Proceedings of the 7 th Iniernatumal Workmg Conference on Stored-product Protection - Volume 2

Study on the factors effecting germinated wheat flour andimproving effect

Wang Ruolan1

Abstract

The factors that influence Brabender maximum viscosity ofgerminated wheat flour and the improving methods werestudied in this paper. Using Response Surface Analysismethod, two factors quadratic regression equation ofmaximum viscosity was obtained. The orthogonalexperiment showed that adding inhibitor to the flour was theimportant factor that influenced maximum viSCOSItyand themost effective method of improving qualities. At the sametime the optimum technological condition m the factors wasobtained. At last adding the inhibitor to the germinatedwheat flour, the regression equation between the maximumviscosity and quantity of inhibitor added was obtained.

Introduction

Wheat IS a kind of crops that have been cultivated and usedwidely. The output of wheat IS about half of the total gramoutput in China every year. Wheat is the main kind of gramin many areas, and wheat flour IS also the raw matenal ofmany traditional foods. So the output and quality of wheatare very important m the national economy and are closelyrelated to the people's dally lifeBecause of the application of advanced science and

technology to the agnculture , especially genetic engineeringand biological engineering, the output of wheat hasincreased in the resent years, but the quality of wheat hasbeen the important question for scientists to study ordiSCUSSIon.Especially when It was cloudy and rainy for dayson end in the harvest seasons, the germinated wheat wasdeveloped widely, the sprouting damage and the flourquality reduction were cntical. ThIS phenomenon was verycommon all over the world. In extremely abnormal years,the extent of sprouting damage may be as high as 15% of atotal production of about 25 million tons m Canada. Similarlevels of sprouting damage have been experienced inAustralia recently. In Europe, sprouting damage of cerealsat relatively high levels occurs almost every year". So does

lZhengzhou Gram College, Zhengzhou 450052, Henan, China

It in China, in 1991 and 1996 the content of germinatedwheat was very high over an extensive area in HenanProvince. Germinated wheat has low flour production, thecolor of flour is gray and dark, the taste of the foods issticky, the elasticity of gluten IS not up to the standard,even the flour can not be processed into steamed bread,noodle, bread, and so on. It brought great damage in grainand food production and also influenced the daily hfe of thepeople.The Brabender maximum viscosity and falling number of

high germinated wheat content were very low (often below200 B. U. or 200s) , the stabilization time of dough was veryshort and resistance extension was very weak. TheImportant reasons of sprouting damage on flour qualities areas follows: the extremely high a - amylase activity, excessmolecules of starch were changed into hydrolysates,maximum VISCOSIty(M. V ) decreased and the taste wassticky. Other factors, such as increased protease activityand protem degradation, the constructions of protein andgluten were damaged, these caused the elasticity of glutenweakness", so the different content of germinated wheat andgermmation time that affect the M. V. was studied here.There are many methods of Improving the wheat's qualities,such as heatmg, peeling and adding enzyme inhibitors, thesemethods were researched by means of the orthogonalexperiment m this paper and the optimum technologicalcondition was obtained.As a result of the high a - amylase and protease activity,

adding the inhibitor to the germinated wheat flour is themost effective improving method. At the same time,investment of this method is low and operation isconvenient, this method IS welcomed in the practice. In1960's it is reported that phytate could inhibit a - amylaseand protease activity", adding inhibitors to germinatedwheat flour to inhibit a-amylase activity" and improving theflour qualities' had got good effects. Because phytate isliquid and It has high corrosiveness, its use IS inconvenientand sometime Its application IS difficult m the practice.Meanwhile the regression equation IS obtained

Materials and Methods

Materials

Wheat: new normal wheat, 13% moisture, M. V. is 570

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Proceedings of the 7th International Worktng Conference on Stored-product Protection - Volume 2

B.U.Cultivation of wheat germtnatwn: soak the normal

whea t in the water for 3 hours at the room temperature,then spread out the wet wheat m the big dish, covered Withwet gauze and put them in the incubator at 25'C to cultivategernunation. After different time, dry the germinatedwheat to the original moisture, mix the germmated wheat indifferent quantities with the normal wheat, so as to obtainthe germinated wheat of. different germmation time andcontent.Germinated wheat flour . mill the germmated wheat into

flour by Brabender experiment mill, called flour A, thesewere the different germinated time and content. Flour B:germinated wheat flour obtained from the null plant, 12.5%moisture, M. V. was 20 B. U. only.

