husk must be removed to process oats for human consumption. Oat structures and relatied chemfil comprments Commercial oat bran is composed of the pericarp, seed coat, aleurone and subaleurone layer (Kent, 1983). The aleurone layer has cuboidal cells with relatively thick cell walls (50-150 pm). The subaleurone layer has more irregular shaped cells and is not present at all points in the kernel. These two outer layers contain a high proportion of aleurone grains and protein bodies. Aleurone grains are protein bodies located exclusively in the aleurone layer and have been shown by selective staining (Fulcher and Wong, 1980; Gates and Oparka, 1982) to differ chemically from protein bodies located in the starchy endosperm. The subaleurone layer of high protein oats 1 8 (> 17% protein) has been shown to contain a large concentration of protein bodies and a lower percentage of starch granules (Fulcher, 1986). Phytin and phenolic compounds are associated with the primary cell wall of the aleurone layer of oats (Yiu, 1986). Beta glucan occurs in the inner cell walls of the aleurone layer and in the cell walls of the adjacent starchy endosperm (Fulcher, 1986). The starchy endosperm of oats contains oat protein bodies and starch granules (Yiu, 1986). Starch in cats is found as simple and compound grains. The oat starch compound grain is composed of two to several polygonal granules. Lipids in oats range from 5 to 9% depending on the variety. Most of the groat lipid is stored in the endosperm in the form of oil droplets. Commercial processing All oats used as human food are commercially processed (Kent, 1983). The enzyme lipase is located almost entirely in the pericarp or outer layers of the groat. The enzyme comes in contact with oat lipids during the milling process. The enzyme lipase hydrolyzes oat lipids which contain high percentages of oleic, linoleic and palmitic fatty acids into glycerol and free fatty acids. The presence of free fatty acids lead' to a bitter flavor in oatmeal. Free fatty acids react with bicarbonate of soda to form sodium salts of fatty acids which have a soapy flavor. The heat treatment known as the Miag process quickly raises the temperature of cleaned oats to 96°-100°C for 2-3 minutes to inactivate lipase and other unwanted enzymes. Lookhart et al (1986) reported on the effect of commercial processing on chemical and physical properties of oat groats. The stages of commercial processing monitored were; dehulling-to give 1 9 “original groats”, drying to yield “dried groats” and steaming and rolling to produce “oat flakes.” Scanning electron micrographs of the de-hulled groat showed rounded compound starch granules with diameters of 3 - 15pm. No fractured granules were seen. The protein bodies were small (diameter 0.5-2.0j.lm) and randomly distributed. The cell walls were intact. Drying resulted in a split groat that had some starch granules fractured into individual granules. The oat flake contained fragmented cell walls that were separated from other cell components. There was an increase in starch fragments into more individual granula than in dried groat; less protein bodies were visible. Browning of the oat flakes was speculated to be caused by the Maillard reaction and from heating. Chang and Solulski (1985) examined the functional properties of oat flour roller milled from Wild and domestic oat groats. The groats were steamed to inactivate oat lipase and kiln dried prior to processing. The steam treatment denatured oat proteins and decreased nitrogen solubility. The proteins were insoluble in the pH range 3 - 6, the range of pH commonly used in foods. Yiu (1986) used fluorescence microscopy to study the effects of processing and cooking on the structural and microchemical composition of oats. The bran, starchy endosperm and germ differed in milling properties, cell structure and chemical contents. The aleurone and subaleurone cell walls were relatively resistant to processing. Microscopic examination indicated that most endospermic cell walls were altered by processing. Many of the cell walls of the subaleurone and aleurone layer remained relatively 20 intact after processing. Mechanical forces did not disrupt the structural association between beta-glucan and the cell walls. Proteins and lipids in the endosperm tissue were most susceptible to processing (Yiu, 1986). Both proteins and lipids changed from distinct structural units to aggregated masses as a result of processing. Rolled oats contained many intact as well as broken starch granules. The broken starch granules were not gelatinized by the processing methods used to prepare rolled groats. Rolled oats had most of the lipid content still within cells that had intact cell walls. Individual lipid bodies could not be detected. Yiu concluded that the loss of lipids in rolled oats due to processing and cooking was insignificant. Oomah (1987) studied the effect of the commercial processing steps of conditioning, drying, cutting and grinding on oat protein solubility and pasting characteristics. Roller milled flour from groats was compared to hammer milled flour from rolled oats. Oat flour prepared from hammer milled rolled oats did not differ significantly in protein content from steel cut oats. Roller milled oat flour contained twice as much protein soluble in distilled water than hammer milled oat flour. The conclusion was that heat treatment, the Miag process, used in preparing commercial oat flour may effect protein solubility. Steam treatment was suggested to cause stable protein aggregates to be formed through non-covalent bonds. Pasting characteristics of oat flours roller mill‘ed from oats in different stages of commercial processing showed few differences (Oomah, 1987). Flours milled from conditioned groats had initial 2 1 swelling at a lower temperature than flour milled from dehulled groats. Oat flours roller milled from groats dried after conditioning had a lower peak torque than flour milled from steel cut oats. The hold torque of flour milled from steel cut oats was also higher than that of flour from dried groats. Differential scanning calorimetry results were that heat treatment of oats raised the starch transition temperature by up to 2°C (Oomah, 1987). The transition peak was narrower by 2-300 and there was a 20% reduction in enthalpy associated with starch gelatinization. The conditioning and drying processes caused a partial denaturation of oat proteins as indicated by an decrease in the transition enthalpy for the protein endotherm. A decrease in denaturation enthalpy has been explained as the increase in the ability of the protein to bind water (Wright, 1984). Eff_ect of Millirrg on Cereal Grains: Milling subjects particles to a combination of two or more of the following forces: compression, impact and/or shear forces (Haque, 1991). A compression or nipping force reduces particle size in a roller mill while impact or blow forces reduce particle size in a hammer mill. Hammer milling: Hammer milling produces a large amount of heat during impact which results in a loss of moisture from grain as evaporation (Haque, 1991). Nishita and Bean (1982) measured a 75°C temperature in hammer milled rice flour. 22 The size of particles produced by hammer mills is dependent on the clearance between the hammer tip and the screen and the diameter of the perforations of the screen (Haque, 1991). Size reduction in hammer milling is caused by the explosion due to the impact of the hammers, cutting by the edge of the hammers and rubbing action or attrition through the perforations of the screen. A disadvantage of hammer milling is that it produces less uniform product size. The particle size of hammer milled rice flour was finer than that of roller milled rice flour as measured by Ro-Tap sieving for 30 minutes (Nishita and Bean, 1982). Higher levels of damaged starch (20%) as measured by percent glucose were produced by hammer milling rice flour, than roller milling (1.2%) (Nishita and Bean, 1982). The level of damaged starch and finer particle size was thought to contribute to a higher alkaline water retention capacity in hammer milled rice flour than in roller milled rice flour. Mar milling; Roller milling subjects particles mainly to shear and compressive forces due to the corrugations on the roller surface and pressure exerted by rolls while pulling particles toward the nip (Haque, 1991). Milling performance in a roller mill is affected by roll diameters, speed and the ratio of the fast roll and slow roller (differential). The greater the roller diameter, the longer the time a particle will be in contact with it and the result is a finer grind. The faster the roll, the larger the amount of particles that pass through the rollers. If there is a great deal of difference between roller speeds more shearing action will occur. 23 The compression action of roller mills yields finer particles for harder grains because they are more brittle. A cereal with a softer grain will tend to flake in a roller mill system. Yamazaki (1959b) reported that the granularity of roller milled flours from soft and hard wheat can be influenced by adjusting the moisture levels of the kernels. The advantage of roller mills is that a more uniform product size is produced than with hammer milling (Haque, 1991). The rolling process does not generate as much heat as hammer milling so less moisture is lost from the particles being milled. Roller milled rice flour had a temperature of 30°C (Nishita and Bean, 1982). One disadvantage to roller milling is that elongated or fibrous materials such as oat hulls may be inefficient to grind (Haque, 1991). Particle Siza Related Properties; Shellenberger (1977) stated an effect of grinding that can be measured and related to kernel hardness is flour granularity. Particle size index is considered the most practical and reproducible method of measuring granularity. Wheat starch granules embedded in the protein matrix of the endosperm are susceptible to damage during grinding and this damaged starch can affect the baking performance of flour. Starch damage increased water absorption in dough, increased gassing power, reduced tolerance to mixing and could be deleterious to bread quality. Starch damage is related to kernel hardness, protein content, tempering conditions and roll pressure. 24 Sullivan et al (1960) examined the relation of particle size to certain flour characteristics. A hard winter wheat was roller milled with an experimental Allis mill 5 times to reduce flour to finer than average granulation by using roll pressure. Extensive air classification (19 times) was used to separate the flour completely into fractions of different particle sizes. The smallest flour fraction, less than 8 microns, had an ash content twice that of the parent flour. The level of ash was said to be determined by the presence of endosperm cell wall material and peripheral cells in the flour fractions. Protein content was highest in the smallest size range of 1 to 16 microns due to protein fragments The influence of milling was stated to be, that finer flour had more broken endosperm cells (Sullivan et al, 1960). An increase in the percent of particles below 55 to 70 microns could cause an increase in the specific surface (specific surface=cm2/cm3) of a flour. Visco-amylogram results were that increased protein content was linked to decreased starch content. If all other factors were equal, a higher protein content resulted in a lower amylograph viscosity. Starch damage was dependent on the type of grinding and was not correlated to the fineness of grind. Weaver et al (1981) investigated the effect of milling on trace elements (iron, zinc, manganese, copper, chromium, nickel) and protein content of oats. Quick cooking oat flakes were prepared from Grade B groats by drying, cutting, steaming and rolling. Oat flour was rolled and ground from the cut, steamed grade B groats. Oat flour and quick cooking oat flakes contained similar levels of 25 protein and mineral concentrations except for iron. Oat flour contained greater (P=0.04) amounts of iron than in oat flakes. Nishita and Bean (1982) used sieve size analysis, scanning electron microscopic examination and the Hunter Color Difference meter to measure particle size in rice flour ground with seven different mills (burr, blade, roller, hammer, pin, turbo or high speed impact). The Hunter Color Difference meter indicated the relative particle size produced by the different mills. Finer flours gave the largest "L" values for whiteness and smallest "b" values for yellowness. Turbo milled rice flour had the largest "L" value (+93.9) and smallest "b" value (+3.4) in agreement with scanning electron microscope examination. Pin and hammer milled flour had, respectively, the second and third highest '"L" values. Visco-amylograph properties were influenced by particle size (Nishita and Bean, 1982). Coarsely ground rice flours produced amylograph pasting curves with initial viscosity increases at 10.500 higher than rice flours with finer particles. Coarser flours had lower peak viscosities and lower viscosities upon cooling than flours containing finer particles. Coarsely ground rice flour did not have the thickening ability of more finely ground flours. The highest alkaline water retention capacity was in flour with the finest particle size and highest percentage of damaged starch, ex. turbo and hammer milled flour (Nishita and Bean, 1982). With the exception of roller milled rice flour, as the percentage of damaged starch increased, alkaline water retention capacity increased. Roller milled rice flour with 1.2% damaged starch retained as much water as pin milled (2x) with 16% damaged starch. 