California State University, Northridge

CALIFORNIA STATE UNIVERSITY, NORTHRIDGE

FOLIC ACID IN EYGYPTIAN VEGETABLES: EFFECT OF DRYING METHOD AND STORAGE ON FOLACIN CONTENT OF t1ULUKHIYAH (CORCHORUS OLITERIUS)

A thesis submitted in partial satisfaction of the requirements for the degree of Master of Science in Home Economics

by Sohair Saad

August, 1980 The Thesis of Sohair Saad is approved:

Tung-Shan Chen, Ph.D., Chairman

California State University, Northridge

i i DEDICATION

To t~y Son Mike

And ~1y Family

; ; ; ACKNOl~LEDGB1ENT

I wish to thank those who worked closely with me and contri buted their knowledge and energy toward the achievement of this goal.

I would especially like to acknowledge the members of my graduate committee:

Dr. ~1arjory Joseph, to whom I owe my knowledge of statistical analysis, and who helped me to solve the statistical problems through out this research. Marjaret Anita King, who taught me initially how to assay for folacin and who gave encouragement and support throughout the duration of this research.

Dr. Tung-Shan Chen, to whom I give special recognition for not only serving as my major advisor, but also for the patience, guidance, and assistance given to me throughout this research.

I would like to thank Seija Hurme, Linh D. Nguyen, and Cliff

Lui, who served as friends and coworkers, and with whom I was able to share ideas which helped to make this project a success.

I gratefully thank my brother, Raed, who acted as a chemistry advisor, consultant, sounding board and optimist throughout this en terprise. And finally I especially thank my mother, Angele Yossef, with out whose patience, understanding, unqualified encouragement, and helping, I would never have attempted nor accomplished this project.

iv TABLE OF CONTENTS

Page

DEDICATION ••• . . . . . iii AC KNOVJLEDGMENTS iv LIST OF TABLES • . . . . vii LIST OF FIGURES • • • viii ABSTRACT ix Chapter

I. INTRODUCTION 1

Objective • 2 Limitations 3 Definition of Terms 3 II. LITERATURE REVIEW Folacin • • • ...... 5 Dehydration . 8 Destruction of Vitamins During Drying and Storage • • • • • • • 10 Mulukhiyah ••••• . . . . 18 III. MATERIALS AND METHODS . . . . . 19

Vegetable • . . 19

~~icroorganism . . . . 19 Ba sa 1 Med i urn • • . . 19 Chemical Reagents . . . . 19

v Chapter Page Equipment • . . . . 20 Methods 20

Sample Preparation . • 20

Drying r~ethods . . . . 20 Storage Test ...... 22 Moisture Determination . . . . 23 Folacin Determination ...... 23 Microbiological Assay Procedure 24 Data Treatment ...... 36 IV. RESULTS AND DISCUSSION Folacin Activities in Mulukhiyah ...... 38 Effect of Drying Methods on Weight Retention and Moisture Content of Mulukhiyah • • 41

Destruction of Folacin During Dehydration 45 Effect of Storage Condition on Folacin Retention in Dried Mulukhiyah ••• 62

V. SUt~MARY, CONCLUSIONS, AND RECOMMENDATIONS 77 REFERENCES ...... 80 APPENDICES A. Preparation of Chemical Solutions and Culture Media ••••••••••• 88 B. Preparation of Hog Kidney Conjugase 90 C. Preparation of Folic Acid STandard Solutions • 93

vi LIST OF TABLES

Table Page

1. Sampling Schedule During Drying of Vegetable by Three Dehydration ~1ethods • . . . . 21 2. Preparation of Assay Tubes for Folic Acid Standard Curve • . • • • • • • • • 28 3. Preparation of Folacin Assay Tubes for Mulukhiyah Samples • • • • • • • • • • • • • • • • • • 35

4. Folacin Content of t~ulukhiyah and Some other Folacin Rich Vegetables • • • • • • • • 39 5. Weight Retention and Changes in Moisture Content of Mulukhiyah During Drying and Process by Three Drying Methods • • • • • • • • • • • • • . • 42 6. Total and Free Folic Acid Content and Retention in Mulukhiyah During Freeze, Tray and Room Drying • • 46 7. Effect of Packaging Condition on Free and Total Folic Acid Retention in Dehydrated Mulukhiyah 69 8. Summary of Tukey's HSD for Comparison of Means of FFA and TFA Retention in Dried Mulukhiyah stored under Different Conditions at Various Storage Times • • • • • . • • . • • . • • • • • • • • • 75

vii LIST OF FIGURES Figure Page 1. Structure of Pteroylglutamic Acid • • • • 6 2. Schematic Diagram of Assay Procedure • • • • • • • 26 3. A Typical Folic Acid Standard Curve of L. casei 11 11 Linear Plot • • • • • • • • • • • • • • 31 4. Semi-log Plot of Folic Acid Standard Curve of L. casei • • • • • • . • • • • • • • • • • • 33 5. Semi-log Plot of Drying Time (hours) Versus Moisture Content g/g solid • • • • • • . • • 44 6. Percent Folacin Ret ntion in Mulukhiyah during Freeze Drying at 32 0 C • • • . • • • • • • • • 49 7. Percent Folacin R@tention in Mulukhiyah during Tray Drying at 50 C • • • • • • • • • • • • • • • 51 8. Percent Folacin Retention in Mulukhiyah during Room Drying at 25° C • • • • • • • • • • • • • 53 9. The Rate of Total Folic Acid Destruction Plotted Against the Percent Moisture Content • • • • • • . 56 10. The Effect of Drying on Folacin Retention in Mulukhiyah by the Three Drying Methods, Percent Total Folacin Retention Is Plotted Against Moisture Content • • • • • • • • • • • • • • • 61 11. Effect of Packaging Condition on TFA Content of Dried Mulukhiyah During Storage at Room Temperature • • • • • • • • . • • • • • • • • • 64 12. Effect of Packaging Condition of FFA Content of Dried Mul ukhiyah !).Iring Storage at Room Temperature • • • • • • • • • • • • • • • • • • 66

viii ABSTRACT

FOLIC ACID IN EGYPTIAN VEGETABLES: EFFECT OF DRYING METHOD AI·JD STORAGE ON FOLACIN

CONTENT OF ~ULUKHIYAH (CORCHORUS OLITERIUS) by Sohair Saad Master of Science in Home Economics

The effect of three drying methods (room, tray, and freeze drying) moisture content, and packaging during storage on folacin con tent of mulukhiyah, which is one of the most common vegetables in Egypt, was investigated.

It was found that fresh mulukhiyah contained 556 meg free folic acid (FFA) and 800 meg total folic acid (TFA) per 100 g fresh weight basis, and room dried mulukhiyah contained 662 meg FFA, and 1138 meg TFA per 100 g dry weight. Therefore, mulukhiyah is an excel lent source of folate in the Egyptian diet. The destruction of folic acid in mulukhiyah was high in all three drying methods studied. The retention of FFA ranged from 34 to

ix 42 percent, and TFA ranged from 42 to 48 percent. Retention of TFA was always higher than that of FFA during drying because 70 percent of the TFA in mulukhiyah is FFA, which is more sensitive to heat than total folacin. Freeze drying method resulted in higher folacin reten tion than the other drying methods. Approximately 10 percent more TFA was preserved by the freeze drying method than that retained by the room drying method while the differences in folacin retention between the tray drying and the room drying methods was 7 percent. The retention of folacin in room dried mulukhiyah during storage varied depending on the packaging method. There was a signifi cant difference (r~O.Ol) in folacin retention between mulukhiyah packed in colored or clear jars under nitrogen or air atmosphere, and length of storage time. There was a significant decrease in folic acid retention with storage time of 48 weeks under all the conditions. Mulukhiyah stored in brown jars packed under nitrogen had the highest retention of folic acid followed by the brown jars with air. The clear jars with nitrogen had similar folic acid retention as that stored in brown jars with air. Severe destruction of folic acid occurred in clear jars with air atmosphere. It is recommended that room drying be continued as a home dehydration method for mulukhiyah because it causes insignificant higher folacin loss than the other methods. Tray drying might be ad opted on a commercial level while freeze drying would be too costly for the purpose of folacin retention. It is also recommended that dried mulukhiyah be stored in colored containers and, if possible, under nitrogen packing.

X Chapter 1 INTRODUCTION A study carried out in countries of the Eastern Mediterranean region has shown that nutritional anemias are the most serious and widespread nutritional disorders in 9 countries of the region including Egypt (Rao, 1974). In the vulnerable groups such as infants and preg nant and lactating women, the proportion of anemic individuals seems to reach 70 to 90 percent. Although iron deficiency anemia is the pre dominant type, other types attributable to deficiencies of folate, vitamin s12 , protein, etc. are also prevalent. Halsted et al. (1969) reported that the anemia of Kwashiorkor in Cairo is usually megaloblas tic and is responsive to a combination of dietary protein with supple mental iron and folic acid. Green vegetables are generally considered to be good sources of folic acid. The only green vegetable consumed in large quantities in Arab Middle East is Jew's mallow (Corchorus oliterius) or mulukhiyah in Arabic (Patwardhan and Darby, 1972). This vegetable is popular all over Egypt. When available in season, it is bought in quantities, and the leaf separated from the stalk, dried, and stored. The dried muluk hiyah is soaked in water, cooked with salt and some sour lime juice, and eaten with bread or rice. Those who can afford it cook the vege table with meat. In spite of its popularity, information on folacin content of mulukhiyah is lacking. Folacin content of foods is greatly affected by conditions

1 2 associated with processing, storage and preparation (Malin, 1975). Since sun drying (or room drying) of vegetables is common in Egypt (Patwardhan and Darby, 1972), better drying methods for nutrient pre servation need to be developed. There are few studies on the effects of dehydration methods and storage conditions on folic acid retention in vegetables found in the literature. The purpose of this study was to generate quantitative data on folacin content of mulukhiyah, which is one of the most common vege tables in Egypt, and to investigate the effect of packaging on the folate retention in mulukhiyah during storage.

Objectives of the Study The objective of this research was to study the effect of drying methods, moisture content, and storage conditions on folacin retention in mulukhiyah, which is commonly used in Egypt. Specifically, three different methods of drying (room, tray, and freeze drying) were compared. The room dried vegetable was stored up to one year in clear and brown glass jars, under either nitrogen or air atmospheres. The free and total folic acid content of mulukhiyah was determined before, during, and after drying processes, as well as during storage.

Hypothesis There is a significant difference in folacin retention in dehydrated mulukhiyah stored in clear versus dark glass jars, and under nitrogen versus air atmosphere. 3

Null Hypothesis There is no significant difference in folacin retention in dehydrated mulukhiyah stored in clear versus dark glass jars, and under nitrogen versus air atmosphere.

Limitations of the Study 1. This study was limited to the examination of three methods of de hydration (room, tra~ and freeze drying). 2. It was limited to the use of one kind of vegetable, namely muluk hiyah. 3. The storage period was limited to twelve months.

Definition of Terms Room-drying. The vegetable was spread on plastic trays in side a room to be dried by air convection at room temperature of 77° F (25° C). Freeze-drying. The water was removed from the vegetable by direct sublimation from the frozen state to the vapor state without passing through an intermediate liquid state. The drying was done under high vacuum at a low temperature. Tray dryirrg. (Home dehydration). The procedure consists of blowing hot dry air over the food to remove the water. It takes from 8 to 12 hours to tray dry food with a temperature of 50° c. Folacin. The comprehensive term for different forms of folic acid. Free folic acid (FFA). The folacin content of vegetables assayed with conjugase treatment. This included mono-, di-, and tri glutamate forms of folacin. 4

Total folic acid (TFA). The folacin content of vegetables determined after treatment with conjugase to convert the more compli cated forms of folacin to the monoglutamate form which is measurable by the assay technique. Chapter II REVIEW OF LITERATURE FOLACIN

Historical The isolation and identification of folic acid is associated with laboratory studies of anemias and growth factors in animals. Stokstad and Manning (1938) described a growth factor for chicks which they named vitamin U. Later this factor was obtained from liver and synthesized by Angier and Stokstad and their associates in 1945 (Angier et al., 1946). The vitamin was named folic acid (L. Folium, leaf) or folacin, because a major source of its extraction was dark green leafy vegetables such as spinach. The reduced form of folic acid has since been discovered. It is folinic acid, first called Citrovorum Factor (CF) because it supplied an essential growth factor for a lactobacillus h· citrovorum (Eigen and Shockman, 1963).

