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
ii
DEDICATION
To
t~y
Son
Mike
And
~1y
Family
;;;
ACKNOl~LEDGB1ENT
I wish to thank those who worked closely with me and contributed 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 throughout 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 enterprise.
And finally I especially thank my mother, Angele Yossef, without 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
• • • viii
LIST OF FIGURES
ABSTRACT
ix
Chapter
I.
II.
INTRODUCTION
1
Objective •
2
Limitations
3
Definition of Terms
3
LITERATURE REVIEW
Folacin • • •
........
Dehydration .
8
Destruction of Vitamins During Drying
and Storage
• • • • • • •
....
Mulukhiyah •••••
III.
5
10
18
MATERIALS AND METHODS
.....
Vegetable
•
..
....
..
~~icroorganism
Ba sa 1 Med i urn • •
....
Chemical Reagents
v
19
19
19
19
19
Chapter
Page
....
Equipment •
20
Methods
20
.•
Drying
....
Storage Test . .
...........
Moisture Determination
....
Folacin Determination . .
....
22
Microbiological Assay Procedure
24
Sample Preparation
r~ethods
........
Data Treatment
IV.
20
23
23
36
RESULTS AND DISCUSSION
Folacin Activities in Mulukhiyah
V.
20
.......
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
SUt~MARY,
REFERENCES
CONCLUSIONS, AND RECOMMENDATIONS
77
.................
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
1.
2.
3.
4.
Page
Sampling Schedule During Drying of Vegetable
by Three Dehydration ~1ethods •
21
Preparation of Assay Tubes for Folic Acid
Standard Curve • . • • • •
• • • •
28
Preparation of Folacin Assay Tubes for Mulukhiyah
Samples • • • • • • • • • • • • • • • • • •
35
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
• • • • • • • • • • • • • . • 42
Drying Methods
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
8.
69
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 • • • • • . • • . • • . • • • • • • • • •
vii
75
LIST OF FIGURES
Page
Figure
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Structure of Pteroylglutamic Acid • • • •
6
Schematic Diagram of Assay Procedure • • • • • • • 26
A Typical Folic Acid Standard Curve of L. casei
11
Linear Plot 11 • • • • • • • • • • • • • •
31
Semi-log Plot of Folic Acid Standard Curve of
L. casei • • • • • • . • • • • • • • • • • •
33
Semi-log Plot of Drying Time (hours) Versus
Moisture Content g/g solid • • • • • • . • •
44
Percent Folacin Ret ntion in Mulukhiyah during
Freeze Drying at 32 0 C • • • . • • • • • • • •
49
Percent Folacin R@tention in Mulukhiyah during
Tray Drying at 50 C • • • • • • • • • • • • • • • 51
Percent Folacin Retention in Mulukhiyah during
Room Drying at 25° C • • • • • • • • • • • • •
53
The Rate of Total Folic Acid Destruction Plotted
Against the Percent Moisture Content • • • • • • . 56
The Effect of Drying on Folacin Retention in
Mulukhiyah by the Three Drying Methods, Percent
Total Folacin Retention Is Plotted Against
Moisture Content • • • • • • • • • • • • • • •
61
Effect of Packaging Condition on TFA Content of
Dried Mulukhiyah During Storage at Room
Temperature • • • • • • • • . • • • • • • • • •
64
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 content 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.
cant difference
(r~O.Ol)
There was a signifi-
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 megaloblastic and is responsive to a combination of dietary protein with supplemental 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).
over Egypt.
This vegetable is popular all
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.
table with meat.
Those who can afford it cook the vege-
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 preservation 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 vegetables 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,
2.
tra~
and freeze drying).
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).
assayed with conjugase treatment.
glutamate forms of folacin.
The folacin content of vegetables
This included mono-, di-, and tri-
4
Total folic acid (TFA).
The folacin content of vegetables
determined after treatment with conjugase to convert the more complicated 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.
been discovered.