26 Burr milled flours, with the coarsest particle size and intermediate level of starch damage, retained the least alkaline water. The Endosperm Separation Index (ESI), is a measure of the milling quality or how easily endosperm is separated from bran flakes (Yamazaki and Andrews, 1982a). Soft and hard wheat cultivars are known to produce patent flours and straight grade flours that differ in particle size distribution (Chadhary et al, 1981, Donelson and Yamazaki, 1972). Chadhary et al (1981) concluded that a flour characteristic related to flour granulation was more influential on cake volume than flour particle size. Gaines (1985) evaluated 219 soft red winter and soft white winter wheat cultivars for associations between particle size, protein content, kernel hardness and other functional properties. Softer wheat kernels were lower in protein content and when milled produced flour with smaller particle size. A smaller straight-grade flour particle size was generally associated with a higher rating for Endosperm Separation Index. Cakes baked with the finer flours had larger volumes and sugar snap cookies had larger cookie spread or diameters. If a soft wheat flour that inherently milled into finer flour particles was subjected to an over milling treatment to increase particle size reduction, the cookies baked with the over milled flour had a smaller diameter. Scalon et al (1988) measured the particle size related properties of flour roller milled from hard red spring wheat farina. The number, type and properties of flour particles were dependent on the manner of fracture and where it occured. The coarse fraction had the highest protein content. The starch granules in the coarse 27 fraction still had wedge protein adhering to the starch granules as in the intact granule. The lower protein content in the fine fraction was thought to be due to a higher percentage of individual starch granules. The fine fraction exhibited greater water absorption. Kurimoto and Shelton (1988) measured the effect of flour particle size on baking quality in yeast bread and on other flour attributes. Flour particle size indicated the degree of fineness and total flour surface area. A hard red spring wheat was roller milled to different particle sizes by adjusting the moisture level of the farina.. Protein content slightly decreased as flour particle size decreased. In wheat flour, protein content and particle size were highly correlated (r=0.96, p< 0.01). There was a significant difference in Hunter Color Difference meter readings only for the 55.9 and 42 um particle size range. Smaller particles resulted in a smoother surface with an increased amount of light reflected off the surface. Medium sized flour particles (62.6 and 55.9 um) produced yeast bread loaves of slightly higher volume and weight. The level of damaged starch (27 and 28 Farrand Units) in the medium flour particle size range may have contributed to the differences in loaf volume and weight. Cookie Flaur Quality Mailhot and Patton (1988) defined flour quality as the ability of the flour to produce a uniformly good and product agreed to by the supplier and the customer. Soft winter wheats are the wheat class preferentially used for cookie flour. Flour specifications have been 28 developed which primarily assist in milling operations. These specifications include ash (0.42-50%), presence of bleaching or maturing agents (none or light Cl2), particle size range (0-125um), starch damage (low as possible) and protein (7.0-9.50/0). Sugar snap cookies are one baking test used by industry to evaluate soft wheat flour suitability for specific baked products because flours meeting these physiochemical requirements may not bake a satisfactory product (Yamazaki, 1969). Sugar snap cookies are evaluated on the basis of cookie spread or diameter with the desired width to thickness (WIT) ratio being 8.0-9.5 (Mailhot and Patton, 1988). Top grain score or the degree of surface break-up is considered optimum if there are “fairly wide cracks somewhat evenly spaced to give uniformly sized islands” (Sollars, 1959). Top grain score is a function of cookie spread. The flour components of protein, starch, lipid and non-starchy polysaccharides all contribute to the baking performance of soft wheat flours. The functionality of flour components can only be determined when the isolation and reconstitution methods do not influence flour properties (Pomeranz, 1988). P_hv§i_ochemical requirements_9f Cooki; Flour Ash or mineral content is sometimes related to the efficiency of the milling process to remove the bran from the endosperm (Mailhot and Patton, 1988). Minerals in the wheat kernel are concentrated in areas adjacent to the bran and bran coat. Flours with higher levels of ash are darker in color because of the fine bran particles. The level of ash must be controlled in cookie flours to 29 accurately perform the MacMichaeI viscosity test on unbleached cookie flours (Brennis, 1965). BleghiaaAgents: Cookie flours are not usually bleached because flour color is not critical in the baked product. Chlorine bleach treatment of soft wheat flour also increases cookie thickness and decreases cookie spread in a direct proportion to the amount of bleach utilized (Brennis, 1965). Commercial bakeries may use chlorinated cookie flours to achieve uniform spread. Donelson (1990) reported that chlorination in the pH 3.90-3.66 range resulted in reduced cookie spread and increased alkaline water retention capacity (AWRC) of the parent soft wheat flour. The loss in cookie spread was due to an increase in the hydration of the starch fraction of the soft wheat flour. _article size: A standardized milling procedure is required if soft wheat particle size is to be an indicator of cookie flour quality (Gaines, 1985). The study of 219 soft red winter and soft white winter straight grade flours found a significantly negative correlation (P=0.001) between flour particle size (mean volume diameter) and cookie diameter. Cookie flours with smaller average particles baked larger cookies. Yamazaki (1959b) had previously concluded that baking characteristics of wheat varieties may be due to granulation differences along with the presence of purified starch tailings and mechanically injured starch. Donelson and Yamazaki (1972) concluded that soft wheat flour particle size is an inherited trait of a wheat variety which is not influenced to a larger extent by crop year. 3O Starch and Starch damaga: Starch comprises about 54-72% of the wheat kernel dry weight depending on variety and growing conditions (Pomeranz, 1988). Particle size distribution or range can be related to starch damage levels in soft wheat flours (Brennis, 1965). The study found flours with a higher percentage of particles over 60 microns in size had a lower percentage of starch damage. The grinding action of mill rolls produces a degree of damage to wheat starch during milling of the wheat kernels into flour. Wheat starch granules are firmly embedded in the protein matrix and this results in physical damage of starch granules. Damaged starch granules swell extensively in cold water and are largely responsible for differences in flour water absorption (Tipples, 1969). In soft wheat, the starch granules are not as strongly held in the protein matrix and this lessens the level of damage. Under uniform milling conditions, soft wheat has finer granulation and a lower absorption properties than that of hard wheat containing the same level of protein (Yamazaki, 1969). Brennis (1965) reported the percentage of starch damage in resulting cookie baking tests was inversely related to cookie WIT ratio. Greenwood (1976) used optical and scanning electron microscopy to show the state of organization of starch granules in cookies and other baked goods. Depending on the type of cookie, the starch granules ranged from being in a swollen state to being in the disrupted state. Lineback and Wongsrikasem (1980) used light and scanning electron microscopy to observe the degree of gelatinization of wheat starch granules in commercially baked sugar cookies. Wheat starch granules had a low degree of deformation and folding 3 1 in sugar cookies. Only 9% of the total starch granules exhibited a loss of birefringence. Enzymatic determination measured 4% gelatinization in starch that had been extracted from the cookies. Abboud and Hoseney (1984) used differential scanning calorimetry on baked sugar-snap cookies and reported the starch to be ungelatinized. P_r_ot_ei_r1: The specification of 7.0 to 9.5% protein in cookie flour is based on functionality. High protein flours tend to cause puffed peaked crown cookies (Brennis, 1965). Additional amounts of sugar and shortening must be incorporated in the formula to produce cookies of acceptable quality. Wheat endosperm proteins have the unique property of forming gluten when hydrated and mixed with water (Pomeranz, 1988). Gluten is a complex of gliaden and glutenin proteins. Gliaden contributes extensibility to a flour dough system while glutenin contributes elasticity. Gluten is the primary structural component of wheat flour dough and enables dough to retain leavening gases. The formula for sugar-snap cookies contains a high concentration of sugar and fat and a low amount of water. Tsen (1976) had identified the need for sugar-snap cookie dough to have tensile strength and extensibility for sheeting. The gluten network in sugar-snap cookies is not extensive, however, the gliaden and glutenin is not functionally inert (Gaines, 1990). Gaines (1990) used dithioerythritol, a chemical which cleaves disulfide bonds and interacts with resulting thiol groups, to influence the rheological properties of sugar-snap cookie dough. The result was a definitive change in the dough consistency and cookie spread. He concluded 32 that the protein precursors to gluten associate in sugar-snap cookies to form a few intra- and intermolecular bonds. The previous evidence for this conclusion was the fact that the level of mixing action and handling of cookie doughs such as rolling can reduce cookie spread (Gaines et al, 1988). Role of other flour components in cookie quality Lipid; Wheat flour lipids comprise an average of 2% by weight of the flour (MacRitchie, 1981). The major components of the non-polar fraction are steryl esters, monoglycerides, diglycerides, triglycerides and free fatty acids. The polar fraction is mostly galactolipids and phospholipids. The non-polar fraction is approximately 50.9% and the polar fraction is approximately 49.1% of the wheat flour lipids (MacMurray and Morrison, 1970). An early study of lipid functionality in flours (Cole et al, 1960) used water saturated n-butyl alcohol to extract essentially all flour lipids. Later research (Finney et al, 1976) reported on the effect of solvents on extracted flours. The study determined that butanol forms a complex with wheat starch and this interaction influences the functional properties of extracted flours. Cookies prepared with water saturated butanol extracted unbleached soft wheat flours had decreased diameters (7.490m vs 8.790m) when compared to the parent flour. The defatted cookies were also browner in color leading to the conclusion that lipids interfered with the browning reaction that takes place during baking. In contrast, Kissel et al (1971) reported that cookies from petroleum 33 ether extracted flours were lighter than normal color and the normal yellow color returned as the percentage of restored lipid increased. Kissel et al (1971) defined “free lipids” as those that are extracted from wheat flour with petroleum ether. Other researchers (Kissel and Yamazaki. 1975: Yamazaki and Donelson. 1976: Clements and Donelson, 1981 and Clements. 1980) defined “free lipids” as those that are extracted from flour with non-polar solvents such as hexane. Regardless of the solvent. restoring lipids to flours resulted in an increase in cookie diameter, and improvement of top grain scores (Cole et al, 1960: Kissel et al 1971: Yamazaki and Donelson. 1976). Kissel et al (1971) defatted flours of four wheat varieties: two soft red (Theme and Blackhawk). one soft white (Avon) and one semihard red (Purkof). . When three to four times the lipid level of the parent flour was added to defatted flours and there was an increase in top grain scores and cookie diameter in all four wheat varieties. For each wheat variety. restoration of an increasing amount of lipid resulted in a larger cookie diameter, a better top grain score and a more intense yellow hue. The conclusion was that addition of free flour lipid could significantly improve the baking performance of poor quality flour. Kissel and Yamazaki (1975) used free lipids from flour to increase the cookie spread and top grain scores of cookies fortified with gluten and soy protein. Yamazaki et al (1979) extracted lipids from soft white and red wheat bran with hexane. The bran lipids had the same cookie diameter increasing effect as flour lipids. A addition of 6% lipids (flour weight basis). increased 34 the cookie spread and top grain scores of cookies baked from a semi- hard red winter wheat with poor cookie spread potential. The phospholipid fraction of wheat flour appears to have the most influence on lipid functionality in sugar-snap cookies. Cole et al (1960) and Clements and Donelson (1981) restored the phospholipid fraction of defatted flours with the result of producing cookies with diameters and top grain scores very similar to cookies baked from the parent flours. The addition of phospholipids from non-wheat sources, soybeans and corn, to defatted flours had an improving effect on cookie spread. top grain and color (Cole et al. 1960). Clements and Donelson (1981) separated hexane extracted flour lipids into ten fractions using preparative thin layer chromatography (TLC). When the flour lipid fraction that corresponded to digalactosyldiglyceride (DGDG) and phosphatidylcholine (PC) was added to defatted flour, cookie spread and top grain scores were very similar to the controls. Restoration of a glycolipid which corresponded to monogalactosyldiglyceride (MGDG) resulted in partial recovery of flour properties. Yamazaki and Donelson (1976) evaluated the external characteristics and the internal appearance of cookies baked from defatted flours. The optimum internal structure was light in color with a well layered crumb and received a score of 9. A rating of 0 was the bottom of the scale used for a cookie with only a top and bottom surface. The average internal score for cookies baked from defatted flours was a 3, indicating the presence of one large coalesced gas pocket and dark and greasy walls. 35 Clements (1980) demonstrated the effect of lipid removal on the internal structure of sugar-snap cookies by a resin embedding method. Four straight grade flours from two different wheat classes, hard and soft. were used in the study. The wheat varieties used were Arthur, a soft red winter. Chris. a hard red spring. Eagle. a hard red winter and Yorkstar, a soft white winter. Cookies baked from hexane extracted soft red winter wheat flour had a 2 cm decrease in diameter while cookies baked from hexane extracted hard red winter and spring wheat flours had a 1 cm decrease. Top grain. which is a consequence of spread, was less desirable than the control for all varieties. The interior of cookies baked from defatted soft wheat had no interior cell walls, possessing only a large pocket enclosed by a thin shell. The free flour lipids appear to stabilize gas cell walls during oven expansion. The weak gluten and high dough plasticity of soft white wheats without the flour lipids contributed to collapsed cell walls instead of retaining expanding gases until the structure solidified. Non-starchy pohrsaccharides: The primary non-starchy polysaccharide of straight grade soft wheat flour are failings. Straight grade soft wheat flour can be fractionated into five components - free lipids. gluten. tailings. starch and water solubles. Tailings are the wheat flour fraction that contains a high level of water soluble pentosans (Yamazaki. 1955). Wheat pentosans are polysaccharides with the pentoses, arabinose and xylose as the major structural component (Campbell. 1972). They are primarily found in and just inside the cell walls of the intact wheat kernel. The concentration of pentosans is higher in bran than in endosperm 36 therefore white flour contains ~ 2-3% pentosans. However. wheat endosperm pentosans are hydrophillic and immobilize free water in doughs. Pentosans can absorb 15 times their own weight in water (Bushuk. 1966). An excessive amount of pentosans reduced the spread of sugar snap cookies (Sollars. 