Louisiana State University LSU Digital Commons

LSU Historical Dissertations and Theses Graduate School

1971 Protein and Amino Acid Content of Rice as Affected by Environmental Modifications. Ruth Martin Patrick Louisiana State University and Agricultural & Mechanical College

Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses

Recommended Citation Patrick, Ruth Martin, "Protein and Amino Acid Content of Rice as Affected by Environmental Modifications." (1971). LSU Historical Dissertations and Theses. 2004. https://digitalcommons.lsu.edu/gradschool_disstheses/2004

This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. 71-29,385

PATRICK, Ruth Martin, 1930- PROTEIN AND AMINO ACID CONTENT OF RICE AS AFFECTED BY ENVIRONMENTAL MODIFICATIONS.

The Louisiana State University and Agricultural and Mechanical College, Ph.D., 1971 Food Technology

University Microfilms, A XEROX Company , Ann Arbor, Michigan

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED PROTEIN AND AMINO ACID CONTENT OF RICE AS AFFECTED BY ENVIRONMENTAL MODIFICATIONS

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College In partial fulfillment of the requirements for the degree of Doctor of Philosophy

in

The Department of Food Science

by Ruth Martin Patrick B .S ., Louisiana State University, 1950 M .S., Louisiana State University, 1951 May, 1971 ACKNOWLEDGMENTS

The author wishes to express special acknowledgment and gratitude to her major professor, Dr. Fred H. Hoskins, for his guidance, encouragement, and sincere interest and understanding during the course of this study.

Sincere gratitude is expressed to Dr. Arthur F. Novak, Head of the Department of Food Science, for granting her an assistantship and laboratory facilities for her research and studies in this department, and also for his counsel and encouragement.

Appreciation is also extended to Dr. Robert Grodner, Dr. William

H. James, Dr. Joseph A. Liuzzo, and Dr. Harvye Lewis, other members of her advisory committee.

The author is indebted to Dr. W. F. Me Knight, School of Home

Economics, for permitting use of facilities for amino acid analyses, I and to Mrs. Charlotte Gandy for technical assistance.

The author is especially indebted to Mr. Emmett Wilson and the

Louisiana Rice Experiment Station for furnishing rice samples and yield data for this investigation.

Above all, the author expresses special gratitude to her husband,

Dr. William H. Patrick, Jr., whose continuous inspiration, encourage­ ment, and love made this course of study possible. She also expresses affection to her four children for their cooperation and understanding during the years of this graduate program. TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS...... ii

LIST OF TABLES...... v

LIST OF FIGURES...... vii

ABSTRACT...... viii

INTRODUCTION...... 1

REVIEW OF LITERATURE...... 3

Nutritional Value of P ro te in s...... 4

Varietal Differences and Environmental Factors...... 6

Protein Fractions...... 10

Amino Acids in Rice Proteins...... 16

Factors Affecting Protein Content of R ice ...... 20

Effect of M illing...... 21

Breeding for High Protein R ice ...... 22

MATERIAIS AND METHODS...... 25

Preparation of S am ples ...... 26

Protein Determination...... 28

Amino Acid Analyses ...... 28

Statistical Analyses ...... 29

RESULTS AND DISCUSSION...... 30

Protein Content...... 30

lii Page

Grain Yields...... 47

Protein Y ield ...... 51

Amino Acid A n a ly s e s ...... 52

Effect of Nitrogen...... 5 7

Effect of M illin g ...... 57

Effect of Year...... 62

Relationship of Amino Acids to Protein C ontent- ...... 64

SUMMARY AND CONCLUSIONS...... 67

SELECTED BIBLIOGRAPHY...... 70

VITA...... 78

iv LIST OF TABLES

T ab le Page

1. Treatments used in experiment...... 27

2. Protein content of brown and milled Saturn, Dawn and Bluebells rice as affected by rate of nitrogen in 196B and 1969 ...... 32

3. Protein content of Saturn, Dawn and Bluebelle rice as affected by environmental variables ...... 33

4. Protein content, grain yield and protein yield of Saturn brown rice as affected by environmental v a ria b le s ...... 35

5 . Protein content, grain yield and protein yield of Saturn milled rice as affected by environmental variab les ...... 36

6. Protein content, grain yield and protein yield of Dawn brown rice as affected by environmental v ariab les ...... 39

7. Protein content, grain yield and protein yield of Dawn milled rice as affected by environmental varia ble s ...... 40

8. Protein content, grain yield and protein yield of Bluebelle brown rice as affected by environmental variab les ...... 43

9. Protein content, grain yield and protein yield of Bluebelle milled rice as affected by environmental v ariables ...... 44

10. Amino acid composition of Saturn milled rice (g amino acid per 16.8 g N ) ...... 53

11. Amino acid composition of Bluebelle milled rice (g amino acid per 16.8 g N) ...... 54

v Table Page

12. Amino acid composition of Saturn and Bluebelle brown rice (g amino acid per 16.8 g N) ...... 55

13. Amino acid composition of Dawn rice (g amino acid per 16.8 g N) ...... 56

14. Amino acid content of comparable rice samples as affected by rate and time of nitrogen application (g amino acid per 16.8 g N) ...... 58

15. Average amino acid composition of protein for 18 samples of milled rice and 7 samples of brown rice (g amino acid per 16.8 g N) ...... 60

16. Amino acid content of comparable samples of brown and milled rice (g amino acid per 16.8 g N) . . . . 61

17. Amino acid content of comparable milled rice samples in 1968 and 1969 (g amino acid per 16.8 g N ) ...... 63

18. Comparative amino acid contents of milled rice (g amino acid per 16.8 g N) ...... 66

vi UST OF FIGURES

Figure Page

1 ♦ Protein content of Saturn brown rice as affected by rate and time of nitrogen application in 1968 and 1969 ...... 37

2. Protein content of Saturn milled rice as affected by rate and time of nitrogen application in 1968 and 1969 ...... 38

3. Protein content of Dawn brown rice as affected by rate and time of nitrogen application in 1968 and 1969 ...... 41

4. Protein content of Dawn milled rice as affected by rate and time of nitrogen application in 1968 and 1969 ...... 42

5. Protein content of Bluebelle brown rice as affected by rate and time of nitrogen application in 1968 and 1969 ...... 45

6. Protein content of Bluebelle milled rice asaffected by rate and time of nitrogen application in 1968 and 1969 ...... 46

7. Relationship between protein content of brown and milled rice ...... 48

8. Relationship of protein content to grain yield of Saturn milled rice in 1969 ...... 50

9. Lysine and threonine contents of rice as affected by various treatments ...... 59

v ii ABSTRACT

Protein and amino acid content of three Louisiana rice varieties as affected by several environmental modifications were studied. Rice samples used were Saturn, Dawn, and Bluebelle varieties grown at the Louisiana Rice Experiment Station at Crowley,

Louisiana during 1968 and 1969. The quantity, method and timing of application of nitrogen fertilizer, and method of seeding were the major treatments used in studying these varieties.

Crude protein content of both brown and milled rice of all varieties and all treatments was determined, and amino acid analyses were determined on selected representative samples.

Nitrogen applied at specified stages in the physiological development of the rice plant produced a marked response in protein content, and in almost every case, protein content was increased by adding nitrogen fertilizer. Protein content varied among the varie­ ties, with Bluebelle generally showing the highest protein values i for both brown and milled rice in both years. Dawn usually had the lowest protein content. Saturn was especially responsive to applied nitrogen with protein increases resulting from both 80 and 120 pound nitrogen rates. For Dawn and Bluebelle varieties, much of the increased protein resulting from nitrogen fertilization was apparently removed during the milling process.

vlii Protein content was much higher in 1969 than in 1968 for all

varieties and all treatments. When all three varieties were con­

sidered , it was evident that more protein was usually found in the

grain when all the nitrogen was applied at seeding (T^), or where

half the nitrogen was applied at seeding and half applied late in the season at the 2 mm panicle stage (Tg).

There was an inverse relationship between grain yield and

protein content in the 1968 and 1969 crops. In 1968, grain yield

was higher and protein content lower than in 1969. This inverse relationship resulted in the yield of protein being relatively constant

for both years.

The yield of protein per acre did not differ as much among treatments, varieties, and years as did protein content and grain yield. Different rates of nitrogen fertilizer had the greatest effect on protein yield. Drill-seeding of rice produced slightly higher

grain yields and a lower protein content than did water-seeding.

Amino acid analyses showed that although some variation in amino acid content was evident, there did not appear to be a marked effect of variety, rate and time of nitrogen application, or method of

seeding on amino acid content, even though there were considerable differences in protein content.

Contents of lysine and threonine, first and second limiting amino acids, respectively, in rice, were markedly reduced during

ix the milling process. Although not statistically significant, a decrease In lysine content accompanied an increase In protein content for both milled and brown rice. There was a large and consistent protein Increase In 1969 compared to 1968, and con­ tents of several amino acids were also greater In 1969.

It was concluded from this study that appreciable increases in protein content and yield of rice can be expected to occur as a result of different rates and times of application of nitrogen ferti­ lizer. Large differences in protein content and yield of rice can also be expected as a result of differences in environmental con­ ditions associated with different seasons. Although some differ­ ences in content of amino acids occurred in this study, these were \ of much less magnitude than were differences in the protein content and y ield .

x INTRODUCTION

Cereals, like other plant foods, are generally considered

inferior in quality to animal foods because many times they do not

contain adequate amounts of the dietary essential amino acids. Rice

contains less protein than other common cereal grains such as wheat

and com, but the quality of rice protein is superior because of a more

favorable balance of essential amino acids. This may explain how

Asians are able to live on a diet made up almost exclusively of rice.

Although rice contains all of the dietary essential amino acids, the

proportions are not ideally balanced, and its quantitative protein

content is low.

Because people will probably continue to eat the same quantity

of cooked rice regardless of its protein content, any increase in this nutrient would enhance the nutritional status of millions, provided the quality of the protein is not lowered.

Rice yields in the United States have doubled during the past decade. Part of this yield increase has been due to the development

of improved varieties which have been more disease resistant, have had greater yield potential, and which have shown greater response to

nitrogen fertilizer. Higher yields have also been due to improved cultural practices, such as more effective weed control, water

1 2

management, and widespread use of nitrogen fertilizer. Increases in

the rate of application of nitrogen and improvements in the timing and

method of application have contributed significantly to the overall

increase in yield. These Improved varieties, higher yields, and

better cultural practices have come about as a result of Intensive

research. No such effort has gone into the search for improved

quantity and quality of protein in rice since protein content is not

a factor in determining economic value on the world market. Recent

emphasis on the need for increasing the protein content of cereals,

however, has stimulated interest in improving protein quantity and

quality of rice through both plant breeding and improved cultural

practices.

Few controlled studies have been reported on the protein and

amino acid content of rice as affected by varietal difference and

environmental factors. The purposes of this study were to determine

(1) the variations in protein content of both brown and milled rice as

affected by different environmental conditions such as rate and method

of application of nitrogen fertilizers, (2) total rice yield and total

protein yield as affected by the different treatments, and (3) the amino acid content of representative samples. REVIEW OF LITERATURE

Protein content of rice (Oryza sativa L.) shows considerable variation due to many factors, such as climatic and environmental factors, types and amounts of fertilizers applied, varietal character­ istics, cultural practices, and degree of milling (Simpson et aj.., 1965).

Watt and Merrill (1963) give as a general representative value for protein content, 7.5 percent for brown rice and 6.7 percent for white, fully milled rice.

Numerous values for crude protein content of rice, however, have appeared in the literature, ranging from 4.6 to 13.4 percent for milled, or white rice and from 6.7 to 13.5 percent for brown or whole rice (Grist, 1959; Juliano, 1966; Parialetal., 1970; Houston and

Kohler, 1970).

With the establishment of the International Rice Research

Institute (IRRI), Los Banos, Laguna, The Philippines, and the concen­ trated effort to find varieties of rice that might possess a genetic character for high protein, screening of thousands of samples for crude protein from the world rice collection revealed 101 varieties that had protein contents from 13.2 to 16.6 percent (IRRI, 1967). Juliano (1968) reported a mean level of 14 percent protein in 12 6 high protein varieties from the world collection.

3 4

McCall and co-workers (1953) reported 15.2 to 19.2 as the percent protein in true bran. Rice polish contained 11.7 to 12.7 percent protein (McCall et a l. , 1951). Besides the milling process, which separates the bran and polish from the grain, many other known factors also significantly influence protein content of rice and will be discussed in greater detail.

Nutritional Value of Proteins

The nutritional adequacy of a protein must include both quantity and quality. One method for determining the quality of a protein is by comparing amino acid composition to total nitrogen content.