Chemical Structure Folic acid consists of three components: a pteridine deriva tive, p-aminobenzoic acid, and L-glutamic acid. The pteridine deriva tive of the basic folacin structure is a 2-amino-4-hydroxy derivative (Rabinowitz, 1960). The moiety composed of the pteridine and p-amino benzoic acid is called pteroic acid. Folic acid is therefore also known as pteroylglutamic acid (Pte Glu). The numbering system for the folic acid molecule (Figure 1)

5 OH COOH ll I N H CO-N--Cli-CH -CH.-COOH srCH2-N8 a . 6 N3"? 4 a 10 1 Aa·2 I . a..,? HaN N 'N ' • i ~------·--~~------''------~~------' Pteridine p -Aminobenzoic Glutamic acid acid L. ~ Pteroic acid .._ -.J T Pteroylglutamic acid Folic acid

Fl8Ure 1. Stmcture of Pteroylglutamic Acid

0'\ is based on the numbering of the pteridine ring system (Rabinowitz, 1960). Other compounds related to folic acid which have been iso lated from natural sources and characterized include: 1. Polyglutamic acid derivatives of folic acid which contain a total of three or seven glutamic acid residues linked by glutamyl peptide bonds. 2. The N10 formyl derivatives of pteroic acid and folic acid. 3. The N5 formyl derivatives of 5,6,7,8, tetrahydrofolic acid (Rabinowitz, 1960).

Physical Properties and Natural Sources Folacin (folic acid - pteroylglutamic acid) is a water soluble vitamin necessary for normal growth, reproduction, prevention and treatment of various types of anemias in man and many other animals and for growth of many microorganisms. The highest concentrations of folacin are found in liver, kidney, yeast and leafy vegetables, and smaller amounts are found in dairy products, cereals, and fruit (Santini eta]., 1964). Approximately 75 to 80 percent of natural folates exist as polyglutamyl conjugates; that is, they are detectable by microbiolog ical assay only after enzyme digestion. About 90 percent of folate in vegetables is present as 5-methyl tetrahydrofolic acid derivatives with the remaining 10 percent or less existing as unreduced pteroylglutamate (Chan et 2}., 1973). Folates are sensitive to light, oxygen, extremes of pH, and heat, especially boiling of foodstuffs, all of which result in loss of 8 folate activity (Rabinowitz, 1960).

Recommended Dietary Allowances The Food and Nutrition Board of the National Research Council (1980) established the Recommended Daily Dietary Allowances (RDA's) for folacin at: 400 meg for adults, 800 meg for pregnant women, 500 meg for lactating women. For children, the RDA's are 300 meg for ages seven to ten years, 200 meg for ages four to six, 100 meg for one to three, 30 meg for infants. These allowances are based on total folacin and take into account the fact that not all polyglutamate forms will be absorbed or utilized by the body.

DEHYDRATION The dehydration processing of food is based on the removal of enough water to lower the availability of water (as defined by water activity, Aw) in order to prevent microbiological deterioration or food poisoning. There are various methods of food dehydration, each approp riate to different products or requirements. In all cases a source of heat is supplied to the food and this helps to evaporate the water. Van Arsdel and Coply (1963) and Charm (1971) have covered the engineering aspects of the various means of drying.

Room Drying Room drying is a natural method of dehydration. Not all food can be room dried. It is low in cost because there is no investment in major equipment. Pieces of food are spread on a plastic tray placed inside a room with a temperature of 25° C (room temperature) and are exposed evenly to the sun for part of the day. Since air convection ' 9 over the food pieces is very low and the temperature is low, the drying process takes 3 to 4 days or longer depending on the product and con dition.

Tray Drying There are many tray drying dehydrators designed for home use. Generally, the procedure consists of blowing hot, dry air over the pieces of food. It takes from 8 to 12 hours to tray dry food \'Ji th a heating temperature of 50° C (Miller et ~., 1975).

Freeze Drying Freeze drying is among the newest methods of food dehydration. It is distinguished from other forms of drying by the presence of fro zen water within the substance during drying. The principle of freeze drying is the removal of water from a substance by direct sublimation from the frozen state to the vapor state, without the water passing through an intermediate liquid state. It takes from 6 to 15 hours to freeze dry food pieces of one centimeter thickness with heating temper atures of 50° to 60° C (Labuza, 1972). The time, temperature, and moisture content relationship during freeze drying has received consid erable attention and has been reviewed by King (1973).

Advantages and Disadvantages of Freeze Drying 1. Freeze drying gives the highest possible qualities, and maintains the highest nutritional values of any drying procedure (Calloway, 1962). This is due to the low temperature held during freeze drying which re duces occurrance of various degradative side reactions. The tempera ture of freeze drying is also usually below the threshold temperature for substantial protein denaturation (King, 1973). 10

2. Retention of shape and color of foods during freeze drying is better when compared to other dehydration methods. The presence of a rigid ice structure in the location where sublimation takes place mech anically prevents shrinkage to any great extent. As a result freeze drying is unique among drying methods by virtue of giving practically no change in volume or physical arrangement of the solid material from that which existed in the frozen state before drying. For example, Malkki and Heinonen (1978) showed that freeze dried onions have a lower shrinkage and a better organoleptic quality than other conventional methods. 3. Freeze dried products have a spongy texture and rehydrate more fully and rapidly than do conventionally dried products (Thomas and Calloway, 1961). This is because frozen cells do not collapse and harden as water vapor is removed. Since this process is expensive and not economic (Goc, 1977) it is reserved for problem foods, such as meats, some vegetables, and products in which large size pieces are desired.

DESTRUCTION OF VITAt•1INS DURING DRYING AND STORAGE Evidence reported leads to the conclusion that a number of factors can contribute to folate losses during food processing and pre paration, and that the losses are generally not a result of an isolated factor. Among the variables most likely to influence folate levels are: contact with water, amount of v1a ter, temperature, exposure to 1 i ght, exposure to oxygen, and length of time of exposure to the above factors. In general, two types of reactions must be considered with respect to nutritional losses during drying and storage of food. The 11

first is the effect of the process itself, such as the effect of temp erature and the reactant concentration on the direct destruction of the nutrient. The second is the interaction between compounds produced during drying or storage of food with various nutrients, rendering the nutrients unavailable biologically.

Destruction During Dehydration Process The dehydration process involves more than just drying of foods. The foods must be sorted, washed, cut into the desired size, and blanched to destroy enzymes. During all these processes, loss of nutritional value can occur. Suchewer _gt £1. (1970) found 30 percent of total folates in French beans and 11 percent of total folates in green peas present in the brine after industrial canning processing. Canning of garbanzo

beans also results in a 25 to 30 percent loss of folates, and Lin~~· (1975) found that this loss occurs during the soaking and blanching steps. Leaching has also been found to account for the major portion of folate losses during industrial processing of pinto bean powders

(~1iller ..Q! .B.]., 1973). The losses of water-soluble vitamins during dehydration processes vary widely. Schroeder (1971) summarized the losses of various water-soluble vitamins during freezing, drying, canning, or milling. His data show anywhere from 0 to 30 percent losses of B-6 in freeze drying of fish, and a 50 percent loss in drying whole milk. Similarly, about a 20 to 30 percent loss of pantothenic acid occurred in freeze dried fish. Bluestein and Labuza (1975) stated that ribo flavin loss in freeze dried chicken was 4 to 8 percent. Twenty percent 12 losses for thiamin, pyridoxin, niacin, and folacin were found in drum drying of bean powders. Spray drying of milk causes about a 10 percent loss of thiamin, while drum drying gave a higher loss of 15 percent.

Karmas g! ~· (1962) showed about a 30 percent loss of thiamin in freeze drying pork, while Calloway (1962) reported 50 to 70 percent of the thiamin level in pork was lost using conventional air drying. Sun dried products have greater losses of nutrients than cabinet dried products (Patil et al., 1978). Mrak and Phaff (1947) showed that loss of ascorbic acid in peaches by sun drying was close to 90 percent, and for tray drying about 80 percent. Also losses of vitamin C in pears were 55 percent in air drying and 30 percent in freeze-drying (Labuza, 1972). Temperature and time of exposure to elevated temperature may affect folate losses from food systems. Biely et £1. (1952) assayed herring meal dried at two temperature levels: low temperature air cur rents of 100-110° C and high temperature commercial flame drying. They found that increased processing temperature caused increased folate losses. There are data that show small or no loss of nutrients during dehydration processing. Bluestein and Labuza (1975) stated that drying processes appear to offer good nutrient retention with the exception of ascorbic acid and beta-carotene. The losses of water-soluble vitamins other than ascorbic acid during drying average approximately 5 percent. For apple flakes there was an 8 percent loss of vitamin C in slicing, a 62 percent loss in blanching, a 10 percent loss during puree prepara tion and only 5 percent in drum drying. 13

Thomas and Calloway (1961) detected no apparent loss of folic acid in dehydrated animal products; however, the initial content of this vitamin was too low to permit an accurate evaluation of its stab ility. It can be concluded that the loss of folic acid by dehydration varies with the drying conditions and the type of product. Little information is available in the literature on there

tention of folic acid in vegetables during drying. Holmes~ E}. (1979) reported that the retention of total folacin during home drying of vegetables and fruits ranged from 46 percent in blanched green beans to 92 percent in unblanched zucchini squash. Destruction of Vitamins During Storage The retention of vitamins during storage depends on time, temperature, moisture content of the product, light, and whether oxygen is present. Therefore, proper food packaging is an important aspect to the basic food processing methods. Light and storage temperature. Many of the deteriorative changes in the nutritional quality of foods are initiated, or acceler ated by light. For example, ascorbic acid losses in milk stored in uncolored glass was 14 times greater than those in brown glass, and milk in blue paper cartons lost 5 times more vitamin C than in red paper cartons. Milk in white polyethylene film lost, after two hours of exposure, 93 percent of vitamin C, but milk in polyethylene over wrapped by black polyethylene lost only 16 percento(Karel and Heidel baugh, 1975). Riboflavin losses were also retarded by the polyethylene film containing black pigments. During distribution of milk, losses of riboflavin and ascorbic acid were lowest when brown bottles were used 14

(Gregory, 1975). It has been shown by several studies that folacin is light sensitive and is subject to photo-degradation and heat destruction. Malin (1975) has reviewed this area. Bloom et ]]. (1944) studied the flourescent properties of Pte.Glu and found it is moderately sensitive to ultraviolet light. Stokstad --et al. (1947) confirmed this finding. they subjected Pte.Glu to sun light and flourescent light, and found that sun light was much more potent in activating Pte.Glu destruction. This degradation proceeded very slowly in artificial light and faster under day light laboratory conditions.

Suchewer et~. (1970) reported that independent of container type (glass or metal) and effect of day light, folic acid present in canned French beans and green peas was stable for 12 months when stored at room temperature. Content of folic acid in tomato juice stored in the dark for 12 months decreased by 7 percent on the average in con tainers of all types, versus 30 percent in orange bottles stored in day light.

Although, f~lacin compounds are light sensitive, it is poss ible that food systems may protect the folates from the influence of light. Hanning and Mitts (1949), evaluated the effect of heat and light on the folacin retention of chicken eggs, and reported that the presence of light did not affect the folacin retention during cooking. It has been suggested that temperature is a factor involved in folate retention not only during processing, but also during food storage (Olson~~., 1947). These researchers reported that fresh vegetables stored at room temperature lost a large amount of folacin p ' 15 within a short period of time, whereas refrigeration and ice storage prevented folate losses for periods of two weeks or more.

In flour stored for 8 weeks, Keagy et ~' (1975) found only 60 percent retention of native folacin when stored at 120° F compared to 85 percent retention when stored at 84° F. In the same study, for tified flour stored for one year at 120° F lost 8 percent of its Pte.Glu activity. There are data however, showing little or no loss of folates during processing and storage at high temperature. Brenner et ~ (1948) detected no significant decrease in folate content of canned foods stored at 70-100° F up to 18 months. Hellendorn --et al. (1971), in investigating the effects of heat sterilization and prolonged storage on folates in canned meats, have shown the folates to be stable under these processes. Some forms of folacin are subject to greater deterioration than others. This has been concluded from research on pure folates in solution at room temperature (O'Broin et ~., 1975), as well as from studies on purified and naturally occurring folates exposed to heat

(Ghitis, 1966; Cooper .!:..t~., 1978). Paine-Hilson and Chen (1979) studied the thermal stability of four folacin derivatives at 100° C.

They found that 5-methyltetrahydrofolic acid (5-CH3-H4 Pte Glu), and tetrahydrofolic acid (H4 Pte Glu) were more labile than the other forms of folate. Chen and Cooper (1979) have found that H4 Pte Glu is ex tremely heat labile and the half life of H4 Pte Glu at 100° C was found to be 2.25 minutes, while that of 5-CH 3-H 4 Pte Glu was 21.4 minutes. The stability of both forms of folate at 100° C was drasti- 16 cally increased in the presence of ascorbate or under a nitrogen at mosphere. These data indicated that degradation of these folates at elevated temperatures was due to an oxidative process requiring the presence of molecular oxygen. These data also suggest the possibility of using nitrogen in storage to protect labile folates in food products. Oxygen. The role of packaging on nutrient stability has been studied extensively. Storage under conditions of low headspace oxygen concentration has been found to increase the stability of ascorbic acid and riboflavin (Waletzkoi and Labuza, 1976). When dehydrated carrot flakes were stored in nitrogen, very little loss of ascorbic acid occurred over a 24 month period. In air packaged carrot flakes, the rate of ascorbic acid loss was very rapid in the first four months, after which no change was found. About 25 percent was lost in this time (Stephens and Mclamore, 1969). Gee (1979) reported that when dried carrots, spinach and tomatoes were stored in air or vacuum at room temperature for 5 to 7 months, the total ascorbic acid, B-carotene and thiamin content were lost more rapidly in air storage.