1959). Yamazaki et al (1977) fractionated three pure variety straight grade wheat flours into five fractions: free lipids. starch, gluten. failings and water solubles. The wheat varieties fractionated were Shawnee, a hard red winter. and Thorne and Blackhawk, soft red winter wheats. The tailings from the three flours had respective alkaline water retention capacities of 165.9%, 193.4% and 266.1%. The tailings. starch and gluten had an additive effect on depressing cookie diameter. Flour moisture: The concept of an “optimized baking test”. in which the performance of a wheat flour is not tested under fixed or arbitrary circumstances was supported by flour moisture studies done by Doescher and Hosnev ( 1985) and Gaines and Kwolek (1982). Doescher and Hosnev (1985) reported flour moisture effected cookie symmetry and top grain or surface cracking. Increasing flour moisture resulted in a decrease in the number of islands on the cookie surface and the size of the cracks between the islands became larger. Flour moisture was found to be more important than total moisture in the cookie formula. Cookies prepared with equal amounts of total water did not have similar cracking patterns. The micro-method Ill formula requires that the amount of water added be adjusted to produce optimum dough consistency. AACC (1983). Gaines and Kwolek (1982) stated that flour moisture 37 levels above 14% are detrimental to micro-method lll cookie top grain. The study examined stickiness and consistency in flour with four levels of flour moisture; 11.5. 12.5, 13.0 and 14.5 percent moisture. ln flours with low moisture content (11.5%). a relatively small change in dough water absorption levels caused relatively large changes in dough stickiness. Cookie spread and top grain score are external characteristics of sugar-snap cookies. Cookie spread is determined by the spreading rate and the setting time (Abboud et al. 1985a). Good quality cookie doughs have a faster spreading rate during baking. Yamazaki ( 1959b) reported that cookie doughs that spread the least have an earlier increase in viscosity during the baking period. Brennis (1965) attributed the rate of viscosity increase to the relative ability of gluten and starch to attract water in the presence of sugar during the earlier stages of baking. Commercial requirements for quality: The level of automation involved in commercial baking of cookies dictates a specific level of uniformity in cookie qualities. Cookie doughs used in commercial bakeries must have an even consistency with the baked cookies being resistant to fracture and crumbling (Fuhr. 1962). The baked cookies must have uniform diameters and thicknesses to give the customer a neat full package. Automated packaging requires uniform cookie size to allow the bottom seam of continuous wrapping machines to have the proper overlap to seal. MATERIALS AND METHODS This research project was composed of three parts. The first part was the production of oat flour from three oat cultivars or varieties. The second part was the evaluation of chemical and physical properties of the flours. The third part was measuring the functionality of oat flour in sugar snap cookies when substituted at two levels for soft wheat flour. QaL_EI_9_l.l.L§_Z Three oat cultivars were used in the study to prepare whole grain oat flour: Mariner, Ogle and Porter. Mariner was donated by Michigan Foundation Seed (East Lansing, Michigan). Ogle and Porter were purchased from Purdue Agricultural Alumni Seed Improvement Association, Inc (Romney, Indiana). The three oat cultivars were dehulled and commercially heat processed to inactivate oat lipase by the Quaker Oats Company (Barrington, Illinois). Raw cats were put through an impact dehuller two times to break groats from the hulls. The. lighter weight hulls, were separated from oats and groats by a pneumatic separator. The groats were heated to 265°C for 7.5 minutes to inactivate oat lipase, dry and toast the greats. Half of the greats from each oat cultivar were steamed and flaked into rolled oats. A description of the processing from raw oats to flakes is given in the Appendix. The batches of oat groats and oat flakes were tested for tyrosinase activity. Tyrosinase activity measurements are used to indicate if residual lipase activity is present in the heat treated oat product. The tyrosinase enzyme is 3 8 39 more heat stable than lipase and a rapid analytical test for tyrosinase activity is available for cat processors (Webster, 1986). Equal amounts of the groats and rolled oat flakes from each cultivar were then milled into whole oat flour. See Figure 1. A Fitzmill model JT (Fitzpatrick Co., Chicago, Ill.) equipped with a screen containing round holes, 4 x 10‘2 inches in diameter, was used to make hammer milled whole oat flour. A Brabender Quadrumat Jr. Mill (C.W. Brabender Instruments, Inc., So. . Hackensack, NJ) was used with a No. 70 gritz gauze reel sifter to make roller milled whole oat flour. The roller milled flours were mixed in a Kitchen Aid Mixer, Model K5SS (thchen Aid Co., St. Joseph, Mi) at speed 4 for 15 minutes to evenly distribute the sifted bran, break and reduction flour fractions. The roller milled and hammer milled flours were bagged in moisture proof polyethylene bags and stored at 2°C. W Two red soft wheat flours (Becker and Compton) and one white soft wheat flour (Caldwell) were donated by the United States Agriculture Research Service (U.S.A.R.S.) Soft Wheat Quality Laboratory (Wooster, Ohio). The three wheat flours were milled as straight grade with no chlorine or bleaching treatment. The flours were bagged in moisture proof polyethylene bags for storage at 2°C. 931m Fleur Composites; Oat-wheat flour composites were prepared by substituting hammer and roller milled oat flours at the 15 and 30 percent level (14% mb) for a soft wheat flour variety. Hammer milled and roller Flour: f iII‘ Figure 1. 40 Mariner Ogle Porter Groat Flake Roller Hammer MHG OHG PHG MHF OHF PHF MRG ORG PRG MRF ORF PRF Experimental Design for Oat Flour Production. 41 milled oat flours were each separately blended with the three soft wheat cultivars to make composite flours. Flour for six batches of cookies was weighed into a glass jar at least 10 times the volume of the flour. Uniform mixing was achieved by rotating the jar in a tumbling motion eight times to the right and then eight times to the left, followed by an additional eight more times to the right (Personal communication, J. Donelson). Composite flour aliquots for each batch of cookies were weighed into pint jars, covered with a screw cap and sealed with Parafilm (American Can Corp Greenwich, Ct.) and stored at 20°C until time of use. _ -o-r' 1:1 -. --:I-. for : _---._ ._ ' The experimental design for measuring the functionality of oat flour in sugar-snap cookies was to separately examine the effects of processing, method of milling and oat cultivar-wheat variety interaction. The experimental design for measuring the effect of processing on cookie quality is given in Figure 2. Only hammer milled whole grain oat flours were used for this sub-study. The gravity feed system of a roller mill does not efficiently feed oat flakes into the rollers. The soft wheat variety was selected based on a preliminary study. The preliminary study used a commercial oat flour, Quaker Oat flour No.1., and the three different soft wheat varieties. A description of the study is given in the Appendix. The soft wheat variety selected for use had produced the best quality cookies on the basis of cookie diameter and top grain scores. The same wheat variety was used to measure the effect of milling on cookie quality. See Figure 3. Only whole grain oat flours 42 Wheat cultivar: Caldwell W Mariner Ogle Porter 9315mm; Groat Flake M Hammer MHG OHG PHG EIQLLL‘. MHF OHF PHF Figure 2. Experimental Design for Effect of Processing. 43 Caldwell Ogle Porter Roller Groat Wheat cultivar: Qat Cultivar: Mariner Tyga at Mill: Hammer QaLEQrm MHG ELQUL MRG OHG ORG Figure 3. Experimental Design for Effect of PHG . pee Milling. 44 from groats were used in this sub-study to eliminate the influence of moist heating from the flaking process. The experimental design for measuring the effect of the interaction of whole grain oat flour and soft wheat flour on cookie qualities is given in Figure 4. Only hammer milled whole grain oat flour from groats was used in this sub-study. The rationale was to increase the applicability to a commercial cookie production system which would more likely purchased hammer milled whole grain oat flour from groats. Ph i l r i f r n fl r The 1000 kernel weight of the cat groats was measured using the method of Glover (1985). The kernel moisture content of all three cultivars was between 6.82 to 7.08 percent. One hundred randomly chosen whole dehulled kernels were weighed. The selection process was replicated six times without replacement for each cultivar. The average weight was then multiplied by 10. The 1000 kernel weight was the average value of six trials. Particle size index of whole grain oat flours was determined in duplicate using a modification of AACC Method 55-30 (AACC, 1989) A No. 100 US screen with 12 rubber sieve cleaners was used on a Ro-Tap Sieve Shaker ( W.S. Tyler, Cleveland, Ohio). Flour color was measured in triplicate with a Hunter Color Difference meter Model D25-PC2 (Hunter Associates Laboratory, Inc. Reston, Va) using Hunter Lab Tile Standard No. 02-30954 (White, L = 92.3, a = -0.9, b = +0.1). The L value represented reflectance ranges from black to white (0 to 100), a value was reflectance ranges from green to red 45 Compton Porter W Becker Caldwell Egrm of oats: Groats Tyga gf mill: Hammer W Mariner Ogle MB OB Flgur: MCA OCA MCO OCO PB PCA PCO Figure 4. Experimental Design for Interaction with Wheat Cultivars 46 (-a to +a) and the b value was reflectance ranges from blue to yellow(-b to +b). Qhamigal Analyses of Cat Flagra: Moisture content was determined in triplicate by AACC method 44-40: Modified Vacuum Oven method (AACC, 1989) Protein content and ash was measured in triplicate by AOAC methods 24.038 and 14.006 (AOAC, 1984) Carbohydrate content was calculated by the difference between the sum of protein, ash, and fat in three dried samples. Lipid extraction procedure: Percentage fat was determined in triplicate by the method of Price and Parsons (1974). A 6.25 9 sample (db) of oat flour and 290 ml of chloroform- methanol-water (1 0:10:09) was placed in a 500 ml separatory funnel. The mixture was swirled to solubilize the flour and initiate extraction. After 48 hours, the lower chloroform layer was filtered through a plug of glass wool into a 500 ml Erlenmeyer flask with a ground glass stopper. A 100 ml volume of chloroform was added to the separatory funnel to replace the original volume every 24 hours for two times. The three volumes of extract were quantitatively transferred to a desiccated, pre-weighed 500 ml evaporating (boiling) flask. The extracts were concentrated to less than 5 ml under vacuum in a 50°C water bath using a Bucchi Rotovapor R rotary evaporator (Bucchi Inc., Switzerland). The rest of the chloroform was evaporated under a hood overnight. The flask containing the extract was dried in a moisture oven at 90-100°C for 45 minutes and then placed in a desiccator containing anhydrous CaSO4 for 45 47 minutes before weighing. The lipids were then redissolved in chloroform, placed in tightly capped glass vials and stored in the _ refrigerator at -4°C until separation into lipid classes. A silicic acid column was used to separate the lipid into classes. The silicic acid was prepared by the method of Hirsch and Ahrens (1958). Fifty grams of 325 mesh silicic acid was washed with four 50 ml portions of anhydrous methanol. The silicic acid was dried and activated by a 24 hour holding period at 100°C. The glass column (30 cm x 2 cm) was prepared by first packing one inch of glass wool into the bottom. A10 gram portion of the dried activated silicic acid was dispersed in chloroform and poured into the glass column. The silicic acid column was allowed to settle for 8 hours with intermittent rinsings of chloroform. Five grams of anhydrous sodium sulfate was placed on the column immediately before use. From 0.4 to 0.6 grams of lipid was placed on the silicic acid column. Fifty ml of each solvent was used to elute the different classes of lipids. Chloroform was used to elute neutral lipids, acetone was used to elute glycolipids and anhydrous methanol was used to elute phospholipids. The elutant from each lipid class was collected in pre-weighed desiccated 50 ml boiling flasks. The solvents were removed by evaporation with a Buchi Rotovapor. The percentage of each lipid class was determined by weighing the desiccated 50 ml boiling flasks and the lipid residue. Total dietary fiber assay: Total dietary fiber was determined in triplicate by the AOAC methods 43.A14 - 43.A20 (AOAC, 1984) with the modification for enzyme activity of the heat 4 8 stable a-amylase. The enzymes were from Sigma (St. Louis, Mo), heat stable or amylase (A-5426), protease (P-3910) and amyloglucosidase (A-9913). A technical bulletin from Sigma described the appropriate modification for the specific lot of the enzymes The phosphate buffer (50 ml) was added to the dried oat flour samples for at least 30 minutes prior to addition of the heat stable or amylase enzyme to allow complete solubilization of the oat flour. 6-glucan Assay: Total p-glucan content was measured in triplicate using the method of Carr et al (1990). See Figure 5. The oat flour samples were dried in a vacuum oven at 90-100°C for 24 hours. A 200 mg sample of oat flour from each cultivar was extracted with refluxing 80% (v/v) ethanol (5 ml) for two 30 minute periods. After cooling, the extracted residues were recovered quantitatively and the supernatants were discarded. The ethanol treated residues were extracted with 1.0 N NaOH (10 ml) at 20°C for 16 hours. The extract was next neutralized by the addition of 1.0 N HCI and centrifuged at 2500 rpm to remove insoluble materials. The supernatant was collected. The pellet was washed with an additional 10 ml of water and centrifuged for a second time. The supernatant from the first and second centrifugation was combined and adjusted to equal volume before assaying for B-glucan. A commercial cellulase, (Sigma C 0901) from P. funiculosum was heat treated to remove any contaminating amylolytic activity. The heat treatment consisted of suspending the crude enzyme (0.40 g) in 10 ml of a 0.05 M sodium acetate-HCI buffer (pH 4.0) for 10 minutes. The enzyme solution was then centrifuged at 2,500 rpm for 49 I Ground Dried SampleJ 5 Refluxing 80% ethanol, 2X 5 Pellet .. Supernatant LDlscard) 5 Alkali extract (1.0 N NaOH. 16 hr) 5 meutralize (1 .o N HCIfl 5 Centrifuge (2,500 rpm, 10 mifl 5 Supernatant up Residue (Wash) 5 5 Centrifuge 42,500 rpm, 10 min , 5 4- Supernatant 5 Assay Total (fl-glucan Figure 5. Quantiative extraction procedure for total B-glucan daterrnination in oat flours. Adapted from Carr at al (1990). 