CHEMICAL ^WD PHYSICAL ^'APIANCE IN COMPOSITION OP THIRTY-PIVE GENOTYPES OP PEARL MILLET
(PENNISETDl^I TYPHOIBJ^S )
by
CHIN-HUA CHEN, B.S. A THESIS IN POOD TECHNOLOGY Submitted to the Graduate Faculty of Texas Tech University in Partial Pulfillment of the Requirements for the Degree of MASTER OP SCIENCE Approved
Accented
December, 1978
P^-Oi^^l
AC OOVniiEDGEMENTS
I am indebted to the chairman of my committee. Dr. Ronald D. Galyean, for his direction of this thesis and to Dr. E. D. Maxson for his help and encouragement. Appreciation is expressed to the other members of the committee. Dr. Daniel R. Krieg, Professor Margarette L. Harden, and Dr. Milton Peeples, for their helpful criticism.
ii TABLE OP CONTENTS
ACKNOlrlLEDGEMENTS ii LIST OP TABLES iv LIST OP FIGURES v I. INTRODUCTION 1 II. LITERATURE REVIEW 3 Physical Properties 3 Chemical Properties 3 Use of Millet in Pood Preparation 9 III. MATERIALS AND METHODS 11 Samples • 11 Physical Analysis 11 Chemical Analysis. 11 Data Analysis l5 IV. RESULTS AND DISCUSSION 16 Physical Analysis. 16 Chemical Analysis. 18 V. SUMMARY AND CONCLUSION 36 LITERATURE CITED 38 APPENDICES k.3
iii LIST OF TABLES
Table 1. Statistical Evaluation for Chemical and Physical Properties in Thirty-Five Different Genotypes of Pearl Millet 17 2. Significant Relationship between Various Chemical and Physical Properties of Thirty- Five Pearl Millet Genotypes 19 3. Pearl Millet Genotypes Ranked for Optimum Production of Selected Chemical Characteristics 2^. i|.. Standard Sugars* Relative Retention Time by HPLC Determination and Rf Value by Thin Layer Chromatography 28 5. Sugar Content {% on Dry Weight Basis) of Thirteen Pearl Millet Genotypes 33 6. Sugar Distribution in Cereal Grains ifo on Dry Weight Basis) 3^1-
iv LIST OF FIGURES
Figure 1. Regression Line of Lipid with Ash in Thirty Five Genotypes of Pearl Millet 21 2. Regression Line of Starch with Protein in Thirty Five Genotypes of Pearl Millet 25 3. Separation of Sugar Extract from Millet Genotype li;3 by HPLC 29 1|.. HPLC Peak Area versus Glucose Concentration 32 CHAPTER I INTRODUCTION
Millet, a term for several genera of small seeded annual grasses, can tolerate infertile land, extreme heat, and low rainfall. It is an important cereal food for people in India, northern China, Africa, and southern Russia. Among the five common genera of millet, pearl millet (Pennisetum typhoides family), is most ijuportant as a food source, and is known as bajra, pencillaria, sajja, or cumbo in India, candle millet or dark millet in Europe, bubrush or Dukhn in Africa, and cattail millet or Mands forage plant in the U.S.A. The yield of millet is usually low compared to other cereal grains, due to being grown iinder poor environmental conditions (Freeman and Bocan, 1973). According to the PAO statistics year book of 1975* 71 million hectares of millet were planted and k.7 million metric tons of the grain were produced in the world. In the developing world, pearl millet probably constituted 70^ of this total millet acreage, Because of the development of hybrids and dwarf types, pearl millet may also become a more important crop for the United States (Burton and Proston, 1966). Millet, a potential crop for wet milling, produces starch similar to corn and sorghum in most respects (Freeman and Bocan, 1973)- Because millet grain does not contain 1 2 gluten, it cannot be utilized as the sole cereal ingredient for preparing white and "leavened" bread. However, other types of bread, cookies, and biscuits have been made exper imentally (Casey and Lorenz, 1977). Unleavened chapaties are made of ground millet flour. Dry parched grain is also eaten as a porridge (Awadalla, 197l|; De-Ruiter, 1972). Several kinds of fermented beverages are made from millet in eastern Europe and Africa (Schery, I963). Biorton, et al. (1972) states that 80 to 90^ of the calories for millions of poorer people in the world are provided by millet grain. In nutrient value, pearl millet is in some respects better than wheat, rice, sorghum, and maize. Millet is therefore a potential source of additional food for our world, in which shortages of food are becoming more common. To best utilize this potential grain source, it is necessary to study and determine in detail the chemical and physical properties of pearl millet. An example of such chemical properties is the free sugar content of the grain. Sugar influences the develop ment of gluten in bread dough and the color and flavor of baked products. Sugar also encourages development of dental caries and possibly cardiovascular disease (Palmer and
Brandes, 197lf). The objective of this study is to provide a direction for selecting suitable genotypes of pearl millet as a food source. Chemical and physical properties of 35 varieties of pearl millet, provided by Dr. Glenn V. Burton, were compared. CHAPTER II LITERATURE REVIEW
Results reported on the physical and chemical proper ties of pearl millet are varied. It is likely that much of the variation is due to variety and to different conditions in growing, harvesting, storage, and methods of analysis.