Although rice contains less protein than wheat or corn, it has a superior biological value. When Sreenivasan (1941) compared the protein of rice, com and wheat in rat feeding studies at the same level of Intake, rice protein had the highest biological value. Mitchell (1924) found that the biological value of brown rice protein was superior to that of corn or oats at the five percent level of Intake. Sure and House (1948) used the nitrogen-balance method to determine the relative biological values of proteins in cereals fed at a five percent protein level. The protein utilization values for milled cereal grains were: rice 75.1, wheat 60.0, rye 63.1, and corn 32.0, with 100 as a basis for compari­ son. Findings for the whole grain were: rice 80.0, wheat 76.1, rye

73.2, corn 78.8, and rolled oats 75.6. Kik (1939) determined 5 biological values for brown rice, white polished rice, rice bran, rice polishings and obtained at the £lve percent protein level values of

72.7, 66.6, 84.9, and 02.9, respectively. Many investigators have reported the biological value of rice protein as the protein efficiency ratio (PER). The PER values for milled rice at protein levels attainable with rice alone range from 1.5 to 2.0 (Sure, 1950; Kik, 1952;

Patwardhan, 1957; Bressani and Valiente, 1962). The proteins of brown rice differ from those of milled rice in their higher content of the soluble proteins, albumins and globulins. The nutritional value and availability then may be expected to differ to some extent. Kik (195 7, 1965) reported PER values for brown rice slightly higher than those for milled rice. There is suggestive evidence that the digestibility of brown rice is a little lower than that of white rice (Houston and Kohler, 1970).

Consumption of large quantitites of brown rice for extended periods of time is not likely due to its strong flavor and because of apparent intestional disturbances (Jones, 1946).

A more detailed discussion of the nutritional adequacy of rice proteins may be found in an extensive review published by Houston and Kohler (1970). An extensive bibliography of experimental nutrition studies dealing with rice protein has been reported by Coons (1968). Varietal Differences and Environmental Factors

Sturgis et al. (1952) studied the effect of nitrogen fertilizers on protein content of rice grown at the Louisiana Rice Experiment Station.

They also studied the variation in protein content of 29 different varieties and selections of rice, and the distribution of protein frac­ tions within these varieties.

Differences in the protein content of the major varieties were highly significant. Analysis of the protein fractions indicated that the water soluble albumins decreased with increasing total nitrogen, and the prolamins and glutelins increased. This varietal difference could be a factor contributing to variation in amino acid composition.

Fertilizer treatments usually increased the yields of rice, but did not significantly increase the percentage of protein nitrogen.

However, when yield was limited by environmental factors, such as late planting and insufficient phosphorous, the effect of the added I nitrogen fertilizer was to increase the protein content rather than the yield of grain.

The nutritional effect of nitrogen on rice usually takes one of two forms: the nitrogen Increases the grain yield without markedly affecting the protein content of the grain, or the nitrogen Increases the protein content with little or no Increase in the yield of grain. \ The first effect occurs when the only limitation on yield is the supply

6 7

of nitrogen Itself, and the second effect occurs when some iactor, or

factors, other than nitrogen limits the yield, and the absorbed nitrogen

therefore becomes available for increased protein storage in the grain.

It should also be recognized that a combination of these two effects

can occur in which nitrogen causes slight increases in both yield and

protein content (Russell, 1950).

Environmental factors such as season and year have caused grain

protein content of a variety to vary from 9.0 to 14.7 percent, a range

of about 6 percent on dry weight basis (IRRI, 1963). Over a period

of years, Kik (1951) found that the average protein content of rice

grown in fertilized plots was not significantly different from that grown

in non-fertilized plots. Kymal (1955) and Bandemer and Evans (1963)

found that the location where rice is grown is an important factor in

protein and amino acid content. Rice varieties grown at the same

location differed widely in their content of essential amino acids,

however, there was greater variation within the same variety grown

at different locations. Moreover, an increase in protein content of a

particular variety, from whatever cause, did not necessarily reflect an

improvement in the essential amino acid pattern. Generally, as the

protein content increased, the content of essential amino acids decreased. This relationship was less definite in the cases of tryptophan and methionine. 8

Michael et aK (1961) found that rice possessed higher glutelin levels only as a result of late fertilization. Increased maturity did not affect the soluble nitrogen (albumins and globulins), but there was increased endosperm nitrogen (prolamins and glutelin). Aleurone protein was globulin, germ protein was albumin plus a small amount of globulin.

It is evident that there are many interrelated factors which are effective in modifying the content of crude protein and amino acids in rice. The usual 6 to 9 percent protein in U.S. milled rice varies not only with variety, but with environmental factors such as season, year, location, soil composition, and cultural practices. Milled rice has been produced with as much as 16 percent protein (IRRI, 1964).

Increase in protein content is possible through fertilization, but the strain of rice must be able to utilize the fertilizer effectively without causing agronomic problems.

Investigations were made at the Maligaya Rice Research Center in Central Luzon, Philippines to study the response of several varie­ ties to different levels of nitrogen fertilizer (IRRI, 1969). Grain yields of most varieties increased significantly with the application of each additional 30 kg N/ha up to 150 kg N /ha. The optimum nitrogen fertilizer level appeared to be 90 kg N/ha during the dry season. In the wet season, yields of all varieties tested generally increased with the addition of 60 kg N/ha. Analysis of the protein content of six lines 9 indicated that, on the average, protein Increased from 7.2 to 9.5

percent as the nitrogen level was increased from 0 to 150 kg/ha.

Different times of fertilizer application were also studied at the Maligaya Rice Research Center. The later applications of

nitrogen fertilizer produced higher protein content in the rice grain than did early applications.

Genetic pattern may be a factor in the differences observed in amino acid content among different varieties at the same location

(Kymal, 1955).

Various projects are underway in several institutions to increase, as well as improve, protein content of rice (IRRI, 1969). One possi­ bility of increasing the protein content is through chemical treatment of the rice plant. Increases in protein content of several crops as a result of applying low rates of the herbicide, simazine (2-chloro-4,

6-bis ethylamino-s-triazine) have been reported. Protein content of several food and forage crops has been increased by as much as 80 percent by treating with trace amounts of simazine, Protein content of peas was increased by 50 percent, and corn and squash, 25 to 40 percent. Simazine increases the plant’s ability to synthesize protein from its carbohydrate content. An experiment.was conducted to deter­ mine the effect of simazine on the protein content of the rice grain when applied to flooded soil at flowering time. Nitrogen fertilizer and four rates of simazine were applied to a California rice variety when the 10 panicles emerged. The simazine Increased the protein content of the brown rice at all four rates of application. Generally, the increase in protein content was higher at lower poll nitrogen levels. Simazine increased the amino acid contents of brown rice without favoring or inhibiting any amino acid except lysine. A decrease in lysine content with an increase in protein content has also been reported in other situations (Kymal, 1955; Juliano et al. , 1964; Cagampang et a l .,

1966). Although simazine increased the protein content of rice, it also increased the sterility of the rice plants, thus lowering the grain yield. The total sugar and starch contents were drastically reduced by the simazine treatment, thus reducing the dry weight without corre­ spondingly reducing protein or nitrogen content. When field experi­ ments were conducted to test the effect of simazine, protein yield was decreased due to sterility of the plants which in turn lowered grain yield. Although the percentage of nitrogen was high, the resulting protein yield per hectare decreased.

Protein Fractions

Early studies on rice protein were chiefly concerned with isolation, characterization, and classification of the protein fractions.

The four fractions, extractable successively by water, dilute NaCl solution, ethanol, and dilute alkali, were designated as albumins, 11 globulins, prolamins, and glutelins, respectively, following the conventional means of classification of Osborne and Mendel (1914).

Rosenheim and Kaijuira (1908) first isolated the glutelin fraction in 1908 and named it oryzenin. Unlike the protein of other cereals, a greater percentage of the protein of rice belongs to this rather uncommon class of alkali-soluble proteins (Jones and Csonka, 1927; Sturgis et a l . , 1952; Cagampang et a l . , 1966). Loza (1953) defined glutelin as the residual protein after successive extractions of rice powder with water, dilute NaCl, and ethanol, rather than as the alkali-soluble extract.

Research in India (Subrahmanyan et a l., 1938; Ramia, et a l.,

1939), Spain (PrimoetaK , 1963), and the U.S. (Sturgis, et al. ,

1952; Hogan et al.., 1964; Normand et al., 1966; Cagampang, et al.,

1966) have shown that the protein distribution in rice is heterogeneous.

The glutelin fraction of rice protein is predominant in the whole grain, milled rice, and rice polish. It is the fraction of lowest nutritional value, containing a small quantity of lysine. Albumin, the water- soluble fraction of highest lysine content, is concentrated in the outer layers, mostly in the bran and germ. Globulins are concentrated in the bran and polish, and prolamin is rather evenly distributed through­ out the grain.

Eighty percent or more of the total protein of milled rice is glutelin (Houston and Mohammed, 1970). The International Rice 12

Research Institute (1968) reported that in the extraction of high protein rices, glutelin fractions comprised 80 percent of total proteins in brown rice and about 91 percent in milled rice. The albumin-globulin fraction was 15 percent in brown and 6 percent in milled rice, and prolamin was 5 percent in brown and 3 perce nt in milled rice.

A compilation by Juliano (1966) showed considerable variation in the amounts of soluble proteins found by various workers. In general, globulins were about 7 to 11 percent of the total proteins, and albumins comprised 0.5 to 6.0 percent.

Houston et a l. (1964) reported the separation of milled rice globulins into two fractions. Recently, Houston and Mohammed

(1970) purified and characterized the major globulin component. It was the less-soluble component of the globulin fraction, and contained

18.1 percent nitrogen. Amino acid analysis revealed a complete absence of lysine and histidine, which is unique among cereal pro­ teins. Glutamic acid and arginine made up 43 percent of the total molecule. High arginine and low tryptophan values agreed with Pence and Elder's (1953) distinction of globulins from albumins. Sulfur- containing amino acids were also found to be quite high. The more- soluble fraction of rice globulins was characterized by a high sulfur content and is currently being studied. 13

Studies by Cagampang et aK (1966) showed that in most cases rice bran had a lower protein content than polish. Indicating that the outermost layers of the rice grain may have not been highest in protein content. More refined milling of successive layers of brown rice by

Primo et aK (1963) and Hogan et al. (1964) demonstrated that the highest protein fraction was not the outermost 5 percent, but the second outermost 5 percent fraction. Protein ratios of various frac­ tions indicated that a major portion of the albumin and globulin were removed during the milling process (Lozsa, 1953; Cagampang, 1966).

Investigators have given considerable attention to the distribu­ tion of protein in cereal grains. Several studies on wheat (Morris e ta l., 1945, 1946), barley (Normand et al., 1965), oats (Portch et a l . , 1968) and grain sorghum (Hubbard et aK , 1950; Normand et aK ,

1965) all report uneven distribution of protein, with high protein bearing layers occurring on the outer portions of most grains. As in rice, the highest protein fraction was not the outermost layer. Normand et al.

(1965) found the fourth fraction of hard wheat to possess the highest percent protein with the second fraction the next highest percent. With soft wheat, however, there was little variation between outer layers and the whole kernel.

From analyses of protein fractions of 29 U.S. rice varieties,

Sturgis et al. (1952) found that albumin, as a percentage of total 14 protein, was negatively correlated to total protein content. Prolamin and glutelin contents were positively correlated with protein levels, but no correlation was found between globulin and protein. Similar changes in the quantity of glutelin and prolamin with a change in protein content have been reported for wheat, barley, and oats

(Michael e ta l., 1961) and for com (Bressani and Mertz, 1958).

Data from extensive studies by Cagampang et al. (1966) indi­ cated that with an increased protein content, glutelin and prolamin fractions doubled, whereas albumin and globulin fractions were only slightly affected. Michael et al. (1961) and Ozaki and Moriyama

(1953) also reported that increased protein content in a rice variety resulted mainly in increased glutelin content.

Taira (1962) has reported the amino acid composition of four main fractions of brown rice and Tamura et al. (1963) those of milled rice. Marked differences in amino acid composition of the four frac­ tions were readily observed. Lysine was concentrated primarily in albumins, cystine in globulins, leucine and proline in the prolamins.