50 10 minutes. The supernatant was transferred into 5 (1 x10 cm) tubes and heated in a 70°C water bath for 1 hour. The tubes containing the supernatant were immediately cooled by placement in an ice bath for 2 minutes. The supernatant was next dialyzed for 16 hours against 2 L of 0.05 M sodium acetate-HCL buffer at 4°C. The enzyme solution was then centrifuged for 30 minutes at 2,500 rpm. The purified enzyme preparation was contained in the supernatant. The enzyme preparation could be stored at 4°C for one week before using in the assay. The enzyme converted the (1-3)(1-4)-6-D-glucan of the oat flour samples into glucose. The percent glucose was measured using the glucose oxidaselperoxidase procedure AOAC Methods 31.240-243 (AOAC, 1984). Cat starch isolation: Oat starch was isolated by alkaline extraction using the method of Paton (1977). See Figure 6. A 100 gram sample of oat flour and 1 L of deionized distilled water (DDW) was slurried for 2 minutes at high speed in a Waring Blender. The pH of the slurry was adjusted to 10.0 with [20% wlv] sodium carbonate. The slurry was heated to 45°C in a circulating water bath with intermittent stirring. The 45°C temperature was maintained for 30 minutes to extract oat proteins and non-starch carbohydrates. The slurry was centrifuged for 30 minutes in a refrigerated (10°C) centrifuge at 5,000 x g. The supernatant containing protein and non- starchy carbohydrates was discarded. The residue was resuspended in DDW. The alkaline extraction and centrifugation was repeated. The twice extracted residue was re-suspended in 500 51 100 g Flour 5 Slurry in Waring Blendor (1 L. Distilled Water, 1 min) 5 pH Adjustment to 10 (20% w/v Sodium Carbonate) 5 Heated Extraction, 45°C, 30 min 5 Centrifuge (5000 x g. 30 min) 5 Re-suspend residue (1 L. Distilled Water) 5 Heated Extraction, 45°C, 30 min 5 Re-suspend residue (500 ml. Distilled Water) 5 Remove Coarse Bran (Pour through No 100 sieve) 5 Centrifuge (1500 x g, 30 min) 5 Remove fine bran layer with spatula 5 Suspend starch in Distilled Water 5 Neutralize starch (2 N HCI) 5 Centrifuge (1500 x g. 30 min) 5 Rewash starch (500 ml Distilled water) 5 Pour through No 200 sieve 5 Freeze dry Figure 6. Oat starch Isolation adapted from Paton (1977). 5 2 ml of DDW. The coarse bran was removed by pouring through a No 100 sieve. The starch milk was centrifuged at 1500 x g for 30 minutes. The fine bran layer which settled above the starch was removed with a metal spatula. The starch was suspended in DDW and neutralized with 2 N HCI. The neutralized starch milk was centrifuged at 1500 x g for 30 minutes. The supernatant was discarded. The starch was re-washad with 500 ml of DDW and filtered through a No 200 sieve. The starch milk was Iyophilizad for 48 hours in a Virtis Unitrap ll freeze dryer at a pressure of 4-6 x 10'2 Torr and tray temperature of 40-50°C. The dried starch was stored in a desiccator over anhydrous CaSO4 until pasting properties were measured. E l' l l l . l' _ Pasting characteristics of the oat flour and starch were determined in duplicate for each sample using a Brabender Viskograph-E (C.W. Brabender Instruments, Inc., So. Hackensack, NJ). The pasting procedure of Chang and Sosulski (1985) was used for a 11% slurry (db) of oat flour. The flour slurry was heated at a constant rate of 1.5°C increase per minute. The pasting procedure of Doublier at al (1977) was used for oat starch with a 9% slurry (db). The starch slurry was heated at a constant rate of 6.0°C increase per minute. The initial pasting temperature or temperature at which the viscosity curve of an amylogram first increased by 10 Brabender Units (BU) was recorded for cat flours and starches. The holding period was 15 minutes for cat flours and 30 minutes for oat starches. The peak hot viscosity was the maximum viscosity of the 5 3 ~ gel during the 96°C holding period. The peak cold viscosity was the maximum viscosity of the gel upon cooling to 50°C. The viscosity after 15 minutes at 96°C was measured for flours while the viscosity after 30 minutes was measured for starches. Alkaline water retention of wheat flours, oat flours and oat- wheat composites was determined in triplicate by AACC method 56- 10 (AACC, 1989). - i r i n n v i A balanced complete block design with three replications of each treatment was used in the preparation of the sugar snap cookies. Room temperature, humidity and barometric pressure was monitored during the baking periods with a Weather Measure Matereograph, Model M701-E (Weather Measure Corp. Sacramento, Ca) Sugar snap cookies were made using AACC Method 10-52 (AACC, 1989). A National nonrecording micromixer was used to mix the cookie dough. A pre-haatad, humidified rotary hearth electric oven (National Manufacturing Co., Lincoln, NE) was used to bake the cookies at 204°C. for 13 minutes. The cookies were cut 6.0 mm in diameter and 0.6 mm thick. Average cookie diameter (cm) was determined using all four cookies from both bakes. The top grain score was assigned by comparing the degree of surface cracking to a set of photographs from the Soft Wheat Quality Lab at Wooster of sugar-snap cookie. standards. The standards represented the scale for top grain score from 0 to 9. See Appendix. A 0 was poor compared to a 9 which was optimum. The color of two cookies from each treatment were measured with a Hunter Color Difference meter Modal D25-PC2 (Hunter Associates Laboratory, Inc. Reston, VA) using 5 4 Hunter Lab Tile Standard No. C2-12403 (Yellow, L = 78.4, a = -3.0, b = +227). A second measurement of color difference was taken after a 60° rotation to calculate an average value for each cookie. Objective measurement of tenderness and crispness was accomplished using a Food Technology Corporation Model TR-5 Texture Recorder (Rockville, Md) equipped with a PTA-300 Force Transducer. Tenderness and crispness determinations were done in duplicate. The standard shear compression call (CS-1) with a range of 11—0 was used to measure tenderness as pounds of force per gram. The single blade shear cell (CA-1) with a range of 3170' was used to measure crispness as pounds of force per square millimeter. Excess cookie dough was stored in sealed polyethylene bags at 5°C for moisture analysis. Moisture retention was determined in duplicate by measuring the difference between moisture in cookie dough and baked cookies crumbs using AACC method 44-40: Modified Vacuum Oven method (AACC, 1989). nn'n El r n i r W Greats of each cultivar weighing 0.03 grams were vacuum dried (25 psi) for 5 hours at 90-100°C and stored in a desiccator containing anhydrous CaSO4. The groats were frozen in liquid nitrogen for five minutes before being cut in half Iatitudinally or longitudinally. The cut groats were sonicatad in 100 % ethanol for 5 minutes to remove cell debris from the cut surface. The samples were stored in a vacuum desiccator for at least 24 hours prior to 55 mounting, to allow the ethanol to completely evaporate. Groats were mounted on aluminum stubs with adhesive tabs. Graphite paint was used to minimize charging. Oat flour fractions from the particle size index determination were also examined using Scanning Electron Microscopy (SEM). Flour fractions (‘overs’ and ‘thrus’ of a No.100 screen) and freeze dried starch samples were each lightly dusted onto separate adhesive tab coated aluminum stubs. The great, flour and starch samples were coated with a 56 nm layer of molecular gold using an Emscope Sputter coater. Samples were stored in a vacuum desiccator containing anhydrous CaSO4. SEM Prgggggra: A JOEL JSM-35C Scanning Electron Microscope was used at an accelerating voltage of 15 W to make electron micrographs of the groat, flour and starch samples. The working distance was 15 and the condenser lens setting was at 400. Polaroid positive/negative Type 665 Film (Polaroid, Cambridge, Ma) was used to record the images. Alaurgga gall wall study: Micrographs of the aleurone and subaleurone cell layer were taken at three position on the kernel using the method of Ewars (1982). Five kernel of each cultivar were examined to determine the variance in cell wall thickness. The cell wall thickness was measured on each micrograph using a ruler. 56 Statiatigal Analyaia gf Data F r r ri i A three factor analysis of variance was performed using SAS (Cary, NC) to determine if any significant differences existed in the main effects of oat variety, form of oats and type of mill for the mean values of flour moisture, protein, ash, total dietary fiber (T DF), lipid, alkaline water retention capacity (AWRC), particle size index and Hunter color difference values (L-valua, a-value and b- valua) . The same three main effects were used in analysis of variance of viscosity characteristics of oat flours and oat starches. The Bonfarroni t tests for differences between the means was also done to calculate minimum significant differences (MSD) at specified probability levels. ki Ii h r ri i The cookie quality studies were designed to test the three null hypotheses: 1. The method of milling does not influence oat-wheat composite flour functionality in sugar snap cookies. 2. The processing step of flaking does not influence oat- whaat composite flour functionality in sugar snap cookies. 3. There is no difference in oat-wheat composite flour functionality in sugar snap cookies when different oat cultivars and soft wheat cultivars are used to make the composite flours. A three factor analysis of variance was calculated to see if significant differences existed between the main effects of oat 57 cultivar, type of mill and level of oat flour substitution. The main effects for the three factor analysis of variance for the second hypothesis were oat cultivar, type of processing (groats vs flakes) and level of oat flour substitution. The three factor analysis of variance for the third hypothesis used the main effects of oat I cultivar, wheat cultivar and level of oat flour substitution. The variables analyzed were cookie diameter, tenderness, crispness, Hunter color difference values (L,a,b) and moisture retention. The Bonfarroni t-tests for differences between the means was also done to calculate minimum significant differences (MSD) at specified probability levels. The correlation procedure was used to calculate Pearson correlation coefficients and associated probabilities between cookie diameter, protein content, lipid content, alkaline water retention and Hunter color difference values. 58 RESULTS AND DISCUSSION firgat and Flaka Analyaes The three varieties of oats had different 1000 kernel weights as illustrated in Figure 7. Porter had the smallest kernel weight while Ogle had the largest kernel weight. Kernel weight will influ- ence proximate analysis results to some degree because a larger great would contain a higher proportion of starchy endosperm and a lower proportion of aleurone cells than a smaller groat. The aleu- rone layer is a single layer of cells which surrounds the starchy endospenn. Table 1 contains means values of moisture for the two oat forms, groats and flakes, that were milled into flour for this study. There was a significant difference in the percent moisture of oat flakes compared to eat groats. Ogle groats and flakes contained a significantly higher level of moisture than groats and flakes of the other two cultivars. Table 1. Composition of oat forms: Means for moisture Moisture Level of Main Effect Classes n Q/o) SiLnificance Oat Form Groats 9 699° Flakes 9 8.713 0.01 Oat Cultivar Mariner 6 7.77b Ogle 6 8.153 Porter 6 763° 0.01 Means in the same main effect having a different superscript are significantly different. 59 30- 24.43 25- 22.91 21.67 __ ‘3 204 - .- a 15- ..l a El 1000KWT. 5 y. 10% O O O , 1- 5- / / 04L“ /A %' MARINER OGLE PORTER OAT CULTIVAR GROATS Figure 7. One Thousand Kernel Weights of Oat Cultivar Greats 6O Oat flakes contained a larger percentage of protein than eat greats but the difference was not significant at the p<0.01 level as seen in Table 2. Processing eat greats into eat flakes subjected the chemical constitutents to elevated temperatures and pressures. The bond between the eat protein bodies and the cell wall material may have been modified by the steam treatment prior to rolling into flakes. Table 2. Composition of oat forms: Means for protein1 Protein2 Level of Main Effect . Classes n (%) Significance Oat Form Greats 9 16.943 Flakes 9 17.3781 n.s. Oat Cultivar Mariner 6 17.97a Ogle 6 16.41° Porter 6 17.10a '3 0.01 1 Dry Weight Basis 2 (N x 6.25) Means in the same main effect having a different superscript are significantly different. T able 2 also shows the Mariner greats and flakes contained a significantly higher percentage of protein than Ogle greats and flakes but was not significantly higher than the Porter greats and flakes. The Ogle greats and flakes contained the lowest percent protein. The percentage of protein contained in Ogle greats could be related to its large kernel size. Youngs (1972) hand dissected greats 61 from five cultivars and two experimental lines of common oats. The results showed that most of the great weight is in the bran and endosperm which also contained the greatest amount of the great protein. Greats with higher protein content generally contained a larger amount of bran protein rather than endosperm protein. The bran weight increased as the total protein of the great increased. In a larger great, the bran or aleurone and subaleurone layer comprise a smaller percentage of the great. Ash is the inorganic residue from the incineration of organic matter (Pomeranz and Melean, 1987). Table 3 shows that eat flakes contained a significantly higher percentage of ash than eat groats. The ash content of the eat greats depended on the mineral contents of the eat cultivar. The ash content of oat flakes could have been affected by the flaking process which disrupted the outer layer of the greats as they passed through the heated rollers. Table 3. Composition of oat forms: Means for ash1 Ash Level of Main Effect Classes n (%) Significance Oat Form Greats 9 195° Flakes 9 2,118 0.01 Oat Cultivar Mariner 6 2.083 Ogle 6 1.81 b Porter 6 2.213 0.01 1 Dry Weight Basis Means in the same main effect having a different superscript are significantly different. 62 Table 3 also shows Mariner and Porter greats and flakes had a significantly higher ash content than greats and flakes of the Ogle cultivar. The percentage of ash contained in the Ogle cultivar could have been related to its large kernel size. The percentage of ash was consistently higher in oat flakes than in oat greats of the same cultivar. Table 4 contains the means and standard deviations of the moisture, protein and ash content of oat cultivar greats and flakes. Mariner and Porter flakes contained a higher percentage of protein than Mariner and Porter greats. Table 4. Thousand kernel weight, means and standard deviations of moisture, protein and ash contents of oat cultivar groats and flakes1 Dry Basis Oat 1000 Cultivar Kernel Wt Moisture Protein2 Ash (9) 96 96 96 Greats Mariner 22.91 7.06 1 0.31 17.81 1 0.64 2.00 1 0.02 Ogle 24.43 7.08 1 0.23 16.44 1 0.27 1.78 1 0.01 Porter 21.67 6.82 1 0.06 16.58 1 0.45 2.06 1 0.03 Flakes Mariner - 8.48 1 0.03 18.12 1 0.72 2.16 1 0.18 Ogle - 9.23 1 0.05 16.38 1 0.23 1.84 1 0.08 Porter - 8.43 1 0.08 17.61 1 0.29 2.35 1 0.19 1 n = 3 2 (N x 6.25) 63 Figure 8 contains scanning electron micrographs of a longitudinal cross section of the endosperm of an Ogle great which was representative of the three oat cultivars used in this study. Figure 8a illustrates the relative size of physical structures in the endosperm section. The cells are elongated and are packed tightly with starch granules and protein bodies. The intact compound starch granules are clearly larger than the protein bodies clustered along the relatively thin endosperm cell walls. The closeness of the association between protein body and endosperm cell wall can be seen in Figure 8b. Micrographs of oat flour frequently showed endosperm cell wall fragments with circular holes where protein bodies had been removed by the milling process. Figure 8c shows the native state of oat starch granules in the great. The individual granules have a rounded surface with five sides that delineate a granula. Fl r An I Three factor analysis of variance determined if there were significant differences in the main effects of type of mill, form of oats and eat variety. The three possible two factor interactions; cultivar and form (c x f), cultivar and mill (c x m), mill and form (m x f), were also examined. The ANOVA tables are located in the Appendix. Analysis of variance means were influenced by significant interactions between the main effects. The interaction of oat form x mill was significant at the p<0.05 level for eat flour moisture content and is illustrated in Figure 9. Hammer milling reduced Figure 8. 