Physical Properties Pearl millet kernels are not covered by lemma or palea, and the caryopsis can be threshed free. Obvoid pearl millet kernels are larger than the other millets. The embryo has a reddish tinge with a greyish-yellow or green color (Leonard and Martin, 1963). Pearl millet kernels are a little less than 10^ of the size of corn kernels, and about one third the size of a sorghum kernel (Freeman and Bocan, 1973; Badi et al., 1976). In a study of i|.5 pearl millet samples of 15 lines and hybrids grown under 3 different soil moisture regions, thousand kernel weight ranged from 2.91 to 8.15 g* and bushel weight of the grain ranged from 25.1 to 29.1|. Kg/bu, (Maxson et al., 1976). As with corn and sorghum grains, pearl millet endo sperm includes translucent (hard) and opaque (soft) portions, and the soft part of millet endosperm contains no protein granules (Badi et al., 1976). k Chemical Properties The reported range of protein in pearl millet, 8 to 23%9 is superior to that of maize, sorghum, and wheat (Sawhney and Naik, 1969; vSwaminathan et al., 1971; Burton et al., 1972). Wheat flour protein can be classified according to solubility characteristics as albumin, glo bulin, proteose, prolamin (gliadin), and glutelin (glutenin) (Osborne, I907). Prolamin and glutelin are insoluble proteins (storage protein), and are packed together in the mature grain (Kent, 1975). In pearl millet, the major pro tein fractions are prolamin and glutelin (Swaminathan et al., 1971). Prolamin concentrated in the endosperm of millet, maize, and sorghum is extremely deficient in lysine. However, prolamin of pearl millet contains a high concentration tryptophan (Sawhney and Naik, 1969; Swamina than et al., 1971; Concon, 1966). The first limiting amino acid in millet is lysine (Biirton et al., 1972; Push- parama et al., 1972). However, pearl millet is higher in lysine and tryptophan than sorghiam, and higher than wheat in tryptophan. The lipid content of pearl millet ranges from 3.0 to 6.5^, with an average of about I|..5^ (Freeman and Bocan, 1973; Goswami et al., 1969; Kent, 1975; Pruthi and Bhatia, I97O; Jellum and Powell, 1971). In contrast, the lipid content in wheat, rye, barley, and rice is 1 to 2%; 2 to 3^ in sorghum; and 5 to 10^ in oat grit (Kent, 1975). 5 Non-polar lipids (triglycerides, free fatty acids, free sterols, and partial glycerides), the predominant lipids of cereal grains, can be extracted by solvents such as petroleum ether or hexane (Pruthi and Bhatia, 1970; Jellum and Powell, 1971). Polar lipids (lipids associated with endosperm of starch) can be extracted with polar solvents, such as chloroform and methanol. These lipids can also be classified as "free" lipids and "bound" lipids (Pruthi and Bhatia, 1970). Free lipids (petroleum ether extracted lipid) are predominantly non-polar lipids. Bound lipids, similar to polar lipids, remain in the residue after free lipids extraction. Pearl millet contains about 5.0^ free lipid, and about 0.^% bound lipid (Pruthi and Bhatia, 1970). The apparent amount and composition of fatty acids in pearl millet are influenced by lipid extracting procedures (Jellum and Powell, 1971; Rogols et al., 1969). The fatty acid profiles of millet resemble those of other vegetable oils, such as corn, sorghum, soybean, and sunflower seed oils (Salomatina and Olifson, 1969). In pearl millet, ash values range from 1.55 to 3-90^ (Burton et al., 1972; Carr, 1961; Goswami et al., 1969), and this grain has greater ash content than wheat, corn, and sorghum. Although calcium and phosphorus content is similar to other cereals (Burton et al., 1972), the iron content of millet is relatively higher (Goswami et al., 6 1970). The ash content of pearl millet can be improved by selection of genotypes (Goswami et al., 1970). Pearl millet contains about 59 to 80^ starch (Burton et al., 1972; Freeman and Bocan, 1973), which is less than wheat, corn, and sorghum. The diameters of millet starch granules range from about 8 to 12 lom and range in shape from spherical to polygonal (Badi et al., 1976). The starch granule size is slightly smaller than that of corn and sorghum (Freeman and Bocan, 1973)- Millet starch comprises 12.0 to 19.55^ amylose and 80.5 to 88.0^ amylopectin (Badi et al., 1976). Except for a slightly lower tendency of cooked pearl millet starch to retrograde or thicken upon cooling, it is otherwise similar to corn and sorghum starch in most characteristics. In thickener applications, there fore, the lower cold-paste viscosity of pearl millet would be considered undesirable (Freeman and Bocan, 1973). Based on the carbon number, monosaccharides can be classified as triose (3 carbon), tetrose {l\. carbon), pen tose (5 carbon), and hexose (6 carbon). Based on the alde hyde or ketone group, monosaccharides can be classified as aldose or ketose. Oligosaccharides usually contain 2 to 9 monosaccharides upon hydrolysis, and are assembled from monosaccharides through glycosidic linkage (Paul and Palmer, 1972). Four out of eight possible D & L- aldopentoses occur naturally, namely D-xylose, D-ribose, and D- and L-arabinose. They are mostly present as constituents of polysaccharides 7 such as hemicellulose, gum, and pectin- Glucose is the only aldohexose naturally present in abundance. Fructose is the only ketohexose present in the free state to a great extent. The most commonly occurring oligosaccharides contain only three monosaccharides, namely D-fructose, D-glucose, and D- galactose. Glucose and fructose occur mainly as sucrose in plants. Rare food sugars that do not commonly occur to any great extent in food include rhamnose, maltose, and raffinose (Shallenberger and Birch, 1975). The total free sugar in millet is 2.01 to 2.70^ of the grain kernel by weight (Uprety and Austin, 197^)- Singh and Popli (1973) reported that the water soluble carbohy drates in high yielding varieties of bajra millet were fructose, glucose, sucrose, maltose, and two large mole cular weight oligosaccharides. The total free sugar contents in barley, corn, rye, and sorghum grain were 2.0, 2.8, 2.7, and 1.2^ respec tively (LaBerger et al., 1973; Uprety and Austin, 1973; Vaisey and Unrau, 1961].; Hirata and Watson, 1967). Glucose, fructose, sucrose, maltose, and raffinose were the free sugars in barley, corn, oat, rye, sorghum, and wheat flour (LaBerger et al., 1973; Cerning-Beroard, 1975; Minakova et al., 1971].; Vaisey and Unrau, 1961].; Hirata and Watson, 1967; Koch et al., 1951)• The free sugars in triticale bran were sucrose (1.78^), raffinose (1.22^), nystose (0.28^), 8 fructosylraffinose (0.35^), and glucofructans (0.98^) (Saunders et al., 1975)« The embryo and endosperm of rice contained sucrose and traces of glucose, and fructose and raffinose as available free sugars (Komogawa and Nikuni, 1959). Since polysaccharides and protein are not usually soluble in 80^ ethanol, the extraction of sugar from food can be performed by using 80^ ethanol as the solvent (Williams and Bevenue, 1951; Mason and Slover, 1971; Palmer and Braiides, 1971*.; Cerning-Beroard, 1975; Conrad and Palmer, 1976). Sugar has also been extracted by refluxing with 70^ ethanol (Koch et al., 1951; Saunders et al., 1975)* The ethanol extraction is clarified with neutral lead acetate (Williams and Bevenue, 1951). After extraction, carbohy drates are then identified qualitatively by paper chromato graphy (Koch et al., 1951; Williams and Bevenue, 1951; Saunders and Walker, 1969), thin layer chromatography (Saun ders and Walker, 1969), liquid chromatography (Linden and Lawhead, 1975), and the anthrone test (Cerning-Beroard,
1975). Quantitative identification of sugars has been accom plished by the copper reduction method (Munson and Walker, 1906), the phenol sulfuric acid method (Dubois et al., 1956; Saunders et al., 1975), paper chromatography (Koch et al., 1951), thin layer chromatography (Saunders and Walker, 1969), gas liquid chromatography (Saunders and 9 vralker, 1969), liquid chromatography (Linden and Lawhead, 1975), and an ion exchange resin method (Hobbs and Lawrence, 1972). More recently sugars have been analyzed by high pressure liquid chromatography (Palmer and Brandes, 1971;; Palmer, 1975; Conrad and Palmer, 1976).
Use of Millet in Food Preparation Kim and Reuter (1968) found that a standard bread dough from non-wheat flour was semifluid, neither elastic nor coherent, and resembled cake batter. One hundred per cent millet meal does not produce good leavened bread. If more than 10 to 30^ of non-wheat flour is mixed in bread dough, improvers such as esters of mono- and di-glycerides, fat, and sugar must be added to improve the dough. Finney et al. (1950), using the micro cookie test, found that both millet and sorgh\im flour produced cookies which were tough and gritty with no top-crack. VJhen 0.6^ unrefined soy lecithin was added to millet flour, cookies with spread characteristics equal to those of soft wheat flour were produced (Badi et al., 1976). Increasing the pH of the cookie dough in millet flour reduced the gritti- ness of the cookies. Adding millet flour made the cookie color darker. Mixing 10^ millet flour with wheat flour gave bis cuits with good sensory qualities in Niger (De-Ruiter, 1972). Gum candy which was tender and transparent was made 10 from modified millet starch (Modi and Kulkarni, 1976). In India chapti is made from millet (Austin et al., 1971). In eastern Europe, a porridge is made of millet (Schery, 1963). European-type beers can be made from millet (Eschen- bach and Jauss, 1976), and in eastern Europe and Africa fermented beverages are made from the grain (Schery, 1963). Pal et al. (1976) reported good malt could be made from millet except that bitter flavors sometimes developed in the product. CHAPTER III MATERIALS AND METHODS
Samples Thirty-five different genotypes of pearl millet, provided by Dr. Glenn W. Burton, were grown in 1976 at the Texas Tech experimental farm at New Deal, Texas, under the direction of Dr. D. R. Krieg of the University Plant and Soil Science Department. Normal dry land farming conditions were used. Sample heads were selected for maturity, threshed by hand, and cleaned with a clipper cleaner and a Satake rice mill. All samples were ground to pass through a 0.01 cm screen with a hammermill. They were stored in sealed glass bottles at room temperature until analyzed.