Instruments

Brabender Amylograph (with cooling water system),Brabender expenment mill, both made in Germany.

Methods

Brabender M. V. measurement: AACC method 22 -10.Peeling: the wheat was soaked in 3% salt solution for 20

min. , then it was peeled by the small rice mill, the peelingtime were 1, 2 and 3 nun. , resp.Heating: Spread the wheat on the piece of dry gauze,

then put them m the incubator With fan for 6 min., thetemperatures were 90'C , 100'C, and 110'C , resp.

Additives

Phytic acid: 70% phytic acid agent, made m Shanghaichemical reagent plant, China.Sodium phytate: 40 - 50% (edible grade), made in

Orient Food Factory, Chengdu, SIchuan province, China.

Results and Discussion

The relationship among the M. V., germinationtime and the germinated wheat content

The M. V. of wheat mainly depends on the content andactivity of a - amylase, while the a - amylase activity isrelated to on the germination time and content, in order tofind the relationship among the M . V., time and content,the experiments were done and the results were shown inTable 1.With germination time and content increasing the M. V.

were decreased, when the time was 48h, content was 25%,M. V. was 22 B. U. only, so damaged the qualities of wheatgreatly.In order to find the duality quadratic regression equation

of M V. , germination time and content.Suppose:

Table 1. Germination time and germina ted wheat ratioaffected the M. V

No. Germinated time Ratio Max. VIscosity(h) (%) (B. U.)

1 24 5 460

2 24 15 390

3 24 25 300

~ 36 5 425

5 36 15 360

6 36 25 250

7 48 5 110

8 48 15 38

9 48 25 22

10 36 15 365

11 36 15 355

y: M. V. (B. U. )ZI: germmation time (h), three levels: 24, 36 and 48.Zz: germinated wheat content (%), three levels: 5, 15

and 25.If XI = (z I - 36) /12, then XI: - 1, 0 and 1;Xz= (zz-15) /10, then Xz: -1, 0 and 1.

Used the central combination design:y = bo + b1Xl + bzxz + b12XIXZ + buXI + bzzx~

the plan of expenment and the results were shown m Table 2.Xl=XZ=X12=O;XI' = X Z' = 6/11 = 0 . 545; y = 297.55Lu =2.xI - n XI = 6; Lzz =2.x~ - nX~ = 6;L1'1' =2.xI' - n XI' = 6 - 11 X 0.545Z = 2.733

Lz'z' =2.x~, - n x~,=6 - 11 x 0.545z = 2.733

L12,IZ =2.xIz - n XIz = 4 - 0 = 4L1,z' =2.xl' XZ' - n Xl'XZ' = 4 - 11 X 0.545z = 0.733

LZ'I' = 0.733

others, LtJ = 0L1y = - 980 Lzy = - 423 L1Zy = 72

L I' y = 1320 - 11 x 0 . 545 x 279.82 = - 355 .90

Lz'y= 1567 - 11xO.545x279.82 = -108.90

then regular equation group:6b1 = -980

6bz = -4234b12 = 72

2.733bll +0.733 bzz= -355.9O.733bll + 2. 733bzz = - 108.9

the solutions: b, = -163.3, bz = -70.5, b12 = 18,bu= -128.8, bzz= -5.3,

bo = 11 - b1Xl' - bzxz'= 279.55 - (- 128.8) x 0 . 545 + 70.5 x 0 . 545= 388.17

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Proceedings of the 7 th Iniernatumal Working Conference on Stored-product Protectwn - Volume 2

Table 2. Calculation of duality quadratic regression.