64 Scanning electron micrographs of structures and chemical components of the oat groat. Scale bar = 10 t1. a) Longitudinal cross section of Ogle great endosperm containing starch granules (S) and protein bodies (P). b) Protein bodies (P) closely associated with endosperm cell wall (EW) in great. 0) Compound starch granules in endosperm cell of oat great. 65 66 8.5 + anon 8-0 ‘ + FLAKE A 7.5 - k H g tit-I 7.0 " D J j... 92 6.5 'I 0 . 2 6.0 1 , 5.5 ‘ 5.0 fl 1 : HAMMER ROLLER MILL Figure 9. Interaction of Cat Form and Mill Type en Oat Flour Moisture Content. 67 moisture to a greater extent in flour milled from groats than in flour milled from flakes. Hammer milled oat flours had significantly lower (p<0.01) moisture contents than. roller milled eat flours as shown in Table 5. Oat flours require a moisture content of less than 11% to prevent growth of mold during storage. Oat flours in the current study had moisture contents ranging from 6 to 8%. Haque (1991) reported impact forces during hammer milling produced a large amount of heat which evaporated moisture from the grain. Table 5. Proximate analysis of oat flours: Means for moisture content. Moisture Level of Main Effect Classes n Q/o) Significance Mill Type Hammer 18 6.68° Roller 18 7.0211 0.01 Oat Form Great 1 8 616° Flake 18 757a 0.01 Oat Cultivar Mariner 1 2 7.073 Ogle 1 2 5.94a Porter 12 6.54° 0.01 Means in the same main effect having a different superscript are significantly different. Table 5 shows eat flours milled from eat flakes were higher in moisture content than eat flours milled from groats. The flaking process required that the moisture content of oat greats be equilibrated to 10%. Oat flakes or rolled oats are commercially 68 packed at a moisture content of about 10.5% (Kent, 1983). The moisture content of Mariner and Ogle flours was significantly higher (p<0.01) than that of Porter oat flours All three interactions; mill x form, mill x cultivar, cultivar x form were significant at the p<0.01 level for protein content. The interactions are illustrated in Figure 10. Roller milling produced groat flours with lower protein contents than hammer milling as seen in Figure 10a. Figure 10b illustrates that the same reduction in protein content was shown for flours from the Mariner and Porter cultivars while the opposite effect was shown for Ogle flours. The difference in protein content of great flours compared to protein content of flake flours from the Porter cultivar was much larger than for flours from the two other oat cultivars as seen in Figure 10c. There was a significant difference in flour protein level due to the effect of the mill type at the p<0.05 leVel as shown in Table 6. The protein content of oat flours milled from oat flakes was significantly higher than oat flours milled from oat groats. Disruption of the outer bran layer during the flaking process did not appear to result in major losses of aleurone or protein bodies from this region. Yui (1986) had reported that eat groat aleurone and subaleurone cell walls were relatively resistant to processing. Table 6 shows the protein contents of Mariner and Porter flours were higher than the protein content of Ogle flours. Mariner and Porter would be considered high protein oat cultivars according to the criteria given by Fulcher (1986) because they contained more than 17 percent protein. The relative protein content among the 69 Figure 10. Interaction of Mill Type, Oat Form and Oat Cultivar on Oat F Iour Protein Content. 7O 152 Q / 15.0 - ’3 J I} z 17.: - u“; . + GROAT '5 -—*— FLAKE 1:: 17.0 - a 1 17.4 - 172 a 17.0 v ‘ HAMMER ROLLER MILL Oat Form x Mill Interaction for Oat Flour Protein Content 199 18.5 - -\ g 13.0 d \. E d I: 17.5 4 —I— MARIMER O , + OGLE E —o—- PORTER 17.0 4 155 u / 15.0 v ' I HAMMER ROLLER MILL Oat Cultivar x Mill Intonation for Oat Flour Protein Content 1910 4 15.5 - g 15.0 -I E ‘ —I— MARINER E 17.5 - —t-— OGLE g , —o—- roman a. mo - 10.5 a / 1 no - GROAT F LAKE OAT FORM Oat Cultivar x Form Interaction for Oat Flour Protein Content 71 three oat cultivars was maintained through the stages of processing and milling. Table 6. Proximate analysis of oat flours: Means for protein content.1 Protein Level of Main Effect Classes n (%) Significance Mill Type Hammer 1 8 17.7851 Roller 18 17.56° 0.01 Oat Form Greats 1 8 17.28° Flakes 18 18.06a 0.01 Oat Cultivar Mariner 1 2 18.343 Ogle 1 2 18.56° Porter 12 18.113 0.01 1 (N x 6.25 ) , Dry Weight Basis Means in the same main effect having a different superscript are significantly different. Analysis of variance means were influenced by the interactions of cultivar x form and form x mill which were significant for oat flour ash content. These interactions are illustrated in Figure 11. The cultivar x form interaction was significant at the p<0.05 level while the form x mill interaction was significant at the p<0.01 level. The flaking process seemed to reduce ash levels in the Mariner cultivar while increasing ash levels in the two other cultivars. Roller milling may have contributed to reduced levels of ash in groats compared to an increase in the level of ash when flakes were similarly milled. 72 Figure 11. Interaction of Mill Type, Oat Form and Oat Cultivar on Oat Flour Ash Content. 73 2.30 2.25 1 2.20 ' 2.15 ‘ J 2.10 " 1 + MARINER ‘ —o— PORTER ASH (%) 2.00 ‘ 1.95 7 / 1.90 I ' fi GROAT FLAKE FORM Oat Cultivar x Form Interaction for Oat Flour Ash Content 2.20 2.15 'I 25 I 2.10 '- m < 205 . + GROAT . + FLAKE 2.00 r . r HAMMER ROLLER MILL Oat Form x Mill Interaction for Oat Flour Ash Content 74 These significant interactions contributed to lack of significance of the main effects of type of mill and form of oats on ash content as seen in Table 7. The means for ash content of oat flours by the type of mill used to grind the oat flours were not significantly different at the p<0.05 level. In wheat flours, ash content and protein content are closely associated because both increase from the inner to the outer part of the wheat kernel (McMasters at al, 1971). There was no significant difference in ash content of oat flours milled from groats or milled from flakes. The ash content of Mariner and Porter flours was significantly higher than the total ash content of Ogle flours. Table 7. Proximate analysis of oat flours: Means for ash content.1 Ash Level of Main Effect Classes 11 (%) Significance Mill Type Hammer 18 2.14a Roller 18 2.09a n.s. Oat Form Greats 1 8 2.133 Flakes 1 8 2.116 n.s. Oat Cultivar Mariner 1 2 2.18al Ogle 1 2 195° Porter 12 2.23a 0.01 1 Dry Weight Basis Means in the same main effect having a different superscript are significantly different. The interaction of form x mill was significant at the p<0.01 level for fat content. A lower percentage of fat was extracted from 75 hammer milled groat flours than from roller milled groat flours as seen in Figure 12. The effect was the same for oat flours milled from flakes but the degree was not as pronounced. A significantly higher percentage of fat was extracted from roller milled oat flours than hammer milled oat flours as seen in Table 8. Oat flours milled from flakes also contained a higher percentage of fat than oat flours milled from groats. Each of the oat flours from an individual oat cultivar contained a statistically different percentage of fat. Porter contained the highest amount of fat while Ogle contained the lewest amount of fat. The analysis results may have been affected by physical properties of the oat flours such as particle size. Yui (1986) reported oat lipid storage was mainly in the endosperm in the form of droplets and the endosperm cell walls of oat groats were disrupted by processing. Table 8. Proximate analysis of oat flours: Means for fat content. Fat Level of Main Effect Classes n (%) SLanificanca Mill Type Hammer 18 7.54° Roller 18 7.7651l 0.01 Oat Form Greats 18 7.56° Flakes 1 8 7.7361 0.01 Oat Cultivar Mariner 12 7.44° Ogle 12 6.880 Porter 12 8.613 0.01 1 Dry Weight Basis Means in the same main effect having a different superscript are significantly different. 76 7.8 k 4‘ 7.7 ' A 7.6 " e: 9 7.5 - E A f 7.4 ' L + HAMMER + ROLLER 7.3 r 7.2 1 A l GROAT FLAKE FORM Figure 12. Interaction of Cat Form and Mill Type on Oat Flour Lipid Content 77 Table 9 shows a higher average percentage of total dietary fiber was measured for roller milled oat flours than for hammer milled oat flours. Oat flours milled from flakes were also determined to contained a higher percentage of total dietary fiber than oat flours milled from groats. Porter eat flours were determined to contain the highest amount of total dietary fiber at a significantly higher level than Ogle or Mariner oat flours. Table 9. Proximate analysis of oat flours: Means for TDF content. TDF Level of Main Effect Classes n (%) Significance Mill Type Hammer 1 8 11 .69° Roller 18 12.803 0.01 Oat Form Greats 1 8 11 .36° Flakes 18 13.138 0.01 Oat Cultivar Mariner 1 2 12.23° Ogle - 1 2 10.98° Porter 12 13.53a 0.01 1 Dry Weight Basis Means in the same main effect having a different superscript are significantly different. Table 10 contains the silicic acid column chromatography results for determining distribution of lipid classes in flours from the three oat cultivars. The recovery rates for the column chromatography were from 91-98 percent. Porter contained a higher percentage of neutral lipids than flours from the two other cultivars. Ogle flours contained a higher percentage of 78 phospholipids than flours from the two other cultivars. There were no significant differences between oat cultivars at the p<0.05 level for any of the three classes of lipids. The distribution of lipid classes was similar to that reported for the Chief cultivar by Price and Parsons (1975). The lipid composition of the Chief cultivar was 72.9 percent neutral lipid, 17.0 percent glycolipid and 10.1 percent phospholipid. MacMurray and Morrison (1970) extracted lipids from wheat flours and determined that the non-polar or neutral fraction was approximately 50.9 percent of wheat flour lipids. Table 10. Distribution of lipid classes in oat flours.1 Neutral lipids 'Glycolipids Phospholipids Oat cultivar (%) (%) (%) Mariner 62.5 1 2.4 27.1 1 6.6 10.3 1 4.2 Ogle 62.3 1 0.4 22.5 1 0.4 15.1 1 0.1 Porter 64.2 1 0.7 24.7 1 0.7 10.9 1 1.4 1 n= 3 Dry Weight Basis Table 11 contains the results for p-glucan analysis of oat flours from the three cultivars. Flour from the oat cultivar Porter contained a significantly higher (p<0.05) percentage of p-glucan (5.32%) than the flours of the two other cultivars. Welch and Lloyd (1989) had reported kernel levels of B-glucan ranging from 3.2 - 6.3 percent. Carr at al (1990) reported ”Quick” rolled oats contained 4.3 percent total p-glucan. Porter eat flours had been determined to 79 contain the highest percentage of total dietary fiber of the three oat cultivar flo'urs. Table 11. Means and standard deviations of Total Dietary Fiber, 6- Glucan Content and Alkaline Water Retention Capacity of Oat flours1 TDF2 B-Glucan AWRC3 Oat cultivar (%) (%) (%) Mariner 12.23 1 1.71 4.73 1 0.06 168.30 1 31.31 Ogle 10.98 1 1.57 4.72 1 0.37 141.90 1 27.74 Porter 13.52 1 1.80 5.32 1 0.12 186.71 1 37.20 1 n= 3 2 Dry Weight Basis, Total dietary fiber in all four types of oat flour. 3 14% moisture basis Table 12 contains the proximate analysis means and standard deviations for each of the twelve types of whole grain eat flours produced by this study. The flours hammer milled from groats consistently had lower moisture levels than their roller milled counterparts. Oat flours hammer milled from flakes had lower moisture contents with the exception of Porter oat flakes. Oat flours milled from flakes contained a higher percentage of protein than flours milled from groats with the exception of Ogle flour hammer milled from flakes. Phyajgal propartiaa gf gat floura Determination of particle size index of whole grain oat flours was most likely effected by the level of fat in oat flours. The oat flours tended to clog the openings of the No 100 US. screen despite 80 0000.006 >0 005050 0 00.0 x 20 N I 0 u c F 00.2 00.0 H 00.9 NF.0 H :0 00.0 H 0N.N 00.0 H 00.0F FN.0 H ¢NK 0x0_u_-s_m 0F.NF 0NF H F.0NF 8.0 H 050 No.0 H 0F.N 00.0 H 00.0F 00.0 H 00.0 890.2,“ NEE N0.F H 00.NF 00.0 H N00 00.0 H 0N.N 9.0 H 0NOF 0N.0 H 00.5 9.0.52: «.0. FR NF.0 H 0F.mF 0N. 0H N0. 0 0F. 0H 0N. N No.0 H 00.: 0F.0 H 0F.0 00.0-2: ..mtoa 00.2 00.F H vaF 00.0 H 00.0 0F.0 H F0.N 00.0 H BKF 0F. 0 H 00. F 30......2m 5.: NF H 00.0 00.0 H 00.\. 00.0 H SF 3.0 H N0.0F 00. 0 H 0N. 0 2090-20”. 00.: 00.0 H 8.3 NF.0 H F00 00.0 H N0.F 0F.0 H 00.0F N0. 0 H 00. n 30.....-2: 00.3. NF.0 H 00.: 00.0 H 00 NF. 0 H F0 F 00 H 2.0F FF. 0 H 0F. 0 890-2: 0.00 um. I 0H0 H 0F.: 00.0 H FON 00.0 H 0F.N 00.0 H N0.0F 00.0 H ER oxm_n_-_2m 00.NF 04.0 H 00.: 00.0 H F: 00.0 H 0F.N 00.0 H RNF 3.0 H :0 $90.20 N0. FF 0.0 H F0.NF NF.0 H 00K :0 H 00.N 3.0 H F0.0F No.0 H NON 9.0.0-5.... 00.NF 0F.0 H N0.0F 00.0 H FUNK 00.0 H mmN FN.0 H 00.0F 00.0 H 00.0 80.0-5.1 80:02 00 00 00 0\0 0\0 0\0 0010 “.0... Fan 20< NEBEm 2306.2 052.... :00 0000 awas. do 0.0000300 0.00090 0:0 memo—2:050: 60 F0 £02000 20.0085 NF 030... 81 the use of the 12 rubber sieve cleaners. Relative particle size of the oat flours was indicated by particle size index and flour color using a Hunter Color Difference meter. The only significant interaction for particle size index was between form and mill type and is shown in Figure 13. There was a greater difference in flour particle size index between flours milled from groats compared to flours milled from flakes. The flaking process appeared to facilitate particle size reduction during milling. The Hunter Color Difference meter measured the amount of light reflected from the sample surface. The sample with smaller flour particles would have a smoother surface than the sample with larger flour particles. The smoother surface would reflect more light and generate higher L-values (brightness) than the surface of a sample comprised of larger flour particles. Kurimoto and Shelton (1988) reported the correlation of the L-value with mean flour particle size determined by a Micro-Trac Particle Size Analyzer was -0.82 (p<0.01) for a hard red spring wheat. Analysis of variance means for Hunter Color Difference values were influenced by significant interactions between the main effects. The interaction of oat form and mill type was highly significant for L-value of oat flours and b-values of oat flours. There was a greater difference in L- and b-values of flours milled from groats than in L-values and b-values of oat flours milled from flakes as illustrated in Figures 14a and 14c. The interaction of cultivar and form was significant at the p<0.01 level for a-value of oat flours. Figure 14b illustrates that Ogle and Porter flours milled from flakes had lower a-values (redness) than flours milled from 82 58 e 56 ‘ v} u". s “I I, 52 j + HAMMER g ——a— ROLLER 3 . 