Little cystine was found in prolamins and glutelins. The amino acid composition of glutelin is not well established (Taira, 1962; Tamura et aju , 1963; Houston et aK , 1970) even though considerable work has been done on this predominant fraction. It is agreed, however, that glutelin is the fraction of lowest nutritional value, and the one most affected by environmental modification. 15

Variations among amino acid contents of different milling frac­

tions of the rice grain reflect differing protein composition. Bran

protein contained much larger proportions of water-soluble albumins

and salt-soluble globulins than did the milled kernel (Juliano, 1966;

Cagampang et a l., 1966). Bran contained the greatest amount of

histidine and was lowest in proline. Glutamic acid decreased markedly

from milled rice to brown rice, whereas aspartic acid was more equally

distributed (Houston, ®Lt aK , 1969). Albumins contained more lysine

(5 to 9 percent) and globulins less lysine (1 to 4 percent) than the

2.5 to 4 percent reported in glutelin.

Extraction of the protein fractions revealed changes in protein

quality brought about by an increase in protein content. The distribu­

tion gradient of amino acids in the protein fractions was reflected in

the chromatograms of milled rice and bran-polish. The two amino acids most affected were lysine and glutamic acid. Bran-polish

protein averaged 16 percent glutamic acid and 5.75 percent lysine,

whereas milled rice had 21.5 percent glutamic and 3.32 percent lysine.

Since there was a more even distribution of protein in high protein rices, the brown rice chromatogram was similar to that for milled rice.

Glutamic acid and lysine contents of brown rice protein were 21.1 and 3.53 percent, respectively. There was a higher percentage of

prolamin in brown rice than in milled rice which was in agreement with previous findings. 16

Protein and free amino acids are concentrated in the outer layers of the rice kernel (Subrahmanyan e £ a l., 1964; Normand et ah , 1965;

Houston et a l., 1968). Free amino acid nitrogen accounts for about one percent of the total crude protein nitrogen. It is highest in the embryo, less in bran, and least in milled rice (Takano and Nozu, 1961).

Alanine, glycine, and glutamic acid are those amino acids generally found in highest concentration. Albumin and globulin fractions are highest in the outer layers, while glutelin is found in highest concen­ tration in the center of the endosperm (Houston, et a l., 1968).

Amino Acids in Rice Proteins

Amino acid composition and balance determine the nutritional adequacy of a protein. Although rice protein contains all the dietary essential amino acids and is superior in biological quality to other cereal proteins, it is not ideally balanced. Comparison of the amino acid composition with the FAO reference pattern shows rice to be appreciably low in lysine. Studies have also demonstrated that lysine is the first limiting amino acid in rice for supporting nitrogen equili­ brium of adult human subjects (Chen et al. , 1966). Some analyses have given low values for the sulfur-containing amino acids (Houston et a l., 1969). Recent data on human requirements (Committee on

Protein Malnutrition, 1963), suggest that several of the FAO figures for individual amino acids in the reference pattern are excessive. 17

Modifications have been made by some investigators such as Kohler

(1966), in which total sulfur amino acid recommendation is lowered from 4.2 to 3.4 g/16 g N. When protein intake is lowered, the adjusted reference pattern shows that threonine is the second limiting amino acid in rice protein. It has been shown that supplementation with lysine and threonine raised the biological value of rice protein markedly (Chen, 1967). Several investigators have confirmed the supplementary value of the synthetic amino acid combinations of lysine and threonine in increased PER (Kik, 1952; Sure, 1955). Howe and co-workers (1965) recently showed that at 7.8 percent protein level, 0.2 percent L-iysine HC1 plus 0.2 percent DL-threonine raised the PER of a rice diet from 1.50 to 2.61.

Only in recent years have extensive studies been made of the amino acid composition of proteins. After 1943, microbiological methods were used to determine amino acid composition and more recently, chromatographic analyses have been possible. Very little data were reported prior to 1950 on amino acid composition of rice, and wide discrepancies exist in data that are now available (Orr and

Watt, 1957).

Compilations of available data on amino acid composition of milled and brown rice and rice products by Juliano (1966) and by

Houston and Kohler (1970) show wide variations. Microbiological analyses on U .S. brans (Schweigert, 1947; Lyman et a 1., 1956; Kik, 18

1956), and recent chromatographic analysed on Spanish and Japanese brans (Tamura and Kenmochi, 1963; Lain and Rodriguez, 1965) also vary. Microbiological analyses on U.S. polishes (Lyman et al. ,

1956; Kik 1956) show appreciable differences as well. Discrepancies are due primarily to differences in hydrolytic conditions and analytical methodology, however varietal and environmental conditions are also important contributing factors (Kohler and Palter, 1967; Houston et al.. ,

1969).

Cagampang et al. (1966) found that with increases in protein content of 8 varieties of milled rice, lysine increased significantly less than the other amino acids. In contrast glutamic acid and tyro­ sine were positively correlated with protein content. These trends for lysine, methionine, and tyrosine were previously reported in 16 varieties of milled rice by Juliano et al. (1964). Kik and Hall (1961) and Kymal, (1955) in determining amino acid content by microbiological assays, found identical trends for lysine and the other seven essential amino acids.

In a study of the protein and amino acid content of several varieties of milled rice and rice by-products, Houston et al. (1969) found that despite a 60 percent variation in nitrogen content, there was relatively minor variation in amino acid content. As protein increased, there were decreases in histidine (r= -0.68**) and lysine

(r= -0.71*) which confirmed an earlier report of this relationship in 19 rice (Cagampang, 1966). In contrast, there were increases in methionine (r= +0.64*), valine (r= +0.42), and tyrosine (r= +0.37) as protein increased.

Ihe X-M rice, from which bran removal is accomplished with a solvent process, did not differ appreciably in amino acid composition from the other milled rices. It was expected that some amino acid values would have been higher due to the fact that the aleurone layer remained intact in this process. Few values differed from the average more than 5 percent, and none by 10 percent.

This relation between lysine and protein is consistent with observed increases in prolamln as total protein increased, since prolamin had the lowest lysine content of the four rice protein fractions.

This finding seems to explain similar negative correlations in com

(Bressani and Mertz, 1958) and wheat (McDermott and Pace, 1960).

Lysine content of rice protein dropped from 4.35 to 3.66 percent as total protein increased from 7.3 to 11.9 percent. Actually, there was a net gain in lysine content of milled rice with increases in protein content. The drop in percentage of lysine was less than that reported for wheat and corn, probably because prolamln is a very minor protein fraction in rice. Lozsa and Koller (1953) substantiated this when they reported that a larger quantity of protein in rice was not associated with a decline in biological quality. 20

Factors Affecting Protein Content of Rice

In selection of new rice varieties, emphasis has been placed on

breeding for higher yield per acre and in maintaining or improving

cooking and processing qualities. The superiority of a variety depends

not only on its yield potential but also on consumer acceptance

(Ghosh et al. , 1968). In the United States there is a coordinated

rice-breeding program conducted cooperatively by the United States

Department of Agriculture and Agricultural Experiment Stations in the

rice producing states of Arkansas, California, Louisiana, Mississippi

and Texas. New varieties developed by rice breeders and released for

commercial production must meet established standards for cooking and processing qualities required for each major grain type. Prior to release, new varieties are tested agronomically and quality-wise for at least 3 years in their likely production area (Webb, 1967).

Unfortunately, little attention has been given to protein content, and the trend has been toward increased plantings of high-yielding, low-

protein varieties. The situation regarding quality of rice protein is even more unsatisfactory. Until recently, almost nothing was known about the relationship between total nitrogen and protein quality of rice.

Protein content of rice in the United States is less of a problem because rice is considered a carbohydrate food rather than a source of

protein. Undemutrltion and malnutrition, however, are currently widespread in many countries where cereals are staple in the diet. 21

With protein deficiency being one of the most urgent nutritional problems today, many workers in India (Ghosh et al. , 1968; Garcha et aK , 1968; Rao, 1969), the Philippines (Juliano, 1966; IRRI, 1963-

1969), United States (Hogan et al.. 1964; Houston et a l.. 1969), and elsewhere are looking for ways to improve both the quantity and quality of protein.

Effect of Milling

Of all cereals, rice alone is eaten predominantly as milled whole grain (Houston and Kohler, 1970). It is generally recognized that degree of milling influences both protein and amino acid content of rice. The loss of protein in brown rice during milling ranged from 11 to 26 percent (Cagampang et a l., 1966). Since albumins and globulins are the major proteins in bran and polish, they are those principally removed during milling and therefore the lysine content is decreased.

Rice germ is usually removed with the bran during milling, and when separated, it has the highest biological value of all rice products with a PER of 2.59 at 5.7 percent protein (Houston and Kohler, 1970).

Cagampang et

A study of the (Hilling fractions of high-protein rice varieties at IRR1 confirmed smaller differences in protein content of brown and milled rice at higher protein levels. A bran-polish recovery of 8 percent from high protein rice as compared to 1 0 percent for low- protein samples reflected greater resistance of high-protein rices to milling. Previous findings had shown that high-protein rices were more difficult to mill and also had a more uniform protein distribution than low-protein rices (IRRI, 1968).

Breeding for High Protein Rice

Research on production of high-protein rices has recently been undertaken at the International Rice Research Institute (IRRI), at the

California Rice Experiment Station, and the Arkansas Rice Experiment

Station. At IRRI (1963) scientists are trying to Improve rice varieties and yields, to determine more specifically reasons for high protein content of various lines, and to demonstrate how high levels can be consistently obtained. Nutritional adequacy of rice protein is not always enhanced by increases in protein content, rather it is sometimes slightly lowered. This anomaly arises from the fact that increases in protein content are mostly in glutelin, the predominant fraction rela­ tively low in lysine, and in prolamln, the fraction containing almost 23 no lysine (Cagampang et aK , 1966). Therefore, lysine Increases at a lesser rate than other amino acids causing a greater amino acid imbalance.

Experience has shown that it is more difficult to increase the relative percentages of specific amino acids in rice than it is to increase the overall protein content of the grain. The discovery by

Mertz et al. (1964) that a mutant gene (Opaque-2) changed the protein composition of com and also Increased lysine content alerted rice researchers to look for a similar possibility.

As the initial phase of a cooperative program of breeding for high protein rice, the IRRI world rice collection was screened for crude protein in order to find high protein varieties (IRRI, 1967). Mean protein content of 7,419 samples including both wet and dry seasons, was 10.5 - 1.6 percent. From these, 101 varieties with at least 13.5 percent protein in each season with a mean level of 14 percent were selected. Amino acid analyses showed a narrow range of values for all amino acids. The ratio of the eight essential amino acids to total amino acids ranged from 0.289 to 0.343. Results indicated that within the protein levels of 13.2 to 16,6 percent, amino acid composition, with the exception of lysine, was essentially independent of protein content. The correlation between protein and lysine to protein was significant (r= -0.249**). Because of the interaction between environ­ ment and protein content, a third planting of the 1 0 1 varieties during the 24

1967-68 dry season was analyzed for protein content to determine which were consistently high-protein varieties. Protein content from three separate plantings gave further evidence that genetically high protein varieties existed (13.5-15.0 percent).

Selected lysine analyses between 2.52 and 5.47 percent for 634 samples with a protein range of 6.7 to 18.2 percent correlated signi­ ficantly (r= -0.49**) with protein content. Only 25 percent of the variation in lysine content of brown rice was attributed to variation in protein content. No high lysine variety has yet been identified

(IRRI, 1968).

Recent research has shown that protein is stored in bodies 1 to

3 microns in size in the rice endosperm, and that protein synthesis takes place in these bodies (Del Rosario et aK , 1968). These results indicated that one of the major differences between high protein and low protein varieties was the higher level of free amino acids occurring in the former, and a greater tendency to Incorporate amino acids into protein than was the case for low-protein varieties. Experiments are now underway to confirm the effect of free amino acids on protein synthesis. This is accomplished by manipulating the nitrogen supply during grain development (IRRI, 1969). MATERIALS AND METHODS

Rice samples used in this study were obtained from the Rice

Experiment Station at Crowley, Louisiana and were harvested during

1968 and 1969. Plantings were part of an experiment designed to study the effect of variety, method of seeding, quantity of nitrogen fertilizer, and method and time of application of nitrogen fertilizer on yield of rice. Varieties used were Saturn, Dawn, and Bluebelle.

Saturn is a medium grain variety, and Dawn and Bluebelle are long grain.

All treatments were replicated four times using a split-plot design with time of application constituting the main plot and nitrogen rates the sub-plots. Separate, but adjacent experiments were used for each variety. All three varieties were used in the water-seeded portion of the experiment, while only Saturn and Dawn were drill-seeded.

Drill-seeding is the traditional method of planting rice, while water- seeding (dropping the seed from an airplane onto a flooded field) now predominates.

