Journal of Cereal Science 24 (1996) 283–290

Biochemical Basis of Kernel Milling Characteristicsand Endosperm Vitreousness of Maize

C. Mestres and F. Matencio

CIRAD-CA, Laboratoire de Technologie des Cereales, Maison de la Technologie, 73 rue J.F. Breton,BP 5035, 34032 Montpellier Cedex 1, France

Received 2 October 1995

ABSTRACTChemical characteristics of 18 normal maize grain samples, having a wide range of physical properties(endosperm vitreousness, kernel friability and milling characteristics), were analysed and related tophysical properties. Measurement of damaged starch showed that starch behaved as a passive fillerin endosperm. Endosperm protein content and class, as determined from extractability propertiesand reversed-phase high-performance liquid chromatography, were correlated with kernel physicalproperties. Endosperm vitreousness and kernel mechanical properties (kernel friability and millingcharacteristics) were related to different components, however. Vitreousness seems to be linked tothe proportion (%) of the two c-zein fractions, whereas friability increased when a-zein contentdecreased and when salt extractable protein content increased. 1996 Academic Press Limited

Keywords: maize, hardness, protein, damaged starch.

In maize, a-zeins may influence kernel vit-INTRODUCTIONreousness2,10–13. Floury endosperm of normal or

The chemical basis of kernel hardness and en-opaque-2 and floury-2 cultivars contains less a-zeins

dosperm vitreousness of cereal grains has long and more water-extractable proteins. c-zein frac-been studied, but remains unexplained: it may tions may also influence maize kernel physicaldepend on various constituents of cereal grains. properties. This fraction has components of Mr 16Since proteins can form a matrix surrounding and and 27 k. The quantity of Mr 27 k c-zein is almostembedding starch granules1–3, they may be the three times higher14,15 for Quality Protein Maizedetermining factor, rather than the discontinuous (QPM), modified opaque-2 cultivars with vitreousstarch phase. Variation in binding forces between endosperm, than for opaque-2 cultivars having com-protein and starch may also be important1. How- pletely floury endosperm. For normal cultivars,ever, the correlation between protein content and however, the quantity of Mr 27 k c-zein remainskernel physical characteristics, when it exists, re- two to three times lower than for QPM cultivars12,14,mains low4–9. even if kernels from normal cultivars are generally

more vitreous than QPM kernels9. Furthermore,the amount of Mr 27 k c-zein does not changesignificantly for most opaque or floury mutants,

: QPM=quality protein maize; except opaque-23. Also Moro et al.16 and Pratt et al.17

RCG=regular and coarse grits; CF=cornmeal flour; did not show clear relationships between kernelFG=flaking grits; ES=enzymatic susceptibility ofhardness, or vitreousness, and c-zein content. Thus,starch; DS=damaged starch; ESN=enzymatic sus-c-zeins are probably not the only factor influencingceptibility of native starch; r=coefficient of correlation;endosperm vitreousness.r2=coefficient of determination; N=nitrogen; SSP=

Most studies have compared normal, opaque, flourysalt soluble protein; RP–HPLC=reversed-phase highperformance liquid chromatography. and QPM cultivars, and their crosses using en-

0733–5210/96/060283+08 $18.00/0 1996 Academic Press Limited283

C. Mestres and F. Matencio284

Table I Proximate analysis and physical characteristics of and flours (CF) were determined using a pilotwhole maize kernels roller mill and the yield of flaking grits (FG) was

obtained using an experimental fragmentation de-Grain chemical and Standardvice.physical characteristics Mean Range deviation

Whole endosperm was obtained by hand dis-Nitrogen content (% db) 1·76 1·23–2·25 0·01 section of kernels. After drying at low temperatureFree lipid content (% db) 4·6 3·9–5·4 0·1 (30 °C), whole endosperm was ground first withAsh content (% db) 1·29 0·89–1·61 0·03

a KT30 disc mill (Falling Number, Stockholm,Vitreousness (%)a 66 50–87 4Specific density (g/cm3)a 1·329 1·251–1·401 0·003 Sweden) with the fine burr at a setting of one andFriability (% db)a 43 39–51 1 then with a Cyclotec 1093 sample mill (Tecator,RCG yield (%)a 69 63–73 1 Hoganas, Sweden), using a screen of fine apertureCF yield (%)a 11 8–16 1 (0·5 mm).FG yield (%)a 37 27–48 nd