2 5° ‘ p. C E 48 1 46 w GROAT FLAKE OAT FORM Figure 13. Interaction of Mill Type and Oat Form on Particle Size Index of Oat Flour. 83 Figure 14. Interaction of Mill Type, Oat Form and Oat Cultivar on Hunter Color Difference Values of Oat Flour. 84 82 In 81 ~ 3 ..I < >. P _I so - —I— HAMMER —O— ROLLER 79 ‘ + ‘ anon FLAKE OAT FORM Oat Form x Mill Interaction for L-value of Cat Flour 0.8 * ——I— MARINER 07 _ + 061.1: ‘ —O— PORTER m 3 < 0.6 - > b < 0.5 - 0.4 '- , 0.3 ‘ ‘ — GROAT FLAKE OAT FORM Oat Form x Oat Cultivar Interaction for a-value of Cat Flour 9.0 + HAMMER 8.8 *- —.— ROLLER Lu 3 as - —l < >. m 3.4 - 8.0 l A l GRMT FLAKE OAT FORM Oat Form x Mill Interaction for b-value of Oat Flour 85 groats. Mariner flours milled from flakes had higher a-values than flours milled from groats. _ A significantly greater percentage of the particles of the roller milled oat flours passed through the openings of the No. 100 US. Screen than hammer milled oat flours as shown in Table 13. The shear and compressive forces of roller milling appeared to yield a larger number of finer oat flour particles than the impact forces of hammer milling. A visual observation of the two types of flours indicated that the roller milled groat flours contained a higher percentage of large sections of pericarp than hammer milled groat flours. A visible percentage of the pericarp did not break into fine pieces when subjected to the shear and compressive forces involved in roller milling. The L-values for roller milled oat flours were significantly higher than for hammer milled oat flours. Roller milled oat flours were lighter in color as shown in Table 13. Kurimoto and Shelton (1988) reported that a- and b-values decreased with decreasing flour particle size in a hard red spring wheat. A positive a-value indicated redness and a positive b-value indicated yellowness. Results of analysis of variance found no significant difference between a-values for hammer milled and roller milled oat flours as shown in Table 13. However, a-values were lower for roller milled flours than for hammer milled flours indicating an agreement with the results previously reported for L- values. Roller milled oat flours had significantly lower b-values or were less yellow than hammer milled flours, in agreement with the L-values for roller milled oat flours and the results reported by Kurimoto and Shelton (1988). 86 Table 13. Mean color differences and particle size index values of oat flours by type of mill Hunter Color Difference1 Particle Size Mlll Type L 3 a 4 b 5 Index2 1%) Hammer 80.70° 0.563 , 8.63a 49.51° Roller 81.633 0.443 814° 55.732 Level of significance 0.01 ns 0.01 0.01 1 n = 18 2 n = 24 3 L values = 0 (black) to 100 (white) 4 a values = positive values indicate redness 5 b values = positive values indicate yellowness Means in the same column having a different superscript are significantly different There was no significant difference in particle size index for oat flours milled from groats when compared to flours milled from oat flakes as shown in Table 14. Oat flours milled from oat flakes had significantly higher L-values than oat flours milled from oat groats. The lower a-values of oat flours ground from flakes indicated an agreement with the L-values. Whole oat flours milled from flakes had significantly lower a- and b-values than oat flours milled from groats. The Hunter Color Difference readings supported the visual observation of screen clogging during flour particle sizing. 87 Lookhart et al (1986) reported browning of oat flakes during the steaming and rolling process. The pre-existing browning of oat flakes may have influenced L-values and b-values when comparing oat flours milled from flakes to eat flours milled from groats. Table 14. Mean color differences and particle size index values of oat flours by form H n r l r Di r n 1 Particle Size Oat Form L 3 a 4 b 5 Index 2 0/0) Groat 80 . 72° 0.582 8.492 52.143 Flake 81 .61a 042° 828° 53.09a Level of siginificance 0.01 0.01 0.01 ns 1 n = 18 2 n = 24 3 L values = 0 (black) to 100 (white) 4 a values = positive values indicate redness 5 b values = positive values indicate yellowness Means in the same column having a different superscript are significantly different Table 15 contains analysis of variance means for comparison by oat cultivar which indicated there was also no significant difference in particle size index. Ogle oat flours had significantly higher L-values than flours from the other two cultivars. There was no significant difference in the a-values of oat flours made from the three cultivars. There was a significant difference between Ogle oat flours and Mariner and Porter oat flours as shown in Table 14. 88 Ogle flours had the lowest mean b-value which agreed with the higher L-value indicating a finer average particle size. There was also a significant difference in b-values measured between flours from Mariner and Porter. Table 15. Mean color differences and particle size index values of oat flours by cultivar. H n rI l r Diff r n 1 Particle Size Oat L 3 a 4 b 5 Index 2 Cultivars (%) Mariner 81 .00° 0.542 8.993 52.962 Ogle 81 .9611 0.44a 7.820 53.273 Porter 80.53c 0.51a 8.353 51.612 Level of iignificance 0.01 ns 0.01 ns 2 n = 12 3 n = 24 4 L values = 0 (black) to 100 (white) 5 a values = positive values indicate redness 5 b values = positive values indicate yellowness 1 Means in the same column having a different superscript are significantly different. The lack of agreement in order between L- and b-values for Mariner and Porter could be attributed to the particle size range within the sample or the residual coloration from the kernel. Kurimoto and Shelton (1988) reported no significant difference in L- values among samples of larger particle size (68 to 55.9 pm). The 89 differences in Hunter L-values were significant when the particle size was from 55.9 to 42.3 um. Table 16 contains the means and standard deviations for particle size index and Hunter color values for oat flours. The range of a-values for Porter oat flours influenced the statistical analysis outcome as shown in Table 15. Figure 15 contains scanning electron micrographs that are representative of the coarse oat flour fractions produced by Particle Size index determination. The coarse oat flour fraction was the 'overs' of a No 100 screen. Flour particles consisted of sections of the pericarp which did not fracture upon milling as shown in Figure 15a and chunks containing sections of the pericarp, endosperm and aleurone layer as seen in Figure 15b. The pericarp section of the flour particle in Figure 15b still has a trichome attached after undergoing the roller milling process. A trichome is a hollow single celled projection of the pericarp. Most domestic eat cultivars have a greater degree of trichome development compared to other cereal grains (Fulcher, 1986). Figure 15c is a micrograph of the coarse flour fraction of the Porter cultivar and shows an intact layer of cuboidal aleurone cells. 90 Table 16. Means and standard deviations of particle size index and color difference data for oat flours. Particle Size Hunter Color Valge§2 Oat Flour Index1 (%) L3 a4 b5 Mariner HM-Groat 48.00 1 1.27 79.8 1 0.5 0.6 1 0.3 9.6 1 0.3 HM-Flake 48.60 1 3.82 81.4 1 0.3 0.7 1 0.1 8.9 1 0.1 RM-Groat 58.50 1 1.27 81.4 1 0.4 0.3 1 0.2 8.7 1 0.1 RM-Flake 56.75 1 7.00 81.3 1 0.2 0.5 1 0.1 8.7 1 0.2 Ogle HM-Groat 48.70 1 0.28 80.7 1 0.2 0.7 1 0.1 8.2 1 0.1 HM-Flake 54.05 1 0.77 82.4 1 0.1 0.3 1 0.3 7.8 1 0.2 RM-Groat 56.75 1 1.34 82.4 1 0.0 0.4 1 0.1 7.6 1 0.1 RM-Flake 53.60 1 4.24 82.3 1 0.3 0.3 1 0.1 7.6 1 0.0 Porter HM-Groat 43.60 1 2.12 79.0 1 0.2 0.7 1 0.1 8.7 1 0 1 HM-Flake 54.10 1 0.56 80.9 1 0.2 0.4 1 0.2 8.4 1 0.1 RM-Groat 57.30 1 4.38 81.0 1 0.2 0.7 1 0.1 8.0 1 0.1 RM-Flake 51.45 1 1.20 81.3 1 0.2 0.3 1 0.1 8.2 1 0.2 1 n=2 2 n=3 3 L values = 0 (black) to 100 (white) 4 a values = positive values indicate redness 5 D values — positive values indicate yellowness 91 Figure 15. Scanning Electron Micrograph of the Coarse Oat Flour Fraction. a) Sections of pericarp. Bar = 100 u. b) Section of pericarp, aleurone layer (A) and endosperm with trichome (T) attached. Bar = 10 p. c) Coarse flour fraction of Porter cultivar with intact aleurone cell (A) layer. Bar = 10 p. 92 93 Viscoamylograph properties of oat flours The interaction of form x mill was highly significant (p<0.0001) for initial paste temperature of oat flours while the interaction of cultivar and mill was significant at the p<0.05 level. Figure 16 illustrates there was a greater difference in initial pasting temperature of hammer milled great flours compared to hammer milled flake flours than there was between roller milled flours from the two different eat forms. Roller milled flour of all three cultivars had lower initial pasting temperatures than hammer milled flours of the same cultivar. However, the difference was greater for Porter and Mariner flours than for Ogle flours. Roller milled oat flour slurries had a significantly lower initial pasting temperature than hammer milled oat flour slurries as shown in Table 17. This result would suggest that roller milling did produce smaller flour particles than hammer milling which is in agreement with particle size index results and Hunter Color Difference data. A finer flour would be expected to have a lower initial pasting temperature because of the greater amount of surface area available for water absorption. Kurimoto and Shelton (1988) stated that a finer particle size allowed water to penetrate into the core of the flour particle faster and a more uniform gel may form more easily. 94 Figure 16. Interaction of Mill Type, Oat Form and Oat Cultivar on Initial Pasting Temperature of Oat Flour. INITIAL PASTE TEMPERATURE ( C) INITIAL PASTE TEMPERATURE ( C) 95 60 50‘ 401 + HAMMER + ROLLER .\a 30 Oat Form x Mill Interaction for Initial Pasting f GROAT FLAKE FORM Temperature of Oat Flours 60 + MARINER . + OQE + PORTER 50 '1 40 1 30 T - I HAMMER ROLLER MILL Oat Cultivar x Mill Interaction for Initial Pasting Temperature of Oat Flours 96 Table 17. Viscoamylograph means for initial paste temperature of oat flour Initial Paste Main Effect Classes n Temperature Level of (OC) Significance Mill Type Hammer 12 52.12 Roller 12 40.5° 0.01 Oat Form Greats 1 2 49.53 Flakes 12 43.1° 0.01 Oat Cultivar Mariner 8 438° Ogle 8 49.6a Porter 8 455° 0.01 Means in the same main effect having a different superscript are significantly different. Oat flours milled from groats had a higher initial paste temperature than oat flours milled from oat flakes as shown in Table 17. This indicated that the process of flaking may have contributed to producing oat flour with a finer particle size when subjected to the same forces during milling. Oat flours from the Ogle cultivar had a significantly higher initial paste temperature than eat flours from the Mariner and Porter oat cultivars. Table 18 contains the means for peak hot viscosity of oat flours. Roller milled oat flours had a higher peak hot viscosity than hammer milled flours but the difference was only significant at the p< 0.1 level. Oat flours from flakes had a higher peak hot viscosity than oat flours from groats but the difference was not significant. Shabakov et al (1980) reported that steam treatment of oat flours 97 increased amylograph peak viscosity. Oat flours from the Ogle cultivar had a significantly higher peak hot viscosity than oat flours from the Mariner and Porter oat cultivars. Table 18. Viscoamylograph means for peak hot viscosity of oat flour Peak Hot Viscosity Level of Main Effect Classes n BU Significance Mill Type Hammer 1 2 1294.9° Roller 12 1384.08 0.1 Oat Form Greats 1 2 1307.7a Flakes 1 2 1371.22 ns Oat Cultivar Mariner 8 1246.5° Ogle 8 1427.22 Porter 8 1344.6a° 0.01 Means in the same main effect having a different superscript are significantly different. The form x mill interaction and cultivar x form interaction were both significant at the p<0.05 level for 15 minute viscosity of oat flours. Figure 17 illustrates that eat flours roller milled from flakes had lower 15 minute viscosities and peak cold viscosities than flours roller milled from groats. Oat flours hammer milled from flakes had higher 15 minute viscosities and peak cold viscosities than flours hammer milled from groats. Mariner flours milled from flakes had lower mean 15 minute viscosity than flours milled from groats. Ogle and Porter flours milled from flakes had higher 15 minute viscosities than flours milled from groats 98 Figure 17. Interaction of Mill Type, Oat Form and Oat Cultivar on 15 Minute and Peak Cold Viscosities of Oat Flour. 15 MINUTE VISCOSITY (BU) Oat Cultivar x 750 S 9. 740 >. I: 8 730 o 1’ > m 720 .— a E a 710 In F 700 Oat Form x 1220 3 1310 E! >. 1300 I: m o 1200 O 1’ > 1200 o .1 o 1210 0 § 1200 u a. 1250 Oat Form x 99 + MARINER + NLE , + PORTER )- b y M I A l GROAT FLAKE OAT FORM Oat Form Interaction for 15 Minute Viscosity of Cat Flours +HAMMER +ROLLER GROAT FLAKE OAT FORM Mill Interaction for 15 Minute Viscosity of Oat Flours 1 + HAMMER r —O— ROLLER I GROAT E FLAKE OAT FORM Mill Interaction for Peak Cold Viscosity of 0a Flours 100 The significant interactions between main effects contributed to a lack of statistically significant differences between means. There was no significant difference between hammer milled and roller milled oat flours for the parameters of viscosity at 15 minutes or peak cold viscosity as seen in Tables 19 and 20. Viscosity after 15 minutes is a measure of starch granule fragility and solubility but in oat flour it is also influenced by B-glucan solubilization. Yiu et al (1987) reported that [fl-glucan was a major contributor to viscosity in a gradually cooked sample of rolled oats. There was no significant difference in the parameters of viscosity at 15 minutes and peak cold viscosity for oat flours milled from eat groats or eat flakes. The oat flours roller milled from flakes tended to have larger variations in peak hot viscosity, 15 minute viscosity and peak cold viscosity. Table 19. Viscoamylograph means for viscosity at 15 minutes of oat flour Viscosity Level of Main Effect Classes n at 15 min Significance BU Mill Type Hammer 12 714.12 Roller 12 728.411 ns. Oat Form Greats 12 718.52 Flakes 1 2 724.08 ns Oat Cultivar Mariner 8 623.16 Ogle 8 811.53 Porter 8 729.1° 0.01 Means in the same main effect having a different superscript are significantly different. 