Brenner~ iJ. (1948) showed that canning, which results in low levels of free oxygen, could decrease folate losses. Malin (1975) concluded that up to 95 percent of the initial folacin in foods may be lost during oxidative heating processes with an average loss of 45 per cent of total folates and 72 precent of free folate. Suckewer et ]]. (1970) reported that the losses of folic acid in tomato juice produc tion were higher on a Yugoslav (70 percent) than on an American line (50 percent). This was attributed to the shorter exposure to heat and oxygen on the latter. The percentage of folacin destroyed during heat 17

processing of milk was greatly affected by the level of residual oxygen in the milk. At a level of 8 mg dissolved oxygen per kg of milk, fola cin was completely lost in a matter of days after processing. At very low oxygen levels minimal losses occurred after 180 days (Rolls and Porter, 1973). Thus, the only really practical protection against oxidation is packing in the absence of oxygen. This may be achieved by packing in vacuum, in nitrogen, (or other inert gas), or by filling the con tainer so completely with compressed dehydrated product that the actual quantity of oxygen remaining in the container is so small that any re sulting oxidation is negligible (Kared and Heidelbaugh, 1975; and Waletzkoj and Labuza, 1976). Moisture content. The lower the moisture content of the de hydrated product the longer is its storage life at high temperatures. Mrak and Phaff (1947) stated that oxygen, moisture content, and sulfur dioxide content all have a considerable influence on storage quality, and their action may be antagonistic under certain conditions. For example, when dehydrated pork was held 7 days at 49° C and at 0, 2, 4, 6, and 9 percent moisture, the losses of thiamin were 9, 40, 80, 90 and 98 percent, respectively (Labuza, 1972). Calloway (1962) concluded that thiamin and ascorbic acid are more resistant to heat damage in the dry state than in liquid media. Losses of these vitamins can be ex pected to decrease as the product approaches dryness, and stability under high temperature storage to be improved as compared with a con ventionally canned moist product. Cart et _gJ. (1976) have studied nutrient stabi 1ity in a 18

specially enriched flour at 9 percent moisture and found very good stability for folic acid, riboflavin, niacin, and thiamin. Flour con taining 13.5 percent moisture retained 100 percent folic acid activity

in 4 weeks when stored at 113° F. Keagy gt al. (1975) also found ex cellent stability of added folacin in all purpose flour at 12.5 percent moisture, even at a temperature of 120° F for one year.

~1ULUKHIYAH Mulukhiyah (or Jew's mallow, Corchorus olitorius) is a stout herb cultivated in Syria and Egypt as a vegetable, and in India and other countries for its jute fiber. Jew's mallow is one of the green vegetables consumed in large quantities in Egypt, and is known as "mulukhiyah" in Arabic (Patwardhan and Darby, 1972). This vegetable is popular all over Egypt. When available in season, it is bought in quantities, and the leaves are separated from the stalk, dried in the sun, and stored. The dried mul ukhiyah is soaked in water, cooked with salt and some sour lime juice, and eaten with bread or rice. Those who can afford it cook the vege table with meat broth. The plant may be grown without difficulty in suitable soils in all warm, moist countries. It grows best in alluvial or clay loam soils retentive of moisture, and where the air is warm and moist during the growing period. The seed is sown in spring, the crop is harvested when in flower, about three months after sowing. The stalks are cut with the knife or sickle, or pulled by hand (Bailey, 1907). Chapter III METHODS AND MATERIAL Material

Vegetable The vegetable used in this study was mulukhiyah, or Jew's mallow (Corchorus olitorius). This vegetable is available fresh during the summer and in dried form throughout the year in local Egyptian stores. To minimize the destruction of folacin due to handling and prolonged standing, the vegetable was planted in the garden and collect ed at the end of June on the same experimental days. The edible part of the plant, which is the leaf, was used for the study.

Assay Microorganism Lactobacillus casei (ATCC 7469) culture was purchased from Difco Laboratories, Detroit, Michigan.

Basal Medium Bacto Folic Acid Casei Medium (Lot No. 630736 and 635971) was purchased from Difco Laboratories, Detroit, Michigan. Instructions for preparation are in Appendix A.4.

Chemical Reagents Maintenance medium, inoculum broth, sterile saline, and phos phate buffered ascorbate solution were prepared as shown in Appendix A. Hog kidney conjugase was prepared for microbiological assay as describ ed in Appendix B. Folic acid standard solutions were prepared as shown

19 20 in Appendix C.

Equipment Used Balance - Mettler H-20 Autoclave - Model STM-E, Type C #45018 Brinkman pH t·1eter, Model 102 Refrigerated Centrifuge - Beckman Model G-21B Refrigerator freezer Tray-dehydrator- Norwalk Model 10, Norwalk Manufacturing Co., Santa Monica, California Freeze-dryer - Labconco Model #75150 Spectrophotometer - Beckman Model 24

METHODS

Sample Preparation The vegetable was washed vJith tap water and dried well on paper towels. The leaves were separated from the stalk and weighed. A sample of fresh vegetable was used to control the experiment and the others were dried by the three dehydration methods described below. No blanching or chemical treatment was used. The sample was withdrawn after each specified period of drying time as shown in Table 1, and the moisture content and the folic acid retention were determined.

Drying Methods Freeze drying. The samples were quick frozen in liquid nitrogen (to accelerate the freezing time), then they were placed on trays in the freeze drier. During drying the shelf temperature was maintained at 33° C, and the vacuum at 200 microns. Samples were re moved for analysis every hour during the first four hours, and every 21

Table 1 Sampling Schedule During Drying of Vegetable By Three Dehydration Methods

Lot Drying Time (in hours) Number Freeze Drying Tray Drying Room Drying

1 1 1 4 2 2 2 8 3 3 3 12 4 4 4 16 5 6 6 20 6 8 8 28 7 10 10 36 8 12 12 48 22

two hours thereafter. Tray drying. The samples were spread out on screened trays and placed in a Norwalk dehydrator. Hot, dry air was blown over the vegetable at moderate speed. The drying temperature was maintained at 50° C. Samples were taken for analysis in intervals similar to that for freeze drying. Room drying. Room drying is a common method of drying in Egypt. It is used, in particular, to dry green vegetables to preserve their color. The samples were spread on plastic trays, and exposed to sunlight part of the day inside the laboratory near the south window at room temperature (25° C). Samples were taken for analysis every four hours for the first twenty hours, then every eight hours until the drying process was completed.

Storage Test For the storage test, ten pounds of fresh vegetables were dried under the same conditions as used in the room drying method men tioned above. The dried vegetable was divided equally into four por tions and packed into four 16 ounce glass jars, two amber and two clear. One of each type of glass jar was flashed with nitrogen for 30 seconds and sealed tightly with a rubber stopper. These nitrogen packed jars were reflashed with nitrogen after every time the jars were opened. The other two jars were capped and the contents stored under atmospher ic air. All the jars were stored at room temperature (25° C) under fluorescent light for up to one year. One gram sample of the dried vegetable was withdrawn for analysis from each jar at the following specified storage time: 0, 1, 23

2, 3, and 4 weeks and 2, 3, 4, 5, 6, 9 and 12 months.

Moisture Determination The AOAC method (AOAC 1975) \'Jas used. Three weighed samp 1es, about two grams each, were placed in pre-dried aluminum dishes and dried in an air oven at 135° C for two hours. The samples were cooled and weighed. The process was repeated every hour until constant weights were reached. Loss in weight was calculated as moisture con tent. Average values were reported. Total solids were calculated from the moisture content.

Folacin Determination Extraction of folates from the vegetable was carried out by the method of Hurdle, Barton and Searles (1968), and revised by Chan, Shin and Stokstad (1973), and Tamura and Stokstad (1973). The assay procedure was essentially that of Waters and Mallin (1961) with re visions reported by Tamura, Shin, Williams and Stokstad (1972).

Extraction of Folates from Vegetable. The following steps outline the procedure followed in obtaining the extraction of folates for ra1.,r and dehydrated vegetables. I. One gram sample of raw or dehydrated vegetable was added to 40 ml of 0.05 M sodium phosphate buffer (Appendix A.5) pH 6.0, containing 0.2 % ascorbate in a Waring blender and homogenized for one minute. The ascorbate protected the folate from destruction during the extrac tion procedure. 2. The homogenate was transferred to a 100 ml graduated cylinder. 3. The blender was rinsed with additional 30 ml buffer. Rinsings were 24

added to the sample and the total volume brought to 100 ml with addi tional buffer. At this point the original vegetable sample was diluted 100 times. 4. The homogenate was transferred to a 250 ml Erlenmeyer flask covered with aluminum foil and autoclaved at 15 psi for 15 minutes to extract the folates. 5. The homogenate was cooled to room temperature and centrifuged in a Beckman refrigerated centrifuge at 16,000 X g and -2° C for 20 minutes. 6. Aliquots of the supernatant, which contained the extracted folates, were stored frozen until ready for assay. Samples of these extracts were used directly in the determin ation of FFA content of the vegetables. Separate samples of these ex tracts were treated with conjugase in the preparation of TFA determina tion. Conjugase treatment. To reduce the higher conjugated forms of folacin to a form available to the assay microorganism, samples of the vegetable extract were treated with hog kidney conjugase. Details of preparation of the conjugase are included in Appendix B.1. One milliliter sample of vegetable extract, 1.0 ml hog kidney conjugase and 8.0 ml acetate buffer 0.2 % ascorbic acid {Appendix B.2) were combined in a test tube, capped, shaken, and incubated at 37° C for six hours. These samples have been diluted one thousand fold at this point (hun dred times during the extraction and ten times during conjugase treat ment).

Microbiological Assay Procedure The microbiological assay of folacin is based on the growth 25

response of Lactobacillus casei to folacin. All the nutrients needed by the microorganism except folacin are provided in excess in the growth medium. Folacin is not present in the medium, and thereby be coMes the growth limiting factor. A standard curve of L. casei re sponse to folacin is prepared by adding known amounts of folacin to the assay tubes which are then inoculated with L. casei cells and allowed to grow for 20 hours at 37° C. The amount of microbial growth is mea sured turbidimetrically and is a function of folate concentration. Un- known concentrations of folacin are then determined by comparison with the standard curve. The assay procedure used in this study included steps to be performed on three consecutive days, as shown in Figure 2. On the first day, h· casei culture was transferred from maintenance medium to inoculum broth (Appendix A.2). This was then incubated at 37° C for 20 hours. This step put the microorganism into an active phase of growth. The inoculum was prepared on the second day by collecting and washing the cells with 0.9 % sterile saline and centrifuging four times. The cell suspension was used to inoculate assay tubes. The tubes were then 0 . incubated at 37 C for 20 hours. On day three, tubes were autoclaved at 15 psi for 5 minutes to stop the growth of the microorganism. Sam- ple tubes were thoroughly mixed on a Vortix mixer, and were then read at 640 nm on a spectrophotometer. This folacin assay was essentially the procedure used by

Waters and Moll in (1961) with revisions reported by Tamura et ~- (1972). 26

Figure 2 SCHEMATIC DIAGRAM OF ASSAY PROCEDURE

1. Transfer the microorganism from maintenance medium to Day One inoculum broth. 2. Incubate the inoculum at 37° C for 18 to 24 hours.

1. Wash the cell 4 times with 0.9 % sterile saline followed by centrifugation. 2. Resuspending the cells in 0.9 % Day Two saline. 3. One drop of cell suspension is added to each assay tube. 4. Incubate the assay tubes at 37° C for 18 to 24 hours.