Various nitrogen application methods included subsurface place­ ment of all nitrogen before seeding (a method designed to provide maximum conservation of nitrogen), broadcast application of all nitrogen prior to first flood (a method commonly used by fanners

25 26 because of Its convenience), and several methods which involved applying half the nitrogen broadcast at first flood and half at various times during the growing season to correspond with given physiological stages of development of the rice plant. Ammonium sulfate at rates of

0, 80 and 120 lb N per acre was used as the nitrogen source. All plots received an amount of phosphate and potash considered adequate for good yields (50 lb P 2 O5 and K2 O per acre). Specific treatments used in this study are outlined in Table 1.

Preparation of Samples

Samples consisting of 1000 grams of cleaned , air-dried rough rice were hulled with a McGill sheller. The hulled rice grains were separated from the loose hulls, and the resulting brown rice was milled and polished with a McGill miller No. 3, according to official government inspection procedures (U.S. Production and Marketing Administration,

1962). Percentage of hulls averaged 20 percent of the rough sample, and milling yields of 67.2, 70.6 and 71.8 percent were obtaine.’ for

Dawn, Bluebelle, and Saturn, respectively.

After hulling and/or milling, replicates of each treatment were packaged, labeled, and stored in air-tight cans. Samples from 1968 were stored at -20 C until after the 1969 harvest when all samples were prepared for simultaneous analyses. 27

T able 1

Treatments used In experiment

Varieties

Saturn (brown and milled)

Dawn (brown and milled)

Bluebelle (brown and milled)

Method of Seeding

Drill seeded

Water seeded

Rate of Nitrogen Fertilizers Used

0 lb per acre

80 lb per acre

1 2 0 lb per acre

Method and Time of Application of Nitrogen

T} Subsurface drilling of all nitrogen at seeding.

T3 Broadcast application of all nitrogen prior to first flood.

Tg Half nitrogen broadcast at first flood and half applied at

elongation of basal intemode, or first Joint.

Tg Half nitrogen broadcast at first flood and half applied when

panicle was 2 mm in length. 28

Four replicates from each treatment were thoroughly mixed, forming a composite sample representing each treatment. A subsample of each composite was hand picked to remove any hull residue, then thoroughly ground in a Waring Blender for approximately four minutes .

This method of grinding produced samples of which 95 percent passed through a 16 mesh sieve, and 5 7 percent passed through a 32 mesh sie v e.

Protein Determination

Approximately 2 g of the well-mixed ground sample was used for estimation of total nitrogen by routine KJeldahl Gunning-Arnold methods

CAOAC, 1965). Duplicate determinations were made on each sample, but in different runs in order to minimize errors. Duplicate values that did not check within two percent were repeated. The conversion factor of 5.95 X N was used for calculating crude protein content. Analyses are reported at 1 2 percent moisture.

Amino Acid Analyses

Amino acid contents of 25 selected samples representing various varieties and treatments were determined by conventional procedures with a Beckman Model 116 Amino Acid Analyzer. Approximately 200 mg of finely ground rice sample was weighed into hydrolysis tubes and

3 ml of 6 N HC1 was added. The acid-sample mixture was frozen under vacuum, thawed, and refrozen under vacuum to remove entrapped air. 29

Samples were hydrolyzed under vacuum at 110 C for 22 hours.

Hydrolyzed solutions were filtered, washed with 40 ml 0,1 N HC1,

and evaporated on a rotary evaporator. A few milliliters of deionized water was added three times to the residue and re evaporated to remove

HC1. Samples were then dissolved in 2 .2 pH buffer, tightly sealed, and frozen for one month until analyzed. Values for isoleucine,

methionine, serine, threonine, and valine were corrected for their incomplete hydrolysis or degradative loss during acid hydrolysis by multiplying by the factors 1.078, 1.034, 1.082, 1.036, and 1.081, respectively {Kohler and Palter, 1967). Results were expressed as grams amino acid per 16.8 g N.

Statistical Analyses

Data obtained for protein content, amino acids, grain yield, and yield of protein were analyzed by analysis of variance and linear corre­ lation analysis (Snedecor, 1956). All comparisons were made at the

5 percent level of probability. RESULTS AND DISCUSSION

Protein Content

Protein contents of brown and milled rice of Saturn, Dawn, and

Bluebelle varieties as affected by rate, time, and method of nitrogen application and method of seeding are reported in Tables 2 and 3 and in Figures 1 through 6 . Data for protein content, grain yield and protein yield of brown and milled rice as a two year average for each variety are reported in Tables 4 through 9.

In almost every case, protein content was increased by the addi­ tion of nitrogen fertilizer. Protein content for milled rice varied from

5.67 percent for Dawn variety receiving 80 pounds nitrogen per acre in 1968 to 7.63 percent for Bluebelle variety receiving 120 pounds nitrogen in 1969. For brown rice, protein content ranged from 6.64 percent for Dawn receiving 80 pounds nitrogen per acre in 1968 to

8.41 percent for Bluebelle receiving 120 pounds nitrogen in 1969.

Bluebelle variety generally showed the highest protein values for both brown and milled rice in 1968 and 1969, while Dawn variety usually had the lowest protein values. Adding either 80 or 120 pounds nitrogen per acre to the crop generally increased protein content more than did any of the other treatments used, although Increases were not always consistent. Saturn variety was especially responsive to nitrogen with

30 31 increases resulting from both 80 and 1 2 0 pound nitrogen rates, with the exception of one case where no Increase was noted. There was much less effect of nitrogen rate on protein content of Dawn and Bluebelle milled rice. The milling process appeared to remove much of the increased protein that resulted from nitrogen fertilization, indicating that much of the increased protein was present in the outer layers.

Other investigators have found that the degree of milling is an impor­ tant factor in determining protein loss in the rice kernel (Kymal, 19 55;

Hogan et a l., 1964; Houston, e t a l . , 1968).

Results from this investigation confirmed that protein content varies widely from year to year. Without exception, protein content for comparable treatments of all varieties was higher in 1969 than in

1968 (Table 2), often as great as one percent or more. The effect of year was so marked that rice from plots receiving no nitrogen in 1969 had higher protein contents than the high nitrogen treatments in 1968.

Kymal (1955) found that protein content of rice varied from year to year when the same variety was grown at the same location.

Juliano and associates (1964) have shown a difference of 4 percent protein in the same rice planted in different seasons.

Two varieties, Saturn and Dawn, were planted by both water- seeded and drill-seeded methods. Bluebelle variety was water-seeded only. For Saturn, little or no difference in protein content of brown or milled rice resulted from different methods of planting (Table 3). 32

T able 2

Protein content of brown and milled Saturn, Dawn and Bluebelle rice as affected by rate of nitrogen in 1968 and 1969

SATURN Brown Milled^ LEVEL OF NITROGEN 1968 1969 1968 1969

0 N 6.49a 7.59a 6.09a 6.53a o to r- -Q * 8 0 1 80 N . b 6.04a 7 - 2 1 b

120 N 7.27b 8.30c 6 . 37fa 7.27b

DAWN Brown 1 Milled,2 1968 1969 1968 1969

0 N 6 . 5la 7.30a 5.84b 0 •94a b

80 N 6 .64a b 7.34ab 5.67a 6.83a

1 2 0 N 7.01b 7.67b 5.89b 7.11b

BLUEBELLE 2 Brown 1 Milled 1968 1969 1968 1969

0 N 6.72a 7.77a 6.43b 6.84a

80 N 6.70a 8 ■ 14a b 5.95a 7 -33a b

120 N 7.32a 8.41b 6-14ab 7.63b

* Water-seeded and drilled-seeded treatment

^Water-seeded treatment only

Values followed by the same letter do not differ significantly at the 5 percent level of probability. 33

T able 3

Protein content of Saturn, Dawn and Bluebelle rice as affected by environmental variables

Saturn Dawn Bluebelle TREATMENT Brown Milled Brown Milled Brown Milled

0 N 7.04a 6 * 41a 6.91a 6.32a 7.25a 6.64a

80 lb N 7 .53b 6.84ab 6.97a 6 . i 8a 7.42a 6.62a

120 lb N 7.78b 7 .i2 b 7.34a 6.41a 7.87a 6.89a

(Tj) All N applied pre plant 1 7.83a 7.13a 7 . 13a 6 . 30a 8 . 0 1 a 7.25a

(T3 ) All N prior to

first flood1 7.47a 6.89a 7.03a 6 .3 la 7.46a 6 .7 lab

(Tg) Half N at first flood,

half at first joint 1 7.44a 6 . 80a 7.00a 6 . 2 1 a 7.19b 6.42b

(Tg) Half N at first flood, half at 2 mm panicle 1 7.88a 7.10a 7.44b 6 . 35a 7.91a 6 .65ab

Water-seeded 7.62a 7.17a 7.34a 6.98a - -

Drill-seeded 7.45a 7.26a 6 . 8 6b 6 . 1 2 b - -

1968 crop 7.02a 6 . 2 2 a 6.74a 5.80a 7.00a 6 . 1 2 a

1969 crop 8 .05b 7.12b 7.47b 6.98b 8 .28b 7 ■ 34b

Values are averages for 1968 and 1969. *80 lb N and 120 lb N treatments only. Values followed by the same letter do not differ significantly (5%). 34

Water-seeding, however, caused a significantly greater protein content in both brown and milled Dawn rice. Higher protein content was asso­ ciated with lower grain yield in water-seeding plantings .

Time and method of application of nitrogen influenced protein content of Saturn (Tables 4 and 5 and Figures 1 and 2). For milled

Saturn rice, applying all the fertilizer at seeding (Tj) was statistically superior to other application treatments in 1969. For brown Saturn rice, both treatments Tj and Tg (half the nitrogen applied at seeding, other half applied late in the season when panicle was 2 mm long) appeared slightly superior to the other two treatments, however the difference was not statistically significant. Differences in protein content of Dawn for the various nitrogen timing treatments are shown in Tables 6 and 7 and Figures 3 and 4. Protein was significantly higher in brown rice for the late season application (Tg), especially in 1968, but the milling process removed most of the increased protein. For

Bluebelle, application of nitrogen at seeding (T^) produced a higher protein content than did the other treatments (Tables 8 and 9 and

Figures 5 and 6 ), Increase in protein content was statistically signi­ ficant for milled rice and approached significance for brown rice.

When all three varieties were considered, it was evident that more protein was found in the grain where all the nitrogen was applied at seeding (Ti), or where half the nitrogen was applied late in the season at the 2 mm panicle stage (Tg). Late season application of nitrogen 35

T ab le 4

Protein content, grain yield and protein yield of Saturn brown rice as affected by environmental variables

Percent Grain Protein protein yield yield TREATMENT % lb/acre lb/acre

0 N 7.04a 3040a 172a

80 lb N 7.53b 4950b 286b

120 lb N 7.78b 5190b 320b

(Tj) All N applied preplant 1 7.83a 5290a 319a

(T3 ) All N prior to first flood* 7 • 47a 4940a 292a

(Tg) Half N at first flood, half at first joint -1 7.44a 5010a 296a

(T9 ) Half N at first flood, half at 2 mm panicle* 7.88a 5060a 306a

Water-seeded 7.62a 4110a 272a

Drill-seeded 7.45a 5070b 305a

1968 crop 7.02a 5230a 302a

1969 crop 8 .05b 4110b 275a

*80 lb N and 120 lb N treatments only.

Values are averages for 1968 and 1969.

Values followed by the same letter do not differ significantly at the 5 percent level of probability. 36

T able 5

Protein content, grain yield and protein yield of Saturn milled rice as affected by environmental variables

Protein Grain Protein con tent yield yield TREATMENT % lb/acre lb/acre

0 N 6 . 4 la 3040a 142a

80 lb N 0•84ab 4950b 224b

120 lb N 7.12b 5190b 243b

(Ti) All N applied preplant 1 7.13a 5290a 253a

(T3) All N prior to first flood * 6.89a 4940a 2 2 2 a

(T6) Half N at first flood,

half at first Joint 1 6.80a 5010a 2 2 2 a

(T9) Half N at first flood, half at 2 mm panicle 1 7 . 1 0 a 5060a 237a

2 Water-seeded 7.17a 4110a 198a

Drill-seeded 2 7.26a 5070b 246b

1968 crop "1 6 . 2 2 a 5230a 225a cr> O G

1969 crop 3 7 .i2 b 4110b (0

J80 lb N and 120 lb N treatments only. 21969 only. 2Water-seeded treatment only. Values are averages for 1968 and 1969. Values followed by the same letter do not differ significantly at the 5 percent level of probability. Figure Percent Protein . rti cnet f aun oroivr. Saturn of rice content Protein 1. plcto i 16 aa 19ti9. ana 1968 ie application r Rate of N of Rate J j fetr o rt aa ie f nitrogen of tine ana rate oy affector: ii g Treatment Timing

10

c *5 i • E 1YDO r f\r f I ,

1 0 8 0 120 l______l OJ Rate of N Timing Treatment OD Figure 2 . Protem content or Saturn millo net.; a-j affected by rate and time of nitrogen application m1 Ju3 and 19t>9. 39

T able 6

Protein content, grain yield and protein yield of Dawn brown rice as affected by environmental variables

Protein Grain Protein content yield yield TREATMENT % lb/acre lb/acre

0 N 6.91a 2860a 157a

80 lb N 6.97a 4790b 267b

120 lb N 7.34a 5180b 302c

(Tj) All N applied preplant 1 7.13a 5040a 289a

(T3 ) All N prior to first flood 1 7.03a 4920a 275a

(Tg) Half N at first flood, half at first joint 1 7.00a 5090a 283a

(Tg) Half N at first flood, half at 2 mm panicle 7.44b 4890a 290a

Water-seeded 7.34a 4450a 269a

Drill-seeded 6 . 8 6b 4670a 272a

1968 crop 6 . 74a 4930a 2 78a

1969 crop 7.47b 4180b 262a

*80 lb N and 120 lb N treatments only.