Physical Analysis Bushel weight was detei*mined by the standard macro method (American Association of Cereal Chemists, 1969)- Thousand kernel weight was determined by weighing one thousand kernels of the millet grains.
Chemical Analysis Moisture was determined by the AACC modified vacuum- oven method; ash was determined by the AACC rapid method; crude fat was determined by Goldfisch extraction method;
11 12 crude protein was determined by micro Kjeldahl method (AACC, 1969). starch was estimated by the Norris method (Appendix I, Norris, 1971). Total sugar was determined by the anthrone method outlined in Appendix II (Sunderwirth et al., 196i|.).
Free sugars in thirteen randomly selected genotypes of pearl millet were identified by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). Although there is no established method for extracting free sugars from millet grain, extraction methods for both total sugar and free sugar are similar, except for clarification and filtration modifications. The method used in this thesis was similar to those of VJilliams and Bevenue (1951) and Palmer and Brandes (197I].). ^liole kernel millet flour (lOg) was extracted with 100 ml ethanol by shaking 1]. hours at room temperature (a small quantity of CaCO^ was added to neutralize acidity). The mixture was centrifuged and 10 ml of 10^ lead acetate was added to precipitate any colloid like materials such as protein. Excess lead ion was then precipitated by adding 10 ml of 10^ potassium oxalate. The solution was centrifuged and the top, clear solution was collected and evaporated by Buechler rotary-vacuum evapor ator using a water bath temperatiire of 50° C. The concen trated fraction was rinsed out repeatedly with small aliquots of distilled water, combined, and made up to 10 ml. The concentrated sugar extract was filtered through Millipore 13 0.22 um filter paper (Millipore Corporation, Bedford, Massachusetts) and analyzed by HPLC and TLC. For the HPLC procedure, equipment was as follows: a Tracer 995 pump (Tracer Instruments, 6500 Tracer Lane, Austin, Texas), Tracer 910 differential refractometer detec tor (RI), with a sensitivity of 10"^ refractive index iinits and attenuation 8-16, a Perkin-Elmer recorder (The Perkin- Elmer Corporation, Main Avenue, Norwalk, Connecticut), chart speed 20 cm/hr; column (u BONDAPAK/carbohydrate, Waters Associates, Maple Street, Milford, Massachusetts), 30 cm x i|. mm ID stainless steel, with bonded phase packing, particle size of 10 microns; injection valve, Rheodyne model 7-10 (Rheodyne, 2809 Tenth Street, Berkeley, California), with model 7-11 sample loop (20 ml); eluent, water-acetonitrile mixture (20:80); flow rate, 1.5-2.0 ml/min at pressures up to 2900 psi. Sugars eluted from the HPLC column were further identi fied by thin layer chromatography. Samples were concentrated, then spotted on silica gel plates. The plates were 20 x 20 cm, precoated with a 250 micron layer of silica gel from the Fisher Company (Fisher Scientific Company, 711 Porlus Avenue, Pittsbiirgh, Pennsylvania). Several solvents were tested, but n-propanol-NH^OH-water (6:2:1) was found most suitable. After separation, sugars were detected by spraying the plates with concentrated sulfuric acid and heating at 100 C Ik for 10 minutes. The Rf value was calculated for each spot by the equation: Distance of sugar migration (cm) Rf = Distance of solvent migration (cm)
Standard solutions were made of 1% and 0.5^ of rham nose, arabinose, fructose, glucose, sucrose, raffinose, maltose, galactose, and melibiose in distilled water. These were selected because they were known to occur in other cereals. After injecting into the HPLC, the unknown sugars were identified by comparing their retention times with those for standard sugars. The spots on thin layer plates were identified by comparing their Rf values with those for standard sugars. The area-to-weight ratios of the chromatographic peaks were used to determine the amounts of sugar separated by HPLC. A compensating polar planimeter was used to measure the area under each peak. By comparing each individual standard sugar peak area with the area for standard glucose, a correction factor was obtained: Ai correction factor K = Ag
Where: Ai = the area (mm^) of standard individual sugar; Ag = the area (mm^) of standard glucose, at same concentration. 15 The formula used to calculate the percentage of detec table free sugar in each sample was:
percent sugar = Ai' x (g) x 10 x 100 Ag X Tw X K
Where: Ai» = the unknown individual sugar area (ram^) of the millet sample eluted from HPLC; (g) = standard glucose concentration (g/ml) injected; Tw = the total weight (g) of pearl millet sample extracted; K = the correction factor.
Data Analysis Analysis of variance was used to test the difference in the bushel weight, lipid, protein, starch, total sugar, and each free sugar among the samples. The relationships between protein and starch, protein and fat, protein and sugar, ash and lipid, or starch and fat were analyzed by the correlation coefficient statistical method of analysis (Snedecor and Cochran, 1967). All data were analyzed for statistical significance by the Statistical Analysis System Program on the Texas Tech University computer. f
CHAPTER IV RESULTS AND DISCUSSION
Data for the physical and chemical characteristics of thirty-five varieties of pearl millet may be found in Appendix III. Information associated with the mono- and oligosaccharides in thirteen different genotypes of pearl millet is shown in Table 5*
Physical Analysis The average thousand kernel weight {TVfK) for the samples in this analysis was 6.1 g. The lowest TWK
(Genotype 191) was 3*6 g and the highest TTA.'K was 8.6 g per thousand kernels (Genotype 125). The range for this evalua tion was 5*0 g with a standard deviation of 1.2 g (Table l). The value for average TWK of 6.1 g is 2.8 g lower than that found by others (Freeman and Bocan, 1973). This difference was probably due to analysis of different millet varieties in the two studies. The average bushel weight of the samples was 26.7 Kg (Table l), compared to a literature value of 27.3 Kg (Maxson et al., 1976). This result is most probably due to differing factors in cultivation and environmental condi tions. The range for the analysis was 5.3 Kg, with a stan dard deviation of 1.1]. Kg (Table l). One way analysis of variance was used to test the hypothesis that there was no 16 17
TABLE 1. Statistical Evaluation for Chemical and Physical Properties in Thirty-Five Different Genotypes of Pearl Millet
Standard Coefficient Variable Mean Deviation Ranp^e of Variation Bushel Weight (Kg) 26.70 l.kk 5.27 5.39 Thousand Kernel Weight (g) 6.09 1.22 5.00 20.10 Moisture (g/lOOg) 7.23 0.73 3.i|.7 10.12 Ash (g/lOOg) 1.95 0.27 1.00 13.79 Crude Protein (g/lOOg) 17.i].2 1.07 5.20 6.12 Lipid (g/lOOg) 6.15 0.98 3.20 15.92 Total Sugar (g/lOOg) 3.01 0.1].3 2.00 11].. 20 Starch (g/lOOg) 61].. 97 3.72 16.30 5.73 18 significant difference among the thirty-five different samples and the results are shown in Appendix IV. The F test at the 0.01 significance level indicated that there was a significant difference among the samples. The high est value obtained here was 28.6 Kg (Genotype 168), the lowest value was 23.3 Kg (Genotype l65. Appendix III).
