Abstract Ten millet cultivars from China and abroad were analyzed for their crude fat， amylose and amylopectin， crude protein and amino acid content. A wide variation was observed in amylopectin contain， ranging from 54.47% to 69.26%， while amylose content ranged from 11.26% to 23.22%. The average crude fat in all cultivates was 3.46%， and most cultivates ranged from 3.1% to 3.7%. Protein contents ranged from 8.61% to 15.54% with a mean value of 11.94%. For amino acid composition， the ten cultivates were particularly rich in leucine， and the mean was 34.57 mg/g. The millet cultivars were also high in glycine， glutamic acid and cysteine. Millet was known to be limiting in the essential amino lysine， and the mean was 5.18 mg/g. Other amino acids， tyrosine， histidine and arginine were also very low in the ten cultivars. In general， significant nutrient composition differences were observed between the different millet cultivars， and A2 （a kind of mother line of the hybrid millet） and hybrid millet registered a higher level of protein and amino acid composition which would be useful in millet breeding.
　　Key words Foxtail millet； Crude fat； Amylose and amylopectin； Protein； Amino acid composition
　　Setaria italica （foxtail millet） is a kind of cereal crop widely grown in Asia with particular significance in semi??arid regions of Northern China. It is also grown on a moderate scale in other parts of the world as a cereal crop for both human food and fodder. Millet is an important crop in the semi??arid tropics with 97% of millet production in developing countries[1]. The crop is favored due to its productivity and short growing season under dry， high temperature conditions. Millet is also rich in protein， mineral and vitamins， and its nutritive parameters are better than common cereals.
　　Cereal grains are the dietary mainstay of mankind and provide man??s energy needs and protein and amino acid needs. As for cereals， they are generally deficient in one or more of the essential amino acids， especially lysine and methionine. Although many studies have been conducted on main cereals such as maize， wheat， rice and soybean[2-3]， Among these four， only soybean protein levels are close to 20%， which can adequate for the lysine requirements of nonruminant. All of these feed stuffs are deficient in methionine， and corn is deficient in tryptophan. The discovery of high protein and high lysine opaque??2 and floury??2 mutant genes in corn[4-5] and Hiproly and Notch??2 mutant genes in barley[6-7] ； developed interest among plant breeders to evolve varieties with better nutritional quality in different cereals. However， the discovery of new sources of plant proteins and amino acid which are of good quality and which can be produced economically， would be desirable in millet. 　　Seed protein content and amino acid composition are known to be different from genus， species and even cultivar， but they can also differ between plants of the same variety according to growth and development condition. Such differences arise from two main groups of factors： genetic and varietals factors[8] . There are few reports in the literature that address the nutrient composition of millet. Since the content of a nutrient may vary considerably depending on conditions， climate and genetic factors， we deemed it worthwhile to also analyze millet seeds from different sources and genotypes. The present work was devoted to assess the seed protein and the amino acid composition for different millet varieties.
　　Materials and Methods
　　Source of plant material
　　Ten cultivars of millet （8 of China local and 2 exotic cultivars） were obtained from Zhangjiakou Academic of Science. ISE120 and Teff are two major cultivars gain from Indian and Africa， Bashang Kanghan was a short growth period cultivar with only 105 growth days， and Hongguzi and Xiaohuangmao are long growth period cultivars with 129 and 125 growth days， respectively. 8311 and Taohua Xiaohonggu are regulated cultivars widely distributed in the North of China. Zhangzagu 5 and Zhangzagu 9 are hybrid millet produced by Zhangjiakou Academic of Science with high yield about 800 to 1 000 kg per 667 m2 on good soils. A2 is a male sterile cultivar used as mother parent of Zhangzagu 5 and Zhangzagu 9.
　　Cultivation conditions
　　The millet was cultivated under uniform cultivation conditions， at the Zhangjiakou Academic of Science. The crops were planted at Zhangjiakou City （Latitude 40??80??N， longitude 113??50??E， and altitude 750 m）， China. The average annual rainfall in the zone was 254 mm. The average annual temperature was 12.8 ??. Fully mature seeds of the proceeding crops were used for the analysis.
　　Pre??treatment of the seeds
　　The fully mature seeds of the proceeding crops were used for the analysis. Seeds harvested from these plants were used for the estimation of seed nutritional ingredient. Plant seeds were ground to a fine powder and dried under vacuum at room temperature until a constant weight was reached， and at last sieved through a 40 mesh sieve.