Values are averages for 1968 and 1969.

Values followed by the same letter do not differ significantly (5%). 40

T able 7

Protein content, grain yield and protein yield of Dawn milled rice as affected by environmental variables

Protein Grain Protein content yield yield TREATMENT % lb/acre lb/acre

0 N 6.32a 2860a 125a

80 lb N 6.18a 4790b 191b

120 lb N 6.41a 5180b 2 11c

(Tj) Ail N applied preplant 1 207 6.30a 5040 a a

(T^) All N prior to first flood 1 6.31a 4920a 196a (Tg) Half N at first flood, half at first joint * 6 .21a 5090a 201 a

(T9 ) Half N at first flood, half at 2 mm panicle * 6.35a 4890a 201a

2 Water-seeded 6.98a 4450a 194a

Drill-seeded 2 6 .1 2 b 4670a 189a

1968 crop"* 5 .80a 4930a 194a

1969 crop"* 6.98b 4180b 194a

180 lb N and 120 lb N treatments only. 2 1969 only. ^Water-seeded treatment only.

Values are averages for 1968 and 1969.

Values followed by the same letter do not differ significantly at the 5 percent level of probability. Percent Protein 10 i 8 i .iQAflWOO -iQAO c WOO g &

8 0 120

Rate of N Timing Treatment figure 4. Protein cunt-:*' re Dv.vr, r.ille rice application in 14oy ana 19o4, 43

T able 8

Protein content, grain yield and protein yield of Bluebelle brown rice as affected by environmental variables

Protein Grain Protein content yield yield TREATMENT % lb/acre lb/acre

0 N 7.25a 3290a 184a

80 lb N 7.42a 4650b 273b

120 lb N 7 .8 7 a 5180b 309b

(Tj) All N applied preplant 1 8.0 1 a 4450a 278a

(T3 ) All N prior to first flood 1 7 .4 6 a 5040a 300a

(Tg) Half N at first flood, half at first joint 7 .1 9 b 4830a 2 78a

(Tg) Half N at first flood, half at 2 mm panicle 1 7.91a 4860a 308a

1968 crop 7 . 00a 4720a 273 a

1969 crop 8 .28b 4270a 285a

*80 lb N and 12 0 lb N treatments only.

All plots water-seeded

Values are averages for 1968 and 1969.

Values followed by the same letter do not differ significantly at the 5 percent level of probability. 44

T able 9

Protein content, grain yield and protein yield of Blue be lie milled rice as affected by environmental variables

Protein Grain Protein content yield yield TREATMENT % lb/acre lb/acre

0 N 6.64a 3290a 149a

80 lb N 6 .62a 4650b 215b

120 lb N 6 .89a 5180b 238b

(Tj) All N applied preplant 1 7 .25a 4450a 2 2 1 a

(T3 ) All N prior to first flood 1 6 .7 lab 5040a 238a

(Tg) Half N at first flood, half N at first Joint 1 6.42b 4830a 219a

(Tg) Half N at first flood,

half at 2 mm panicle 1 6 • 6 Sab 4860a 228a

1968 crop 6 . 1 2 a 4720a 2 1 0 a

1969 crop 7 • 34b 42 70a 226a

*80 lb N and 120 lb N treatments only.

Values are averages for 1968 and 1969.

Values followed by the same letter do not differ significantly at the 5 percent level of probability. iao . rti cnet t ieto rw ■ a retn y ae n tm o nitrogen of time dnu rate by ■ artecten aa urown biuebtmo ut content Protein 5. Figaro Percent Protein 10 9 6 8 7 5 plcto i 19 ana 63 in application 1 ______2 T 120 0 8 0 —1968 A W A W F Q i _ aeo Tmn Treatment Timing N of Rate 19o9. i I ______t i 6 t T6 Ti i

10

! • ■iQAflUIUU 1909 (Is i 7

80 120 T’ * * , Rate of N Timing Treatment ^ Figure 6. Protein content of Blueo 'Jl'1 ;n;.l atfpcteo oy rate anc time cf nitrogen application in 19t>8 and1969. 47 has been suggested as a means of increasing protein content of the grain, but little experimental verification of this effect is available.

Kik and Hall (1961) showed in a greenhouse experiment with one sample that late season application of nitrogen increased the protein content of the grain as compared to earlier application.

Protein content of milled rice was highly correlated with protein content of brown rice (Figure 7), although the correlation coefficient

(r= +0.827) was not as high as has been reported by some other investigators. Because of the close association found between protein contents of brown and milled rice, some investigators use only brown rice for evaluating protein content (Juliano, 1968).

Grain Yields

Grain yields for the three varieties as affected by the various treatments are shown in Tables 4 through 9. As expected, the addition of nitrogen produced large increases in yield with the biggest increase coming from the application of 80 pounds of nitrogen. An additional yield increase usually resulted from the 120 pound application. Dif­ ferences in grain yield due to different timing applications of nitrogen were not significantly different for individual varieties. The larger field experiment from which these samples were taken, however, con­ tained several other treatments. When these other treatments were considered, there were significant differences in yield as a result of nitrogen timing, with the treatment receiving nitrogen at seeding Percent Protein in Milled Rice iue . eainhp ewe poen otn o bon n milled and brown ot content protein between Relationship 7. Figure 6 8 9 5 7 5 rice. 6 Percent Protein in Brown Rice Brown in Protein Percent r—*-0.827 7 8 9 48 0 1

49

producing higher yields than most of the top-dress treatments (Wilson

and Peterson, 1969).

These results are in agreement with the findings of Patrick and

associates (1967) who concluded that the best utilization of applied

nitrogen and highest yields of rice were obtained when all nitrogen

was applied several inches deep at seeding rather than from top-dress

applications.

Grain yields were lower and protein contents higher in 1969 than

in 1968. Although the reason for higher protein content in 1969 is not

definitely known, it is likely that the relatively adverse environmental

conditions in 1969 had a pronounced effect on both yield of rice and

protein content. The yield of rice was higher in 1969 than in 1968,

resulting in there being little difference in per acre yield of protein in

the two years. The cool growing season and increased insect damage

caused a limitation on yield in 1969, and resulted in nitrogen being

present in the plant in a larger concentration than was needed. In

1968, the extra nitrogen was utilized in grain production and conse­ quently did not show up in increased protein content.

When all varieties and treatments were considered separately, no general relationship was shown between grain yield and protein content. Saturn milled rice in 1969 showed a highly significant correlation coefficient (r= +0.706) between grain yield and protein content (Figure 8 ), This was not a typical response and occurred only in 1969 when yields were generally low. However, the correlation for 7 » I c iue . eainhp f rti cnet ogan il of yield grain to content protein of Relationship 8. Figure F 2000 8 aun ild e i 1969. in nee milled Saturn o o +0.706 - r YlekM bs per acre per bs YlekM 4000 4000 GO X o 6000 50 Saturn in 1968 was also positive and approached significance.

Apparently, Saturn may be quite different from most other varieties and responds to increased nitrogen supply by both an increase in yield and an increase in protein content. Because of the prevalent belief that protein content and grain yield are inversely related, there was concern at IRRI that the high yield of the recently developed IR 8 variety might be obtained at the sacrifice of protein content. Analyses of 1R8 and traditional varieties grown in the Institute (IRRI, 1968) indicated that even with its much higher yields IR 8 had a protein level comparable to that of traditional varieties.

Protein Yield

Protein yields for brown and milled rice of Saturn, Dawn, and

Bluebelle varieties were markedly increased with each increment of applied nitrogen (Tables 4 to 9). For Saturn and Bluebelle, protein yields were significantly greater with the 80 pound rate of nitrogen.

The 120 pound rate brought an additional, although non-significant protein yield increase. For Dawn, protein yield was significantly increased by both levels of nitrogen. Timing of nitrogen application had no significant effect on protein yield for any variety.

Although protein yields of drill-seeded Saturn brown and milled rice were higher than for water-seeded, only the protein yield of milled rice was significantly higher. Little or no difference in protein yield 52 was noted due to method of planting Dawn rice. Protein yield of milled rice for all varieties was markedly less than protein yield of brown rice due primarily to milling loss.

Effect of season caused little difference in protein yield. As indicated previously, percent protein in the 1969 rice was much higher than that in 1968, however there was little or no difference in yield of protein for the two y^ars. In most cases where grain yields were higher, protein content was lower, thus giving a fairly constant protein yield.

Amino Acid Analyses

Data for 16 amino acids and ammonia found in 25 rice samples selected from three varieties studied are reported in Tables 10 through

13. Samples represent both brown and milled rice, and were selected to show the effect of variety, year, and rate and time of nitrogen appli­ cation on amino acid composition. Amino acids are reported as actual nitrogen recovery. Nitrogen recovered accounted for 73.3 to 99.2 percent of total protein nitrogen. Most values ranged from 75 to 88 percent. Tryptophan was not included. Considerable variation in nitrogen recovery has been reported, and the recovery values in this study were not consistently as high as was reported by Houston et al.

(1969). 53 Table 10

Amino acid composition of Saturn milled rice (g amino acid per 16.8 g N)

1968 1969 0-N 80-N 120-N 120-N 120-N 120-N 120-N J T t * 1 Tl t 9 Tl J-6 Tl 2 AMINO ACIDS W 5 .1 w .s . W .S. W.S. W.S. w .s . D.S . 2 Lysine 3.23 3.38 3.04 3.33 3 .43 3.22 3.35

Histidine 2.51 2.95 2.33 2.61 2.85 2.60 2 .6 8

Ammonia 1.45 1 . 8 8 1.51 1.56 1.64 1.76 1 . 8 8

Arginine 7.45 4.47 7.28 8 . 2 0 7.83 5.88 7.99

Aspartic Acid 6.92 7.08 7.18 7.47 7.38 7.55 8 . 72

Threonine 2 .6 8 3 .39 3.14 2 .78 1.89 3.34 3 . 77

Serine 4.12 4.37 4.27 4.51 2 .80 4,64 5 .42

Glutamic Acid 9 .54 13.92 13.33 12 .93 11. 63 13.52 17. 30

Proline 3.60 3.55 3. 74 3.68 3. 75 3.63 4. 64

Glycine 3.81 4.04 3.93 4.21 3.94 4.03 4.93 Alanine 4 .28 4.38 4.46 4.68 4.59 3.66 5 .63

Valine 4.44 5.25 5.08 5.29 5.49 5.16 6 . 70

Methionine 1 . 6 6 2.32 1.76 2 .71 2 . 2 1 1.80 2.27

Isoleucine 3.15 3.59 3.27 3 .45 3.56 3.47 4.47

Leucine 5.85 6.28 5.90 6 . 2 2 2.55 6.06 7.65

Tyrosine 1.61 1 . 8 6 1.44 2.44 1.90 1.75 2.67 Phenylalanine 3.50 3.56 3.32 3.77 3.96 2.96 4.38

% N Recovered 73.3 8 0 .1 78.8 83.9 75.0 78.8 99 .2

% Crude Protein 6.07 6.14 6 . 6 8 6.23 7.76 7.19 7.44

* Water-seeded.