1. Autoclave the assay tubes at 15 psi for 5 minutes. Day Three 2. Read optical density through spectrophotometer. 27

Maintenance of microorganism. The h· casei (ATCC 7469) culture was maintained in a maintenance medium (Appendix A.1). A series of stabs of the culture into the maintenance medium followed by 20 hours of incubation at 37° C in an incubator was repeated monthly to regenerate the organism. The stabs were then held at 0-4° C until used for monthly transfers to new stabs. Preparation of inoculum. Tubes containing inoculum broth were prepared as described in Appendix A.2. These tubes were stored frozen until needed. After thawing the inoculum broth, the micro organism was transferred from a stab culture to the inoculum broth with an inoculating needle. This was then incubated at 37° C for 20 hours. The growth was then harvested by centrifugation at 1000 X g for 10 minutes at -2° C, and the supernatant discarded. The cells were washed with 5 ml 0.9 % sterile saline and centrifuged again. The washing and centrifugation procedure was repeated three times. After the final centrifugation, the supernatant was discarded and the cells suspended in 10 ml sterile 0.9% saline (Appendix A.3). One drop of the suspension from a 5 ml disposable pipette was used for inoculation of each culture tube. Standard curve. The following steps outline the procedure followed in establishing a standard curve, and Table 2 shows the pre paration of culture tubes for the standard curve. In order to control day to day variability of the organism, a standard curve was establish ed each day when the experiment was conducted. Each concentration of the standard curve was prepared in triplicate. 1. 2.5 ml basal medium (Appendix A.4) was added to each of the culture Table 2 PREPARATION OF ASSAY TUBES FOR FOLIC ACID STANDARD CURVE

Pte Glu Phosphate L. casei Pte Glu standard ascorbate Basal inoculum concentration solution* buffer+ medium++ added (ng/5 ml) (ml) (ml) (ml) (one drop)

0 0 2.5 2.5 No 0 0 2.5 2.5 Yes 0.1 0.2 2.3 2.5 Yes 0.2 0.4 2.1 2.5 Yes 0.3 0.6 1.9 2.5 Yes 0.4 0.8 1.7 2.5 Yes 0.5 1.0 1.5 2.5 Yes 0.6 1.2 1.3 2.5 Yes 0.8 1.6 0.9 2.5 Yes 1.0 2.0 0.5 2.5 Yes 1.2 2.4 0.1 2.5 Yes

* Assay solution C, See Appendix C.3 + 0.05 M phosphate buffered ascorbate solution (0.1 %), See Appendix A.5 ++ Difco Bacto Folic Acid Casei medium, See Appendix A.4

N (X) 29 tubes. This folic acid free growth medium provided all other nutrients required for the growth of L. casei. 2. 0.05 M phosphate buffered ascorbate solution (0.1 %) (Appendix A.5) was added in each assay tube in specific amounts, as shown in Table 2, to protect the folacin from oxidation during the assay. 3. The culture tubes were covered with aluminum foil and autoclaved for five minutes at 15 psi as a terminal sterilization step. 4. Pte.Glu solution C (Appendix C.3) was added to the culture tubes in calculated volumes to give a final concentration range of 0.1 to 1.2 ng/tube (5 ml). 5. Assay tubes were inoculated with L. casei cell suspensions that had been washed free of folacin and suspended in sterile saline. 6. Assay tubes were incubated at 37° C for 20 hours. They were then autoclaved 5 minutes at 15 psi to stop the growth. 7. The microbial gorwth was determined quantitatively by measuring the optical density of the incubated tubes at 640 nm using a Beckman spec trophotometer Model 24 equipped with a sipper system. Deionized water was used for calibrating the spectrophotometer and as a reference solu tion for reading the optical density of the inoculated and uninoculated blanks. An inoculated blank was then used to calibrate the instrument again and was the reference solution for reading optical density of all samples. The means of the optical densities for each triplicate folic acid concentration were plotted against the known concentration of folic acid. A typical standard curve is shown in linear plot in Figure 3, and in a semi-log plot in Figure 4. The standard curve of L. casei 30

Figure 3. Typical folic acid standard curve. This figure shows a typical 11 L. casei Folic Acid 11 standard growth curve. -Assay tubes were prepared in tripJicate. After twenty hours incubation at 37 C, optical densities were read at 640 nm against distilled water on a Beckman, Model 24 Spectrophotometer, equipped with a sipper system. The mean optical densities ± one standard deviation for each folic acid concentration was plotted. 31

1.6 r-----r----..,.-----...... -----....------. -

E c: 1.2 0 ~ f-"" - f- -(./) z UJ 0 _, 0.6 <( u f- a.. 0 0.4

0.2

0 0.5 1.0 1.5 2.0 2 .or:· PteGiu CONCENTRATION (ng) 32

Figure 4. A semi-log plot of folic acid standard curve of L. casei. In this plot optical density is-linear while concentration is on a logarithmic scale. The curve is a sigmoid. 33

1- <( 0.7 0:::: . 1-z UJ 0.5 u z 0 u 0.3

0 Q) a..-

Qj~~~~--~~~~~~~~----~--~ 1.5 1.3 1.1 0.9 0.7 0.5 0.3 Oj ·O.D AT 640 nm 34

linear plot is similar to that reported by Waters and ~1ollin (1961), and the semi-log plot is similar to that reported by Tamura et~. (1972). Assay of mulukhiyah samples. The procedure was the same for all samples whether or not they were treated with conjugase. Frozen samples of mulukhiyah extract were thawed, then diluted with appropri ate volumes of 0.05 M ascorbate phosphate buffer (Appendix A.5). Table 3 shows the volumes of sample extract, ascorbate buffer and basal med ium that were pipetted into each tube. (Note: In Table 3, 11 100X 11 means that the sample extract of the vegetable was not diluted before addition to the tubes; 11 1000X 11 means that 1 ml of lOOX sample extract was diluted with 9 ml of ascorbate phosphate buffer and then added to the tubes in appropriate volumes, etc.). This range of dilution was tried initially to determine the proper dilution range at which the assay could be carried out. It was found that the mulukhiyah samples could be assayed in the 10,000X dilution and that further dilution was unnecessary. Two concentrations (0.2 and 0.5 ml) of the lO,OOOX dilu tion sample were micropipetted in triplicate into sterilized assay medium. After the assay tubes were prepared, they were covered \vith aluminum foil and autoclaved for five minutes at 15 psi, and then cooled to 37° C. Each assay tube was inoculated and incubated follow ing the same procedure used for the standard curve. Folate concen tration of the experimental samples was determined by comparing their optical density to that of the standard growth curve, and the results were expressed as ng of Pte.Glu equivalents per tube. Maintenance of glassware. Maintenance of glassware is 35

Table 3 PREPARATION OF FOLACIN ASSAY TUBES FOR MULUKIYAH SAMPLES

Sample Vegetable Ascorbate Basal dilution* extract phosphate medium (ml)+ buffer (ml) (ml)++

0.1 2.4 2.5 0.2 2.3 2.5 100 X 0.5 2.0 2.5 1.0 1.5 2.5 0.1 2.4 2.5 0.2 2.3 2.5 1,000 X 0.5 2.0 2.5 1.0 1.5 2.5 0.1 2.4 2.5 0.2 2.3 2.5 10,000 X 0.5 2.0 2.5 1.0 1.5 2.5 0.1 2.4 2.5 0.2 2.3 2.5 10,000 X 0.5 2.0 2.5 1.0 1.5 2.5

* Vegetable extract and conjugase treated vegetable extract were dilu ted 100, 1,000, 10,000, and 100,000 times by the addition of ascor bate phosphate buffer (0.05 M). +At each dilution level, aliquots of 0.1, 0.2, 0.5, and 1.0 ml were further diluted by the addition of ascorbate phosphate buffer and basal medium to a final volume of five milliliters. ++The buffer used was 0.05 M phosphate buffer with 0.1 % ascorbic acid just prior to use. 36 important to avoid contamination by folacin remaining from a previous experiment. After use, all glassware was soaked in detergent for several hours and washed with a brush three times. It was found that the presence of heavy metal contaminants in the water may inhibit the growth of the test organisms. Therefore, washing was followed by rinsing 6 to 8 times with hot water and then rinsing with additional deionized water three times. The glassware was allowed to air dry inverted. Sterile glassware was prepared by routinely autoclaving all glassware for 20 minutes at 15 psi.

Data Treatment Folate content calculation. The data collected were com puted in terms of folic acid content in mcg/g of wet weight basis, as well as in dry weight basis. The mean and standard deviation of six replications in fresh and dried mulukhiyah in dry weight basis were also computed by a programmable computer calculator (t~odel TI 55, Texas Instruments). Folate retention calculation. The sample folacin mcg/g solid was used to compute the percent retention of folate during both drying and storage. The folic acid content of fresh mlulkhiyah was considered to be 100 % for drying, and the content of that in room dried mulukhi yah at the beginning of the storage period was considered to be 100 % for storage. Statistical analysis. The repeated treatment (F Test) of variance was used to determine the effect of packaging condition and storage time on folic acid content (Joseph and Joseph, 1975) at 0.01 level. Following this analysis, Tukey's Honestly Significant 37 differences test (Roscoe, 1975) was used to determine which groups of means were significantly different at 0.01 probability level. Chapter IV

RESULTS AND DISCUSSION

Folacin Activities in Mulukhiyah As shown in Table 4, raw mulukhiyah contained 556 meg free folic acid (FFA) and 800 meg total folic acid (TFA) per 100 g of the fresh vegetable. The room dried vegetable was found to contain 662 meg of FFA and 1138 meg of TFA per 100 g dry weight. When compared with other vegetables known to be good sources of folacin (Table 4), mulu khiyah is significantly higher in folacin content. For example, mulukhiyah is four times as high as spinach, and eight times as high as broccoli in its total folic acid content. In this study it was also found that 70 percent of the TFA in fresh mulukhiyah and 58 percent of the TFA in dried mulukhiyah is FFA (Table 4). Percentage values of FFA in TFA of some green vege tables were calculated from data compiled by Perloff and Butrum (1977), and are also presented in Table 4. In raw green leafy vegetables such as romaine lettuce, cabbage, and spinach, 33, 50, and 62 percent of TFA, respectively, is FFA. In other raw non-leafy vegetables such as cauliflower, green beans, asparagus, and broccoli, the percentage of FFA in TFA is 56, 75, 91, and 97, respectively. Some forms of folacin are subject to greater destruction during food processing than others. This has been concluded from re search on pure folates in solution at room temperature

38 39

Table 4 FOLACIN CONTENT OF MULUKHIYAH AND SOME OTHER FOLACIN RICH VEGETABLESa

Folacin content Free Vegetable (mcg/100 g ed i b1 e Eorti on f Tota 1 X 100 Free Total (%)

~1ulukhiyah, raw 556 ± 61c BOO :I: 55c 70 Mulukhiyah, room dried 662 .± 74c 1138 ±. 62c 58 Spinach, raw 119 193 62 Spinach, cooked 60 91 66 Broccoli flower, raw 102 105 97 Romaine lettuce, raw 60 179 34 Brussel sprouts, raw 55 78 71 Cabbage, raw 33 66 50 Asparagus, raw 58 64 91 Green beans, raw 33 44 75 Cauliflower, raw 31 55 56

a Data other than for mulukhiyah are taken from Perloff and Butrum ( 1977). b Dried mulukhiyah was based on dry weight basis, vegetables other than dried mulukhiyah were based on fresh weight basis. c Values are the means .± standard deviation of the mean from nine replicates. 40

(O'Broin et 2]., 1975) as well as from studies on purified and natural ly occuring folates exposed to heat (Ghitis, 1966; Cooper _g,! .21·, 1978).

Chen and Cooper (1979) reported that tetrahydrofolic acid (H 4 Pte Glu) is extremely heat labile. The half-life of H4 Pte Glu at 100° C was found to be 2.25 minutes, while that of DL-N-5-methyltetrahydrofolic

acid (5-CH3H4 Pte Glu) was 21.4 minutes. It has also been established that the predominant forms of folacin in most foods are polyglutamates

and mono- and diglutamates (free folates) of H4 Pte Glu, 5-formyl tetrahydropteroylglutamic acid (5-CHOH 4 Pte Glu), 5-CH 3H4 Pte Glu, and 10-formyl-tetrahydropetroylglutamic acid (10-CHOH 4 Pte Glu) (Paine Wilson and Chen, 1979). Since the major form of folacin in fresh mulukhiyah is free (70%), the destruction of folacin is therefore ex- pected during dehydration and storage process. Free folate activity in food could be over estimated due to the presence of naturally occurring conjugases within the extract of the sample (Malin, 1977). The total folate activity; therefore, is recommended for use as the best indicator of the vitamin level for subsequent changes in the level brought about in processing. The results of this study show that mulukhiyah is an excell ent source of folate in the Egyptian diet. One serving of the fresh mulukhiyah (leaves), approximately 50 g, would contain 400 meg of TFA, and one serving of the dried vegetable (25 g) would contain 283 meg of TFA. The RDA for adults as established by the Food and Nutrition Board of the National Research Council (1980) is 400 meg. Since mulukhiyah is among the most common vegetable in the Egyptian diet, more research is needed to determine the effect of the cooking process on folic acid 41

retention in mulukhiyah. The fresh vegetable is usually prepared by washing, setting aside in an air current to dry, separating the leaves from the stalk, shredding by a special curved knife, and then boiling for approximately 5 minutes. The dried mulukhiyah, on the other hand, is crushed and then boiled for approximately 5 minutes.