　　Analysis of crude fat， amylose， amylopectin， and crude protein contents
　　Crude fat content was analyzed by the Soxhlet extraction method with absolute ethylether as solvent following the Chinese official methods of analysis （Chinese stand GB5009.6??85）. 　　Amylose and amylopectin contents were measured according to the Chinese National standard method GB7648??87. The values were calculated according to the absorbance at 620 nm（OD620） and OD550-OD740， respectively.
　　The dried seeds were powdered to pass through a 40 mesh sieve and defatted with n??hexane （Soxhlet， 6 h）. The total protein contents （N??6.25） were determined by the micro??kjeldahl method.
　　Analysis of amino acid composition
　　For the analysis of total amino acid composition with the exception of， asparagine（Asn）， glutamine （Gln） and alanine （Ala）， protein hydrolysis was performed before analysis. Amino acids were extracted from dry seed flour （150 mg） and hydrolyzed with 6 N HCl in a sealed tube at 104-110 ?? for 24 h. After the completion of hydrolysis， HCl was removed in vacuous. Tryptophan （Trp） content was determined separately. As to the Trp analysis， 450 ??l of 4.67 M KOH containing 1% （w/v） thiodiglycol were added to each sample. Hydrolysis was performed in plastic tubes within an evacuated ampoule at 110 ?? for 24 h. The hydrolysis was analyzed by an amino acid analyzer （model L??8900； Tokyo， Japan）.
　　Results
　　Seed amylose and amylopectin content
　　From Fig.1， it could be seen that the millet amylopectin content ranged from 54.47% to 69.26%， while amylose content ranged from 11.26% to 23.22%. Teff had the lowest amylopectin content only of 54.47% and also had a rather high amylose content （20.94%）. Taohua Xiaohonggu had the highest amylopectin content of 69.26%， and the amylose content was 14.35%. 8311 had the highest amylose contain of 23.22% and amylopectin content of 56.36%. The average of amylopectin in all cultivars was 63.27%， and most cultivars ranged from 60% to 77%. As for amylose， the average was 17%， and most cultivars ranged from 14% to 20%.
　　Seed crude fat content
　　The crude fat content of millet is summarized in Fig.2. Total fatty acid content of millet was relatively low. The average of crude fat in all cultivars was 3.46%. A2 had the highest fat content of 4.47%， while Teff had the lowest fat content only of 2.18%， and most cultivars ranged from 3.1% to 3.7%.
　　Seed crude protein content
　　The millet corresponding to different sources and characters showed a very large variation in protein content： from 8.61 to 15.54% and with an average of 11.94% （Fig. 3）. In general， significant differences were observed between the different cultivars of millet. Low protein content was found in abroad sample Teff （8.61%） from Africa. Medium protein contents were in the range of 10.94%-11.49， including ISE120 （gained from Indian）， two regulated millet cultivars （8311 and Taohua Xiaohonggu）， and three cultivars with different growth periods， Bashan Kanghan， Hongguzi and Xiaohuangmao. The high protein levels were gained in two hybrid millet cultivars Zhangzagu 5 （14.27%）， Zhangzagu 9 （12.81%） and A2 （15.54%）. 　　Seed amino acid composition
　　Because of the impossibility to distinguish glutamine from glutamic acid and asparagine from aspartic acid， and alanine was nearly destroyed during acid hydrolysis， the values of glutamine， asparagine and alanine were therefore not reported. The results showed that the different cultivars had a large amino acid range.The different ranges of amino acids were as follows： aspartic acid ranged from 6.28 to 35.39 mg/g with a mean content of 16.51 mg/g， threonine from 1.99 to 10.32 mg/g with a mean content of 4.94 mg/g， serine from 2.65 to 13.33 mg/g with a mean content of 6.74 mg/g， glutamic acid from 12 to 63.68 mg/g with a mean content of 31.15 mg/g， cysteine from 11.47 to 39.89 mg/g with a mean content of 22.98 mg/g， glycine from 9.42 to 64.15 mg/g with a mean content of 33.29 mg/g， valine from 6.51 to 25.64 mg/g with a mean content of 14.19 mg/g， methionine from 0.99 to 9.2 mg/g with a mean content of 6.1 mg/g， isoleucine from 1.94 to 17.65 mg/g with a mean content of 8.98 mg/g， leucine from 7.79 to 75.66 mg/g with a mean content of 34.57 mg/g， tyrosine from 2.134 to 5.03 mg/g with a mean content of 3.56 mg/g， lysine from 3.82 to 8.21 mg/g with a mean content of 5.18 mg/g， histidine from 1.4 to 11.1 mg/g with a mean content of 4.49 mg/g， argnine from 2.07 to 6.78 mg/g with a mean content of 3.56 mg/g， proline from 3.89 to 44.62 mg/g with a mean content of 17.94 mg/g， and tryptophane from 2.34 to 11.89 mg/g with a mean content of 8.7 mg/g. The ten millet cultivars were particularly rich in leucine， and the mean leucine content of the ten cultivars was 34.57 mg/g. The millet cultivars were also high in glycine， glutamic acid and cysteine. Millet was known to be limiting in the essential amino lysine. The mean lysine content of the ten cultivars was only 5.18 mg/g. Other amino acids such as tyrosine， histidine and arginine were also very low in the ten millet cultivars.