The reduced form of folic acid has since
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 derivative, 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
COOH
OH
N3"?4
12
HaN
Aa· I
N
'
•
ll I
CO-N--Cli-CHa-CH.-COOH
.
N
H
srCH2-N
8
a
10
6
. a..,?
'N
~------·--~~-----------''-----------------~~------------------'
i
Pteridine
p-Aminobenzoic
Glutamic acid
acid
~
L.
Pteroic acid
.._
T
-.J
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 isolated 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.
3.
The N10 formyl derivatives of pteroic acid and folic acid.
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 microbiological 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.
can be room dried.
major equipment.
Not all food
It is low in cost because there is no investment in
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 condition.
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 frozen 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 temperatures of 50° to 60° C (Labuza, 1972).
The time, temperature, and
moisture content relationship during freeze drying has received considerable 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 reduces 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 mechanically 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 preparation, 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 temperature 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
The foods must be sorted, washed, cut into the desired size,
foods.
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 currents 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 preparation 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 stability.
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 theretention 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 accelerated 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 overwrapped by black polyethylene lost only 16 percento(Karel and Heidelbaugh, 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 containers 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-CH 3-H 4 Pte Glu), and
tetrahydrofolic acid (H 4 Pte Glu) were more labile than the other forms
of folate.
Chen and Cooper (1979) have found that H4 Pte Glu is extremely 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 atmosphere.
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 percent of total folates and 72 precent of free folate.
Suckewer et ]].
(1970) reported that the losses of folic acid in tomato juice production 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 container so completely with compressed dehydrated product that the actual
quantity of oxygen remaining in the container is so small that any resulting 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 conventionally 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 collected 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 phosphate buffered ascorbate solution were prepared as shown in Appendix A.
Hog kidney conjugase was prepared for microbiological assay as described 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.
blanching or chemical treatment was used.
No
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
Number
Freeze Drying
1
2
1
2
1
2
3
3
3
4
4
4
5
6
8
10
12
6
6
7
8
Drying Time (in hours)
Tray Drying
Room Drying
8
10
12
4
8
12
16
20
28
36
48
22
two hours thereafter.
Tray drying.
The samples were spread out on screened trays
and placed in a Norwalk dehydrator.
vegetable at moderate speed.
50° C.
Hot, dry air was blown over the
The drying temperature was maintained at
Samples were taken for analysis in intervals similar to that
for freeze drying.
Room drying.
Egypt.
Room drying is a common method of drying in
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 mentioned 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 atmospheric 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.
and weighed.
The process was repeated every hour until constant
weights were reached.
tent.
The samples were cooled
Loss in weight was calculated as moisture con-
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 revisions 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 extraction 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 additional 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 determination of FFA content of the vegetables.
Separate samples of these ex-
tracts were treated with conjugase in the preparation of TFA determination.
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 (hundred times during the extraction and ten times during conjugase treatment).
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.
first day,
h·
casei culture was transferred from maintenance medium to
inoculum broth (Appendix A.2).
hours.
On the
This was then incubated at 37° C for 20
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.
cell suspension was used to inoculate assay tubes.
0
.
incubated at 37 C for 20 hours.
The
The tubes were then
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.
Day Two
Resuspending the cells in 0.9 %
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.
frozen until needed.
These tubes were stored
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.
washing and centrifugation procedure was repeated three times.
The
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 preparation of culture tubes for the standard curve.
In order to control
day to day variability of the organism, a standard curve was established 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
concentration
(ng/5 ml)
0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.8
1.0
1.2
Pte Glu
standard
solution*
(ml)
0
0
0.2
0.4
0.6
0.8
1.0
1.2
1.6
2.0
2.4
Phosphate
ascorbate
buffer+
(ml)
2.5
2.5
2.3
2.1
1.9
1.7
1.5
1.3
0.9
0.5
0.1
L. casei
Basal
medium++
(ml)
inoculum
added
(one drop)
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
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 spectrophotometer Model 24 equipped with a sipper system.