No Xl Xz Y XIXZ Xl' XZ' XlY XzY XlZY Xl' Y «c» yZ

1 -1 -1 460 1 1 1 -460 -460 460 460 460 2116002 -1 0 390 0 1 0 -390 0 0 390 0 1521003 -1 1 300 -1 1 1 -300 300 -300 300 300 900004 0 -1 425 0 0 1 0 -425 0 0 425 1806255 0 0 360 0 0 0 0 0 0 0 0 1296006 0 1 250 0 0 1 0 250 0 0 250 625007 1 -1 110 -1 1 1 110 -110 -110 110 110 121008 1 0 38 0 1 0 38 0 0 38 0 14449 1 1 22 1 1 1 22 22 22 22 22 48410 0 0 0 0 0 0 0 0 0 0 0 13322511 0 0 0 0 0 0 0 0 0 0 0 126025~ 0 0 3075 0 6 6 -980 -423 72 1320 1567 1099703

the regression equation:Y = 388.17 -163 .3XI -70 .5xz + 18xl Xz -128 .8xI - 5 .3x~using Zl, Zz, taken the place of Xl' Xz:y= -113.81 +48.95z1 -lO.86zz +0.15zlzZ -O.89zI -0.05z~variance analysis and regression significance test:Lyy = ~Yz - n"iF = 1099703 - 11x 279. 55z

Lyy = 240072.77bILly + bzLzy = -163.3 x ( - 980) + ( -70.5) x ( - 423)

= 189955.5b12LlZy = 18x 72 = 1296bnLI,y + b22Lz'y = ( -128.8) x ( - 355.9)+ ( - 5.3) x ( -108.9)

= 46417.09u = bILly + bzLzy + b12LlZy + buLI,y + bzzLz,y

Table 3. Variance analysis.

=237568.59central experiments No.5, 10 and 11,Qe = 360z + 365z + 3552 - 3 x [(360 + 365 + 355) -:-3JZ= 50QL= Lyy- U - Qe= 2454 18checked the tables:Fo 05(2,2) = 19.00 F« 05(1,2)= 18.51 Fo 05(3,2)= 19.16Fo 01 (2,2) =99.00 Fo 01 (1,2) =98.50 Fo 01 (3,2) =99.17R = (U ~ Lyy)ll2 = (237568.59-:-240072.77)112 = 0.9948when a = 0.05, N, = 5, Nz= 5, checked the table Fa = 5.05R, = [5Fa-:-(5+5FJJl12= [5x5.05~(5+5x5.05)Jll2

= 0.9136because R >Ra, SO regression is significance (Table 3) .

Vanance sources Sum square Degree of freedom Average sum square F Sigmficance

linear term 189855.50 2 94927.80 3797.10 * *mutual term 1296 1 1296 51.84 *quadratic term 46417.09 2 23208.55 928.34 * *residua 2454.18 3 818.06 32.72 *error 50 2 25sum 40072.77 10

Study on improving effects of different methods

Except for germmation time and contents, improvingmethods and conditions also influenced the M. V , here usedthe orthogonal Iexperiment of 5 factors and 4 levels,researched M. V. The factors and levels were in Table 4.The additrve was phytate , addmg quantities were 0.1 ml,

0.3 ml and 0.5 ml m 300 ml of flour solution, these wereconcentration levell, 2 and 3 in Table 4 Usmg orthogonaltable LI6(45), results and analysis were m Table 5

Depending on R (rn Table 5), the order of the factorsmfluenced the M. V. was:Primary • Secondary

A DEC BThe order of different methods was: D, E and C, addmg

the additive was the most effective improving method, whenthe M. V of flour was m 400 - 500 B. U., the good flourqualities can be obtained, so the factor D should choose thelevel 3, here the average M. V. was 470.5 B. U.; theimproving effects of factor E at levelland level 2 were

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Proceedings of the 7 th International Worktng Conference on Stored-product. Protectwn - Volume 2

almost equal, level 1 was more econormcal, so to chose level1 was reasonable; Factor C should choose level 2, theaverage M. V. was 475.5 B. U. The optimum technologicalcondition of three methods was CzD3El.