　　Fig. 3 showed very differences in the amino acid content between the ten cultivars. Interestingly thing was there was a intuitively linear relationship. It is interesting that there was an intuitively linear relationship among the amino acid content of different varieties， that is， when the amino acid content of a variety is high， most of the other amino acids in this variety are also rich. The A2 specie contained greater amounts of most of the amino acids reported with the exception of isoleucine， tryptophane and histidine. While the Teff specie from Africa contained less amounts of most of the amino acids with the exception of lysine， proline and arginine. The two hybrid millet cultivars also had more amounts of most the amino acids. 　　Relationship between seed protein content and amino acid level
　　Generally， amino acid composition varied according to protein level. Relationship between seed amino acid composition and seed protein content in Table 1 showed correlation coefficients （r） and degrees of significance of the relationship between concentrations of amino acids and seed protein content. Data from three replications were used in calculating correlation coefficients. Very significant positive correlation between seed protein content and amino acid concentrations were found in isoleucine （r=0.003） and phenylalanine （r=0.009）. The correlation was significant for glycine （r=0.015）， proline （r=0.012）， serine （r=0.014）， methionine （r=0.020）， threonine （r=0.023）， glutamic acid （r=0.018） and aspartic acid （r=0.022）， but were not significant （P>0.05） for isoleucine， tryptophane， cystine， tyrosine， histidine， lysine and arginine. For all amino acid concentrations， no negative correlation with seed protein content was found.
　　Discussion
　　Breeding programs for S. italica （foxtail millet） have lagged behind those of the major crops. However， in the past few years the importance of millet and other small grain millets in the agriculture of semi??arid regions of the world has been underscored[9] . Millet grains can be cooked in the same manner as rice and has many food applications such as breads， cakes， rolls， noodles， etc. It is also an important staple food in India and Northern China[10] .Foxtail millets are valued for the high degree of tolerance to soil moisture stress， good nutritional value， substantial palatable straw as fodder. They also have good seed storage quality and are used as famine food. Although breeding for high yield is the primary objective of millet breeders， the improvement of the nutritional quality of these cereal crops should also be an important consideration given the continued world??wide prevalence of malnutrition. The main purpose of this study was to assess the qualitative and quantitative aspects of the nutrient content of millet seeds.
　　Foxtail millet is a cereal grain with a moderate protein content （about 11%-12%）. Protein variability quite large and low （about 9%） as well as very high （over 20%） has been recorded by Moss¨?[11] . Other scholars have also found substantial differences in the nutrient composition of varieties of finger millet[12-13] ， while there has been little information to date concerning the nutrient content of the foxtail species. This study indicates that there are wide variations of protein content from 8.61% to 15.54% of different millet cultivars. In this paper， low protein contents is 8.61% （Teff） from Africa and high protein content is 15.54% （A2） from a male sterile line in China. The variability is over 180%. Fig. 1 also shows different sources of millet have different protein contents. In general， most millet has medium protein contents in the range of 10.94%-11.49%， and the hybrid millet and their mother parent have the highest protein content than others. Factors including location， level of nitrogen （N） application， variety and year of harvesting and genotype have been found to influence content of millet[14] . Despite the fact that many different parameters influence the final content of protein in plant seeds， our results show that genotype having a greater influence on protein content. However， the environmental components such as location and N application in the variability of protein content need further study. 　　Our results are in general agreement with previous report that millet is generally higher in the essential amino acids such as lysine， methionine， and leucine[15] . Several authors have noted differences in amino acid composition within and among millet cultivars[16] . In most of these millet varieties， the contents of lysine， leucine and tyrosine are even higher than that in rice and maize. The analysis of most millet seeds such as A2， Zhangzagu 5 and Zhangzagu 9 indicates a more desirable amino acid composition than rice and maize. However， all of these millet cultivars are deficient in Lys， Arg and Tyr. As with most cereal foods， this limits their nutritional value.