Deionized water
was used for calibrating the spectrophotometer and as a reference solution 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:
0
1.2
~
""
f-
<t: 10
>f-
-
( ./)
zUJ
0
_, 0.6
<(
u
f-
a..
0 0.4
0.2
0
0.5
1.0
1.5
2.0
PteGiu CONCENTRATION (ng)
2 .or:·
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
2
1-
<(
0::::
0.7
.
1-
z 0.5
UJ
u
z
0
u 0.3
0Q)
-
a..
Qj~~~~--~~~~~~~~----~--~
1.5
1.3
1.1
0.9
0.7
·O.D AT 640 nm
0.5
0.3
Oj
34
linear plot is similar to that reported by Waters and
~1ollin
and the semi-log plot is similar to that reported by Tamura
(1961),
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 appropriate volumes of 0.05 Mascorbate phosphate buffer (Appendix A.5).
Table
3 shows the volumes of sample extract, ascorbate buffer and basal medium 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
dilution*
100 X
1,000 X
10,000 X
10,000 X
Vegetable
extract
(ml)+
0.1
0.2
0.5
1.0
0.1
0.2
0.5
1.0
0.1
0.2
0.5
1.0
0.1
0.2
0.5
1.0
Ascorbate
phosphate
buffer
(ml)++
2.4
Basal
medium
(ml)
2.3
2.0
1.5
2.5
2.5
2.5
2.5
2.4
2.5
2.3
2.0
2.5
2.5
1.5
2.5
2.4
2.5
2.3
2.0
1.5
2.5
2.5
2.5
2.4
2.5
2.3
2.0
2.5
2.5
2.5
1.5
* Vegetable extract and conjugase treated vegetable extract were diluted 100, 1,000, 10,000, and 100,000 times by the addition of ascorbate 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
After use, all glassware was soaked in detergent for
experiment.
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.
inverted.
The glassware was allowed to air dry
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 mulukhiyah 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), mulukhiyah 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
Vegetable
raw
Mulukhiyah, room dried
Spinach, raw
Spinach, cooked
Broccoli flower, raw
Romaine lettuce, raw
Brussel sprouts, raw
Cabbage, raw
Asparagus, raw
Green beans, raw
Cauliflower, raw
~1ulukhiyah,
a
Folacin content
Free
(mcg/100 g ed i b1e Eorti on f Tota
1 X 100
(%)
Free
Total
556 ± 61c
662 .± 74c
119
60
102
60
55
33
58
33
31
BOO :I: 55c
1138 ±. 62c
193
91
105
179
78
66
64
44
55
70
58
62
66
97
34
71
50
91
75
56
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 naturally 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-CH 3H4 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-formyltetrahydropteroylglutamic acid (5-CHOH 4 Pte Glu), 5-CH 3H4 Pte Glu, and
10-formyl-tetrahydropetroylglutamic acid (10-CHOH 4 Pte Glu) (PaineWilson and Chen, 1979).
Since the major form of folacin in fresh
mulukhiyah is free (70%), the destruction of folacin is therefore expected 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 excellent 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 temperature) 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
Drying Time
(hr)
0
1
2
3
4
6
8
10
12
16
24
32
36
Freeze dr~ing at 33° C
\<Iei ght H 0 g H 0
2
reten2
tion
% g solid
Tra~ dr~ing at 50° C
weight
reten- H20 g H20
tion
% g solid
Room dr~ing at 25° C
weight H 0 g H 0
2
reten2
tion
% g solid
100
66.1
40.8
30.8
29.0
27.7
27.7
25.5
25.6
100
54.0
39.3
32.0
28.2
26.5
26.0
25.5
25.4
100
74.6
61.6
37.7
17.5
12.4
8.3
8.3
0.4
0.8
2.94
1.60
0.61
0.21
0.14
0.09
0.09
0.01
0.01
-
-
-
-
-
-
-
-
-
-
-
-
74.6
53.0
35.4
21.0
9.9
4.2
2.3
0.7
0.0
2.94
1.13
0.55
0.26
0.11
0.04
0.02
0.003
0.0
-
-
-
-
-
-
74.6
2.94
72.1
64.8
1.84
52.9
52.0
2.08
40.1
36.0
30.1
27.8
27.8
36.7
29.4
15.6
8.6
8.6
0.58
0.42
0.19
0.09
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
2
0
_J
0
tl)
1
.8
0>
.6
"
30
20
10
A
~
0
o Freeze Drying
0
o Tray Drying
40
Room Drying
(J)
........