Chemical Analysis The moisture in these samples averaged 7*2^, with a standard deviation of 0.7^ (Table l). Moisture content can be affected by drying during handling and by the moisture determination method. The average ash content for the 35 millet genotypes was 2.0^. The highest ash content was 2.5^ (Genotype 122), and the lowest was 1.5^ (Genotype I36 and 197), with a range of 1.0^ and standard deviation of 0.3^ (Table l). These values are similar to values of 1.58 to 2.^.6% reported by Goswami et al. (1970) and 2.2^ by Burton et al. (1972). According to Watt and Merrill (1963), the percentage of ash in wheat, corn, brown rice, and sorghum was determined to be 1.7, 1.2, 1.2, and 1.7^, respectively. These values are lower than the ash percentage in this report. Regression analysis (Table 2) indicated a significant linear increase of ash with increasing amount of lipid (r = 0.31;.). The high mineral content in millet whole kernel flour may catalyze the rancidity of lipid. It is suggested that this result may 19
OJ iH • CM i + C3 C^ G <\J •oH •H . o GO © H •H CO 43 CO O O 00 %i 1 + lb Pu ' ,a ® O 00 K-H 00 < 43 -d- a tJ (d . vO 05 ® ?s OJ c^ +3 CF^ o . H OH iH r^ Q] 00 ® O (D iH 11 II •H a
10.0 oto o 7.5 rH w 5.0 TJ •H a •H 2.5
1.6 1.8 2.0 2.2 2.h
Ash (g/lOOg) Pig. 1. Regression line of lipid with ash in thirty-five genotypes of pearl millet. 22 of its amino acid profile and high protein content. Be cause of the low gluten content in millet, good, high-rising bread can not be made from this grain. However, quantities of millet are directly consumed in some coiintries (Schery, 1963). A one way analysis of variance was used to test the hypothesis that there was no significant difference in the protein content among 35 different pearl millet. The hypo thesis, however, was rejected at the 0.01 level of signifi cance (Appendix IV). Among the 35 pearl millet genotypes analyzed here. Genotype 191]-, 122, ll].2, and 195 had the highest protein and lowest starch and lipid contents (Table 3). The average lipid content of the 35 millet genotypes was 6.2^ (Table 1). The range was 3*2^ with a standard deviation of 1.0^. Literature values for lipid in pearl millet were 1]..5^ (Burton et al., 1972), 5.0^ (Pruthi and Bhatia, 1970), both of which were lower than the average value in this study. The lipid contents of winter wheat (1.8%), field corn (3.9^), brown rice (1.9^), and sorghum (3.3^) (Watt and Merrill, 1963), are all lower than lipid content in millet. This finding agrees with Burton et al. (1972). One way analysis of variance was used to test the hypothesis that the difference of lipid content among 35 samples was not significant. The P test at the 0.01 level of significance indicated that there was a significant difference among the samples (Appendix IV). Pearl millet genotypes containing the highest lipid content with minimum 23 protein and starch content were I76, 131, 18I, and 190 (Table 3). Fatty acid composition in pearl millet is similar to other vegetable oils (Salomatina and Olifson, 1969). In this respect, pearl millet is a good source for extraction of polyunsattirated vegetable oils, if efficient extraction methods and equipment can be developed. Average starch content in the 35 millet genotypes was 65.0^ (Table l). The highest starch content was 71.1*.^ (Grenotype 165), and the lowest was 55*1^ (Genotype 122). The range was 16.3^ and the standard deviation was 3.7^ (Table 1). These results are similar to that of Freeman and Bocan (1973). They reported that Tiflate millet con tains 58.8^ and Tift 23 DB millet contains 61]..6^ starch; as compared to 72.0^ and 73-8^ for commercial corn and sorghum, respectively. Millet thus contains less starch than wheat, corn, and sorghum. A one way analysis of variance was used to test the hypothesis that the differ ence in starch content among 35 samples was not significant. The hypothesis was rejected at the 0.01 level of signifi cance (Appendix IV). There was a negative linear relation ship (r = -0.1].2) between starch and protein, as shown in Figure 2 and in Table 2- This indicates that higher starch content is accompanied by relatively lower protein content in pearl millet. Poor starch-protein separation makes starch yields from pearl millets lower than those usually 24
TABLE 3. Pearl Millet Genotypes Ranked for Optimum Production of Selected Chemical Characteristics
Rank abode
1 G 165 G 176 G ll].6 G 191]. G l6l
2 G 167 G 131 G 197 G 122 G 172
3 G 166 G 181 G 123 G ll].2 G 179
I|. G 169 G 190 G 150 G 195 G l59
^Maximum crude protein and starch content, minimum lipid content. ^Maximum lipid content, minimxam crude protein and starch content. ^Maximum starch, minimum lipid and protein content. ^Maximum crude protein content, minimum starch and lipid content. ^Maximum crude protein, lipid, and starch content. 25
Y = 102.1].8 - 2.12 X • . r = -0.i].2 70 0 •*- • to % o 65 > rH ^^—*^ • \ bO 60 • ^^ 1 1
Cd » 43 SS
Gn {.— / 1 - -J 1
16 17 18 19 20
Protein (g/lOOg) Pig. 2. Regression line of starch with protein in thirty five genotypes of pearl millet. 26 obtained with corn or sorghiam using the same procedures (Freeman and Bocan, 1973). Carefully choosing the low protein-high starch genotype and adjusting the starch separation equipment in a modern wet-milling plant might overcome the above problem. Genotype ll].6, 197, 123, and 150 have the maximiam starch, and minimum lipid and protein content among the 35 pearl millet samples. There are con flicting reports on the retrogradation properties of millet starch during cooling (Badi et al., 1976; Freeman and Bocan, 1973). These are the properties which influence starch applications as thickeners. Further research on pearl millet starch properties is therefore recommended.