Two commercial regular grit samples were alsoa Data of Mestres et al.18 used to develop the protein extraction procedurend: not determined. using salt solution; these were ground by one pass

through a pin mill (Alpine, Duisburg, Germany)giving flours called cornmeal 1 and 2. They werethen re-milled using a Cyclotec 1093 sample milldosperm vitreousness as a criterion of kernel millingusing screens of coarse and fine apertures (1·0 andcharacteristics. However, these two characteristics0·5 mm, respectively).are not closely related6,7,9,18. Therefore, in breeding

maize for grain quality, it appears to be moreimportant to focus on the chemical basis of the Proximate analysismilling characteristics of maize kernels, rather thanon their endosperm texture, and to compare normal Proximate composition (water, nitrogen, free lipidcultivars having a wide range of milling char- and ash contents) of maize grains were performedacteristics, rather than cultivars with opaque or floury as described elsewhere9.genes. This was the aim of this study. Chemicalcharacteristics, especially starch properties and pro-

Starch and damaged starchtein distribution, were determined for kernels of18 normal cultivars and correlated with milling Starch and damaged starch contents were de-characteristics. termined on ground products passing through a

315 lm sieve using enzymic methods19. Starchcontent was determined after complete digestionEXPERIMENTALof starch by thermostable alpha-amylase at 85 °C

Materials then glucoamylase at 55 °C. Enzymic susceptibilityof starch (ES, % starch basis) was defined asEighteen maize grain samples were analysed. They the proportion (%) of starch being hydrolysed towere collected from several seed farms in West glucose by glucoamylase at 37 °C for 2 h. DamagedAfrican countries and the French West Indies. starch (DS) was calculated using the formula:They had various genetic backgrounds: five were

ecotypes; seven composites; one a population; twovarieties; and three were single hybrids. They were DS (%)=100×

ES−ESN100−ESNcharacterised previously in terms of endosperm

vitreousness and dry-milling characteristics (Tablewhere ESN was the ES of native maize starch.I)18. Endosperm vitreousness was measured by the

relative proportion of vitreous endosperm area of100 kernel cross-sections or evaluated by the spe- Protein fractionationcific density of kernels (determined using a Helium

Protein fractionations were performed on wholepycnometer). Friability was defined as the pro-endosperms.portion (%) of ground corn kernels passing through

a 315 lm sieve. Dry milling characteristics weremeasured on 2·5 to 5 kg samples: the yields of Procedure 1. A procedure derived from that of

Landry and Moureaux11 was used. Extractionsregular and coarse grits (RCG) and of cornmeal

Maize kernel characteristics 285

were performed in 10 ml centrifuge tubes (0·5 g of formed at 210 nm and peak areas were expressedas mV·s on dry basis.ground endosperm with 6 mL solvent) using a

rotating agitator. Three successive extractionswere performed for each step. The first was over-night (about 18 h), and the other two for 3 h. The RESULTS AND DISCUSSIONsolvents used, in sequence, were, 0·5 NaCl then

Proximate analysisultrapure water for step 1, 70% (v/v) propan-2-olthen 60% (v/v) propan-2-ol for step 2, 60% (v/v) As previously reported9, maize cultivars of tropicalpropan-2-ol containing 1% (v/v) 2-mercapto- origin vary greatly in kernel proximate com-ethanol for step 3 and borate buffer, pH 10 position (Table I), even with no mutants for high(0·0125 Na2B4O7 and 0·01 NaOH) with 0·5 protein or lipid content. Kernel physical propertiesNaCl and 1% (v/v) 2-mercaptoethanol for step 4. were not correlated with free lipid and ash con-Step 1 was performed at 4 °C, and the others tents, contrary to previous results9. Kernel nitrogenat 30 °C. The three supernatant solutions (after content correlated poorly (r=0·49) with specificcentrifugation at 3000 g for 10 min) from each density and did not correlate with endospermstep were pooled into Kjeldhal digestion tubes. vitreousness. Kernels with low nitrogen contentsResidual solids were also rinsed into Kjeldhal tubes appeared more friable: nitrogen content correlatedwith distilled water. After evaporating most of the negatively with friability and CF yield (r=−0·70liquid (by air drying at 120 °C), nitrogen was and−0·74 respectively) and positively with yieldsdetermined by the Kjeldhal method. All ex- of RCG (r=0·56) and of FG (r=0·73). Thus,tractions and nitrogen determinations were per- protein content was the only component linked toformed in triplicate. grain physical properties, but it explained only

half the variation in these characteristics (r2). Also,previous results9 showed protein content not toProcedure 2. A simpler method was used to quant-be correlated with endosperm vitreousness andify salt extractable proteins. Ground endospermmilling performance.(100 mg) was suspended in 0·5 NaCl (1·5 mL) in