Effect of Drying Methods on Weight Retention and Moisture Content of Mulukhiyah Table 5 shows the weight retention and changes in moisture content of mulukhiyah during drying process by three dehydration methods: freeze, tray and room drying. Drying temperature was 50° C for tray drying, up to 33° C for freeze drying and 25° C (room temp erature) for room drying. The loss of moisture was very rapid during the first 4 hours in freeze and tray drying, and the first 16 hours in room drying, after which time very little change in moisture content was found. The drying process was completed when the equilibrium drying condition was established. This is obtained when food approaches its normal equilibrium relative humidity. As this happens, it begins to pick up molecules of water vapor from the drying atmosphere as fast as it loses them. Equilibrium is established when the rates of these two processes are equal. The equilibrium state was reached after 6 hours in tray drying, 8 hours in freeze drying and 32 hours in room drying. The residual moisture content at the equilibrium state was 4.2, 8.3, 8.6 percent in tray, freeze, and room drying, respectively (Table 5). Figure 5 shows a semi-log plot of drying time versus moisture content. Straight lines were obtained up to the points when the I I Table 5 WEIGHT RETENTION AND CHANGES IN MOISTURE CONTENT OF MULUKHIYAH DURING DRYING PROCESS

Freeze dr~ing at 33° C Tra~ dr~ing at 50° C Room dr~ing at 25° C \

0 100 74.6 2.94 100 74.6 2.94 100 74.6 2.94 1 66.1 61.6 1.60 54.0 53.0 1.13 2 40.8 37.7 0.61 39.3 35.4 0.55 3 30.8 17.5 0.21 32.0 21.0 0.26 4 29.0 12.4 0.14 28.2 9.9 0.11 72.1 64.8 1.84 6 27.7 8.3 0.09 26.5 4.2 0.04 8 27.7 8.3 0.09 26.0 2.3 0.02 52.9 52.0 2.08 10 25.5 0.4 0.01 25.5 0.7 0.003 12 25.6 0.8 0.01 25.4 0.0 0.0 40.1 36.7 0.58 16 ------36.0 29.4 0.42 24 ------30.1 15.6 0.19 32 ------27.8 8.6 0.09 36 ------27.8 8.6 0.09

.p. 1'0 43

Figure 5. Semi-log plot of drying time (hour) versus moisture content g/g solid 44

DRYING TIME, HRS 10 20 30 40

2 A ~ Room Drying

0 0 o Freeze Drying _J 0 1 tl) .8 0 o Tray Drying 0> .6 (J) " .4 ...... z w 1- z .2 0u w cr .1 ~ .08 ~ .06 0 2 .04

.02~--~----~----~----~--~----- 0 5 10 15. DRYIN.G TIME, HRS 45 equilibrium conditions were reached. This finding is similar to the drying curves of most high moisture content vegetables, where there is initially a high rate of water removed followed by a falling rate that sharply decreases during the final stage of drying. Generally, at the beginning of drying, and for sometime thereafter, water continues to evaporate from the food pieces at a rather constant rate, as if it were drying from a free surface. This is referred to as the constant rate period of drying. This is followed by an inflection in the drying curve which leads into the falling rate period of drying (Potter, 1978). The changes in moisture content of mulukhiyah during tray and freeze drying followed the pattern of water removal as that in carrot dices during dehydration (Potter, 1978) and in dehydrated taro by tray and freeze drying (May ~~0, 1977). In this pattern, as shown in Figure 4, the majority of water was removed in 3 to 4 hours and the remaining moisture up to the equilibrium state took almost the same amount of time to remove. Room drying required more time to com plete because of the low temperature, and the slow air convection over the vegetable.

Destruction of Folacin During Dehydration The changes in TFA and FFA content of mulukhiyah during dry ing are presented in Table 6. Figures 6, 7, and 8 show the plots of percent folacin retention versus drying time for freeze drying, tray drying, and room drying, respectively. The destruction of FFA and TFA in all drying methods was very rapid at the beginning of the process. For example, the retention of FFA and TFA in freeze drying during the first 4 hours were 44 % and 49 %, respectively (Figure 6). The 46

Table 6 TOTAL AND FREE FOLIC ACID CONTENT AND PERCENT RETENTION IN MULUKHIYAH DURING FREEZE, TRAY, AND ROOM DRYING

Free folacin Total folacin Drying Time content * % content % (mcg/g solid) ret. (mcg/g solid)* ret.

Freeze drying

0 21.89 .± 2. 40 100 31.50 ± 2.17 100 1 16.30 ± 2.57 75 25.70 .± 0. 68 82 2 11.93 .± o. 93 55 19.52 ± 0. 68 62 3 9.93.± 0.55 45 16.59 ± 2.07 53 4 9. 70 .± o.13 44 15.56 ± 0.38 49 6 9.38 ± 1.99 43 13.90 .± 1.12 44 8 9.23 .± 0.65 42 15.17 ± 1.65 48 10 8.89.± 0.78 41 14.49.± 1.52 46 12 8. 94 .± 1. 38 41 14.62 ± 2.46 46

Tray Drying 0 21.89 .± 2. 40 100 31.50 ± 2.17 100 1 15.36 .± 1.45 70 22.00.±1.79 70 2 12.24 .± 1.84 56 17.77 .± 1.69 56 3 10.23 ± 1.10 47 15.92 .± 1.13 50 4 9.26.±1.12 42 14.43 ± 1.39 46 6 8.75 ± 0.85 39 13.86 ± 0.46 44 8 8.27 .± 0.79 38 13.18 .± 2.10 43 10 7.78 ± 0.26 35 12.92 ± 0.88 41 12 7. 88 .± 0.36 36 12.62 .± 0.58 40

* Values are the means and standard deviations of the mean from 6 replications. 47

Table 6 (Continued) TOTAL AND FREE FOLIC ACID CONTENT AND PERCENT RETENTION IN MULUKHIYAH DURING FREEZE, TRAY, AND ROOM DRYING

Free folacin Total folacin Drying Time content % content % (mcg/g solid)* ret. (mcg/g solid)* ret.

Room Drying 0 21.89 ± 2.40 100 31.50 ± 2.17 100 4 16.45 ± 4.18 75 24. 69 .± 1. Bl 78 8 12.77±2.42 58 18.88 ± 0. 92 60 12 10.22 ± 1.88 50 16.07 .± 1. 49 51 16 9.49 .± 1.57 43 16.32 ± 1.46 52 24 8. 03 ± 1.83 37 14.53 ± 0.56 46 32 7.46 ± 1. 20 34 13.04 ± 1.75 41 36 7.24 ± 0.81 33 12.45 ± 0.68 40

* Values are the means ± standard deviations of the means from 6 re plications. 48

• I

Figure 6. Percent folacin retention ~n mulukhiyah during freeze drying at 32 C. 49

100~------~--~~--~------~--

z ° Free Folic Acid 0 80 1--z ~::&To ta I Folic Acid w 1-- w 70 ~ z u 60 <( ....J 0 LL 50 1--z w 0 u 40 ~ w c... 30

20~--~--~----~--~----~--~~ 0 2 4 6 8 10 12 DRYING TIME, HRS 50

Figure 7. Percent folacin retention in mulukhiyah during tray drying at 50o C. 51

100~--~--~----~--~--~~-----

90 z 0 o Free Folic Acid 0 80 z1- A :A Total Folic Acid w 1- w 70 0::: z u 60 <( --' 0 u.. 50

1- . I z A w 40 /::r ---:6 u 0 0:: w a.. 30

2 4 6 8 10 12 DRYING TIME,. HRS 52

Figure 8. Percent folacin retentioB in mulukhiyah during room drying at 25 C. 53

z 0 o Free Folic Acid 0 80 zt- A A Total Fo I ic Acid w t- w 70 0::: z u 60 <--' 0 L1.. 50 t-z w u 40 0::: w 0.. 30

20~--~--~----~--~----~--~~ 0 6 12 18 24 30 36

DRYING TIME I HRS 54 corresponding retention after 4 hours of tray drying was 42 % and 46 % (Figure 7), and after 12 hours of room drying, 47 %and 51 %, respec tively (Figure 8), after which time the rate of destruction of folic acid in all three drying methods started to slow down. At the equili brium state, mentioned above, the retention of FFA was 42 % after 8 hours in freeze drying, 40 % after 6 hours in tray drying, and 34 % after 32 hours in room drying. The corresponding retention of TFA was 48, 44, and 41 percent, respectively. The only data available that compare folacin retention in vegetables by various dehydration methods were those reported by

Holmes et ~. {1979). They reported that the percent TFA retention for unblanched green beans, tomato puree, zucchini squash, raspberry 1eat her, and boysenberry 1eat her dried by home food dryer \

Rate of folic acid destruction. The rate of TFA destruction was determined as a function of moisture content, where: % folic acid destruction rate = % moisture Figure 9 shows the rate of TFA destruction plotted against the percent moisture content. As shown, the rate was very high at the high 55

Figure 9. The rate of total folic acid destruction plotted against the percent moisture content. 56 ' I

. I ON :::c 2.1 \ D-·-· -·-o Freeze Drying *- ' \ \ \ o--__;_o Tray Drying \ \ \ \ 1::..- ____ -.A Ro o m Dry i n g \

li I . I ,, . .p 'A'-.' . ./f u.. ' ...... / I 0 ' I ' ' I ' ' ' 'A-__ - -tlI

QL-----~---~----L---~--~----~--~ 70 60 50 40 30 20 10 0 PERCENT MOISTURE 57 moisture level, then slowed down as the vegetable approached the equilibrium state, the rate of destruction then increased again. The rate of TFA destruction in freeze and tray drying be haved similarly and had the lowest rate at 25 % and 15 % moisture con tent, respectively. In room drying, the rate of TFA destruction was higher than in tray and freeze drying up to the 51 % moisture content, and then the rate became lower with a minimum rate occurring at 22.5 % moisture level (Figure 9). From these results, as well as for other water-soluble vita mins, it appears that any deviation from the equilibrium state (i.e. water level at which vitamin destruction rate is lowest) would result in an increase of destruction rate. The rate of destruction of reduced ascorbic acid, for example, increases as the moisture content and water activity increase (Kirk~ al., 1977). In the present study; however, water activity was not measured. Lee and Labuza (197~) have inter preted the increase in destruction rates to be the results of dilution of the aqueous phase, which results in a decreased viscosity, and thus increased mobility of reactants. Calloway (1962) stated that too little moisture, as well as too much, is detrimental to both quality and stability of dehydrated foods. The moisture content which corresponds to a theoretical monomo lecular layer of adsorbed water (according to the Brunauer-Emmett Teller equation) is said to be both the maximum allowable and minimum desirable amount. Water in excess of this amount is essentially free and promotes such defects as caking, browning and hydrolysis. Drying below this level increases susceptibility to oxidation. In the case of 58 sweet potatoes and carrots, for example, excess moisture promotes loss of ascorbic acid, while drying below the monolayer value results in oxidation of beta-carotene to beta-ionone. In this study, as shown in Figures 6, 7, and 8, retention of TFA was always higher than FFA. Total folic acid retention in all three dehydration methods ranged from 41 to 48 percent, and that of FFA ranged from 34 to 42 Percent at the equilibrium state. It has been previously established that loss of FFA appears to be greater than that of TFA in food processing and cooking (Perloff and Butrum, 1977). In a study by Taguchi et al. (1973), loss of fola cin was measured in nineteen foods after they had been boiled. After 5 minutes of boiling, 10 to 50 percent of FFA remained, and 20 to 90 percent of TFA remained. After food had been boiled for 15 minutes, only 50 to 10 percent FFA remained and 20 to 40 percent TFA remained.

Huskisson et ~· (1970) studied twenty-eight foods for folacin reten tion after they had been cooked. In their study, mean retention was 27 percent for FFA and 55 percent for TFA. Other data were cited by Per loff and Butrum (1977) showing that TFA retention was higher than FFA in frozen green beans, yellow beans, and sweet potatoes. For example, raw green beans contained 33 % FFA and 44 % TFA, whereas frozen green beans contained 8 % FFA and 33 % TFA. No conditions of process were reported. It is important to notice that in cooking the temperature involved is high (100° C), but the time of heat exposure is short, while in the present study the time interval for drying is long and temperature is low (between 25° to 50° C). In this time-temperature 59 relationship, it seems that the effect on folacin destruction is similar whenever either variable dominates. Holmes et al. (1979) reported that the percent retention of free folacin in home dehydrated unblanched green beans, tomatoes, zucchini, raspberries, and boysenberries was greater than that of total folacin. However, the results of the present study do not agree with that reported by Homes et al. These differences could be due to the fact that fresh mulukhiyah contains a high percentage of free folic acid (70 %). It has been established that free folacin is more sensi tive to heat (Perloff and Butrum, 1977); oxidation (Cooper et £1., 1978); sunlight and artificial light (Stokstad et £1., 1947); and chemical environment (O'Broin !1 ~., 1975) than total folacin. To compare the effect of drying on folacin retention in mulukhiyah by the three drying methods, percent total folacin retention is plotted against moisture content as shown in Figure 10. Tray drying has better folacin retention than room drying. Freeze drying, on the other hand, resulted in the highest folacin retention of the three methods. Calloway (1962) reported that, in general, freeze drying gives the highest possible quality and maintains the highest nutrition al value of any drying procedure. Based on this study, it can be stated that freeze drying is more favored with respect to folacin retention than the other conventional methods of drying studied. Although freeze drying has gained acceptance as the method of drying which will generally produce a product of the highest quality in comparison to other common methods of drying, it is an expensive method of drying. King (1973) cited that 60

Figure 10. The effect of drying on folacin retention in mulukhiyah by the three drying methods, percent total folacin retention is plotted against moisture content. ' . 61

z 90 0 1- z LU ~ 80 a::: z lJ 5 70 0 u..

_ _J

...... z 6. ~:::. Room Drying ~50 o----o Freeze Drying 0:: w a... o o Tray Drying

4 Q '----..L-----'------L_.__ L __.._...... __--:--___. 3.0 2.5 2.0 1. 5 l.O 0.5 0

MOISTURE CONTENT, 9/9 SOLID 62 the cost per pound of water removed is in the range of 10 to 30 cents. By virtue of its position as a high-cost, high-quality drying method, freeze drying has found its application for specialty items, where the quality gain offsets the costs. Since freeze drying is more expensive than other methods and is not readily available to the average Egyptian family, room dry ing is recommended for household application. Tray drying, on the other hand, is more practical for commercial application.