The total free carbohydrate content of tested millet genotypes averaged 3*0^, with a standard deviation of 0.1].^ and a range of 2.0^ (Table l). The highest value was 1]..3^ and the lowest value was 2.li% (Appendix III). These results were higher than those reported by Uprety and Austin (1972), who found values for total sugars in pearl millet of 2.01 to 2.70^. Pearl millet thus contains a similar amount of total sugar (2.21 to 3.1|-7^) as corn (Uprety and Austin, 1973), and higher contents than sorghum (2.8^) (Hirata and Watson, 1967). One way analysis of variance was used to test the hypothesis that there was no significant differ ence of total sugar content among 35 millet samples. As the hypothesis was rejected at the 0.05 level of signifi cance, there was a significant difference of total free 27 sugars among the 35 samples (Appendix IV). Statistical analysis indicated that total free sugars and protein were negatively correlated (r = -0.29, Table 2). Nonenzymatic browning problems may thus occur if high protein pearl millet genotypes were selected for baking processes. Pree sugars in 13 randomly selected pearl millet genotypes were analyzed by HPLC and TLC. As free sugars were extracted under acid-free conditions, their content and concentrations were not increased by the acid hydrolysis of starch. The relative retention times and Rf values for the standard sugars arabinose, fructose, glucose, galactose, sucrose, melibiose, and raffinose are shown in Table I|.. HPLC sugar peaks were identified by comparison to retention times of standard sugars eluted xmder the same conditions. TLC spots were identified by comparison with known sugar Rf values. A typical HPLC separation of free sugars ex tracted from pearl millet is shown in Figure 3. Glucose, fructose, sucrose, and raffinose were separated and identi fied in all pearl millet genotypes examined. Some samples contained arabinose and galactose. Thin layer chromato graphic results confirmed those obtained by HPLC. Other reports (Shallenberger and Birch, 1975; Hirata and Watson, 1967; Cerning-Beroard, 1975) have shown that glucose, fruc tose, sucrose, and raffinose are contained in almost every other cereal grain. Shallenberger and Birth (1975), re ported that raffinose crystallizes easily. In the concen- 2Q
TABLE 1].. Standard Sugars' Relative Retention Time by HPLC Determination and Rf Value by Thin Layer Chromatography
Relative Retention Rf Value (TLC) Time (HPLC) vSugars a b c d
Arabinose 0.36 0.57 0.75 0.73 Fructose 0.29 0.55 0.88 0.82 Glucose 0.27 0.1].9 1.00 1.00
Galactose 0.21]. -- 1.06 1.10 Sucrose 0.22 0.25 1.59 1.95
Melibiose 0.10 0.15 2.50 --
Raffinose 0.08 0.13 3.i|.l --
^TLC: Solvent = n-propanol/NHhOH/HoO (6:2:1); silica gel plates, 20 x 20 cm, 250 microns silica gel. ^TLC (Hay et al., I963): Solvent = 1-butanol/acetic acid/water (2:1:1); silica gel plates. °HPLC: Solvent = acetonitrile/water (80:20); flow rate =1.5 cm/min; detector = RI; attenuation = 8x; glucose retention time about 8 min. ^HPLC (Conrad and Palmer, 1976): Solvent = acetoni trile/water (85:15); flow rate =2.0 ml/rain; detector = RI; attenuation = difference in relative retention time of about 0.10 yields useful separation; glucose retention time about 12 min. 29
0 10 15 20 25 30 35 Time (minutes)
Pig. 3. Separation of sugar extract from mill et Genotype ll].3 by HPLC: Solvent = water/acetonitrile (80:20); flow rate =1.5 cm/min; recorder atten- uation 8x temperature = 25^ C; peak identific ation, 1 = water front; 2,3,14. unknowns; 5 = fructose; 6 = glucose; 7 = galactose ; 8 = sucrose; 9,10,11 = unknowns; 12 = raffinose. 30 trated sugar extract of pearl millet in this study, raffinose was easily crystallized and dissolved with difficulty. The absence of maltose in the samples indicates a lack of amylase activity during the sugar extraction of pearl millet. Similar results were reported by Saunders et al. (1975) in their analysis of the sugars in triticale bran. Singh and Popli (1973), using water to extract sugar from pearl millet, found maltose, fructose, glucose, su crose, and two higher molecular weight oligosaccharides. Their use of water as solvent, without CaC03 to neutralize acidity, may have permitted acid hydrolysis of starch, resulting in the production of maltose. Galactose found in pearl millet sugar extract may be produced by enzymatic hydrolysis of raffinose. Since ara binose is a constituent of hemicellulose, it may occur in the free state in small amounts (Shallenberger and Birch, 1975). The millet flour analyzed in this study consisted of whole kernel pearl millet, and therefore the presence of arabinose is reasonable. The HPLC peaks eluted between sucrose and raffinose (PigTxre 3), were believed to be glucofructans, such as melibiose. These results are similar to the observations of Mason and Slover (1971)* Plant foods are primary sources of free sugars in the diet. Recent evidence shows that sugar encourages develop ment of dental caries and possibly cardiovascular disease. 31 It is thus very important to analyze the individual sugars in order to measure dietary intake. Sugars are the elementary constituents for the first stage of fermentation in baking products. Because of the importance of free sugars in nonenzymatic browning reac tions, yeast fermentation, nutritional value, and sweet flavor, sugar components were quantitated by HPLC. The linear response of glucose concentration versus recorder peak area is shown in Figure 1].. The correction factors for arabinose, fructose, sucrose, galactose, and raffinose were O.7I, 1.09, 1.10, 0.81]., and 1.19, respec tively. The distribution of individual sugars for the 13 millet samples is shown in Table 5* The results were similar to those for free sugar distribution in freeze-dried corn, rye flour, waxy and regular sorghum, and wheat floior, as shown in Table 6 (Cerning-Beroard, 1975; Vaisey and Unrau, 1961].; Hirata and Watson, 1967; Koch et al., 1951). The detectable sugar concentration by HPLC was not the same as the total sugar concentration obtained by the anthrone procedure (Table 5). Unknown sugar concentrations were therefore obtained by subtracting total known sugar contents from total sugar determined by the anthrone procedure. There were significant differences in carbohydrate content among the different genotypes. Appendix IV shows the analysis of variance for quantitated values of fructose, glucose, and sucrose, respectively. The average dry weight concentrations 32
