Jianshuang QI,Shuanggui TIE,Xiaohua HAN,Runqing YUE,Shufeng YAN,Caixia LU

The Cereal Crops Insititute of Henan Academy of Agricultural Sciences,Zhengzhou 450002,China

Responsible editor:Nanling WANG Responsible proofreader:Xiaoyan WU

Maize(Zea mays Linn.Sp.)is a grain crop with the largest planting area in China,and is also one of larger planting area crops worldwide.Annual maize kernel output all over the world has been about 8.41×108ton,and annual maize output in China has already broken through 2.0×108ton,it is mainly used as the fodder of animal husbandry.However,the content of protein in common corn kernel is low,especially the content of lysine,what’s more,lysine is one of the necessary nutrient elements for the growth and development of livestock and poultry,and it can not be synthesized in vivo but from food intake[1].Common maize,as the fodder of monogastric livestock,must be mixed with industrial lysine and a large amount of protein feed like soya-bean cake,which not only increases the cost of the fodder of animal husbandry,but also causes immense waste of grain due to its low digestibility.According to the experiment,special compound feed for livestock and poultry taking quality protein maize as raw material was prepared,it had great agvantages in the ratio of feed to meat,raising period and feeding cost[2-6].America is the birthplace of high-lysine maize and and the earliest research country in world[7].Middle America,South America and some countries in Africa also carried out some studies on quality protein maize and its utilization,and invented multiple varieties of quality protein maize in succession.CIMMYThas begun the study on high-lysine maize since 1965,and introduced endosperm modifier gene in the middle of 1970s,modified the soft endosperm of o2maize into semitransparent stereoplasm or semihard endosperm,thereby improving the yield and the resistance to disease[8].Since 1972,our country has introduced a batch of QPM Pools and group from CIMMYT,such as Pool33,Pool34,Pob69 and Pob70,etc.,and has begun the study on quality protein maize[9-12].Therefore,the rapid determination of lysine content in maize kernel has become the key breeding technique for quality protein maize.

In the breeding process of quality protein maize,the lysine content in the selected germplasm of high-lysine maize needed to be detected,however,due to the limited project expenditure,it was impossible to send all materials to National Food Quality Supervision and Inspection Center,which greatly limited the development of the work,finally,only visual estimation combined with the experience of breeders or limited chemical detection analysis was carried out,of course,the detection efficiency was low and it was difficult to meet the requirements of breeding[13].Hitachi L-8900 Automatic Amino-acid Analyzer,as the product with post-column derivation technology,its principle is that after the separation,sample mixtures exude from the chromatographic column with mobile phase,then mix with derivatization reagent in the response detector and carry out derivative reaction,after that reenter the detector to detect its response signal,finally quantifying by standard curve and calculating the contents of all amino acids.The method only needs fewer materials,moreove,its detection efficiency is high.In this experiment,nine high-lysine maize kernels and two kernels of common maize hybrid were used as experiment materials,and quantitative determination of lysine contents in high-lysine maize kernels and common maize kernels was carried out using Hitachi L-8900 Automatic Amino-acid Analyzer,to know the effect of the Analyzer in distinguishing the lysine contents between common maize kernels and high-lysine maize kernels,thereby providing a basis for further detecting lysine contents in quality protein maize kernels.

Materials and Methods

Experimental materials

Nine high-lysine maize kernels and two kernels of common maize hybrid were used as experiment materials,and lysine contents in these maize kernels were measured.The kernels of the experimental samples were provided by the Cereal Crops Insititute of Henan Academy of Agricultural Sciences.

The instrument and reagents

Amino-acid analyzer:Hitachi L-8900 Automatic Amino-acid Analyzer.

Standard amino-acid mixture sample(produced by Japan Ajinomoto Company):2 ml sample was taken from amino-acid calibration mixture ampoule which contained 18 components,then it was diluted to 50 ml solution by 0.02 N-hydrochloric acid solution.

Ninhydrin reagent:39 g ninhydrin was dissolved in 979 ml dipropylene glycol monomethyl ether,then 81 mg sodium borohydride was added,after that the mixture was bubbled for 30 min using nitrogen.That should be kept at low temperature.