Effect of Storage Condition on Folacin Retention in Dried Mulukhiyah

Room dried mulukhiyah was stored under f~orescent light up to 12 months at room temperature (25° C), packed under either air or nitrogen. The moisture content was 8.6 % as determined at the end of the room drying process. Brown jars with air (BA), brown jars with nitrogen (BN), clear jars with air (CA), and clear jars with nitrogen (CN), were compared in Figures 11 and 12 where TFA and FFA content (meg/g) were plotted as a function of storage time. The destruction of TFA under all storage conditions exhibited similar patterns with storage time (Figure 11). During the first weeks of storage the destruction of TFA was very rapid, then slowed down before leveling off; however, the inflection point for each curve was different. The destruction of TFA in BN stabilized the earliest at the end of 4 weeks, then very little change occurred. BA and CN were very similar in their destruction pattern of total folic acid. Both stabi lized at the end of 24 weeks, whereas the destruction of TFA in CA was the highest and stabilized later at the end of 36 weeks. 63

Figure 11. Effect of packaging condition on TFA content of dried mulukhiyah during storage at room temperature. 64

__. 0 9' e-·---e Brow·n Jar, N· V) 2 0---<> Brown Jar. A! R ~ ., v Clear Jar. N ~8 2 E b-··-·-A Clear Jur, AIR • - z1- 7 w 1-66 u \~ 0 5 u '· <{ u 4 __. 'A...... -6 0 ...... U- ··A-·· __. 3 -·· ~ ---··--··~··A <( 1- 8 2

t--__._-...£-_~_...... ---1--' ..1..--11----A---~---'_.,j 0 8 16 24 32 40 48 STORAGE Tltv\E, WEEKS 65

Figure 12. Effect of packaging condition on FFA content of dried mulukhiyah during storage at room temperature. 66

e-·-·-· Brown Jar, N2 -0 .....1 6 ·o-- --o Brown Jar, AIR· 0 V) v v Cleor Jar, N2 ~ .b-··-··.-A Clear Jar, AIR CJ) v 5 -E 1- z ..__L!..J z 4 0 u Q u <{ 3 u .....1 0 ·. ._ U- '~ ·· ...... 0 w 2 A'·· -o--- w ...... --- - u..~ A A--·· .. - --- . -··-··--A 1 0 8 24 32 40 48

TII\~E I WEEKS 67

As shown in Figure 11, the highest content of TFA in mulu khiyah during storage was found in BN, next was both BA and CN. CA had the lowest TFA content. At the end of the storage period TFA con tents were 6.1, 4.6, 4.2, and 2.9 micrograms per gram of dry weight for BN, BA, CN, and CA, respectively. The destruction pattern of FFA was similar to that found in TFA. The destruction of FFA was very rapid in the first weeks, then the rate of destruction gradually slowed down with different rates, dependent on storage conditions (Figure 12). BN stabilized the earli est, at the end of 4 weeks, and had the highest content of FFA. BA and CN were very similar and stabilized at the end of 20 weeks. The rapid destruction of FFA in CA continued up to 24 weeks, after which time it slowed down. CA had the lowest content of FFA throughout the storage period. Free folic acid contents in dried mulukhiyah samples at the end of the storage period were 3.0, 2.1, 1.7, and 1.3 micrograms per gram of dry weight for BN, BA, CN, and CA, respectively. Therefore, it can be stated that stability of folacin in stored, dried mulukhiyah varied widely with conditions of storage. The destruction of free and total folic acid acid was affected by the con dition of packaging (air or nitrogen). Folic acid content in jars packed with nitrogen was higher than that in jars packed with atmos pheric air. Also folic acid content in brown jars was higher than that in clear jars under both air or nitrogen. No significant differences, however, were found between BA and CN. These results show that the destruction of folic acid by oxygen is in the same order of magnitude as that by light (Figures 11 and 12). That is to say that auto- and 68 photo-oxidation have the same magnitude of effect in folic acid des truction. A similar pattern in folic acid destruction during storage of flour was reported by Keagy etJ8, (1975). The native flour folacin content decreased early during storage. This decrease did not depend on storage temperature, but stabilized later at a level dependent on temperature. Other data were reported by Malin (1977), where the greatest rate of TFA destruction during frozen storage of Brussel sprouts at -21° C occurred during the first 67 days of storage. Then very little change occurred. The percent retention of FFA and TFA in dried mulukhiyah stored in brown and clear jars, under nitrogen or atmospheric air, was calculated and presented in Table 7. The storage of dehydrated mulukhiyah resulted in a mean of 45, 32, 25, and 20 percent retention of FFA in BN, BA, CN, and CA, respectively. The corresponding TFA retention was 54, 40, 37, and 26 percent at the end of the storage period. The percent retention of TFA in BA, BN, and CN during the first 16 weeks of storage was very much similar to that of the FFA, after which time the retention of TFA was higher than that of FFA (Table 7). This observation could be explained by: the stability of FFA and TFA are the same in the dry state under these storage condi tions. TFA in CA was always higher than FFA throughout the storage period. No systematic studies that compare the retention of FFA and TFA during storage have been reported. Most of the studies on the effect of storage conditions found in the literature were either the 69 f .

Table 7 EFFECT OF PACKAGING CONDITION ON FREE AND TOTAL FOLIC ACID RETENTION IN DEHYDRATED MULUKHIYAH

FFA Retention (%) TFA Retention (%) Storage Brown bottle Clear bottle Brown bottle Clear bottle time air N2 air N2 air N2 air N2

0 100 100 100 100 100 100 100 100 1 day 89 86 71 87 96 91 78 88 3 day 81 83 67 76 83 80 71 79 5 day 67 72 56 74 72 71 66 72 2 week 62 67 53 69 66 67 63 69 3 week 62 69 50 59 60 65 57 4 week 56 65 41 50 57 60 53 58 8 week 53 61 37 49 54 60 49 53 12 week 49 59 35 46 56 38 49 16 week 44 55 29 42 49 56 38 47 20 week 41 53 26 36 47 57 34 46 24 week 39 51 24 32 46 57 32 43 36 week 31 48 24 28 40 57 27 39 48 week 32 45 20 25 40 54 26 37 70

TFA or the FFA. It was also found that the retention of FFA and TFA in BA and BN at the beginning of the storage period was very similar. This could be attributed to gas entering the BN jar during withdrawal of the sam ples for analysis. Therefore, it is recommended for future experiments to use smaller samples in separate jars, that is to eliminate the poss ibility of gas entering the jars. As was noted before, loss of TFA in dried mulukhiyah during 6 months of storage at room temperature (77° F) was substantial and ranged from 43 to 68 percent in all storage conditions (Table 7). These data are higher than those reported by Augustin et al., (1978). They reported that the loss of TFA in stored potato for 8 months at 35-45° F ranged from 17-40 percent; however, no other conditions were reported. The higher loss of TFA in the present study could be due to the higher storage temperature (77-85° F). It has been reported by Olson et al. (1947) that fresh vegetables stored at room temperature lost large amounts of folacin within a short period of time, whereas refrigeration and ice storage prevented folate loss for a period of 2 weeks or more. In the study by Keagy et al. (1973) on the stability of native folic acid in flour during storage at three temperatures, it was shown that the natural folic acid in the flour is fairly stable at 84° F, with 86 % retention in 12 months, but showed progressively in creasing losses as the temperature increased. Retention of 78 % in 4.5 months at 100° F, and 62 % in one month at 120° F were reported. The retention of TFA in the present study is lower than that reported 71

by Keagy et al. (1973). This can be explained by the differences in the chemical composition and type of light used. Mulukhiyah was stored under flourescent light, whereas flour was stored under warehouse con ditions. It has been reported that folic acid is sensitive to light (Stokstad et al., 1947). Retention of TFA in dried mulukhiyah in all the storage con ditions ranged from 54 to 26 percent (Table 7). These results are lower than those reported by Suchewer et al. (1970). They showed that retention of folic acid in tomato juice stored in the dark for 12 months was an average of 93 % in containers of all types, versus 70 % in orange bottles stored in daylight. This difference could be ex plained by the possible higher ascorbic acid content in tomato juice than in dried mulukhiyah. Thus, the natural vitamin C in tomato ap pears to be sufficient to exert an anti-oxidative effect on folate activity. It has been established that both indigenous and added as corbic acid exhibit a protective effect on the folate activity of UHT milk, and that added ascorbic acid (60 mg/liter) was sufficient to protect the folate in milk during UHT processing and subsequent storage at 20° C for 60 days (Gregory, 1975). It has also been reported by Malin (1977) that higher retention of TFA in Brussel sprouts during storage is due to high vitamin C content. Few data are available that compare the effect of the type and color of containers on the stability of folic acid during storage. However, numerous investigations have shown the effect of type and color of container on other water-soluble vitamins. For example, ascorbic acid losses in milk stored in uncolored glass are 14 times 72

greater than those in brown glass, and that milk in blue paper cartons loses 5 times more vitamin C than in red paper (Karel and Heidelbaugh, 1975). Gregory (1975) has reviewed the effect of type of containers on the ascorbic acid destruction in milk. When homogenized milk was stor ed in glass or plastic containers and exposed to a cool white fluores cent light at 7° C, there was a rapid decrease in the ascorbic acid content (from 13 to 1.5 mg/1) during the first 48 hours. In fibre board containers, the loss was more gradual and only reached 16 % in 144 hours. He concluded that during distribution of milk losses of riboflavin and ascorbic acid were lowest when brown bottles were used. The present study also shows that retention of FFA and TFA was higher in jars packed with nitrogen than in jars packed under at mospheric air in both brown and clear jars. This destruction is due to auto-oxidation. Chen and Cooper (1979) reported that the stability of some forms of folate at 100° C was drastically increased in the presence of ascorbate or under nitrogen atmosphere. These data indi cated that degradation of these folates at elevated temperatures is due to an oxidative process requiring the presence of molecular oxygen. These data also suggest the possibility of using nitrogen storage to protect labile folate in food products. There were no published data that compare the effect of pack aging on folic acid destruction in dehydrated vegetables. However, there are data showing the effect of oxygen on folic acid destruction in foods processed by heating. For example, canning, which results in low levels of free oxygen in foods, has been shown to be a method that eliminates oxygen and decreases folate losses during storage. Suchewer 73

et 2}. (1970) stated that losses of folic acid in tomato juice pro duction were higher on a Yugoslav (70 %) than on an American line (50 %). This was attributed to the shorter exposure to heat and oxygen on the latter. Rolls and Porter (1973) also observed increased folacin retention as a result of eliminating oxygen during processing of milk. They reported that pasteurization processes resulting in very low oxygen levels, such as evaporative cooling, suffer little folacin loss up to 180 days of storage. In contrast, milk containing higher levels of dissolved oxygen ~tJas reported to suffer large folacin losses within a fe~tJ days. The role of packaging on the stability of other water-soluble vitamins has been investigated extensively. Storage under conditions of low headspace oxygen concentration has been found to increase the stability of ascorbic acid and riboflavin (Ualetzkoi and Labuza, 1976). When carrot flakes were stored with nitrogen, very little loss of as corbic acid occurred over a 24 month period. When stored in air, the rate of loss was very rapid in the first 4 months, after which no change was found (Stephens and Mclamore, 1969). Gee (1979) stored dried carrots, spinach, and tomatoes in the dark, in air or in nitrogen and found total ascorbic acid, thiamin and beta-carotene were lost more rapidly in air storage than under other conditions. The repeated treatment (Factorial Designs) of variance (Joseph and Joseph, 1975) was used to determine the effect of storage time, and storage condition on folic acid retention. It was found that there were significant differences in FFA and TFA retention (p< 0.01) , between the color of container (brown and clear jar), the storage 74 condition (atmospheric oxygen and nitrogen), and storage time. There fore, the working hypothesis was accepted. Following this analysis, Tukey's Honestly Significant Diff erence test (Roscoe, 1975) was used to determine which groups of means were significantly different. Table 8 presents a summary of Tukey's HSD for comparison of means of FFA and TFA retention in dried mulukhi yah stored under different conditions at various storage times. The folic acid retention at four storage periods were compared as follows: week 1 and 4, week 1 and 24, week 1 and 48, week 4 and 24, week 4 and

48, and v1eek 24 and 48. Significant differences (p< 0.01 in FFA retention were observed between week 1 and 4, 24 , 48 week 4 and 48 in BA, week 1 and 24, week 4 and 48 in BN, week 1 and 24, week 4 and 24 in CA, and weeks 1 and 4, weeks 4 and 48 in CN. The corresponding difference in TFA was found between weeks 1 and 24, weeks 4 and 48 in BA, weeks 1 and 48 in BN, weeks 1 and 24, weeks 4 and 24 in CA, and weeks 4 and 24 in CN. These data indicate that storage time affected the stability of folic acid. Within the experimental sample at a given storage time, significant differences in FFA and TFA retention between the storage conditions were not observed until week 4. After week 4 the storage condition significantly affected the stability of folic acid. For example, in week 24 there was a significant difference (p~ 0.01) between BA, BN, and CA, and between BN, CA, and CN in FFA retention and between BN and CA in TFA retention. In week 48 there was a sig nificant difference between BA, BN, and CA, and between BN and CA in FFA retention and between Btl, CN, and CA in TFA retention (Table 8). 75