0.15 o 0.10 ©
od © 0.05
0 0.08 0.12 0.16 0.20
Concentration (mg/ml) Fig. 1].. HPLC peak area versus glucose concen tration. Chart speed =10 cm/hr; range = 8x; ambient temperature; u bondapak column; solvent = acetonitrile/water (80:20). 33
cH ^ Cd cd vO O^lA-d-O -d-C^vOvO VOUNNO VO 00 43 to © ...... a, tH CO (\JC\J r^cnfOir^OJr^OJ (\I<\J CM ce\ 4J O © o (d vOOO"lAr^<^<^O^HO OO^OJ H 43 © jg CO (HrHCMrHHiHOiHrHrHOrHCM p iH •H © CQ f>> O © © © © iH G O O OC^^O^COvO"lAIV-rH OH. 43 Cd •H Od CO CdC\Jr^C\JHC\IHOcv>cd-=T G ©
o 0000000000000 "-^ '—• •—• £ cd^-^ o © © © > Pi r$ G G 43 cd cH r-i |H U G bO © CO CO CO cd © CO © > > > G P4 rH O 00 i O I 5o^ to^ I -d- H © H Cd i I X «M G fill I • I P 0 0 . U CO CO CO OS
O © £ 00 -d- .H © -d- O -d- rH © ^ 00 I vO •H 00 O o. 00 © ® © I I I © ed o rH o Q cd H CM in o u 43 EH EH xi •H
Od rf CyJ r-i O 00 O 00 © O O O r-i rH . o • . E: O O o o o fi ^ n Od Od 3 00 G r^Xi c^ c^ XA H o iH •H >*p to O 00 . • . Cd O od © o o o o
(d © © s \A r-i -d- O © t>>o o O -d- o . • • rH . O O CM o O cd C G Pi \A O vO O © O CM s CM OJ xn NO c^ •H N O . . . . 43 © o O iH o O^ H :3 © "O I 1 1 I © -d- C> rH £ r-i O O CM G o UN ai CO c^ 43 43 Cd 00 o od u I 00 © H PQ G Cd © I od od Od 43 •H to (0 © © 43 CO 03 cd ^ © © O •H ^1 o © •H 00 © 00 00 O © Cd o (0 00 © © © O O o > w w 00 o CO 00 00 G 00 o TJ CJ o Cd p, -H 43 o o o •H o ai Xi O o 1^ PQ to od G p o 43 iH to G fH G (A © £ CO IK EH k o 35 of glucose, fructose, and sucrose were 0.1].l, 0.25, and 0.50^. Significantly different (p < O.05) means of fructose, glucose, and sucrose for the tested millet varieties are shown in Table 5- Genotype 192 contained the lowest and Genotype 166 and ll].6 contained the highest fructose. Genotype I76 contained the lowest concentration and Genotype 1[].6 and 166 contained the highest glucose concentration. Genotype 166 contained the lowest concen tration and Genotype 157 contained the highest sucrose. Thus, Genotype li].6 contained the highest concentrations of both fructose and glucose. In applying pearl millet as a food processing source, this information may be of value in determining baking parameters and nutritional value. CHAPTER V SUMMARY AND CONCLUSION
The objective of this study was to provide basic information which may lead to selection of suitable geno types of pearl millet for use as a food soiirce. The study was also conducted to determine the amount of mono- and oligosaccharides in pearl millet. Chemical and physical properties of 35 different varieties of pearl millet were analyzed and compared. Analysis of the 35 pearl millet genotypes yielded the following average results: bushel weight, 26.7 Kg; thousand kernel weight, 6.1 g; moisture, 7.2^; ash, 2.0%; crude protein, 17.1].^; lipid, 6.2^; total sugar, 3.0^; starch, 65.0^, respectively (Table l). One way analysis of variance for the differences in bushel weight, protein, lipid, starch, total sugar, and free sugar content among the samples were done. There were significant differences in constituents among the samples (Appendix IV). Regres sion analysis showed that ash and lipid were positively correlated (r = 0.31].); crude protein and starch were nega tively correlated ( r = -O.ij.2); crude protein and sugars were negatively correlated (r = -0.29). There were no significant relationships between protein and fat, and starch and fat. Pree sugars determined in 13 pearl millet 36 37 samples were fructose, glucose, sucrose, and raffinose. Some pearl millet genotypes contained galactose or ara binose. Pearl millet is a good source of protein, lipid, ash, and starch. The reasons are as follows: 1. Pearl millet contains more protein than wheat, sorghum, rice, and corn, and its amino acid profile is better than most cereal grains in some respects. 2. Pearl millet contains fatty acids that are similar to other vege table oils, and lipid content is higher than other cereal grains. 3* In pearl millet, the ash and iron content is higher than sorghum, wheat, corn, and brown rice. 1].. Al though starch in pearl millet is lower than sorghum and corn, its properties are similar to the starch in those cereal grains. Thus, pearl millet is a good source for food, due to its high content of protein, lipid, and ash. If proper equipment is developed, starch and fat can be extracted from pearl millet for food uses. LITERATURE CITED
Adrian, J., and Jacquot, R. 1961].. Le sorgho et les mils en alimentation humaine et animals. Centre Recherches sur la Nutrition Du C.N.R.s. Bellevne (Seine-ET-Olse) Vigot Preres Editeurs, Paris, p. 189. American Association of Cereal Chemists. 1969. Approved methods of the AACC. The Association: St. Paul, Minnesota. Austin, A., Hanslas, V.K., Singh, H.D. and Ram, A. 1971. Protein and Chapti-making properties of some improved bajra (Pearl Millet) hybrids and varieties. Bulletin of Grain Technol. 9: 2l].7. Awadalla, M.Z. 1971].. Native Egyptian millet as supplement of wheat flour in bread. Z. Technology Study. Nutr. Rep. Inter. 6:69. Badi, S.M., Hoseney, R.C. and Casady, A.J. 1976. Pearl millet I. Characterization by SEM, amino acid analy sis, lipid composition, and prolamine solubility. Cereal Chem. 53: 1].78. Badi, S.M., Hoseney, R.C. and Finney, P.L. 1976. Pearl millet II. Partical characterization of starch and use of millet flour in bread making. Cereal Chem. 53: 718. Burton, G.W. and Proston, J.C. 1966. Inheritance and util ization of five dwarfs in pearl millet (Pennisetum typhoides) breeding. Crop Sci. 6: 69. Burton, G.W., Wallace, A.T. and Rachie, K.O. 1972. Chemi cal composition and nutritive value of pearl millet grain. Crop Sci. 12: l87. Carr, W. R. 1961. Observation on the nutritive value of traditionally grotind cereals in southern Rhodesia. Brit. J. Nutr. 15:339. Casey, P. and Lorenz, K. 1977* Millet functional and nutri tional properties. The Baker Digest. 2: k$. Cerning-Beroard, J. 1975. A note on sugar determination by anthrone method. Cereal Chem. 52: 857. 38 39 Concon, J.M. 1966. Corn Conference. Corn Refiners Asso ciation, Inc., V^ashington, U.S.A. p. 67. Conrad, E.G. and Palmer, J.K. 1976. Rapid analysis of carbohydrates by high-pressure liquid chromatography. Food Technol. 