2 Drill-seeded. 54

Table 11

Amino acid composition of Bluebelle milled rice (g amino acid per 16.8 g N)

1968 1969 0-N 80-N 120-N 120-N 120-N 120-N 120-N

Tl Tl t 6 T9 Tl Tg AMINO ACIDS W.S. W.S. W.S. W.S. W.S. W.S. W.S. Lysine 3.35 3.40 2.95 3.19 3.21 3.00 2.49

Histidine 2 . 6 6 2 .46 2.31 2.40 3 .03 2.37 1.99

Ammonia 1.91 2.18 1 . 8 6 1 . 8 8 1.91 1.60 1 . 6 6 Arginine 8.14 7.27 6.42 7.07 8.24 7.42 5.12 Aspartic Acid 7.79 7.92 6.81 6.87 8.25 7.95 6.78 Threonine 3.55 3 .30 2.98 2.96 3.56 3.53 2.96 Serine 5.12 4.20 3.75 3.96 4.36 4.70 4.11 Glutamic Acid 9.81 14.03 12.85 12.85 14.23 14.98 13.18

Proline 3.82 4.04 3.56 3.64 4.04 3.78 3 .6 6

Glycine 4.42 4.05 3.69 3.71 5.38 4 .02 3.89 Alanine 4.83 4.56 4.14 4.11 5.04 4.52 4.42 Valine 5.57 5.35 5.14 5.07 5.77 5.50 4.99

Methionine 2.57 1.91 1.55 1.80 2 . 1 2 2.04 1.39 Isoleucine 3.52 3.60 3.35 3.46 3.56 3.63 3.40 Leucine 6.35 5.95 5.53 5.78 6.35 6.25 5.71

Tyrosine 2.37 1.25 0.92 1 . 2 0 1.38 2.09 1.17

Phenylalanine 3.86 3.42 3.31 3.40 3.69 3.68 3.16

% N Recovered 83.7 82.9 74.7 77.1 88.4 85.1 73.4

% Crude Protein 6.39 6 . 1 1 6.62 5.80 5.97 8.35 7.51 55 Table 12

Amino acid composition of Saturn and Bluebelle brown rice (g amino acid per 16.8 g N)

SATURN - 1968 BLUEBELLE 0-N 120-N 120-N ON 120-N 120-N

Tl T 1 Tl Tl AMINO ACIDS W.S, W.S. D.S.* W.S. w .s . w .s . 1968 1968 1969** Lysine 3.90 3.63 3.44 3.50 3 .25 3.29 Histidine 2.90 2.81 2.64 2.45 2.45 2.69 Ammonia 1.74 1.80 1.70 1.64 3.12 1.73 Arginine 7.72 7.82 7.33 7.28 6.98 8.04 Aspartic Acid 7.79 7.86 7.40 7.58 7.01 7.20 Threonine 3.30 3.58 3.05 3.27 4.96 2.51 Serine 4.58 5 .00 3.28 4.23 4.22 4.12 Glutamic Acid 12.75 15.26 12.38 12.43 13 .53 12.95 Pro line 4.40 3.95 3.59 3.64 3.49 3.65 Glycine 4.42 4.51 4.19 4.38 3 .94 4.13 Alanine 4.80 5.07 4.60 4.93 4.35 4.61 Valine 5.21 5.55 5.73 5.64 5.18 5.21 Methionine 1.89 1.79 1.84 1.72 1.94 1.93 Isoleucine 3.37 3.44 3.55 3.31 3.41 3.29

Leucine 5.98 6 . 2 2 5 .8 8 6 . 0 1 5 .78 6 . 0 1

Tyrosine 1.52 1.34 2.05 1.89 1.42 2.07 Phenyla la nine 3.44 3.73 3.57 3.55 3.26 3.14

% N Recovered 83.7 87.6 80.6 81.4 82.2 80.4

% Crude Protein 6.45 7.86 6.79 6 . 8 6 7.19 8.33 56

Table 13

Amino acid composition of Dawn.rice (g amino acid per 16.8 g N)

1968 1969 Milled Brown Milled O-N 120-N 0-N 0-N 120-N Tg T9 AMINO ACIDS W.S. w .s. w .s. W.S. w .s.

Lysine 3.40 3.28 3.26 2.94 3 .24

Histidine 2.51 2.57 2.59 2.16 2 .60 Ammonia 1.9i 1.78 1.56 2.16 1.83

Arginine 7.90 7,19 7.44 6 . 62 7.78

Aspartic Acid 8 . 0 0 7. 10 7.56 - 8.31 Threonine 3.21 3.11 3.38 - 3.64 Serine 4.44 4.26 4.27 - 4.62 Glutamic Acid 14.93 13. 14 14.12 - 14. 17 Pro line 3.42 3.76 3.53 3.44 3.94

Glycine 4.08 3.93 4.15 3 .6 8 4.38 Alanine 4.13 4 .74 4.32 3.88 4.93

Valine 5.61 5.39 6 . 0 1 5.31 5.77 Methionine 2 .30 2.05 2.03 2.27 2.32 Isoleucine 4.02 3.58 3.71 4. 17 3.96 Leucine 7.00 6.50 6.74 7.26 6.80 Tyrosine 2.34 1.42 1.61 1.85 2 .23 Phenylalanine 4.78 3.77 4.06 4.37 3.84

% N Recovered 8 8 . 2 81.4 84.4 - 89.1

% Crude Protein 5.84 5.81 6.74 7.02 7.21 57

Although some variation in amino acid content was evident, there did not appear to be a marked effect of variety, rate of nitrogen,

« time of nitrogen application, or method of seeding on amino acid content, even though there were considerable differences in the protein content.

These results are in agreement with those obtained by Houston et al. (1969) who found that despite a 60 percent variation in protein content, there was relatively little variation in amino acid content of milled rices.

Effect of Nitrogen

In general, increased levels of nitrogen fertilizer did not increase lysine content (Table 14 and Figure 9). Actually, in most cases, lysine content was lower where 1 2 0 pounds nitrogen was applied.

Threonine showed a response similar to lysine. Glutamic acid, on the other hand, was usually considerably higher with increased nitrogen fertilization.

Effect of Milling

There was a considerable loss of crude protein during the milling process. Because of the heterogeneous distribution of the proteins in the rice kernel, with this loss of protein there was also a corresponding loss of certain amino acids (Tables 15 and 16). Not only did the outer layers of the rice kernel contain higher percentages of protein, but the 58 Table 14

Amino acid content of comparable rice samples as affected by rate and time of nitrogen application^ (g amino acid per 16.8 g N)

AMINO ACIDS 0-N 80-N, T: 120-N, 120-N, Tg

Lysine 3.29 3.39 3.00 3.27

Histidine 2.59 2.71 2.31 2 .82

Arginine 7.80 5.87 6.85 8 . 2 2 Aspartic Acid 7.36 7.50 7.00 7.86 Threonine 3. 12 3.35 3.06 3.17 Serine 4.62 4.29 4.01 4.44

Glutamic Acid 9.70 13.98 13.09 13.58

Proline 3.71 3.80 3.65 3.86

Glycine 4.12 4.05 3.81 4.80

Alanine 4.56 4.47 4.30 4.86

Valine 5.01 5.30 5.11 5.53

Methionine 2 . 1 2 2 . 1 2 1 . 6 6 2.42

Isoleucine 3.34 3.60 3.31 3.51

Leucine 6 . 1 0 6 . 1 2 5.72 6.29 Tyrosine 1.99 1.56 1.18 1.91

Phenylalanine 3.65 3.49 3.32 3.73

Crude Protein 6.23 6.13 6.65 6 . 1 0

^Data used are averages of the following treatments used in comparison: Saturn milled, water-seeded, 1968 and Bluebelle milled, water-seeded, 1968. iue . yie n henn cnet o ne s fetd y aiu treatments. various by affected as nee of contents threonine and Lysine 9. Figure Qrams Amino Acid per 16.8 grams N I yieTroie yie henn Lsn Threonine Lysine Threonine Lysine Threonine Lysine ______Brown i i ______-1969 r- i i ______I 60 Table 15

Average amino acid composition of protein for 18 samples of milled rice and 7 samples of brown rice (g amino acid per 16.8 g N)

AMINO ACID MILLED BROWN

Lysine 3.19 - 0.228 3.46 * 0.219

Histidine 2.53 ± 0.253 2.65 ± 0.157 Ammonia 1.76 ± 0.182 1.90 - 0,504 Arginine 7.13 ± 1.04 7.52 ± 0.336

Aspartic Acid 7.53 ± 0.573 7.49 ± 0.283 Threonine 3.16 ± 0.438 3.44 ± 0.697

Serine 4.33 ± 0.548 4.32 ± 0.347

Glutamic Acid 13.52 ± 1 .6 0 13 .35 ± 0.970

Pro line 3.77 ± 0.278 3.75 ± 0.299

Glycine 4.12 t 0.424 4.25 ± 0.184

Alanine 4.50 ± 0.444 4.67 ± 0.261

Valine 5.38 ± 0.442 5.50 ± 0.294

Methionine 2.05 ± 0.342 1 . 8 8 ± 0.096

Isoleucine 3.62 ±0.322 3.44 ± 0.136

Leucine 6 . 1 1 ± 1 . 0 2 6.09 ± 0.294

Tyrosine 1.77 ± 0.493 1.70 t 0.279

Phenylalanine 3.71 ±0.445 3.55 ± 0.282 61 Table 16

Amino acid content of comparable samples of brown and milled rice1 (g amino acid per 16.8 g N)

AMINO ACIDS BROWN MILLED Mean difference

Lysine 3.57 3 .14 S

Histidine 2 .65 2 ,45 NS

Arginine 7.45 7.32 NS

Aspartic Acid 7.56 7.18 NS

Threonine 3.78 3.09 NS

Serine 4.51 4.32 NS

Glutamic Acid 13.49 11.38 NS

Proline 3.87 3.68 NS

Glycine 4.31 3 .96 NS

Alanine 4.83 4.43 NS

Valine 5.15 5.06 NS

Methionine 1.84 1.89 NS

Isoleucine 3 .38 3.32 NS

Leucine 6 . 0 0 5.91 NS Tyrosine 1.54 1.59 NS

Phenyla la nine 3.50 3.50 NS

% Crude Protein 7.09 6.44

1Data used are averages of the following treatments: Saturn, water- seeded, 0-N, 1968; Bluebelle, w ater-seeded, 0-N, 1968; Saturn, water-seeded, 120-N Ti, 1968; Bluebelle, water-seeded, 120-N Tj. 62

protein also contained higher percentages of the water-soluble albumin

than did the milled kernel. As the protein fraction albumin is reported

relatively high in lysine, it would be expected that total protein in the

outer layers would contain a higher percentage of lysine than does the

predominant protein, glutelin, found largely in the endosperm (Jullano,

1966; Cagampang, 1966; Houston et al_. , 1968).

In this Investigation, contents of both lysine and threonine,

first and second limiting amino acids, respectively, in rice, were

markedly reduced during the milling process (Figure 9). Houston et al.

(1969) found similar results for lysine content of Calrose, a California

short-grain rice. Table 15 shows the average values for amino acid

contents of 18 samples of milled and 7 samples of brown rice.

Average contents of lysine, histidine, arginine, threonine, glycine,

and valine decreased upon milling.

Effect o£ Year

There was a large and consistent protein increase in 1969 com­

pared to 1968 (Tables 2 and 17). Amino acid contents were also

generally greater In 1969 (Table 17). Based on relatively few samples,

aspartic acid, valine, and tyrosine were significantly higher in 1969,

and the difference in several others, including lysine, approached

significance. Threonine was one of the few amino acids that showed

little or no difference between years (Figure 9). 63

Table 17

Amino acid content of comparable milled rice samples in 1968 and 19691 (g amino acid per 16.8 g N)

AMINO ACIDS 1968 1969 Mean difference Lysine 3.09 3 .22 NS2

Histidine 2.40 2.61 NS

Arginine 6.96 7.68 NS

Aspartic Acid 7.03 7.88 S3

Threonine 3.08 3.02 NS Serine 4.10 4.04 NS

Glutamic Acid 13.11 13.59 NS

Pro line 3. 69 3.82 NS

Glycine 3 .85 4.11 NS

Alanine 4.45 4.68 NS

Valine 5.20 5.59 S

Methionine 1.79 2 .19 NS

I s ole u cine 3.40 3.72 NS

Leucine 5.90 5 .20 NS

Tyrosine 1.26 2.07 S

Phenylaline 3.47 3.83 NS

% Crude Protein 6.37 7.77 S

1 Treatments used in comparison were: Saturn milled, water-seeded, 120-N, Tj; Bluebelle milled, water-seeded, 120-N, T^; Dawn milled, water-seeded, 120-N, T9 2Mean difference nonsignificant. 3Mean difference significant. 64

Relationship of Amino Acids to Protein Content

The relationship between amino acid and protein content was studied for 25 samples. Correlation coefficients between each of the amino acids and protein content were determined, but no significant relationship was observed.

Although not statistically significant, a decrease in lysine content accompanied an increase in protein content for both milled and brown rice. Histidine and leucine followed the same pattern.