Table 40 Factors and levels.

Factors Levels

A: germmation time (h) 0 24 36 48B: germmated wheat content/ %) 5 15 25 35C: Heating C'C ) 20 90 100 110D: Additive (concentration levels) 0 1 2 3E: peeling (nun) 0 1 2 3

Table 50 Results and analysis of orthogonal experiment.

No. A B C D( concentration E M V.(h) (%) C'C ) levels) (min) (B. U. )

1 0 5 20 0 0 5702 0 15 90 1 1 6803 0 25 100 2 2 7604 0 35 110 3 3 8705 24 5 90 2 3 7606 24 15 20 3 2 7507 24 25 110 0 1 4808 24 35 100 1 0 6109 36 5 110 1 2 46010 36 15 100 0 3 40011 36 25 90 3 0 42012 36 35 20 2 1 30013 48 5 100 3 1 46014 48 15 110 2 0 40015 48 25 20 1 3 42016 48 35 90 0 2 300Kl 2880 1990 1670 1492 1662 200Kz 2600 1892 1902 1800 1660 62K3 1580 1710 1970 1882 2012 50

K. 354 1822 1872 2240 2080 42kl 720 497.5 417.5 373 415.5kz 650 473 475.5 450 415k3 395 427.5 492.5 470.5 503

14 88.5 455.5 468 560 520R 631.5 70 75 187 105

The relationship between the additive quantities andthe M. V.

According to the experiments, addmg the inhibitor wasthe most effective improving method in three methods, sostudied further that additive influenced the M. V. furtherhere, built the polynomial regression equation between M.V. and adding quantities.

For convenient application in practice, taking sodiumphytate as enzyme inhibitor through changing the addingquantities could observe the changing of the M. V., theresults were in Table 6.

Table 60 Inhibitor adding quantities and M V.

Flourt g) Inhibitorf g) M V. (B. U.)

50505050505050

0.300.400.500.600.700.800.90

95160220270340460610

The increasing of M. V. along with the content ofInhibitor quantities, in order to build the polynomialregression equation of them,supposed: z was mhibitor quantities,

y was (M V -;-100),and y = Co + Clz + Czzz + ~Z3 + C4Z4

order: x = (z - 0.30) -;-0 . 1 + 1 ;so, Xl =1, xz=2, x3=3, x4=4, xs=5, x6=6, x7=7;supposed.: y = bo = bl <PI(x) + bz<Pz(z ) + b3 <1>3(X)+ b4<P4(x),then n = 7,checked the relative tables, the calculations and results werehsted in Table 7.Lyy = "J.ykZ - n f? = 85.52 - 7 x 3.079 = 19.158

U =2.b~ty= 19.717 Q = Lyy- U = 0.013vanance analysis was listed in Table 8.Checked the tablesF 0 os ( 1 ,2) = 18 . 15 FOOl ( 1 ,2) = 98. 50

when n = 7, there is:<Pl(x) = X - 4 <pz(x) = xz- 8x+12<P3(x) = x3- 12xz+41x - 36

y = 3.079+0.809(x - 4)+0.091Cxz- 8x+12)+0.158(x3- 12x2+41x - 36)-4.753+6.559x - 1 805xz+0.158x3

-4.753+6.559[(z - 0.30)-;-0.1 + 1]- 1.805[ (z - 0.30) -;-0.1 + 1]z+ 0.158 [ (z - 0.30) -;-0 . 1 + 1 J3- 18.606 + 101. 879z - 18. 998zz + 1. 580z3

Accordmg to the regression equation of adding quantityand M V., could decide the adding quantities m theImproving different germmated wheat. So we can apply thisimproving method to practice and guide practice.