.4
z
w
1-
z
0
.2
u
w
cr
.1
~ .08
~ .06
0
2 .04
.02~--~----~----~----~--~-----
0
5
DRYIN.G
10
TIME, HRS
15.
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.
period of drying.
This is referred to as the constant rate
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 trayand 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 drying 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
Drying Time
Free folacin
content * %
(mcg/g solid)
ret.
Total folacin
%
content
(mcg/g solid)*
ret.
21.89 .±
16.30 ±
11.93 .±
9.93.±
9. 70 .±
9.38 ±
9.23 .±
8.89.±
8. 94 .±
Freeze drying
0
1
2
3
4
6
8
10
12
Tray Drying
0
1
2
3
4
6
8
10
12
2. 40
2.57
o. 93
0.55
o.13
1.99
0.65
0.78
1. 38
100
75
55
45
44
43
42
41
41
31.50 ±
25.70 .±
19.52 ±
16.59 ±
15.56 ±
13.90 .±
15.17 ±
14.49.±
14.62 ±
2.17
0. 68
0. 68
2.07
0.38
1.12
1.65
1.52
2.46
100
82
62
53
49
44
48
46
46
21.89 .± 2. 40
15.36 .± 1.45
12.24 .± 1.84
10.23 ± 1.10
9.26.±1.12
8.75 ± 0.85
8.27 .± 0.79
7.78 ± 0.26
7. 88 .± 0.36
100
70
56
47
42
39
38
35
36
31.50 ± 2.17
22.00.±1.79
17.77 .± 1.69
15.92 .± 1.13
14.43 ± 1.39
13.86 ± 0.46
13.18 .± 2.10
12.92 ± 0.88
12.62 .± 0.58
100
70
56
50
46
44
43
41
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
content
%
(mcg/g solid)* ret.
Total folacin
content
%
(mcg/g solid)*
ret.
4
8
21.89 ± 2.40
16.45 ± 4.18
12.77±2.42
100
75
58
31.50 ± 2.17
24. 69 .± 1. Bl
18.88 ± 0. 92
12
16
24
10.22 ± 1.88
9.49 .± 1.57
8. 03 ± 1.83
50
16.07 .± 1. 49
60
51
32
36
7.46 ± 1. 20
7.24 ± 0.81
43
37
34
33
16.32
14.53
13.04
12.45
± 1.46
± 0.56
± 1.75
± 0.68
52
46
41
40
Drying Time
Room Drying
0
100
78
* Values are the means ± standard deviations of the means from 6 replications.
48
.I
• I
Figure 6. Percent folacin retention ~n
mulukhiyah during freeze drying at 32 C.
49
100~------~--~~--~------~--
° Free
z
0
1--
80
~::&To ta I Folic Acid
zw
1--
w
~
z
u
Folic Acid
70
60
<(
....J
0
LL
50
1--
z
0
w
u 40
~
w
c...
30
20~--~--~----~--~----~--~~
0
2
4
6
8
DRYING TIME, HRS
10
12
50
Figure 7. Percent folacin retention in
mulukhiyah during tray drying at 50o C.
51
100~--~--~----~--~--~~-----
90
z
0
1-
80
zw
1-
w
0:::
z
u
0
o Free Folic Acid
A
:A
Total Folic Acid
70
60
<(
--'
0u.. 50
1-
z
w
A
u0:: 40
. I
/::r
0
---:6
w
a..
30
2
4
6
8
DRYING TIME,. HRS
10
12
52
Figure 8. Percent folacin retentioB in
mulukhiyah during room drying at 25 C.