Table 8 SUM~1ARY OF TUKEY 1 S HSD FOR COMPARISON OF MEANS OF FFA AND TFA RETENTION IN DRIED MULUKHIYAH STORED UNDER DIFFERENT CONDITIONS AT VARIOUS STORAGE TIMES

Storage Mean FFA retention i%1* Mean TFA retention (%)* condition 1 4 24 48 1 4 24 48 week weeks weeks weeks week weeks \"'eeks weeks Brm1n jar with air (BA) 67axyz 56 ax 39ay 32 a xz 72axyz 57az 46ay 40acxz Brown jar with nitrogen (BN) 72 bxyz 65bz 51 a by 45bYZ 71bXYZ 60b 57b 54bz Clear jar with air (CA) 56cxyz 41bz 24 ab yz 20 ayz 65cxyz 53cy 32bxy 26ba~y Clear jar with nitrogen (CN) 74dxyz 50cxz 32bx 25bxz 72dxyz 58dxy 43cxy 37bxy

*Mean followed by same letters (a,b,c, or d) within the same column are significantly different at to 1 % level. Means followed by same letters (x,y, or z) within the same line are significantly different at the 1 % level according to Tukey•s Test. 76

In general, these results indicate that storage time as well as storage condition are important factors in the stability of folic acid present in dry mulukhiyah. Chapter V SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

The effects of drying method (room, tray, and freeze drying), moisture content, storage time (up to 48 weeks), and storage condition (clear and brown jars, packed under either nitrogen or air) on folacin retention in mulukhiyah were studied. The FFA and TFA contents of mulukhiyah were determined before, during, and after drying processes, as well as during storage. The results show that mulukhiyah is an excellent source of folate in the Egyptian diet. Fresh mulukhiyah contained 556 meg FFA and 800 meg TFA per 100 g. The room dried vegetable was found to con tain 662 meg of FFA and 1138 meg of TFA per 100 g dry weight basis. One serving of the fresh mulukhiyah would contain 400 meg of TFA, and one serving of the dried vegetable would contain 283 meg of TFA. Since the RDA for folacin for adults, as established by the Food and Nutri tion Board of the National Research Countil {1980) is 400 meg, mulu khiyah can make a major contribution in fulfilling the folacin require ment. The destruction of folic acid in mulukhiyah was high in all three drying methods studied. The retention of FFA ranged from 34 to 42 percent, and TFA ranged from 42 to 48 percent. Retention of TFA was always higher than that of FFA. This higher destruction of free folic acid in mulukhiyah during the drying process could be attributed to the

77 78 form of folic acid in mulukhiyah (70 % of the TFA is FFA), and the fact that free folacin is more sensitive to heat than total folacin. The freeze drying method resulted in higher folacin reten tion than the other drying methods. However, the differences found in folic acid content between freeze, tray and room dried mulukhiyah were small. Therefore, it can be stated that freeze drying is more favored with respect to folacin retention than the other conventional methods of drying used in this study, next to it is the tray drying. Since freeze drying is more expensive than the other methods, and is not readily available to the Egyptian family, room drying is recommended for household application. Tray drying, on the other hand, is more practical for commercial application. The retention of folacin in room dried mulukhiyah during storage varied depending on the storage conditions. There was a sig nificant difference (p~O.Ol) in folacin retention between packaging color, packaging gas, and the length of storage time. There was a significant decrease in folic acid retention with storage time of 48 weeks under all conditions. The rate of folic acid destruction was very rapid in the first 4 weeks, after which the rate slowed down de pending on storage conditions. Storage in brown jars packed under nit rogen had a higher retention of folic acid than the others, next to it was the brown jar with air. Brown jars with air and clear jars with nitrogen had very much similar folic acid retention. This suggests that auto- and photo-oxidation have the same magnitude of effect in folacin retention. Severe destruction of folic acid was found when clear jars with air were used. This appears to be due to both auto- 79 and photo-oxidation in effect. The percent retention of TFA in BA, BN, and CN during the first 16 weeks of storage was very similar to that of the FFA, after which time the retention of TFA was higher than that of FFA. TFA in CA was always higher than FFA throughout the storage period. Since mulukhiyah is among the most common vegetable in the Egyptian diet, more research is needed to determine the effect of the cooking process on folic acid retention in mulukhiyah. Further studies also are needed to determine the exact form of folate in mulukhiyah. Based on the information derived from this study, it is re commended that a study to determine the kinetics of folic acid losses during dehydration, constant temperatures and storage at various mois ture contents be conducted. Also, more research should be done to com pare the rate of folic acid destruction in other dehydration methods, and the rate of destruction of the different forms of folic acid. REFERENCES

80 REFERENCES

AOAC, 1975. Official Methods of Analysis 12th Ed. Association of Official Analytical Chemists, Washington, D.E. p. 130. ~gier, R.B., Boothe, J.H., Hutchings, B.W., Mowat, J.H., Smeb, J., Stokstad, E.L.R., Subbarow, Y., Waller, C.W., Cosulich, D.B., Fahrenbach, M.F., Hultquist, ~1.D., Kuh, E., Northey, E.H., Seeger, D.R., Sickels, J.P., and Smith, J.~1.jr. 1946. 11 The Structure and Synthesis of the Liver!:_. casei Factor ... Science, 103: 667-69.

Augustin, J., Johnson, S.R., Teitzel, C., Toma, R.B., Shaw, R.L., True, R.H., Hogon, J.M. and Deutsch, R.~1. 1978. 11 Vitamin Composition of Freshly Harvested and Stored Potatoes ... Journal of Food Science, 43 (5): 1566-70.

Bailey, L.H. 1907. Cyclopedia of American Agriculture. Vol. II New York, The Macmillan Company; London Macmillan & Co. Ltd., p. 282- 283 and 507.

Biely, J., ~1arch, B., and Tarr, H.L.A. 1952. 11 The Effect of Drying Temperature on Folic Acid Content of Herring ~1eals. 11 Science, 116 (9): 259-50.

Bloom, E.S., Vandenbelt, J.~·1., Binkley, S.B., O'Dell, B.L., and Pfiffner, J.J. 1944. 11 The Ultraviolet Absorption of Vitamin Be and Xanthopterin ... Science, 100: 295-97.

Bluestein, P.~1., and Labuza, T.P. 1975. 11 Effects of Moisture Removal on Nutrients ... Nutritional Evaluation of Food Processing. pp. 289- 323. Edited by Harris, R.S., and Karms, E. 2nd Ed. l4estport, Conneticut: The Avi Publishing Company, Inc.

Brenner, S., Hodicka, V.O., and Dunlop, S.G'. 1948. 11 Effect of High Temperature Storage on the Retention of Nutrients in Canned Food. 11 Food Technology, 2: 207-21.

Calloway, D.H. 1962. 11 Dehydrated Foods. 11 Nutrition Review, 20: 257-60.

/chan, C., Shin, Y.S., and Stokstad, E.L.R. 1974. 11 Studies of Folic Acid Compounds in Nature. 111, Folic Acid Components in Cabbage ... Canadian Journal of Biochemistry, 51: 1917-23.

Charm, S.E. 1971. Fundamentals of Foods Engineering. Westport, Conneti cut: Avi Publishing Co.,

81 82

/chem, T.S., and Cooper, R.G. 1979 11 Thermal Destruction of Folacin: Effect of Ascorbic Acid, Oxygen and Temperature ... Journal of Food Science. 44: 713-716.

Cooper, R.G., Chen, T.S., and King, M.A. 1978 11 Thermal Destruction of Folacin in Microwave and Conventional Heating ... Journal of American Dietetic Association, 73: 406-410. Cort, W.M., Borenstein, B., Harly, J.H., Osadea, M., and Scheiner, J. 1976. 11 Nutri ent Stability of Fortified Cerea 1 Products. 11 Food Technology, 30 (4): 52-62. -- ~igen, E., and Shockman, G.D. 1963 11 The Folic Acid Group. 11 Chap. 7.5 in Analytical Microbiology, I, pp. 431-488 Edited by Kavanaugh, F.J. New York: Academic Press. /Food and Nutrition Board, 1980: Recommended Daily Dietary A11 owance. Ninth revised edition. Washington, D.C. National Academy of Sci.

Gee, M. 1979. 11 Stability of Ascorbic Acid, Thiamin, and B-Carotene in Some Low Temperature Dried Vegetables. 11 Lebensmittel Wissenschaft Und - Technologie, 12 (3): 147-49. Through Food Science and Tech nology Abstract, (1980).

Ghitis, J. 1966. 11 The Labile Folate of ~1ilk. 11 American Journal of Clinical Nutrition, 18 (6): 452-57.

Goc, A. 1977. 11 A Method for Cold Drying of Food Products. 11 Bulletin Technique d'en Forwation, 322: 497-99, 516. Through Food Science and Technology Abstract, 11 (1). {1979).

Gregory, J.F. and Kirk, J.F. 1978. 11 Assessment of Storage Effects on Vitamin B6 Stability and Bioavailability in Dehydrated Food Sys tems ... Journal of Food Science, 43: 1801-15.

Gregory, ~1.E. 1975. 11 Water Soluble Vitamins in Milk and Milk Products. 11 Journal of Dairy Research, 42: 197-216.

Hanning, G., and ~1itts, M.L. 1949. 11 Effect of Cooking of Folic Acid Content of Eggs. 11 Journal of the American Dietetic Association, 25 (3): 226-28. Harris, R.S., and Von Loesecke, H. 1960. Nutrition Evaluation of Food Processing. Westport, Conneticut: Avi Publishing Co.

llellendorn, E.W., DeGroat, A.P., Van Der ~1ijll Dekker, L.P., Slump, P., and ~Jill iams, J.J.L. 1971. 11 Nutritive Value of Canned Meals ... Journal of the American Dietetic Association, 58 (5): 434-41. 83 ~ '

Halsted, C.H., Sourial, N., Cuindi, S. 1969. 11 Anemia of Kwashoirkor in Cairo: Deficiency of Protein, Iron and Folic Ad:d. 11 The American Journal of Clinical Nutrition, Vol. 22 (10): 1371-82. Holmes, Z.A., Miller, L., Edwards, M., and Benson, E. 1979. 11 Vitamin Retention During Home Drying of Vegetables and Fruits ... Home Economics Research Journal, 7 (4): 258-64. --

/t1urdle, A.O.F., Barton, D. and Searles, I.H. 1968. 11 A Method for Measur ing Folate in Food and its Application to a Hospital Diet. 11 American Journal of Clinical Nutrition, 21: 1202.

Huskisson, Y.J. and Retief, F.P. 1970. 11 Folate Content of Foods ... South African Medical Journal, 28 (12): 362-63. ~oseph, ~1.L. and Joseph, H.D. 1975. Essentials of Research Methodology and Evaluation for Home Economist. Plycon Press. pp. 93.

Karel. ~-1., and Heidelbaugh, N.D. 1975. 11 Effects of Packaging on Nutri tion ... In the Nutritional Evaluation of Food Processing, 2nd Ed. pp. 412-62. Edited by Harris, R.S., and Karmas, E. Westport Conn eticut: The Avi Publishing Co.

Karmas, E., Thompson, J.E. and Peryam. D. B. 1962. 11 Thiamin Retention in Freeze Dehydrated Irradiated Pork. 11 Food Technology, 16: 107. Keagy, P.t1., Stokstad, E.L.R., and Fellers, D.A. 1975. 11 Folacin Stab ility During Bread Processing and Family Flour Storage. 11 Cereal Chemistry, 52: 348-56. Kirk, J., Dennison, D., Kokoczka, P., Heldman, D. 1977. 11 Degradation of Ascorbic Acid in a Dehydrated Food System. 11 Journal of Food Sci., 42 (5): 1274-79. King, C.J. 1973. 11 Freeze Drying ... Chapter 6 in Food Dehydration, pp. 161-62. Edited by Van Arsdel, W.B., Coply, M.F. and Morgan, M.A. 2nd Ed., Vol I. Westport, Conneticut: Avi Publishing Co. Labuza, T.F. 1972. 11 Nutrient Losses During Drying and Storage of De hydrated Foods. 11 CRC Critical Review in Food Science and Tech nology, 3: 217-40.

Lee, S.H., and Labuza, T.P. 1975. 11 Destruction of Ascorbic Acid as a Function of Water Activity ... Journal of Food Science, 40: 370-73.