Ninhydrin buffer solution:204 g lithium acetate was dissolved in 401 ml dipropylene glycol monomethyl ether,followed by 123 ml glacial acetic acid,then 336 ml water was added for dissolving.

5% ethanol:50 ml ethanol was taken to dissolve in 900 ml distilled water.

Chromatographic conditions

Separation column:the filler of 4.6 mm×60 mm column was 3 μm sulfoacid cationic resin separation column,exchange resin was Hitachi dedicated ion exchange resin.Column temperature:50 ℃; sample volume:20 μl.

Reaction column:4.6 mm ID×40 mm; filling material:emery inert material.

Sample pretreatment

Grinding sample The sample was ground into powder,and then sieved with 60-mesh sifter.(Matters needing attention:all kernels were ground into powder,and some powder on the sifter should be reground and resieved,all sample powder must be the mixed powder of all kernels).

Table1 The mass fraction of lysine in maize kernels

The determination of water in the sample[14]2-10 g sieved sample was taken and put in the oblate weighing bottle (or small Petri dish),the bottle cap was aslant placed on the side of bottle,then the sample was put in the drying oven with the temperature of 101-105 ℃for 2-4 h,then it was taken out and lidded,followed by cooling in dryer for 0.5 h,finally weighing.After weighing,the sample was reheated for 1 h,and then cooled for 0.5 h,finally,weighing again.These processes were repeated until the differences between the former and the later were less than 2 mg,then the water content in the sample can be calculated.

Weighing and hydrolyzing0.2 g dried sample was got and mixed with 5 ml (6 mol/L)HCl,then the mixture was filled with nitrogen,and followed by tube sealing,finally the mixture was put into 110 ℃dryer for hydrolyzing for 22 h.

Neutralizing,filtering (common filter papers can be used),and metering volumeThe hydrolysate was taken out for cooling,5 ml (6 mol/L)NaOH was put in 100 ml volumetric flask,the hydrolysate was filtered into the volumetric flask,buffer solution with the pH of 2.2 (0.02 mol/L diluted hydrochloric acid)was used for washing hydrolyzing tube,then filtering,finally the volume was metered to 100 ml.

RefilteringBefore the sample was put into the Analyzer,it should be shaked up and refiltered again with 0.45 μm filter membrane.

Putting the sample into the analyzerPutting the sample into the Analyzer in accordance with the stated steps.

Results and Analyses

Mixed sample and amino-acid chromatogram of maize sample

The chromatograms of mixed sample and maize sample of 18 amino acids were in Fig.1 and Fig.2,respectively.

Peaks in the Fig.1 and Fig.2 were caused by the detection results of twochannel wavelength(440 and 570 nm).Because proline was different from other amino-acid color materials,its generated compound was yellow substance,and its absorption peak was at 440 nm.However,other amino-acids generated hyacinthine DYDA,and the absorption peak was at 570 nm.

The detection of high-lysine maize sample

In this experiment,lysine analysis was carried out for nine high-lysine maize kernels and two kernels of common maize hybrid.Based on three repeated tests,the results showed that the lysine contents of high-lysine maize kernels were among 0.34% -0.42%,while of common maize kernels were among 0.24%-0.25%,and the difference was significant.The results were in line with the lysine contents detected by Zhengzhou Branch of National Maize Quality Supervision and Inspection Center,and also coincided with the detection results of highlysine maize kernels held by Hunter et al.in 2002[15].

Conclusions and Discussions

The experimental results showed that using Hitachi L-8900 Automatic Amino-acid Analyzer to determine lysine contents in maize kernels can well distinguish common maize kernels and high-lysine maize kernels,moreover,the determined results are efficient and reliable.Compared with general chemical detection method,the Analyzer needs fewer maize kernels,this not only reduces the detecton workload,but also saves many breeding materials,thus it is an ideal method for detecting lysine contents for quality protein maize breeding; furthermore,the method is less affected by human factor and has good reproducibility,moreover,it is rapid and simple to determine multi-amino acids,so it is a relatively ideal method to qualitatively and quantitatively detect lysine content in maize kernel.However,the reagents for the Analyzer have been imported reagents recently,so the price has been high.Based on this,this study will try using domestic reagents instead of imported reagents,which will greatly reduce the detection cost of amino acids,to provide new and effective detection means for greatly breeding high-lysine maize germplasms.

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