53
z
0
o Free Folic Acid
t-
A
A
0 80
zw
t-
w
Total Fo I ic Acid
70
0:::
z 60
u
<
--'
0L1.. 50
t-
zw
u0::: 40
w
0..
30
20~--~--~----~--~----~--~~
0
6
12
18
DRYING TIME
24
I
HRS
30
36
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 %, respectively (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 \<Jere 68, 70,
92, 49, and 65 percent, respectively.
In the present study, the re-
tention of TFA in mulukhiyah by tray drying (home dryer) was 41 %.
This is lower than those reported by Holmes for the vegetables mentioned above.
These differences in folic acid retention; however, are
likely to be a function of food composition, form of folic acid, moisture content, and physical differences (i.e. leafy versus non-leafy
vegetables).
Rate of folic acid destruction.
The rate of TFA destruction
was determined as a function of moisture content, where:
rate =
% folic acid destruction
% 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
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'-.
' ' . ............
u..
''
0
/
./f
I
I
''
I
' ' 'A- __ - -tlI
QL-----~---~----L---~--~----~--~
70
60
50
40
30
20
PERCENT MOISTURE
10
0
I
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 behaved similarly and had the lowest rate at 25 % and 15 % moisture content, 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 vitamins, 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).
water activity was not measured.
In the present study; however,
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.
below this level increases susceptibility to oxidation.
Drying
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
1978); sunlight and artificial light (Stokstad et
chemical environment (O'Broin !1
~.,
£1.,
£1.,
1947); and
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.
has better folacin retention than room drying.
Tray 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 nutritional 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
......
6.
~50
o----o Freeze Drying
z
0::
w
a...
o
~:::. Room
o Tray
Drying
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 drying 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
11
__.
0
9'
V)
e-·---e Brow·n Jar, N·2
0---<> Brown Jar. A! R
v Clear Jar. N 2
b-··-·-A Clear Jur, AIR
.,
~
~8
-
E
•
1-
zw
7
1-
66
u
0
u
5
<{
\~
'·
u 4
__.
0
U__. 3
'A..... .....-6.....
.......
-··- ··A-··
~
---··--··~··A
<(
1-
8
2
1
......... ---1--'
t--__._-...£-_~_
0
8
..1..--11----A---~---'_.,j
16
24
32
STORAGE Tltv\E, WEEKS
40
48
65
Figure 12. Effect of packaging condition on
FFA content of dried mulukhiyah during storage
at room temperature.
66
0
.....1
e-·-·-·
v
v Cleor Jar, N2
.b-··-··.-A Clear Jar, AIR
~
CJ)
-
N2
·o-- --o Brown Jar, AIR·
6
0
V)
v
E
Brown Jar,
5
1-
z
L!..J
..__
z
4
0
u
Q
u 3
u
<{
.....1
·.
0
Uw
w
2
._
'~ ·· .........
A'·· ..........
~
A
u..
1
0
8
A--··
- --..
24
TII\~E
--o--- ---
I
0
-
. -··-··--A
32
40
WEEKS
48
67
As shown in Figure 11, the highest content of TFA in mulukhiyah during storage was found in BN, next was both BA and CN.
had the lowest TFA content.
CA
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.
CN were very similar and stabilized at the end of 20 weeks.
BA and
The rapid
destruction of FFA in CA continued up to 24 weeks, after which time it
slowed down.
period.
CA had the lowest content of FFA throughout the storage
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 condition of packaging (air or nitrogen).
Folic acid content in jars
packed with nitrogen was higher than that in jars packed with atmospheric air.
Also folic acid content in brown jars was higher than that
in clear jars under both air or nitrogen.
however, were found between BA and CN.
No significant differences,
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 destruction.
A similar pattern in folic acid destruction during storage of
flour was reported by Keagy etJ8, (1975).
content decreased early during storage.
The native flour folacin
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 conditions.
period.