2 mL Eppendorf centrifuge tubes, and agitated for1 h at 4 °C. The tubes were then centrifuged at12 000 g and 4 °C for 10 min, and the protein Damaged starchcontents of the supernatants were determined by a

Damaged starch was measured on eight samples,colorimetric method20 using bovine serum albuminrepresentative of the variability in endosperm vit-(A-8022, Sigma, St Louis, U.S.A.) as reference.reousness and milling characteristics of the 18We checked that salt extractability was not in-maize grain samples. Damaged starch varied fromfluenced by sample particle size using cornmeal2·2% to 5·1% (starch basis). There was a highlysamples 1 and 2 that had proportions (%) of overssignificant inverse correlation (r=−0·93) betweenon the 250 lm sieve ranging from 11 to 45%.kernel friability and damaged starch: more friableExtraction was complete when overs of the 250 lmkernels had less damaged starch after grinding,sieve were less than 45%. Re-ground endospermse.g. wheat21,22. In other words the more friable theof the 18 samples in this study had less than 12%grain the more intact (undamaged) were the starchovers at 250 lm sieve.granules in the milled products. The kernel appearsto be a composite material, in which starch is a

Procedure 3. Samples (300 mg) were extracted for filler; in harder grain matrices, the filler is more2 h with 70% (v/v) ethanol/5% (v/v) 2-mer- damaged by mechanical stress. Indeed, freeze-captoethanol/0·5% (w/v) sodium acetate at room etching experiments show fractures through starchtemperature in 2 mL Eppendorf tubes as in pro- granules of hard wheats but around the granulescedure 2. The suspension was then centrifuged at in soft grains4.5000 g for 5 min at room temperature. Alcohol-extractable proteins were separated on a Vydac(Hesperia, California, U.S.A.) RP-C18 column Fractionation of endosperm proteins(25 cm×4·6 mm, 5 lm particule size, 300 A poresize)13, also using a Vydac 218FSK54 guard col- As in previous results11, nitrogen (N) content was

slightly lower (13%) for endosperm than for wholeumn. Spectrophotometric detection was per-

C. Mestres and F. Matencio286

Table II Nitrogen and protein repartition in whole Partial correlation coefficients between friabilityendosperm and amounts of N in fractions 1 (salt-extractable

N) and 4 (G2-Glutelins) appeared significant atNitrogen and protein Standardthe 6% and 5% levels, respectively. One-third offraction Mean Range deviationthe N in fraction 1 is non-protein N, however11.

Nitrogen content of whole 1·54 0·98–2·17 0·01 Therefore, we also quantified salt extractable pro-endosperm (% db) teins by a colorimetric method (procedure 2). TheProcedure 1a

amount of salt extractable protein was then highlySalt extractable N 0·12 0·10–0·14 0·01Zein 0·63 0·23–1·04 0·02 and positively correlated with kernel friabilityG1-Glutelins 0·24 0·18–0·36 0·02 (Table III). A multiple regression described 71%G2-Glutelins 0·10 0·06–0·12 0·01 (r2) of the variation in kernel friability (1):Residue 0·42 0·35–0·51 0·01

Procedure 2Friability (%)=37·2−2·4×F2+48×SEP (1)Salt extractable protein 0·32 0·26–0·41 0·01

Procedure 3b

where F2 was the amount of N in zein fractionb-zeins 27·4 11·7–42·1 0·4Mr 27 k c-zein 108 38–135 8 and SEP the salt extractable protein content ofMr 16 k c-zein 41 14–72 3 the endosperm.a-zeins 470 160–660 30 Furthermore, the correlation coefficient with

friability was 0·91 when the SEP results werea N content expressed as percentage of endosperm drymatter. Fractions were labelled according to Landry and expressed as relative amounts of endosperm pro-Moureaux11. tein content (Fig. 1). Maize grain friability in-

b mV∗s/mg (db), RP–HPLC peaks were labelled according creased as the relative amount of salt extractableto Paulis and Bietz28.protein increased. A comparison can be made withwheat, for which a protein class, called friabilin,has been observed to be associated with endospermsoftness25–27.kernels (Tables I and II). Salt extractable N (frac-