8: 81].. De-Ruiter, J. 1972. "Summary of an inquiry on work done in the field of composite flour". Institute for Cereal, Flour, and Bread, TNO, Wageningen. The Netherlands. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, P. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350. Eschenbach, R. and Jauss, H. 1976. Foundation of breweries in African developing countries. Brawwelt. 116: 1396. Finney, K.F., Morris, V.H. and Yamazaki, W.T. 1950. Micro versus macro cookie-baking procedures for evaluating the cookie quality of wheat varieties. Cereal Chem. 27: 1].2. Freeman, J.E. and Bocan, B.J. 1973. Pearl millet: a poten tial crop for wet milling. Cereal Sci. Today. 18: 69- Goswami, A.K., Sehgal, K.L. and Gupta, B.K. 1970. Chemical composition of Bajra grains. 1. African entries. Indian J. Nutr. and Dietet. 6: 287. Goswami, A.K., Sharma, K.P. and Gupta, B.K. 1969. Chemical composition of Bajra grains. 2. American entries. Indian J. Nutr. and Dietet. 6: 291. Hay, G.W., Lewis, B .A. and Smith, P. 1963- Thin layer chromatography in the study of carbohydrates. J. Chrom. 11: 479. Hirata, Y. and Watson, S.A. 1967. Cited in Starch Chem istry and Technology. 2. Industrial Aspects. Academic Press, New York. p. 29* Hobbs, J.S. and Lawrence, J.G. 1972. The separation and quantitation of carbohydrates on cation exchange resin colxamn having organic counterions. J. Chrom. 72: 3311. Jellum, M.D. and Powell, J.B. 1971. Patty acid composition of oil from pearl millet seed. Agron. J. 63: 29. Kent, N.L. 1975. Technology of cereals with special refer ence to wheat (2nd). Pergamon Press, p. 1].3. Kim, J.C. and Reuter, O.D. 1968. Bread from none wheat flours. Pood Technol. 22: 867. Koch, R.B., Geddes, W. P. and Smith, P. 1951. The carbohy drates of gramineae. I. The sugars of the flour of wheat (Triticum vulgare). Cereal Chem. 28: l].2lj.. Komogawa, A. and Nikuni, Z. 1959. Changes in sugar content during the germination of rice seed. Appl. of Ion- Exchange Chromatography. Nippon, Nogikagaku, Kaishi. p. 33. La-Berger, D.E., MacGregor, A.W. and Meredith, W.O.S. 1973. Changes in the free sugar content of barley kernels during maturation. J. of the Institute of Brewing. 79: 1].71. Leonard, W.H. and Martin, J.H. 1963* Cereal Crop., Mac- millan Co., New York. p. 605. Linden, J.C. and Lawhead, C.L. 1975* Liquid chromatography of saccharides. J. Chrom. 105: 125. Mason, B.S. and Slover, H.T. 1971. A gas chromatographic method for the determination of sugars in food. J. Agr. Pood Chem. 19: 551. Maxson, E.D., Krieg, D.R. and Hutcheson, R. 1976. Milling and physical properties of pearl millet grown under varying moistiipe stress conditions. Proc. Amer. Assoc, of Amer. Chem., New Orleans, Oct. Minakova, Yu.A., Popov, M.P. and Starodubtseva, A.I. 1971].• Izvestiya vysshikh Uchebnykh Zavedenii. Pishchevaya Tekhnologiya. 1: 52. (U.S.S.R.) Modi, J.D. and Kulkarni, P.R. 1976. Use of modified ragi starch in food preparation. J. of Food Sci. and Technol., India. 13: 1].6. Munson, L.S. and Walker, P.H. 1906. The unification of reducing sugar methods. J. Amer. Chem. Soc. 28: 663. Norris, J.R. 1971. Chemical physical and histological characteristics of sorghum grain as related to wet milling properties. Ph.D. Dissertation. Texas A & M University. College Station, Texas. Osborne, T.B. 1907. The proteins of the wheat kernel. Carnegie Institute, Washington, Publish No. 81].. Pal, A., Wagle, O.S. and Sheorain, V.S. 1976. Some enzyma tic studies on bajra (Pennisetum typhoides) and barley (Hordum vulgare) during malting properties. J. of Pood Sci. and Technol., India. 13: 75. Palmer, J.K. 1975. A versatile system for sugar analysis via liquid chromatography. Anal. Letters. 8: 215. Palmer, J.K. and Brandes, W.B. 1971;. Determination of sucrose, glucose, and fructose by liquid chromato graphy. J. Agr. Pood Chem. 22: 709. Paul, P.O. and Palmer, H.H. 1972. Pood theory and appli cation. John Willey & Sons, Inc. p. 36. Pruthi, T.D. and Bhatia, I.S. 1970. Liquid in cereals. 1. Pennisetum typhoides. J. Sci. Pood Agr. 21: 1].19. Pushpamma, S., Parrish, D.B. and Deyce, C.W. 1972. Im proving protein quality of millet, sorghum, and maize diets by supplementation. Nutr. Rep. Inter. 5: 93. Rogols, S., Green, J.E. and Hilt, M.A. 1969. Starch- complexed lipid: Differences in extraction with various solvents. Cereal Chem. i].6: I8I. Salomatina, L.O. and Olifson, L.E. 1969. Chemical composi tion and physical properties of millet oil. Mas- lozhirovaya Promyshlennost. 35: 9. (U.S.S.R.) Saiinders, R.M., Betschart, A.A. and Lorenz, K. 1975- The sugars of triticale bran. Cereal Chem. 52: i\.72. Saunders, R.M. and Walker, H.G. 1969. The sugars of wheat bran. Cereal Chem. lj.6: 85- Sawhney, S.K. and Naik, M.S. 1969- Amino acid composition of protein fraction of pearl millet and the effect of N fertilization on its protein. Indian J. of Genetics and Plant Breeding. 29: 395. Schery, R.W. 1963- Plants for man. Prentice-Hall, Inc., Englewood Cliffs, New Jersey. Shallenberger, R.S. and Birch, G.G. 1975. Sugar chemistry. Westpoint, Connecticut, The Av. Publish Company, Inc. p. 1].6. Singh, R. and Popli, S. 1973* Amylose content and amylo- lytic studies on high yielding varieties of bajra (Pennisetum typhoides). J. of Pood Sci. and Technol., India. 10: 31. Snedecor, G.W. and Cochran, W.G. 1967. Statistical method: Snedecor and Cochran. The Iowa State University Press. Ames, Iowa, U.S.A. Sunderwirth, S.G., Olson, G.G. and Johnson, G. 1961].. Paper chromatography-Anthrone determination of sugars. J. Chrom. 16: I76. Swaminathan, M.S., Naik, M.S., Kaul, A.K. and Austin, A. 1971. Choice of strategy for the genetic upgrading of protein properties in cereals, millet, and pulses. Indian J. Agr. Sci. l\X: 393- Uprety, D.C. and Austin, A. 1972. Varietal difference in the nutrient composition of improved bajra (pearl millet) hybrids. Bulletin of Grain Technol. 10: 2i].9. Uprety, D.C. and Austin, A. 1973. Chemical composition of some improved maize hybrids and composite. Bulletin of Grain Technol. 11: 121].. Vaisey, M. and Unrau, A.M. 1961].. Flour composition, chemi cal constituents of flour from cytologically synthe sized and natural cereal species. J. Agr. Pood Chem. 12: 1].. VJatt, B.K. and Merrill, A.L. 1963- Composition of food. U.S.D.A. Agricultural Handbook. No. 8. Williams, K.T. and Bevenue, A. 1951. The chromatographic examination of sugars in wheat flour. Cereal Chem. 2: 1].16. Year Book of Food and Agricultural Statistics. 1975* Pood and Agriculture Organization of the United Nations in Rome. 20: 70. APPENDICES