There was a tendency for threonine, glutamic acid, valine, isoleucine, and tyrosine to increase as the protein content increased. Several investigators (Kymal, 1955; Juliano et a± ., 1964; Caqampang et a l . .

1966; Houston et a l., 1969) have reported significant decreases in lysine content as protein content increased. In an extensive study of rice protein and amino acid composition of high and low protein rices conducted at the International Rice Research Institute (IRRI, 1969), protein of rice samples differed only in their lysine content. It should be kept in mind that even though the amino acid content of total protein decreases with an increase in protein content, the amount of the amino acid per unit weight of grain usually shows a net increase as a result of the overall increase in protein content. Such was the case in this study where there were definite differences in protein content due to several of the treatments (timing of nitrogen application and 65 year), but no significant decrease in lysine content of the protein as a result of these treatments.

Values for amino acids obtained in this study are within the range of amino acid values reported by other investigators using both auto­ matic analyses and microbiological methods of assay (Table 18). Only the value for phenylalanine was lower than the range of values reported.

Complete agreement cannot be expected because of the many different variables involved in separate investigations. A few of the factors causing variation in amino acid composition are differences in varieties, location, year, degree of milling, cultural practices (such as fertilization), and other environmental conditions (Kymal, 1955;

Brandemer and Evans, 1963; Normand et a l . , 1966).

Differences in methodology, particularly of the hydrolysis procedure, is a principal cause of variation in amino acid results

(Kohler and Palter, 1967; Houston et a l., 1969). There is a real need for cooperative research on analysis of rice for amino acid composition.

Perhaps a standardized hydrolysis and automated procedure would eliminate many of the questions of differences in methodology and make possible a more accurate comparison of results obtained by various investigators. Table 18

Comparative amino acid contents of milled rice (g amino acid per 16.8 g N)

______METHOD OF ASSAY______Column chromatography ______Microbiological FAO This Houston Cagampang Juliano Normand Bandemer Chancel Kik Kymal Pattern Study et al. et al. et al. and Evans AMINO ACID (1963) (1969) 19 6 6) (1964) (1966) _ (1963) (1962) (1969) (1955)

Lysine 4.2 3.2 3.7 3.0-5.0 4.0 3.7 3.7 3 .4 -3 .6 4.8 3.0-4.5

Histidine 2.5 2.4 2 . 1- 2 .9 2 . 6 2 . 8 2 . 2 2 . 2 - 2 . 6 2 . 8 -

Arginine 7.1 8.7 7.0-9.4 8.9 9.0 1 0 .1 8 .4-9.5 8.5 -

Threonine 2 . 8 3.2 3.7 3.1-4 .6 4.0 3.8 2 . 6 3 .4 -3 .6 5.7 2.8-4.7

Valine 4.2 5.4 6 . 8 3 . 6- 6 . 6 6 .2 5.9 4.5 5.8-6 .6 7.1 6 . 2 - 8 .5

Cystine - 2.7 0.3-1.5 1 .1 1 .8 1 . 6 2 . 2 - 2 .5 1.7 -

Methionine 2 . 2 2 . 1 3.0 0 .7 -2 .6 2 . 0 - 2 . 2 1 . 2 - 1 .7 3.1 0.9-1.5

Isoleu cine 4.2 3.6 4.9 3.8-5.2 4.7 4.1 2.9 4.0-4.5 5.7 3.4-5.4

Leucine 4.8 6 . 1 8.4- 7 .0 -9.8 9.5 8.4 7.1 8 .3-9.0 9.9 3.1-10.5

Tryptophan 1.4 - - 1 . 0 - 1 .5 - 1.4 -- 2.4 1.6-3.0

Phenylalanine 2 . 8 3.7 5.5 4.6-7.1 6 .1 5.4 4.5 5.2-5.7 4.5 3.9-6.7

% Crude Protein 5 .7-7.6 6.2-14.3 7.0 5.8-8.9 (dry wt) SUMMARY AND CONCLUSIONS

Protein and amino acid contents of three rice varieties as affected by various environmental conditions were studied. In almost every case, protein content was increased by adding nitrogen fertilizer.

Protein content varied among the varieties, with Bluebelle generally showing the highest protein values for both brown and milled rice in 1968 and 1969. Dawn usually had the lowest protein content.

Saturn was especially responsive to applied nitrogen with protein increases resulting from both 80 and 120 pound nitrogen rates.

For Dawn and Bluebelle varieties, much of the Increased protein resulting from nitrogen fertilization was apparently removed during the milling process.

Protein content was much higher in 1969 than in 1968 for all varieties and all treatments. When all three varieties were considered, it was evident that more protein was usually found in the grain when all the nitrogen was applied at seeding (T^), or where half the nitrogen was applied at seeding and half applied late in the season at the 2 mm panicle stage (Tg).

There was an inverse relationship between grain yield and protein content in the 1968 and 1969 crops. In 1968, grain yield was higher and protein content lower than in 1969. This inverse relationship

67 68 resulted in the yield of protein being relatively constant for both years.

The yield of protein per acre did not differ as much among treatments, varieties, and years as did protein content and grain yield. Different rates of nitrogen fertilizer had the greatest effect on protein yield. Drill-seeding of rice produced slightly higher grain yields and a lower protein content than did water-seeding.

Amino acid analyses showed that although some variation in amino acid content was evident, there did not appear to be a marked effect of variety, rate and time of nitrogen application, or method of seeding on amino acid content, even though there were considerable differences in protein content.

Contents of lysine and threonine, first and second limiting amino acids, respectively, in rice, were markedly reduced during the milling process. Although not statistically significant, a decrease in lysine content accompanied an increase in protein content for both milled and brown rice. There was a large and consistent protein increase in 1969 compared to 1968, and contents of several amino acids were also greater in 1969.

Results of this study showed that appreciable increases in protein content and yield of rice can be expected to occur as a result of different rates and times of application of nitrogen fertilizer.

Larger differences in protein content and yield of rice can also be 69 expected as a result of differences in environmental conditions associated with different seasons. Although some differences in amino acids occurred in this study, these were of much less magnitude than were differences in the protein content and yield.

It would be of enormous benefit to extend these studies over several years to ascertain trends in the effect of environmental con­ ditions on composition of rice. Also of importance would be studies aimed at elucidating inherent differences and characteristics of protein and amino acid content of the rice genetic pool as influenced by these same environmental variables. SELECTED BIBLIOGRAPHY

Association of Official Analytical Chemists. 1965. Official Methods o_f Analysis. 10th ed. The Association: Washington, D. C.

Bandemer, S. L. and R. J. Evans. 1963. Nutrients in seeds. The amino acid composition of some seeds. J. Agr. Food Chem. 11:134.

Barber, S. 1967. Third Conference on Intern. Problems of Modem Cereal Processing and Chemistry, Inst, fur Gertreideverarbeitung, Berghalz-Rehbrucke, German Democratic Republic, June 11-17.

Bressani, R. and E. T. Mertz. 19 58. Studies on com proteins. IV. Protein and amino acid content of different com varieties. Cereal Chem. 35:227.

Bressani, R. and T. Valiente. 1962. All-vegetable protein mixtures for human feeding. VII. Protein complementation between polished rice and cooked black beans. J. Food Sci. 21:401.

Cagampang, G. B. , L. J. Cruz, S. G. Espiritu, R. G. Santiago, and B. O. Juliano. 1966. Studies on the extraction and composition of rice proteins. Cereal Chem. 43:145.

Chen, S. C. S., C. Kies, and H. M. Fox. 1966. Nitrogenous factors affecting nutritional value of rice protein for human adults. Fed. Proc., 25, No. 2, Part 1:300.

Committee on Protein Malnutrition. 1963. Evaluation of protein quality. National Academy of Sciences - NRC Pub.. 1100. Washington, D.C.

Coons, C. M. 1968. Selected references on cereal grains in protein nutrition. Human and experimental animal studies of major and minor cereals, 1910-1966. U.S. Dept. Agr. Pub. ARS 61-5. Washington, D. C.

Del Rosario, Aurora R., Vivian P. Briones, Amanda J. Vidal, and B. O. Juliano. 1968. Composition and endosperm structure of develop­ ing and mature rice kernel. Cereal Chem. 45:225.

Garcha, J. S. and A. K. Chopra. 1968. Amino acid composition of different varieties of rice (Qrvza satlval grown in the Punjab. J. Nutr. Dietetics 5:13. 70 71

Ghosh, A. K,, M. Naimudeen, and S. Govindaswami. 1968. Study on the quality characteristics of rice. I. Ten varieties from Bihar and four high-yielding varieties from Taiwan. Gryza, Vol. 5, No. 1.

Grist, D. H. 1959. Rice . 3rd ed. London: Longmans, Green and Co.

Hogan, J. T., F. L. Normand, and H. J. Deobald. 1964. Method for removal of successive surface layers from brown and milled rice. RiceJ. 67(4):27.

Houston, D. F., A. Mohammad, and Norma E. Alfonso-Herna nde z. 1964. High-sulfur seed proteins. Cereal Chem. 41:427.

Houston, D. F., T. Iwasaki, A. Mohammad, and L. Chen. 1968. Radial distribution of protein by solubility classes in the milled rice kernel. J. Agr. Food Chem. 16:720.

Houston, D. F., Marian E. Allis, and G. O. Kohler. 1969. Amino acid composition of rice and rice by-products. Cereal Chem. 46:527.

Houston, D. F. and G. O. Kohler, 1970. Nutritional properties of rice. National Academy of Sciences - NRC, Washington, D. C.

Howe, E. E., G. R. Jansen, and E. W. Gilfillan. 1965. Amino acid supplementation of cereal grains as related to the world food supply. Am. J. Clin. Nutr. 16:315.

Hubbard, J. E., H. H. Hall, and F. R. Earle. 1950. Composition of the component parts of the sorghum kernel. Cereal Chem. 2 7:415.

Ignacio, C. C. and B. O. Juliano. 1968. Physicochemical properties of brown rice from Oryza species and hybrids. J. Agr. Food Chem. 16:125.

International Rice Research Institute (IRRI) Annual Report. 1963. Los Banos, Laguna, Philippines.

International Rice Research Institute (IRRI) Annual Report. 1964. Los Banos, Laguna, Philippines.

International Rice Research Institute (IRRI) Annual Report. 1965. Los Banos, Laguna, Philippines. 72

International Rice Research Institute (IRRI) Annual Report. 1966. Los Banos, Laguna, Philippines.

International Rice Research Institute (IRRI) Annual Report. 1967. Los Banos, Laguna, Philippines.

International Rice Research Institute (IRRI) Annual Report. 1968. Los Banos, Laguna, Philippines.

International Rice Research Institute (IRRI) Annual Report. 1969. Los Banos , Laguna , Philippines .

Jones, D. B., A. Caldwell, and K. D. Widness. 1948. Comparative growth promoting values of the proteins of cereal grains. J. Nut. 35:639.

Jones, J. W,, L. Zeleny, and J. W. Taylor. 1946. Effect of parboiling and related treatments on the milling, nutritional, and cooking quality of rice. U. S. Dept. Agr. Circular No. 572 . Washington, D. C.

Juliano, B. O., Compiler. 1966. Physicochemical data on the rice grain. Intern. Rice Res. Inst. Tech. Bull. No. 6 . Manila.

Juliano, B. O., Gloria B. Cagampang, L. J. Cruz, and R. G. Santiago. 1964. Some physicochemical properties of rice in Southeast Asia. Cereal Chem. 41:275.

Juliano, B. O. , G. M. Bautista, J. C. Lugay, and A. C. Reyes. 1964. Studies on the physicochemical properties of rice. J. Agr. Food Chem. 12:131.

Juliano, B. O., C. C. Ignacio, V. M. Panganiban, and C. M. Perez. 1968, Screening for high protein rice varieties. Cereal Sci. Today 13:299.

Kik, M. C. 1939. The biological value of the proteins of rice and its by - products . Cerea 1 Chem. 16:440.

Kik, M. C. 1940. The nutritive value of the proteins of rice andits by-products. II. Effect of amino acid additions on growth. Cereal Chem. 17:473.

Kik, M. C. 1941. The nutritive value of the proteins of rice and its by-products. III. Amino acid content. Cereal Chem. 18:349, 73

Kik, M. C., and R. R. Williams. 1945. The nutritional improvement of white rice. Nat. Res. Council Bull. 112, p. 76.

Kik, M. C. 1956. Nutrients in rice bran and rice polish and improve­ ment of protein quality with amino acids. J. Agr. Food Chem. 4:170.