For milling performances, trends similar to thosetion 1) averaged 8% of total N; zein (fraction 2),for friability exist (Table III). For example, the41%; G1-Glutelins (fraction 3), 16%; G2-Glutelinscorrelation coefficients for the relationship be-(fraction 4), 6%; and sediment, 27%.tween milling product yields and the amounts ofNeither the total nor the partial correlationN in the zein fraction and sediment were significantcoefficient (after removing total nitrogen contentas a whole, but not when the effect of total Nas a variable) was significant for the relationshipcontent was removed. Also, partial correlationbetween endosperm vitreousness (or specific dens-coefficients were significant for the relationshipity) and N recoveries (% db) of the different frac-between the yield of flaking grits and salt ex-tions (Table III).tractable protein content or amount of N in G2-On the other hand, friability correlated withGlutelins fraction.the amount of N in the zein fraction (significant

We have, therefore, demonstrated a relationshipat 1% level) and to a lesser extent (significant atbetween kernel milling characteristics and salt5% level) with unextracted N (Table III). Theseextractable protein content. For zein classes, how-fractions, which contain most of the endospermever, no clear conclusion could be drawn. Indeed,N, were correlated highly with total endosperm Neven if fraction 2 were almost pure a-zeins, somecontent (correlation coefficient of 0·93 and 0·72,a-zeins are also present in fractions 3 and 4.respectively). Partial correlation coefficients (afterFraction 3 also contains b-, c- and d-zeins andremoving total nitrogen content variable) withfraction 4 contains c-zein14,23.friability were, however, insignificant. Thus, the

amounts of these protein classes influence kernelfriability inversely, but these proteins have no Separation of alcohol-extractable proteins withspecific role. For example, the zein fraction mainly RP–HPLCcomprises a-zeins14,23 located within protein bodiessurrounded by b- and c-zeins24. Thus, a-zeins, as Zein classes were identified by retention times13

according to the classification of Paulis and Bietz28.they do not come into contact with the endospermcell contents, may reinforce the protein matrix, Depending on cultivar, b-zeins appeared as a

single peak at 24·5 min, for 13 samples, but alsobut cannot act as a ligand.

Maize kernel characteristics 287

Table III Correlation coefficients between physical properties of kernels and protein or nitrogen distribution of theirendosperm as determined using procedures 1 and 2 (all correlation coefficients were calculated with 18 maize samples, exceptfor FG yield, 12 samples). Partial correlation coefficients after removing total nitrogen variable are indicated in parentheses.

Specific RCGVitreousnessa densitya Friabilitya yielda CF yielda FG yielda

Nitrogen content of whole 0·31 0·41 −0·69∗∗ 0·55∗ −0·72∗∗ 0·64∗endospermProcedure 1

Salt extractable Nb 0·14 −0·02 −0·03 0·03 −0·21 0·17(−0·01) (−0·26) (0·46) (−0·31) (0·22) (−0·02)

Zeinb 0·45 0·36 −0·66∗∗ 0·43 −0·65∗∗ 0·75∗∗(0·05) (−0·05) (−0·05) (−0·26) (0·10) (0·53)

G1-Glutelinsb 0·07 0·40 −0·34 0·46 −0·43 0·29(−0·02) (0·33) (−0·22) (0·39) (−0·35) (0·04)

G2-Glutelinsb 0·02 −0·13 0·12 −0·08 −0·05 0·36(−0·09) (−0·31) (0·50∗) (−0·32) (0·28) (−0·62∗)

Residueb 0·02 0·35 −0·50∗ 0·54∗ −0·54∗ 0·10(−0·32) (0·10) (0·01) (0·25) (0·05) (−0·39)

Procedure 2Salt extractable proteins −0·09 −0·18 0·29 −0·01 0·02 −0·36

(−0·21) (−0·35) (0·74∗∗) (−0·24) (0·38) (−0·60∗)

a Physical characteristics of whole maize kernels were determined previously18.b N content expressed as percentage of endosperm dry matter. Fractions were labelled according to Landry and Moureaux11.∗ Significant at P<0·05.∗∗ Significant at P<0·01.

that some presented both alleles for b-zein char-acter. Thus, the combined areas of both b-zeinpeaks (24·5 and 24·9 min) are reported as peak 1(Table II). c-zeins (Mr 27 k and Mr 16 k) wereclearly identified as peaks 2 and 3, respectively(26·3 and 27·1 min). The multiple peaks between38 and 58 min were characteristic of a-zeins (Mr

19 k and Mr 22 k); they were integrated as a wholewith a single linear baseline.