Lin, K.C., Luh, B.S., and Schweigert, B.S. 1975. 11 Folic Acid Content of Canned Garbanzo Beans. 11 Journal of Food Science, 40: 562-65. ~lin, J.D. 1975. 11 Folic Acid. 11 World Review of Nutrition and Dietetics, 21: 198-223. 84

~1alin, J.D. 1977. 11 Total Folate Activity in Brussel Sprouts: the Effects of Storage, Processing, Cooking and Ascorbic Acid Content ... Journal of Food Technology, 12: 623-32.

t1alkki, Y. and Heinonen, S. 1978. 11 Freeze drying of High Aroma Onions. 11 Journal of Sci. A ric. Soc. Finl., 50 (2): 125-36. In Chemical Abstract, 90 2 1979.

Moy, J.H. Wang, N.T.S, and Nakayama, T.O.M. 1977. 11 Dehydration and Pro cessing Problems of Taro. 11 Journal of Food Science, 42 (4): 917-20.

~1iller, C.F., Guadagni, .D.G, and Kon, S. 1973. 11 Vitamin Retention in Bean Products: Cooked, Canned, and Instant Bean Powders. 11 Journal of Food Science, 38: 493-95. Miller, M.W., Frank, H.W., and George, K.Y. 1975. Drying Foods at Home. University of California: Division of Agricultural Science.

Mrak, E.~1., and Phaff, H.J. 1947. 11 Recent Advances in the Production and Handling of Dehydrated Fruits. 11 Food Technology, 1: 147. O'Broin, J.D., Temperley, I.J., Brown, J.P., and Scott, J.M. 1975. 11 Nutritional Stability of Various Naturally Occurring Monoglutamate Derivatives of Folic Acid. 11 American Journal of Clinical Nutrition, 28 (5): 438-44.

Olson, O.E., Burris, R.H., and Elvehiem, C.A. 1947. 11 A Preliminary Re port on the Folic Acid Content of Certain Foods. 11 Journal of the American Dietetic Association, 23 (1): 200-03.

Paine-~~ilson, B.P. and Chen, T.S. 1979. 11 Thermal Destruction of Folacin: Effects of pH and Buffer Ions. 11 Journal of Food Science, 44: 717-22.

Patil' v.R.' Kulkarni' D.N.' Kulkarni' K.' and Ingle, u.~-1. 1978. 11 Effect of Blanching Factors on Quality and Durability of Sun Dried and De hydrated Fenugreek (Methi). 11 Indian Food Packer, January-February: 43-9. Patwardhan, V.N. and Darby, W.J. 1972. The State of Nutrition in the Arab Middle East. Nashville, Tennessee: Vanderbilt Univ. Press.

·Perl off, B. P. and Butrum. R.R. 1977. 11 Folacin in Selected Foods. 11 Journal of the American Dietetic Association, 70 (2): 161-71. Potter, N.N. 1978. Food Science. 3rd Ed. pp. 234-36. Westport, Conneti ·cut: Avi Publishing Co. ~abinowitz, J.C. 1960. 11 Folic Acid." Chapter 2 in The Enzymes, pp. 185- 252. Edited by Boyer, P.O., Lardy, H., and t1yrbeck, K. 2nd ed. Nev-1 York: Academic Press. 85

Rao, S.K. 1974. 11 Malnutrition in the Eastern Mediterranean Region. 11 World Health Organization Chronical, 28: 172-77. Rolls, B.A., and Porter, J.W.G. 1973. 11 Some Effects of Processing and 11 Storage on the Nutritive Value of ~·1ilk and Milk Products. Proceedings of the Nutrition Society, 32: 9-15. ~oscoe, J.T. 1975. Fundamental Research Statistics for the Behavioral Sciences. 2nd Ed. Holt, Rinehart and Winston, Inc. lsantini, R., Brewster, C., and Butterworth, C.E. Jr. 1964. 11 The Dis tribution of Folic Acid Active Compounds in Individual Foods. 11 American Journal of Clinical Nutrition, 14 (4): 205-10.

Schroeder, H.A. 1971. 11 Losses of Vitamins and Trace Minerals Resulting from Processing and Preservation of Food. 11 American Journal of Clinical Nutrition, 24 (5): 562-72.

Stephens, T.S. and ~kLamore, T.A. 11 Preparation and Storage of De hydrated Carrot Flakes. 11 Food Technology, 12: 1600-02.

'Stokstad, E.L.R., Fordham, D., and DeGrunigen, A. 1947. 11 The Inactiva tion of PGA by Light. 11 Journal of Biological Chemistry, 167: 877-78.

Suckewer, A., Bartnik, J., and Seconiska, B. 1970. 11 Influence of Tech nological Processes and Conditions of Storage on Content of Folic Acid in Some Vegetable Products 11 Rocziniki Panstwowego Zakled Hegieny, 21 (6): 619-29. Through Food Science and Technology . Abstracts, 3 (7) 1971. ~mura, T., Shin, Y.S., Williams, M.A. and Stokstad, E.L.R. 1972. 11 Lactobacillus casei Response to Pteroylglutamates. 11 Analytical Biochemistry, 49: 517. lramura, T., and Stokstad, E.L.R. 1973. 11 The Availability of Food Fol ates in Man. 11 British Journal of Haematology, 25: 513. Taguchi, H., Hara, K., Hasei, T., and Sanada, H. 1972. 11 Study on the Folic Acid Content of Foods I: Folic Acid Contents of Various Foods. 11 Vitamin, 46 (6): 313-38. Through Food Science and Tech nology Abstracts, 6 (1) 1974.

Taguchi, H., Hara, K., Hasei, T. 1973. 11 Study of Folic Acid Content of Foods II: Loss of Folate in Foods by Boiling. 11 Vitamin, 47 (1): 21-5. Through Food Science and Technology Abstracts, 5 (12) 1974.

Thomas, ~1.H. and Calloway, D.H. 1961. 11 Nutritional Value of Dehydrated Food. 11 Journal of the American Dietetic Association 86

Van Arsdel, H.B., and Coply, ~1.F. 1963. Food Dehydration, Vol. 1, Westport, Conneticut: Avi Publishing Co.

l~aletzkoi, P., and Labuza, T.P. 1976. 11 Exce1lerated Shelf Life Testing of an Intermediate r1oisture Food in Air and in an Oxygen-Free Atmosphere ... Journal of Food Science, 41: 1338. /waters, A.H., and r~ollin, E.L. 1961. "Studies on the Folic Acid Ac tivity of Human Serum." Journal of Clinical Pathology, 14: 335-44. APPENDICES

87 APPENDIX A PREPARATION OF CHEMICAL SOLUTIONS AND CULTURE MEDIA

A.1 Maintenance Medium - The environment in which the L. casei stabe were maintained was prepared following the methods of Stokstad (1968). The following chemicals were combined in a 500 ml beaker:

Difco Yeast Extract 1. 0% 2.0 grams Dextrose 0.5% 1.0 grams Difco Bacto Agar 1. 5% 3.0 grams

Sodium Acetate 3H20 0.5% 1. 0 grams Deionized Hater 200 ml

The solution was heated while stirring unti 1 it reached the boiling point, at which time it became clear. The beaker was promptly removed from the heat to prevent the solution from boiling over. Ten milliliter aliquots were pipetted into culture tubes. The tubes were capped and autoclaved for 15 minutes at 15 psi. The tubes were cooled in a vertical position and refrigerated for up to 10 weeks.

A.2 Inoculum Broth - The medium for subculture of L. casei from the maintenance medium was prepared following the methods of Stokstad (1968). The following solutions were pipetted into culture tubes:

Pte.Glu Solution C 2.0 ml Phosphate Buffer+ 0.1% Ascorbate 0.5 ml Bas a 1 t-1ed i urn 2.5 ml

88 89 f '

The culture tubes were capped, autoclaved for five minutes at 15 psi, and cooled. Each tube contained 1 ng of Pte.Glu.

A.3 Sterile Saline - Sterile saline was used to wash the L. casei cells free of folacin and to suspend the cells for inoculating. It was prepared by dissolving 0.9 qrams sodium chloride crystals in 100 ml deionized water. The solution was autoclaved for 15 minutes at 15 psi before use.

A.4 Basal Medium - Folic Acid Casei Medium (Difco Labs) was prepared weekly by suspending 94 grams medium in one liter deionized water. The solution was heated to boiling and allowed to boil 2 to 3 minutes with continuous stirring. The basal medium, without auto claving, was stored in a refrigerator (small quantity for immediate use) or in a freezer (large quantity divided into small quantities for longer period of time).

A.5 Phosphate Buffered Ascorbate Solution - A 0.05 M sodium phosphate buffer, pH 6.1 was first prepared using:

NaH Po g 2 4 27.17 Na 2HP04 10.75 g Distilled Water 4 liters

The pH was adjusted to 6.1 with HCl and NaOH. One hundred milli grams percent (100 mg %) ascorbic acid was added to the buffer no more than four hours before use for the purpose of protecting the folate activity from destruction by oxidation during the assay. APPENDIX B PREPARATION OF HOG KIDNEY CONJUGASE

B.1 Deatils of the procedure were first reported by Eigen and Shockman (1963). The following steps outling the procedure followed in establishing the preparation of hog kidney conjugase:

1. Fresh kidneys were obtained from a slaughter house. 2. Two hundred grams of fresh, defatted hog kidney were chopped and homogenized in a Waring blender containing 3 volumes (about 700 ml) of cysteine hydrochloride buffer, 0.3 % (2 x 10-2 M) at pH 5.4. 3. The suspension was poured into 1000 ml Erlenmeyer flask. The flask was stoppered 4. The suspension was autolysed under a layer of toluene for 2 hours at 37° C in an incubator. 5. The foam was discarded, and the suspension was filtered through glass wool. 6. The filtrate was centrifuged at 0° C for about 20 minutes at

1,000 X g. 7. Fat that floated on top was removed and the supernatant re centrifuged at 0° C for 30 minutes at 4,000 x g. 8. The pH of the supernatant was adjusted to 4.5 with HCl. 9. The supernatant was treated with 30 g Dowex 1-X8 (chloride form) in an ice bath for 1 hour with occasional stirring

90 91

(folic acid was removed by the resin). 10. The mixture was centrifuged at 2,000 x g for 30 minutes. (Steps 9 and 10 were repeated to remove folic acid further from supernatant.) 11. The supernatant was tested for folic acid. 12. When the supernatant (conjugase) was free from folic acid it was stored in 50 ml aliquots in the frozen state (-20° C). 13. Gel chromatograph was used to purify the supernatant if it still contained folic acid, as follows: 1. Fine Sephadex G-25 was suspended in 0.1 M acetate buffer containing 0.2 % ascorbate at pH 4.8. 2. Gel was poured into a glass column to at least 20-25 em height. 3. After the gel settled down (the top should be flat) the enzyme solution was poured as soon as the solvent level dropped to the top of the gel. (Approximately 2 inches height volume of solution was purified each time.) 4. After the layer of enzyme dropped, buffer was always added to maintain a head of 1 inch. It was possible visually to see the separation on the column. The enzyme fraction was brown in color and passed through the column quickly. The folic acid was a yellow color layer and passed slowly 5. The enzyme was collected and stored frozen (in 10 ml ali quots).

B.2 0.1 M Acetate Buffer Preparation (pH 4.7) -The 0.1 M acetate buf- 92 fer used for the conjugase treatment of samples and for conjugase purification was prepared as follows:

0.1 M Sodium acetate - 13.6 g Na0Ac·3H2o dissolved in 1 liter of distilled H2o 0.1 M Acetic acid - 6 g (5.7 ml) acetic acid was diluted with distilled water to 500 ml

The sodium acetate solution was titrated with the acetic acid solution to a pH of 4.7. (Note: the pH of NaOAc is about 8.0; it took 700 ml of acetic acid solution to adjust 1 liter of NaOAc to pH 4.7.) One gram ascorbic acid (0.2 %) was added to acetate buffer for use in conjugase purification. APPENDIX C PREPARATION OF FOLIC ACID STANDARD SOLUTIONS

Folic acid standard solutions were prepared as follows:

C.1 Solution A (25 mcg/ml)

1. Twenty-five milligra~s Pte Glu (folic acid) crystals (ICN Pharmaceuticals, Inc., Life Sciences Group, Cleveland, Ohio) were precisely weighed out. 2. Crystaline folic acid was dissolved in 100 ml of a 0.01 N NaOH solution containing 20% ethanol (See C.4). 3. Enough 0.01 N NaOH with 20 % ethanol was added to the above solution to bring the volume to one liter. 4. Ten milliliter aliquots were pipetted into foil-wrapped test tubes, flashed with nitrogen, stoppered, and stored until used.

C.2 Solution B (25 ng/ml) One milliliter of solution A was diluted precisely to one liter with 0.01N NaOH containing 20 % ethanol (See C.4) and stored in a freezer. This solution had to be used within 2 weeks.

C.3 Solution C (0.5 ng/ml)

Two ~illiliters of solution B were diluted precisely to 100 ml with 0.05 M phosphate ascorbate (0.1 %) buffer (See A.5).

C.4 0.01 N NaOH Containing 20 % Ethanol

210 ml 95 % EtOH (~1C/B Denature ethyl alcohol) were combined with

93 94

790 ml of distilled water (total = 1 liter), and 12.5 ml of 0.08 N NaOH (MC/B, SX 607) was added.