I. Starch Determination II. Total Free Sugar Determination III, Chemical and Physical Properties of Thirty Five Different Pearl Millet Genotypes IV. Analysis of Variance Tables
k3 APPENDIX I: STARCH DETERMINATION
A commercial enzyme (Diazyme L-lOO) was added to samples which quantitatively converted starch to glucose. After hydrolysis of starch to glucose, an enzyme-buffer- chromogen is added to develop brown color for glucose determination. A 150 to 200 mg grain sample was weighed and placed in autoclavable polyprophlene centrifuge tubes (32 X 61]. mm). Twenty ml distilled water was added and the starch was allowed to gelatinize in a boiling waterbath for 3 to 5 minutes. The tubes were then autoclaved 1; hours at 121^ C. Tubes were removed and cooled. Twenty- five ml of acetate buffer (I3.6 g CH3COONa'3 H2O + 6.0 ml glacial acetic acid/2.0 1) and 1.0 ml Diazyme L-lOO were added and incubated for 30 minutes in a 52^ C waterbath. After centrifuging, 2.0 ml of the supernatant liquid was pipetted into a 100 ml volumetric flask. Distilled water was added to volume. One ml of the above dilution was pipetted into a cuvette and placed in a waterbath at 37 C. At 30 second intervals, 1.0 ml of enzyme-chromogen-buffer was added to each cuvette. After exactly 30 minutes the cuvettes were removed and absorbance of the mixture was determined at I].50 nm. Concentration of starch was deter mined by comparison to a glucose standard curve (Norris,
1971)- APPENDIX II: TOTAL PREE SUGAR DETERMINATION
Free sugars in 250 mg samples were extracted with 50 ml 1 N ^2^% ^y shaking for Ij. ho\irs at room tempera ture. Ten ml of Anthrone solution (0.5 g Anthrone reagent + 280 ml H2O + 660 ml concentrated ^2^% + 10 g thiourea) was added to 1 ml of the supernatant liquid in a glass tube. The tube was placed in a water bath at 100° C for 30 minutes. The absorbance was read at 620 ram in a Bausch & Lamb Spectronic 20 colorimeter. Concentrations of total sugars were determined by comparison to a glucose standard curve (Sunderwirth et al., 1961].). Xio EH rH ONXAfv^r^cD^CM H^rH CDXAO 0\At--CM^I>-vO CMU>0 O^ W 43—' XA5SSS2S:S^-^I:1®**=^-^^^'^*-^*^OCM'XA^ ^^ CO uAsuvtivOsOsOvOsOlAvOsO r-vO vO vO ND vD >0 t^vO I^vO NO Co vO
H rH Pi Od Cd'-^ OU^^O rH CM ^-0^00 O r-CM C^CMCO rH^Arj 13 CM cv-s CM CD-d-lV C^OO rH CM 00 O rH ONsO-d-f^Jd<0 NO O^ r-IV CM rH P c^-d-^o c^ C-IANO ^f^^o -d^o vo c^\A.d^ r-iA-d-tcNiAvo XA c^ t- w © -H \A"lAC^00<^r^O rr>\AvO r<^C^ONC^vO C^ cnoo o^ CM 00 H <^ iH rH EH 'O ©'—» 3 43^ B JH O—' ?i^ ^^^^^^ C^C^sOlAsOvO E^OOOOvO t^oo t^c--oocooo w VVJrHrHrHrHrHHrHiHrHrHHrHiHrHrHrHrHrHfHrHiHrHrHrH EH O CO w M XOiO CM rH I>-OE^lA-d"E^rH f-H O ^<^C^r^C^^OvO C^C^sO O rH EH CMCMCMCMr-lrHrHrHCMrHCMHCMCMCMHOJiHrHrHrHiHrHCMCM O E © •-I < cnsOlAiH C--NO-dNO iH O vO "Lf^-d-0 O CM^vOlAO O^O-d-CMOO o 43^ M 00 v-* r^ r«- c^ o^so vO 00 vo 00 t^irv i>- c^vo oo c^ c^so vo f-vo c^ t^oo c^ CO •H O P is; <: CJ iH-P CO Od © .£{ r-i CvJsOlAC^CM>--d-1jr\CM^I>-l>-"lAmO H C^CM00CM.d O C!5 oo-d-rHtAO c^-d-^-u^lACMU^c^aJ^Aooo H-d--d--d-vooo o vo © •i}''^ 00 C^XAiHsO C^CUjd^0O_d-rHvOlANO0O CMl/\fnc\JCM\AC^rHO ,i:} to to 00 .HW vO f-UNiAoo vo i>-c«-c^c^iAr*-t^-d-i>-"iAiAt^<^oooooo r--c^^ M G © P P. 43 OJ mlAvO H r^-d^D C>-CM r^vD CO O C--O^ H CVJlAvO C^OOOO CM O CM CM CM CM <^c^<^r^fnjd-d--d--d-"LA'lAXANONOvDvONOsDvD C^C^ G rHrHrHrHHrHrHHrHpHiHrHrHHrHHHrHrHpHrHrHrHiHrH AH s \0000^00 H Pi Cd 00"-^ o^\D t»-\D fnJd•^*-^*-c^oo 43 tO^ 00 O TJ—' r^CMCMCMc^r^CMCMCsJCM •H EH CO 00 Cd Xt •D •H' I rH-d-rH m C^ CM <> C^ H-d" •H NO i>- c^ c*-vO NO VO -d"^ -d" © © •H r-i t^QO-d-o ^-c^Jd © p. I 43 l/XvO CT^ H O rH CM -d-tTS |>- O r»- r^ c^oo o^ o^ o^ o^ o^ o^ c r-ir-ir-ir-ir-ir-ir^r-ir-ir-i w APPENDIX IV: ANALYSIS OP VARIANCE TABLES TABLE 1. Analysis of Variance of Bushel V^eight in 35 Pearl Millet Genotypes Source of Sum of Degrees of Mean P Variation Squares Freedom Square Ratio Bushel Weight 637-78 3k 18.75 60.52^ Experimental Error 11.00 35 O.3I ^Significant at the 0.05 probability level; «JH«-Significant at the 0.01 probability level. k9 TABLE 2. Analysis of Variance of Lipid Content in 35 Pearl Millet Genotypes Source of Sum of Degrees of Mean P Variation Squares Freedom Square Ratio Lipid 62.61]. 3k 1.8i|- 6.3l].-J«J- Experimental Error 10.00 35 0.29 •Js-Significant at the 0.05 probability level; ^^Significant at the 0.01 probability level. 50 TABLE 3. Analysis of Variance of Protein Content in 35 Pearl Millet Genotypes Source of Sum of Degrees of Mean P Variation Squares Freed can Square Ratio Protein 76.11 3k 2.21]. 71]-.67^ Experimental Error O.9O 35 O.O3 ^Significant at the 0.05 probability level; ^HtSignificant at the 0.01 probability level. 51 TABLE 1].. Analysis of Variance of Starch Content in 35 Pearl Millet Genotypes Source of Sum of Degrees of Mean F Variation Squares Freedom Square Ratio Starch ll].09.30 3k k'^'k^ 1.90^^n- Experimental Error 1530.50 70 21.86 -s^Significant at the 0.05 probability level; ^HJ-Significant at the 0.01 probability level. 52 TABLE 5. Analysis of Variance of Total Sugar Content in 35 Pearl Millet Genotypes Soiirce of Sum of Degrees of Mean F Variation Squares Freedom Square Ratio Total Sugar l8.1|lj- 3k O.^k 1.8^ Experimental Error 21.20 70 O.3O ^^Significant at the 0.05 probability level 53 TABLE 6. Analysis of Variance of Glucose Content in 13 Pearl Millet Genotypes Source of Sum of Degrees of Mean F Variation Squares Freedom Square Ratio Glucose 0.78 12 0.065 7.22^ Experimental Error 0.12 13 0.009 ^Significant at the 0.05 probability level; •JHS-Significant at the 0.01 probability level. 51^ TABLE 7. Analysis of Variance of Fructose Content in 13 Pearl Millet Genotypes Source of Sura of Degrees of Mean F Variation Squares Freedom Square Ratio Fructose 0.75 12 0.063 l5.75-«-^^ Experimental Error 0.05 13 O.OOi}. ^Significant at the 0-05 probability level; ^JJ-Significant at the 0.01 probability level. 55 TABLE 8. Analysis of Variance of Sucrose Content in 13 Pearl Millet Genotypes Source of Sum of Degrees of Mean P Variation Squares Freedom Square Ratio Sucrose 1.50 12 0.125 25.004Hf Experimental Error 0.06 13 0.005 -x-Significant at the 0.05 probability level; -x-Js-Significant at the 0.01 probability level.