101 Table 19 shows the three oat flours had significantly different viscosities at 15 minutes. Ogle oat flours had a significantly higher viscosity at 15 minutes than the two other cultivars. Mariner oat flours had a significantly lower viscosity than Porter flours. Enzyme analysis results shown in Table 11 had indicated Porter oat flours had the highest B-glucan content among the three cultivars. Table 20 contains viscoamylograph means for peak cold viscosity. There was no significant difference in peak cold viscosities for Porter and Ogle oat flours but Mariner oat flours did have significantly lower viscosities than the two other oat flours. Table 20. Viscoamylograph means for peak cold viscosity of oat flour Peak Cold Level of Main Effect Classes n Viscosity Significance BU Mill Type Hammer 12 1276.011 Roller 12 1283.33 ns. Oat Form Greats 1 2 1278.33 Flakes 1 2 1281.08 ns Oat Cultivar Mariner 8 1156.5° 09le 8 1364.43 Porter 8 1318.1a 0.01 Means in the same main effect having a different superscript are significantly different. Table 21 contains the means and standard deviations of visco- amylograph parameters for the twelve oat flours. The oat flours 102 Table 21. Viscoamylograph means and standard deviations for eat flours1 Initial Peak hot Viscosity Peak cold pasting viscosity after 15 min viscosity Oat flour temp °C BU BU BU Mariner HM-Groat 56.4 1 0.6 1175.0 1 77.8 625.0 1 7.1 1142.5 1 3.5 HM-Flake 43.5 1 0.6 1268.5 1 9.2 615.0 1 7.1 1153.5 1 12.0 RM-Groat 37.3 1 2.3 1325.0 1 66.5 673.5 1 9.2 1202.5 1 17.7 RM-Flake 38.0 1 2.1 1217.5 1 236.9 579.0 1 86.3 1127.5 1 102.5 Ogle HM-Groat 58.3 1 3.8 1315.0 1 35.3 751.0 1 41.1 1350.0 1 56.6 HM-Flake 48.7 1 1.1 1470.0 1 14.1 847.5 1 46.0 1372.5 1 10.6 RM-Groat 47.1 1 0.2 1443.0 1 9.9 822.5 1 3.5 1370.0 1 0.0 RM-Flake 44.4 1 0.3 1481.0 1 69.3 825.0 1 7.1 1365.0 1 7.1 Porter HM-Great 58.1 + 24 1216.0 1 33.9 706.0 1 19.8 1265.0 1 21.2 HM-Flake 47.6 1 30 1325.0 1 7.1 740.0 1 14.1 1372.5 1 3.5 RM-Groat 39.7 1 05 1372.5 1 31.8 733.0 1 4.2 1340.0 1 14.1 RM-Flake 36.3 + 1 1 1465.0 1 35.3 737.5 1 10.6 1295.0 1 134.3 1n=2 103 roller milled from flakes tended to have larger variations in peak hot viscosity, 15 minute viscosity and peak cold viscosity. Viapp-amylpgraph propefliea of pat atarches The visco-amylograph properties of oat starches that had been isolated from the twelve oat flours were measured. Oat flour viscosity may have been effected by flour particle size and non- starchy carbohydrates. Scalon at al (1988) described flour as a heterogeneous collection of particle sizes. That study separated roller milled wheat flour into coarse (91-136um) and fine (<53pm) fractions and reported greater water absorption in the fine fraction when compared to the coarse fraction. The fine fractions also had reduced anthalpies(A H) of starch gelation when compared to corresponding composite flours. The conclusion was that starch in the fine fraction was less crystalline than in the coarser fractions due to starch damage during milling. The major non-starchy carbohydrate that may have influenced oat flour viscosity is 6-glucan. As previously stated, Yiu et al (1987) reported that B-glucan was a major contributor to viscosity in a gradually cooked sample of rolled oats. Isolation of starch from oat flour and measurement of oat starch visco-amylograph properties was one approach to measure the effect of milling on physical properties of oats. The eat starch extraction procedure of Paton (1977) used sodium carbonate to adjust the pH of the slurry to be heated and to prevent chemical gelatinization. The process of milling wheat flour has long been known to result in mechanical damage to wheat starch (Alsberg and Griffing, 104 1925; Pulkki,1938). Kent (1983) stated that the degree of mechanical damage to starch granules in soft wheats is not as great as that produced in hard wheats. Oat groats are a softer grain than wheat kernels. No published literature was found that discussed susceptibility of a compound starch granule to damage during milling. There were no significant interactions for viscoamylograph parameters of oat starches as indicated by Tables 98-102. Table 22 shows there was no significant difference in initial paste temperatures of oat starches extracted from hammer milled oat flours compared to eat starches extracted from roller milled flours. Oat starch extracted from Ogle flours had a significantly lower initial paste temperature than oat starches from Mariner and Porter oat cultivars. Table 22. Viscoamylograph means for initial paste temperature of oat starch Initial Paste Main Effect Classes n Temperature Level of (DC) Significance Mill Type Hammer 12 88.211 Roller 12 88.08 ns. Oat Form Greats 12 87.82 Flakes 1 2 88.5a ns Oat Cultivar Mariner 8 88.63 Ogle 8 862° Porter 8 89.53 0.01 Means in the same main effect having a different superscript are significantly different. 105 Table 23 shows there was no significant difference in peak hot viscosity for oat starches extracted from hammer milled oat flours compared to eat starches extracted from roller milled flours. Oat starches extracted from flours milled from groats were not statistically different from starch extracted from flours milled from flakes. There was no statistically significant difference between the starches from the three oat cultivars in the viscoamylograph parameter of peak hot viscosity. Table 23. Viscoamylograph means for peak hot viscosity of oat starch Peak Hot Viscosity Level of Main Effect Classes n BU Significance Mill Type Hammer 12 754.23 Roller 12 744.0a ns Oat Form Greats 1 2 742.431 Flakes 1 2 755.73 ns Oat Cultivar Mariner 8 741.48‘ Ogle 8 737.13 Porter 8 768.7a ns Means in the same main effect having a different superscript are significantly different. Oat starches extracted from hammer milled oat flours had a significantly higher viscosity at 30 minutes than those from roller milled flours as shown in Table 24. Hot viscosity behavior seemed to indicate that starch granules from roller milled oat flours were more fragile. A greater reduction in flour particle size during roller 106 milling may have also influenced the pasting properties of the compound oat starch granules. Roller milling subjected the compound oat starch granules to shear and compressive forces. Table 24. Viscoamylograph means for viscosity at 30 minutes of oat starch Viscosity Level of Main Effect Classes 11 at 30 min Significance BU Mill Type Hammer 12 488.311l Roller 12 474.1° 0.01 Oat Form Greats 12 486.2a Flakes 1 2 476.22 ns Oat Cultivar Mariner 8 437.6° Ogle 8 511.9a Porter 8 494.12 0.01 Means in the same main effect having a different superscript are significantly different. MacArthur and D‘Appelonia (1979) milled the hard spring red wheat Waldron into flour with a Brabender Quadramat Jr. flour mill and a Miag Pilot flour mill. Wheat starch separated from the roller milled flour had an initial pasting temperature of 825°C compared to 84°C for the Miag milled product. The wheat starch isolated from roller milled flour had a lower peak viscosity, a lower viscosity at 15 minutes and a lower viscosity upon cooling to 50°C. The peak cold viscosity of starches extracted from hammer milled eat flours was also higher than that measured for oat starches extracted from roller milled oat flours as seen in Table 25. 107 Porter oat starches had the highest peak cold viscosity while Mariner oat starches had the lowest peak cold viscosities. All three starches were significantly different from each other in the peak cold viscosity parameter. Table 25. Viscoamylograph means for peak cold viscosity of oat starches Peak Cold Level of Main Effect Classes n Viscosity Significance BU Mill Type Hammer 1 2 1308.78 Roller 12 1247.0° 0.01 Oat Form Greats 1 2 1290.73 Flakes 1 2 1265.02 08 Oat Cultivar Mariner 8 1181 .4c Ogle 8 1286.2° Porter 8 1366.02 0.01 Means in the same main effect having a different superscript are significantly different. There was no difference in any of the pasting parameters at the significance level of p<0.01 for oat starches extracted from groats was compared to eat starches extracted from flakes. This result would imply that the flaking process that groats were subjected to did not significantly affect oat starch viscoamylograph properties. Yiu (1986) had reported some compound oat starch granules being broken into individual starch grains by the flaking process that produced rolled oats. _ -.. W-‘h" 108 Table 26 contains the means of the viscoamylograph parameters for the twelve oat starches. The range of initial paste temperature for Mariner and Porter oat starches was from 87.2 - 91.500. This was higher than the range for initial paste temperatures of oat starch extracted from high nitrogen oats by the same procedure previously reported by Paton (1977). Oat starch from four different oat cultivars had initial paste temperatures in the range of 65.0 - 70.0°C. MacArthur and D'Appolonia (1979) compared eat and wheat starch and reported initial pasting temperatures for oat starches from three different cultivars ranged from 81-83.5°C compared to 82.5-84.00C for wheat starch. The wheat starch used in the study was from the hard red spring wheat, Waldron. The oat starches exhibited a higher peak viscosity, 15 minute viscosity and viscosity upon cooling to 50°C than the wheat starch used in the MacArthur and D'Appolonia study. Scanning electron micrographs of three representative alkaline extracted oat starches are shown in Figure 18. Compound oat starch granules with varying degrees of loss of individual granules could be observed in samples from all twelve types of starch. Intact oat starch granule shapes in all three oat cultivars varied from elongated to rounded as seen in Figure 18a from Mariner roller milled groat flour and in Figure 18b from Porter roller milled groat flour. No attempt was made to determine the ratio of intact granules as seen in Figures 18a, b, d to individual granules as seen in Figure 18c which was from Mariner hammer milled flake flour. The micrographs demonstrate the inherent difficulty of separating oat Table 26. for oat starches1 109 Means and standard deviations for viscoamylograph data Initial Peak hot Viscosity Peak cold Oat pasting viscosity after 30 min viscosity starch temp BU BU BU oC Mariner HM-Groat 87.8 1 0.4 767.0 1 4.3 455.0 1 0.0 1181.5 1 65.8 HM-Flake 89.2 1 0.3 734.0 1 36.8 430.0 1 0.0 1220.0 1 99.9 RM-Groat 88.6 1 0.1 747.5 1 17.7 445.5 1 7.8 1189.0 1 43.8 RM-Flake 88.6 1 0.5 717.0 1 7.1 420.0 1 0.0 1135.0 1 21.2 Ogle HM-Groat 85.7 1 0.1 729.0 1 12.7 527.5 1 6.4 1333.5 1 4.9 HM-Flaka 87.6 1 1.4 740.5 1 13.4 520.0 1 0.0 1315.0 1 21.2 RM-Groat 85.6 1 0.9 702.0 1 4.2 501.5 1 2.1 1271.5 1 54.4 RM-Flake 86.0 1 0.1 777.0 1 7.1 498.5 1 29.9 1225.0 1 7.1 Porter HM-Groat 89.2 1 0.1 758.5 1 13.4 502.5 1 3.5 1407.5 1 10.6 HM-Flake 89.9 1 1.5 796.0 165.0 495.0 1 14.1 1395.0 1 21.2 RM-Groat 89.5 1 0.5 750.5 114.8 485.0 1 14.1 1361.5 1 4.9 RM-Flake 89.4 1 1.8 770.0 1 2.8 494.0 1 8.5 1300.0 1 70.7 1n=2 llO Figure 18. Scanning electron micrographs of freeze dried alkaline extracted oat starch granules (SG). Scale bar = 10 p. a) Mariner oat starch isolated from hammer milled groat flour b) Porter oat starch isolated from hammer milled groat flour Protein body (P) c) Mariner oat starch isolated from hammer milled flake flour d) Ogle oat starch isolated from hammer milled flake flour. 111 112 protein from oat starch. The size and shape of the oat protein bodies enable them to fit into intact starch granules that have lost a few individual granules as shown in Figure 18b and d. 5 The mean percent protein in alkali extracted oat starches is given in Table 27. Analysis of variance indicated there was a significant difference at the p<0.01 level in protein content of starches extracted from oat flours milled from groats when compared to starches extracted from oat flours milled from flakes. There was no significant difference in protein content between starches by milling method or eat cultivar. Oat starches extracted from oat flours milled from flakes may have contained more damaged compound granules that provided indentations in which protein bodies could lodge. Table 27. Means and standard deviations of percent protein in alkali extracted oat starches1 Oat Cultivar Type of starch2 Mariner Ogle Porter HUG 0.04 1 0.06 0.18 1 0.17 0.02 1 0.03 I-IVF 0.35 1 0.05 0.36 1 0.04 0.47 1 0.08 FIVE 0.10 1 0.08 0.00 1 0.00 0.04 1 0.06 FM: 0.37; 0.03 0.42 1 0.03 0.25; 0.05 2 HMG= Hammer milled groats, HMF= Hammer milled flakes RMG= Roller milled groats, RMF = Roller milled flakes 113 Figure 19 are scanning electron micrographs of the gelatinized groat oat starch slurry after freeze drying. The honeycomb-like structure as shown in Figure 19a and b is composed of cells with a similar five sided shape as the individual oat starch granules. Paton (1977) had reported a sponge like texture in refrigerated oat starch gels. Figure 190 is ground freeze dried Ogle hammer milled groat starch and Figure 19d is Porter hammer milled groat starch. The freeze dried oat starch gels maintained the honeycomb like structure and tended to fracture into smaller segments instead of granular particles after grinding with a mortar and pestle. Alkalina watar ratantipp papapitiaa pf flppra Alkaline water retention capacity is a measure of the ability of a flour to retain water when subjected to centrifugal force. The test was introduced by Yamazaki in 1953. His results were that alkaline water retention capacity for soft wheat flours was highly negatively correlated with sugar-snap cookie diameter. The hydration characteristics of a flour influenced its performance in sugar-snap cookies. The interaction of oat cultivar x mill was significant at p<0.05 for AWRC and is illustrated in Figure 20. The lines are not parallel with the difference in AWRC between hammer and roller milled flour being greater for Porter and Mariner oat flours compared to Ogle flours. Table 28 shows the means for alkaline water retention capacities (AWRC) of oat flours. The mean AWRC of roller milled oat flours was significantly higher than the AWRC of hammer milled oat flours. With the exception of Porter hammer milled flake flour, the 114 Figure 19 Scanning electron micrographs of freeze dried oat starch gels. Scale bar = 100 p. a) Mariner hammer milled groat starch gel b) Mariner hammer milled groat starch gel 0) Gelatinized Ogle hammer milled groat starch after grinding d) Gelatinized Porter hammer milled oat starch after grinding. 115 Iarch 'oat arch AWRC (%) Figure 20. 116 220 + MARINER —*— OGLE 200 1 + PORTER 1801 1601 140 1 120 5 . f HAMMER ROLLER MILL Interaction of Mill Type and Oat Cultivar on Alkaline Water Retention Capacity (AWRC) of Oat Flour. 