TFA in CA was always higher than FFA throughout the storage
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
Table 7
EFFECT OF PACKAGING CONDITION ON FREE AND TOTAL
FOLIC ACID RETENTION IN DEHYDRATED MULUKHIYAH
Storage
time
0
1
3
5
2
3
4
8
12
16
20
24
36
48
day
day
day
week
week
week
week
week
week
week
week
week
week
FFA Retention (%)
Brown bottle Clear bottle
air
air
N2
N2
100
89
81
67
62
62
56
53
49
44
41
39
31
32
100
86
83
72
67
69
65
61
59
55
53
51
48
45
100
71
67
56
53
50
41
37
35
29
26
24
24
20
100
87
76
74
69
59
50
49
46
42
36
32
28
25
TFA Retention (%)
Brown bottle Clear bottle
air
air
N2
N2
100
96
83
100
91
80
100
78
72
71
66
60
57
54
67
65
60
60
56
56
57
57
57
54
66
63
57
53
49
38
38
34
32
27
26
49
47
46
40
40
71
100
88
79
72
69
58
53
49
47
46
43
39
37
f
.
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 samples for analysis.
Therefore, it is recommended for future experiments
to use smaller samples in separate jars, that is to eliminate the possibility 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 increasing 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 conditions.
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 conditions 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 fluorescent 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 atmospheric air in both brown and clear jars.
auto-oxidation.
This destruction is due to
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 packaging 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 production 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.
of dissolved oxygen
a
fe~tJ
~tJas
In contrast, milk containing higher levels
reported to suffer large folacin losses within
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 ascorbic 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 Difference 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 mulukhiyah 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 re-
tention 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.
example, in week 24 there was a significant difference
(p~
For
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 S HSD FOR COMPARISON OF MEANS OF FFA
AND TFA RETENTION IN DRIED MULUKHIYAH STORED UNDER
DIFFERENT CONDITIONS AT VARIOUS STORAGE TIMES
1
Storage
condition
Brm1n jar
with air (BA)
Mean TFA retention (%)*
Mean FFA retention i%1*
48
48
1
24
4
1
24
4
week
weeks weeks weeks week weeks \"'eeks weeks
67axyz 56 ax
Brown jar
with nitrogen
(BN)
72 bxyz
Clear jar
with air (CA)
65bz
56cxyz 41bz
Clear jar
with nitrogen
74dxyz
(CN)
50cxz
39ay
32 a xz 72axyz 57az
46ay 40acxz
51 a by 45bYZ 71bXYZ 60b
57b
24 abyz 20 ayz 65cxyz 53cy
32bxy 26ba~y
32bx
54bz
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.
and 800 meg TFA per 100 g.
Fresh mulukhiyah contained 556 meg FFA
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 Nutrition Board of the National Research Countil {1980) is 400 meg, mulukhiyah can make a major contribution in fulfilling the folacin requirement.
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.
always higher than that of FFA.
Retention of TFA was
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 retention 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.
nificant difference
(p~O.Ol)
There was a sig-
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 depending 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 recommended that a study to determine the kinetics of folic acid losses
during dehydration, constant temperatures and storage at various moisture 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.
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80
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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 3H 20
Deionized Hater
0.5%
1. 0 grams
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 re-
moved from the heat to prevent the solution from boiling over.
Ten milliliter aliquots were pipetted into culture tubes.
tubes were capped and autoclaved for 15 minutes at 15 psi.
The
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
The culture tubes were capped, autoclaved for five minutes at 15
psi, and cooled.
A.3
Each tube contained 1 ng of Pte.Glu.
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 2Po 4
27.17 g
Na 2HP0 4
10.75
Distilled Water
4 liters
g
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.
f
'
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
7.
X
g.
Fat that floated on top was removed and the supernatant recentrifuged 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 aliquots).
B.2 0.1 MAcetate Buffer Preparation (pH 4.7) -The 0.1 Macetate 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·3H 2o dissolved in 1
liter of distilled H2o
0.1 MAcetic 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
NaOH (MC/B, SX 607) was added.
=
1 liter), and 12.5 ml of 0.08 N