Kik, M. C. 1957. The nutritive value of rice and its by-products. Ark. Exp. Station Bull. No. 589, Fayetteville.

Kik, M. C. and V. L. Hall. 1961. The nutritive value of rice. Ark. Farm Research, Agr. Exp. Stat. Vol. 10, No. 4, p. 11.

Kik, M. C. 1965. Nutritional improvement of rice diets and effect of rice on nutritive value of other foodstuffs. Ark. Agr. Exp. Station Bull. No. 698.

Kohler, G. O. 1967, In World Protein Resources, ed. by A. M. Altschul. Washington, D. C.: American Chemical Society, p. 247.

Kohler, G. O. and Rhoda Palter. 1967. Studies on methods for amino acid analysis of wheat products. Cereal Chem. 44:512.

Kymal, P. Krishna. 1955. Variation in amino acid content of rice varieties. Ph.D. Dissertation. La. State Univ., Baton Rouge.

Lain, E. R. and E. S. Rodriguez. 1965. Subproductos del arroz. I. Contenido en principios lnmediatos. II. Contenido en amino acides. III. Calidad proteica - Indices de Mitchell. Block Y Oser. Rev. Nutr. Animal 3:92.

Lozsa, A. and K. Roller. 1953. Estimation of the biological value of rice proteins. Acta Physiol. Acad. Sci. Hung. 5:477.

Lyman, C. M. , K. A. Kuiken, and F. Hale. 1956. Essential amino acid content of farm feeds. J. Agr. Food Chem. 4:1008.

McCall, E. R., C. L. Hoffpauir, and D. B. Skau. 1951. The chemical composition of rice. A literature review. U.S. Dept. Agr. Publication AIC-312 . Washington, D. C.

McCall, E. R., J. F. Jurgens, C. L. Hoffpauir, W. A. Pons, Jr., S. M. Stark, A. F. Cucullu, D. C. Helnzelman, V. O. Cirino, and M. D. Murray. 1953. Composition of rice. Influence of variety and environment on physical and chemical composition. J. Agr. Food Chem. 1:988. 74

McDermott, E. E, and J. Pace. 1960. Comparison of the amino acid composition of the protein in flour and endosperm from different types of wheat, with particular reference to variation in lysine content. J. S cl. Food Agr. 11:109. McElroy, L. W ., D. R. Clandirin, W. Lobay, and S. I. Pethybridgo. 1949. Nine essential amino acids in pure varieties of wheat. J. Nut. 37:329. Mandal, B. B. and I, C. Mahapatra. 1968. Studies on the cultural and manurial requirements of some varieties of rice. Oryza, J. of Assoc, of Rice Res. Workers. Vol. 5, No. 1. Merrill, D. F. 1958. Composition of cereal grains and forages . National Academy of Sciences - NRC Pub. No. 585. Washington, D. C.

Mertz, E. T., L. 6 . Bates, and O. E. Nelson. 1964. Mutant gene that changes protein composition and increases lysine content of maize endosperm. Science 145:279. Michael, G., B. Blume and H. Faust. 1961. Die Eiweissqualitat von Kornen verschiedner Getreidearten in Abhangigkeit von Stickstoffversorgung und Entwicklungzustand.2. Pflanzenernaehr. Dueng. Bodenk. 92:106-116. Mitchell, H. H. 1924. The biological value of proteins at different levels of intake. J. Biol. Chem. 58:905. Morris, V. H ,, T. L. Alexander, and Et D. Pascoe. 1946.Studies of the composition of the wheat kernel. II. Distribution of ash and protein in central and peripheral zones of whole kernels. Cereal Chem. 23:540. Morris, V. H., T. L. Alexander, and E. D. Pascoe. 1945. Studies of the composition of the wheat kernel. I. Distribution of ash and protein in center sections. Cereal Chem. 22:351. Normand, F. L., D. M. Soignet, J. T. Hogan, and H. J. Deobald. 1966. Content of certain nutrients and amino acids pattern in high-protein rice flour. Rice J. 69:13.

Normand, Floyd L ., Joseph T. Hogan, and Harold J. Deobald. 1965. Protein content of successive pheripheral layers milled from wheat, barley, grain sorghum, and glutinous rice by tangential abrasion. Cereal Chem. 42:359. 75

Orr, M. L. and B. K. Watt. 1957. Amino acid content of foods. Home Economics Res. Report No. 4. ARS, U. S. Dept. Agr. Washington, D. C.

Osborne, T. B. and L. B. Mendel. 1914. Nutritional properties of proteins of maize kernel. J. Biol. Chem. 18:1.

Ozaki, K. and M. Moriyama. 1953. The nitrogen metabolism of rice plant. I. Protein content and quality of hulled rice from the plant to which nitrogen was applied at heading. Nippon Dojo- Hiryogaku Zasshl 23:146.

Palmiano, Evelyn P., A. M. Almazan, and B. O. Juliano. 1968. Physicochemical properties of protein and developing and mature rice grain. Cereal Chem. 45:1 .

Parial, Lucila C ., L. W. Rooney, and B. D. Webb. 1970. Use of dye-binding and biuret techniques for estimating protein in brown and milled rice. Creal Chem. 47:38.

Patrick, W. H. , Jr., F. J. Peterson, J. E. Seaholm, M. D. Faulkner, and R. J. Miears. 1967. Placement of nitrogen fertilizers for rice. La. Agr. Exp. Station Bull. No. 619,

Patwardhan, V. H. 1957 . Nutritive value of cereal and pulse proteins. Cited from L. Voris, ed. National Academy of Sciences - NRC Pub. No. 843. Meeting protein requirements and their fulfillment in practice. Proceedings of a Conference in Princeton, United States, 1955, Rome. 1957.

Pence, J. W. and A. H. Eider. 1953. The albumin and globulin proteins of wheat. Cereal Chem. 30:2 75.

Portch, S., A. F. Mackenzie, and H. A. Steppler. 1968. Effect of fertilizers , soil drainage, class and year upon protein yield and content of oats. Agron. J. 60:672 .

Primo, E., A. Casas, S. Barber, and C. Benedito de Barber. 1962. Factors de calldad del arroz. V. Determinacion de una capa externa diferenciada en el arroz cocido. Revista de Agroquimica y Technologla de Alimentos 2:130.

Prlmo, E., A. Casas, S. Barber, and C. Benedito de Barber. 1963. Factors de calidad del arToz. IV. Distribucion del nitrogeno en al endospernio. Revista de Agroquimic y Technologla de Alimentos 3:22. 76

Ramiah, K. and Mudaliar C. Rajasekhara. 1939. The proteins in the rice grain and the development of the aleurone layer. Ind. J. Agr. S ci. 9:39 .

Rao, M. V. 1969. Recent studies in production technology of high- yielding dwarf indica rice varieties. Oryza, Vol. 6 , No. 2.

Rosenheim, O. and S. Kajiura. 1908. The proteins of rice. J. Physiol. 36:54.

Russell, Sir E. John. 1954. Soil Conditions and Plant Growth, 8th e d . Longmans, Green and Co. New York.

Schweigert, B. S. 1947. Amino acid content of feeds. I. Leucine, valine, isoleucine, and phenylalanine. J. Nutr. 33:553.

Shoup, F. K. , C. W. Deyoe, L. Skoch, M. Shamsuddin, J. Bathurst, G. D. Miller, L. S. Murphy, and D. P. Parrish. 1970. Cereal Chem. 47:266.

Simpson, J. E ., C. R. Adair, G. O. Kohler, E. H. Dawson, H. J. Deobald, E. B. Kester, J. T. Hogan, O. M. Batcher, and J. V. Halick. 1965. Quality evaluation studies of foreign and domestic rices. Tech. Bull. No. 1331. ARS, U. S. Dept. Agr., Washington, D. C.

Skoch, L. V., C, W. Deyoe, F. K, Shoup, J. Bathurst, and D. Liang. 1970. Protein fractionation of sorghum grain. Cereal Chem. 47:472.

Snedecor, G. W. 1956. Statistical Methods, 5th ed. The Iowa State University Press, Ames, Iowa.

Sreenivasan, A. 1941. Quality in rice. Calcutta Med. J. 38:107. Abstracted in Chem. Abs . 36:3858.

Stevenson, C. K. and C. S. Baldwin( 1969. Effect of time and method of nitrogen application and source of nitrogen on the yield and nitrogen content of corn. Agron. J. 61:381.

Sturgis, F. E., R. J. Miears, and R. K. Walker. 1952 . Proteinin rice as influenced by variety and fertilizer levels. La. Agr. Exp. S ta. Bull. No. 466:1-27. 77

Subrahmanyan, V. , A. Sreenlvasan, and H. P. Das Gupta. 1938. Studies In quality of rice. I. Effect of milling on the chemical composition and commercial qualities of raw and parboiled rice. Indian J. Agr. Sci. 8:459.

Sure, B. 1950. Nutritional improvement of cereal grains with small amounts of food of high protein content. Ark. Agr. Exp. Station Bull. No. 493.

Sure, B. 1955. Effect of amino acid and vitamin Bj 2 supplements on the biologic value of protein in rice and wheat. J. Am. Dietet. Assoc. 3 1:1232.

Sure, B. and F. House. 1948. The relative biological values of pro­ teins in cereal grains . J. Nut. 36:595.

Taira, H. 1962. Studies on amino acid contents in plant seed. II. Amino acid pattern of seed protein fractions of Gramineae. (English translation) Botan. Mag. (Tokyo) 75:273.

Takano, K. and M. Nozu. 1961. Proc. Crop Sci. Soc. (Japan), 29: 216.

Tamura, S. and K. Kenmochi. 1963. Studies on amino acid content of rice. III. Distribution of amino acid in rice grain. (English translation) Nippon Nogeikagaku Kaishi 37:753.

Tamura, G ., T. Tsunoda, J. Kirimura, and S. Miyazawa. 1952. J. Agr. Chem. Soc. (Japan), 26:480.

Tsukasa, A. 1960. Med. J. Fukuoka 51:1217. Data from B. O. Juliano, 1966, IRRI Tech, Bull. No. 6 .

U. S. Production and Marketing Administration. 1962. Rice Inspection Manual. AMS GR Instruction No. 918-2, (revised).

Watt, B. K. and A. L. Merrill. 1963. Composition of foods. Agr. Handbook No. 8 , revised. U. S. Dept. Agr., Washington, D. C.

Webb, B. D. 1967. Cooking and processing qualities required of rice varieties in the United States - Their evaluation in rice breeding programs. International Rice Commission Newsletter, Special Issue p. 116.

Wilson, Emmett and F. J. Peterson. 1969. Timing and midseason nitrogen at developmental stages of the rice plant. 61st Annual Progress Report, Rice Exp. Station, La. State Univ. VITA

Ruth Martin Patrick was born jn Darnell, Louisiana on

November 4, 1930. Sho was graduated from the public high school

in lake Providence, Louisiana in 194b. She attended Northeast Junior

College (L.S.U.) in Monroe, Louisiana and earned highest honors. In

September, 1948, she entered Louisiana State University, and was

granted a Bachelor of Science degree in Home economics m June, 1950.

She received the Master ol Science degree in Pood and Nutrition in

August, 1951 from L .S.U .

The author worked as a member ol the nutrition research staff of

the Louisiana State University Home Lconomics Department from Septombe

1951 until July, 1953, and served the; same department as an assistant

professor in 1954, and for the academic year 1966-19 67.

During 1958-59, the author completed requirements tor teacher

certification in home economics, chemistry, biology and general science

at L.S.U.

In September, 1967, she was accepted as a graduate assistant

in the 1'ood Science Department of Louisiana State University, and

was awarded a National Science Foundation Traineeship1970. in

The author is married to William H. Patrick,Jr., Professor of

Agronomy atL.S.U. , and they are the parents of two daughters, Terry

and Carol, and two sons, Billy and Henry. 79

She is a member of Phi Kappa Phi, Sigma Xi, Iota Sigma Pi, Phi

Upsilon Omicron, Phi Lamda Pi, Gamma Sigma Delta, the Institute of Food Technologists, and the American Society of Cereal Chemists.

In November, 1970 the author accepted a position with the L.S.U,

Cooperative Extension Service as an Extension Assistant (Nutiition).

She is currently a candidate for the Degree of Doctor of Philosophy. EXAMINATION AND THESIS REPORT

Candidate: Ruth Martin Patrick

Major Field: Food Science

Title of Thesis; Protein and Amino Acid Content of Rice as Affected by Environmental Modifications.

Approved:

Major Professor and Chairman

Dean ill the tiraduato St lux11

EXAMINING COMMITTEE:

A ■

iu - !V

CL .7. YLnra

Date of Examination:

May 12. 19 71