117 roller milled oat flours had the largest standard deviations for AWRC. Table 28. Means for alkaline water retention capacities (AWRC) of oat flours AWRC1 Level of Main Effect Classes n (%) sgnificance Mill Type Hammer 1 8 143.63 Roller 18 187.6° 0.01 Oat Form Greats 1 8 145.2° Flakes 18 186.1a 0.01 Oat Cultivar Mariner 1 2 168.3° Ogle 1 2 141.90 Porter 12 186.72 0.01 1 14% moisture basis Means in the same main effect having a different superscript are significantly different. Particle size is related to the water absorption of flour (Mailhot and Patton, 1988). A flour with finer average particle size would be expected to have greater hydration capacity due to increased surface area. The results of the physical (Hunter L-value and PSI) and functional (AWRC) tests agreed leading to the conclusion that roller milled oat flours contained finer flour particles than hammer milled oat flours. Oat flours milled from oat flakes had a significantly higher AWRC than oat flours milled from groats. Although there was not a significant difference in Particle Size Index between flours milled from groats and flours milled from flakes from Table 14, there was 118 a highly significant difference in alkaline water retention capacities. The size of the oat flake flours particles combined with the lipid content of the oat flours may have influenced Particle Size Index resuhs. Yamazaki (1969) stated that wheat and flour properties that appear to be cultivar related include flour granularity, absorption, viscosity and cookie spread potential. All three oat cultivars had significantly different AWRC when compared to each other. Porter oat flours had the highest AWRC while Ogle had the lowest AWRC of the three oat flours. There was disagreement between physical (Hunter L-value and PSI) and functional (AWRC) test results. Porter had the lowest Hunter L-value and the smallest Particle Size Index. Particle Size Index results may have been influenced by lipid content of the Porter oat flours. As previously reported in Table 8, Porter oat flours contained a significantly higher percentage of lipid than the other two oat cultivars. Table 29 contains the means and standard deviations of alkaline water retention capacities of the twelve types of oat flours produced in this study. The means of the roller milled oat flours were consistently higher than their hammer milled counterparts. With the exception of Porter hammer milled flake flour, the roller milled flours had the largest standard deviations for AWRC. 119 Table 29 Means and standard deviations of alkaline water retention capacities of oat flours 1 2 Oat Cultivars Type of mill Mariner Ogle Porter and eat form Hammer milled Groat 130.5 1 1.8 111.6 1 0.8 132.6 1 3.1 Flake 162.4 1 3.9 136.0 1 1.0 188.6 1 12.1 Roller milled Groat 166.3 1 4.4 135.5 1 3.5 194.6 1 4.0 Flake 213.9 1 5.8 184.5 1 3.0 231.0 1 3.3 1 n= 3 2 14% moisture basis nnin lrnMir f rn IIWII: The aleurone and subaleurone cell wall widths were measured at the three positions on a Iatitudinally out great or kernel as shown in Figure 21. Position 1 was adjacent to the crease area, position 2 at a 90° angle to the crease and position 3 was distal to the crease. The latitudinal cut was made half way between the base and tip of the great to avoid the oat germ at position 3. It was thought necessary to examine the oat aleurone and subaleurone cell walls at three specific locations because of previous studies by Pomeranz (1972) and Ewars (1982). The microstructure of a oat great from the cultivar Orbit, was previously studied by Pomeranz (1972). He reported that the distal side of the oat groat contained two lines of rectangular shaped aleurone cells while cells adjacent to the crease area were almost elliptical in shape. Pomeranz (1972) had also observed an increase 120 Figure 21. Scanning electron micrograph of Porter groat identifying the three positions on oat groat cross section at which measurements were taken of aleurone and subaleurone cell wall width. Scale bar = 1000 u 121 in aleurone cell size at the germ end of the oat groat. Ewars (1982) reported that mean cell growth in all layers occurred halfway between the base and tip of evergreen needle leaves. A difference in cell shape may have meant possible differences in cell well width so measurements were taken at the three distinctive positions. Figures 22a, b, c are scanning electron micrographs of transverse latitudinal sections of groats from the cultivar, Mariner. Figure 22a was taken at position one and shows rectangular shaped aleurone cells. The center aleurone cell in this micrograph is filled with aleurone grains. The aleurone cell walls that are perpendicular to the outer cell walls are relatively small when compared to the thickness of the cell walls that separates the aleurone layer from the starchy endosperm. The average cell well width at this position was 4.46;: At the top of the micrograph, two smaller cells packed with aleurone grains can be seen. These observations agree with Pomeranz (1972) and Bechtel and Pomeranz (1981) that two lines of aleurone cells may be found in portions of the great. Figure 22b was photographed at position two on a Mariner great. The cells are still predominantly rectangular in shape but one cell is observed to be slightly elliptical in shape in the aleurone layer. In the subaleurone layer, two cells both still packed with aleurone grains have different shapes, one oval and the second triangular. Oval shaped subaleurone cells were observed in three of the five groats at position two. The average aleurone cell wall width at position two was 4.280, slightly smaller than cell wall width at position one. Figure 22c, also taken at position two, l22 Figure 22. Scanning electron micrographs of aleurone and subaleurone cell wall on latitudinal cross section of Mariner groat. Aleurone cell (a), aleurone grains (ag), Subaleurone cells (sa), Endosperm cells (a) Scale bar = 100 a) Position 1 b) Position 2 c) Position 2 (1) Double layered aleurone cells at Position 3 123 124 illustrates the variation in cell shapes and arrangements of double layered cells in this oat cultivar. Figure 22d was photographed at position three on a Mariner great. The aleurone cells are rectangular in shape but have a tendency to be slightly oblique. A double layer of aleurone cells were observed in two of the five Mariner groats at position three. This position had the thickest aleurone cell walls, 4.6811. Aleurone cells located at position one on a Ogle groat are shown in Figure 23a. The cell walls at position one had an average width of 3.080, the smallest thickness among the three positions. The cells were mostly rectangular in shape and no subaleurone layer was present in the five randomly selected groats Figure 23b shows aleurone cells located at position two on a Ogle great. The aleurone cells at this position had a variety of shapes, some samples having rectangular cells while others were cubodial in appearance and perpendicular to the outer edge of the great. The aleurone cell walls in this section had an average width of 3.380, the largest thickness among the three positions. Figure 23c was photographed at position three on a Ogle great. In contrast with position two, the cells are perpendicular to the outer edge of the great and strongly rectangular in shape. One small oval subaleurone cell is shown in the micrograph. An average cell wall width of 3.140 was measured at this position in Ogle groats. Figures 24a, b, c and d are scanning electron micrographs of Porter groats. Figure 24a was taken at position one where the average aleurone cell wall width was 3.730. The cells had more 125 Figure 23. Scanning electron micrographs of aleurone and subaleurone cell walls on latitudinal cross section of Ogle groat. Aleurone cell (a), aleurone grains (ag) Subaleurone cells (sa), Endosperm cells (9) Scale bar = 10 u a) Position 1 b) Position 2 c) Position 3. lOI 127 rounded corners with less of an rectangular shape when compared to Mariner groats (Figure 22a) at the same position. Figures 24a and b indicates the Porter cultivarhad similarly shaped rounded cells at positions one and two. An average aleurone cell well width of 3.71 p was measured at position two. No sub- aleurone layer was observed in any of the five groats examined in this study. The aleurone grains appeared to be closely adhered to the cell walls when compared to aleurone grains seen in Figure 22a (Mariner) and Figure 24a (Porter). Figure 240 shows a rectangular shaped subaleurone cell that was tightly packed with aleurone grains at position three. The cell walls located between the aleurone layer and subaleurone layer were of comparable thickness to the well located between the sub- aleurone layer and the starchy endosperm. There was no consistent presence of a subaleurone layer at this position on Porter groats among the five samples. However, Figure 24d illustrates a section at position three that consisted of five consecutive double layered aleurone cells. The average cell wall measurement for Porter groats was 4.06m at position three. Table 30 and Figure 25 show that examination of five randomly chosen groats from each cultivar revealed that there was no consistent relationship in aleurone cell wall thickness among these three oat cultivars. No aleurone cell wall position on the oat great was consistently larger or smaller than another aleurone cell wall at a specific position. The three oat cultivars had different aleurone cell wall thicknesses in greats of approximately the same weight. The Mariner oat cultivar had the largest average aleurone cell wall 128 Figure 24. Scanning electron micrographs of aleurone and subaleurone cell wall on latitudinal cross section of Porter groat. Aleurone cell (a), aleurone grains (ag) Subaleurone cells (sa), Endosperm cells (9) Scale bar = 101.1 a) Position 1 b) Position 2 0) Position 3 d) Double layered aleurone cells at Position 3 129 130 thickness of the three cultivars. Ogle oat cultivar had the smallest average aleurone cell wall thickness of the three cultivars. Table 30. Means and standard deviations of aleurone cell wall measurements1 at three positions on the oat groat. Mean cell Oat Position Position Position well width Cultivar 1 2 3 mm (x10-3) Mariner 4.46 1 1.05 4.28 1 1.31 4.68 1 1.60 4.47 Ogle 3.08 1 0.90 3.38 1 1.13 3.14 1 0.83 3.20 Porter 3.73 1 1.30 3.71 1 0.97 4.06 1 1.14 3.83 1n=5 Table 31 contains proximate analysis results for protein content of groats and total dietary fiber content for all oat flours from that cultivar. The great protein percent varied with the average aleurone cell wall width. Mariner groats contained the highest percent of protein among the three cultivars while having the largest aleurone cell wall thickness. Ogle groats contained the smallest percentage of protein while having the smallest aleurone cell wall thickness. Fulcher (1986) observed that high protein cultivars tend to have subaleurone cell walls that are four to five times thicker than endosperm cell walls. 131 El POSITION1 El POSITION 2 5 POSITION 3 .3 :_ 1;: Ph.”\\\:\\\\\\\\\\\\\\\ III/IIIIIIIIIIIIII \\\\\\\\\\\\\\\\\\ as.» _ 1 _ xxx xxxxxxxx wasnxxx xx x xxxxxxxx xx xxx xxxxxxxx xTxVVxxxurvxxxx/le s .7//////////// \ I \ 2.. ///////////////////A 5 4 3.0 2 1 0 8.2 .0 EE 13:5 .555 $00 PORTER OGLE OAT CU LTIVAR MARINER Figure 25. Aleurone cell wall measurements 132 Table 31. Means and standard deviations of great protein content, total dietary fiber content of oat flours and aleurone cell wall widths. Oat Groat Total Dietary Average Cell Cultivar Protein1 Fiber2 Width (%) (%) mm (x 10'3) Mariner 17.81 1 0.64 . 12.23 1 1.71 4.47 1 0.20 Ogle 16.44 1 0.27 10.98 1 1.57 3.20 1 0.16 Porter 16.58 1 0.45 13.52 1 1.80 3.83 1 0.20 1 n = 3, Dry weight basis 2 n=12 Total Dietary Fiber in all four types of oat flour, dry weight basis As previously mentioned in the results and discussion section concerning comparison of oat forms used in this study, the size of the great may be influencing protein content. Ogle had the largest 1000 kernel weight of the three oat cultivars. Youngs (1972) reported that in five oat cultivars and two experimental lines of common eats, the endosperm weights varied inversely and the bran weights directly with the great protein concentration. Oat bran being composed of the two outermost layers plus the aleurone and subaleurone cells contain a high percentage of oat protein in the form of aleurone grains and protein bodies. Bechtel and Pomeranz (1981) reported that aleurone grain contents could partially be digested by proteases. Figure 26a and b show the two forms of oat protein found in the aleurone and subaleurone cells; aleurone grains 133 and protein bodies. The spherical protein bodies are shown to be located in clusters along the inner wall of the aleurone cells in Figure 26b and c. The preliminary oat flour study, documented in the Appendix, measured the percentage of protein in commercial whole grain oat flour fractionated by sieving. The coarse oat flour fraction or 'overs' of a US. No 54 screen was predominantly composed of the aleurone and subaleurone cell fragments. This coarse flour fraction from commercial oat flour was measured to contain 24 percent protein. Youngs (1972) concluded that bran usually contains almost double the protein concentration of the starchy endosperm or about half the total groat protein. Wood and Fulcher (1978), Fulcher and Wong (1980), Wood (1980, 1981,1982), Fulcher and Wood (1983) and Wood et al (1983) used chemically specific fluorescent dyes and light microscopy to identify location and quantify levels of 6-glucan in oat cell walls. In this study the aleurone cell walls were measured because Wood (1989) and Fulcher(1989) identified p-glucan, a major component of soluble fiber in oats, to be present in large amounts in the thick aleurone and subaleurone cell walls. Fulcher (1989) also stated that the properties of the bran (pericarp, seed coat, aleurone and sub- aleurone layer) most influence the quality characteristics of oats. Table 31 contains the results of total dietary fiber analysis on oat flours milled from groats and flakes by both kinds of mills. The level of total dietary fiber does not appear to vary with the aleurone cell wall width. Wood at al (1983) using Calcofluor and Congo red identified the endosperm cell walls as the major reservoir of 6- 134 Figure 26. Scanning electron micrographs of cross section of Mariner oat groat showing aleurone grains and protein bodies located in the aleurone cells. a) Tightly packed aleurone grains (AG). Scale bar = 100 b) Cluster of protein bodies (PB) located along inner walls of aleurone cell. Scale bar = 10 p c) Protein bodies located along inner walls of aleurone cell. Scale bar = 10 u. I35 136 glucan in oats. Although endosperm walls located in the mid or inner endosperm are smaller in width than aleurone cell walls, endosperm cell walls comprise a larger percentage of the oat great. In the mature great, the starchy endosperm contributes between 55.8 and 68.3% of the weight (Youngs, 1972; Youngs and Peterson, 1973). Fulcher (1986) theorized that the presence of 6-glucans in the oat aleurone layer may substantially enhance'the water binding capacity of oat bran and support its role as a source of dietary fiber. mm r : The results of the aleurone cell wall study indicated an agreement with previously published observations of the diversity of aleurone cell shape at different locations on the great. There was also agreement with the relative amount of protein and aleurone cell wall thickness. The lack of agreement between cell wall thickness and total dietary fiber content may have been influenced by the presence of double layers of cells in the aleurone layer. When this occured the great was contributing additional amounts of total dietary fiber in the form of B-glucan and this may have affected the theorectical relationship. HICHIGRN STRTE UNIV. LIBRRRIES Ill" 9 1827 312930101