Inhibition mechanism analysis

Phytate is non-competitiveness inhibitor of a - amylase.Phytate and a - amylase can form compounds, and thehydrolysis of 'inhibitor/enzyme/substrate' was difficult, soinhibited hydrolysis of starch molecules. Phytate was also

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Proceedings of the 7 th Internatwnal Working Conference on Stored-product Protection - Volume 2

inhibitor of protease, it can resist hydrolysis and damage of the stable factor of ex- amylase, ca + + was chelated, and ex-protein m 'germinated wheat, improve the gluten

,s amylase activity was inhibited greatly.

qualities", meanwhile it can chelate metal ions strongly, as

Table 7. Calculation of polynomial regression.

x Ch (e ) 'hex) <P.3(x) CP4 (x) Y yZ

1 -3 5 -1 3 0.95 0.902 -2 0 1 -7 1.60 2.563 -1 -3 1 1 2.20 4.844 0 -4 0 6 2.70 7.295 1 -3 -1 1 3.40 11.566 2 0 -1 -7 4.60 21.167 3 5 1 3 6.10 37.21

t.;= CPt (k) Yk 22.65 7.65 0.95 -0.45 ~Y =21.55 85.52St 28 84 6 154 n=7

b, = LtY/St 0.809 0.091 0.158 -0.003»i; 18.324 0.696 0.150 0.001 Y =3.079A.t 1 1 1/6 7/12

Table 8. Vanance analysis.

Sources of sum square Sum square Degree Average F Significance

hnear term 18.324 1 18.324 2819.1 * *quadratic term 0.696 1 0.696 107.08 * *cubic term 0.150 1 0.150 23.077 *

quadruplet term 0.001 1 0.001 0.167error 0.013 2 0.0065sum 19.158 6

Sodium phytate can hydrolysize into phytate after addingwater, so adding It into flour can obtain the satisfactoryimproving effects.In addition, phytate inhibition effects also are related to

pre-incubation time and pH m solution, when pre-incubationtime was 1 h, pH was below 7, can obtain the good results".

Conclusion

The main factors that influenced M. V. of germmated wheatflour were germmation time and germinated wheat contentThe duality quadratic regression equation of M. V. andgermmation time, content was:Y = -113.81+48.95zI- 1O.86zz+0.15zIzz- 0.89zI

- 0.05z~In three improving methods (heating, adding mhibitor

and peeling), adding inhibitor was the most effectiveimproving method and very convenient in practice.Adding sodium phytate can improve Brabender Maximum

viSCOSItyof germinated wheat flour, and changing the addingquantity can adjust M. V., the polynomial regressionequation between M. V. and the adding quantity was:Y = -18.606 + 101. 879z - 18.998zz + 1. 580z3

References

1. Cawley, R. Wand Mitchell, T. A. , 1968. Inhibition ofwheat ex- amylase by bran phytic acid. Journal of ScienceFood and Agnculture, 19, 106.

2. Fei Rongchang, 1997. Design and data Processing ofexperiments. ed by Wuxi University of light industry.

3. Li Weili and Wang Ruolan, 1993. Utilization of physicacid as ex- amylase inhibitor in improving flour quality ofgerminated wheat. Journal of Zhengzhou Grain College.2,50.

4 S. S. Deshpande and Munir Cheryan, 1984. Effects ofphytic acid, Divalent cations, and their interactions on ex- amylase activity. Journal Food Science, 49, 516.

5. U. ZaWIStoWSka, J. Langstaff and W. Bushuk. 1988.Improvmg effect of a natural ex- amylase Inhibitor on thebaking quality of wheat flour containing malted barleyflour. Journal of Cereal Science, 8, 207.

6. Wang Ruolan and Li Weih. 1992. Study on decreasing ex- amylase activity and improving the flour quality ofgerminated wheat. Journal of Zhengzhou Grain College,2,43.

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