Extraction and RP–HPLC separation were du-plicated for three samples; the coefficient of vari-ation of the methodology was Ζ10% for each ofthe four peak areas. a-zein multiple peaks ac-5

54

382Salt extractable protein content (% protein content)

Fri

abil

ity

(% d

b)

42

50

46

3 4

counted for 65 to 80% of total chromatogram area(Table II), consistent with previous results3,13. GreatFigure 1 Relationship between maize kernel friability andvariability in the proportions (%) of c-zeins wassalt extractable protein content of endosperm (expressed as

percentage of total protein content, N∗6·25, of endosperm); observed: the relative ratio of Mr 16 k c-zein con-the correlation coefficient is 0·91. tent to that of Mr 27 k c-zein ranged from 0·2 to

2.Significant total and partial correlation co-

as a doublet (24·5 and 24·9 min, Fig. 2), for five efficients were observed between c-zein contentssamples. This is consistent with results of Wilson and endosperm vitreousness (or specific density,and co-workers23 and Dombrink-Kurtzman3, who Table IV). Multiple regression using areas of Mrfound b-zeins with two retention times in different 27 k c-zein peak (P2) and of Mr 16 k c-zein peakinbreds; these two b-zeins behave as if their genes (P3) explained 53% (r2) of the variation in en-were allelic at the same locus23. The 18 cultivars dosperm vitreousness (2):varied widely in genetic background, and nonewas inbred; it is quite understandable, therefore, Vitreousness (%)=51·5+0·28×P2−0·44×P3(2)

C. Mestres and F. Matencio288

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Figure 2 RP–HPLC chromatograms of alcohol-extractable proteins from endosperms of two cultivars. Total zein wasextracted with 70% (v/v) ethanol, containing 5% (v/v) 2-mercaptoethanol and 0·5% (w/v) sodium acetate, and analysed usinga C18 column with a non-linear 25–64·4% acetonitrile gradient for 60 min. Peaks were labelled according to Paulis and Bietz28.

This tends to confirm the positive relationship firming that this protein class is not directly re-sponsible for the flouriness phenotype3.between Mr 27 k c-zein and vitreousness as already

observed for floury opaque-2 lines and QPM cul- As with the fractionation procedure, the a-zeincontent of endosperm, as determined by RP–tivars14,15. Furthermore, Pratt and co-workers17

found a negative correlation between Mr 16 k c- HPLC, correlated significantly with kernel millingcharacteristics. But a-zein content was also highlyzein and specific density for one population of

crosses between floury and flint cultivars. But the significantly correlated (r=0·94) with endospermN content; thus, partial correlation coefficientsa-zein content of endosperm (peak 4 area) was not

correlated with endosperm vitreousness con- between a-zein content and kernel milling char-

Maize kernel characteristics 289

Table IV Correlation coefficients between physical properties of kernels and amounts of alcohol-extractable endospermproteins as determined by RP–HPLC (all correlation coefficients were calculated with 18 maize samples, except for FG yield,

12 samples). Partial correlation coefficients after removing total nitrogen variable are indicated in parentheses.

Specific RCGVitreousnessa densitya Friabilitya yielda CF yielda FG yielda

b-zeinsb −0·22 0·18 −0·31 0·31 −0·42 0·11(−0·41) (0·01) (−0·02) (0·10) (−0·18) (−0·12)

Mr 27 k c-zeinb 0·61∗∗ 0·71∗∗ −0·64∗∗ 0·50∗ −0·58∗ 0·61∗(0·55∗) (0·64∗∗) (−0·43) (0·28) (−0·32) (0·44)

Mr 16 k c-zeinb −0·51∗ −0·24 0·26 −0·01 0·47∗ 0·23(−0·50∗) (−0·21) (0·24) (0·07) (0·56∗) (0·11)

a-zeinsb 0·41 0·42 −0·65∗∗ 0·40 −0·70∗∗ 0·60∗(0·35) (0·12) (0·03) (−0·41) (−0·08) (0·20)

a Physical characteristics of whole maize kernels were determined previously18.b RP–HPLC peaks were labelled according to Paulis and Bietz28.∗ Significant at P<0·05.∗∗ Significant at P<0·01.

chromatography and scanning electron microscopy.acteristics were not significant. The only significantJournal of Cereal Science 19 (1994) 57–64.partial correlation coefficient was observed be-

4. Simmonds, D.H. Chemical basis of hardness and vit-tween Mr 16 k c-zein content and coarse meal reousity in the wheat kernel. The Bakers Digest Octoberand fine flour yield. This probably reflects the (1974) 16–29.correlation between vitreousness and Mr 16 k c- 5. Manoharkumar, B., Gestenkorn, P., Zwingelberg, H.

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