　　In summary， the results reported here clearly demonstrate the availability of a different gene pool for plant breeders to select for the improvement of the nutritional value of foxtail millet. The two hybrid millet cultivars produced in Zhangjiakou Academic of Science have reasonable and well??balanced compositions. Protein and amino acid content of male sterile line A2 originated in China may be possible to increase the content of lysine and other essential amino acids in millet by selective cross??breeding of the cereal.
　　References
　　[1] MCDONOUGH CM， ROONEY LW. The millets Handbook of Cereal Science and Technology[M]. In： Kulp， K.， Ponte， Jr.， J.G. （Eds.）， Marcel Dekker， Inc.， New York， 2000.
　　[2] EDWARD G. HAGOP， SUHAILA A. YOUNIS， HISHAM A. SHAHATHA. Proteins and amino acids of some local varieties of rice seeds （Oryza sativa L.）[J]. Plant Foods for Human Nutrition， 1990， 40 （4）： 309-315.
　　[3] YOICHI KITA， YUMI NAKAMOTO， MASAKAZU TAKAHASHI， et al. Manipulation of amino acid composition in soybean seeds by the combination of deregulated tryptophan biosynthesis and storage protein deficiency[J]. Plant Cell Rep， 2010， 29：87-95.
　　[4] MERTZ ET， BATES LS， NELSON OE. The mutant gene that changes protein composition and increases lysine content of maize endosperm[J]. Science， 1964， 145：279-280.
　　[5] NELSON OE. The second mutant gene affecting the amino acid patterns of maize endosperm[J]. proteins Science， 1965， 150：1469-1470.
　　[6] MUNCK L， KARLSON KE， HAGBERG A. A selection and characterization of a high protein high lysine variety from the world barley collection[J]. In： Barley Genetics??II （ed），Nilan R Washington State Univ. Press. Pullman. p.， 1971， 544.
　　[7] BANSAL HC. A new mutant induced in barley[J]. Curr Sci， 1970， 39：494-495. 　　[8] MOSSE?? J. BAUDET J. Crude protein content and amino acid composition of seeds： variability and correlations[J]. Qual Plant Plant Foods， Hum Nutr， 1983， 32： 225-245.
　　[9] YANG XS， WANG LL， ZHOU XR， et al. Determination of Protein， Fat， Starch， and Amino Acids in Foxtail Millet [Setaria italica （L.） Beauv.] by Fourier Transform Near??Infrared Reflectance Spectroscopy[J]. Food Sci. Biotechnol， 2013， 22（6）： 1495-1500.
　　[10] MOHAMED TK， ISSOUFOU A， FATMATA T， et al. Effect of enzymatic hydrolysis on the functional properties of foxtail millet （Setaria italica L.） proteins[J]. Int. J. Food Sci. Tech.， 2010， 45： 1175-1183.
　　[11] MOSS？ J， BAUDET J， HUET JC. Relationships between amino acid composition and nitrogen of foxtail （Italian） millet （Setaria italica） grain of different varieties [J]. Sci. Food Agric.， 1989， 46 （4）： 383-392.
　　[12] KUMAR KK， PARAMESWARAN PK. Characterisation of storage protein from selected varieties of foxtail millet [Setaria italica （L） Beauv][J]. Sci. Food Agr， 1998， 77： 535-542.
　　[13] MOHAMED TK， ZHU K， ISSOUFOU A， et al. Functionality， in vitro digestibility and physicochemical properties of two varieties of defatted foxtail millet protein concentrates[J]. Int. J.Mol. Sci， 2009， 10： 5224-5238.
　　[14] EJETAG M， HASSEN M， MERTZ ET. In vitro digestibility and amino acid composition of pearl millet （Pennisetum typhoides） and other cereals （pepsin digestibility/protein fractionation/protein quality）[J]. Proc. Natl. Acad. Sci. USA， 1987， 84：6016-6019.
　　[15] BARBEAUW E， HILU KW. Protein， calcium， iron， and amino acid content of selected wild and domesticated cultivars of finger millet[J]. Plant Foods for Human Nutrition， 1993， 43： 97-104.
　　[16] LIU JK， TANG X， ZHANG YZ， et al. Determination of the volatile composition in brown millet， milled millet and millet bran by gas chromatography/mass spectrometry[J]. Molecules， 2012， 17： 2271-2282.