CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
CHANGES IN FOLACIN LEVELS DURING COMMERCIAL
PRODUCTION OF TWO CALIFORNIA CHARDONNAY WINES
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Home Economics
by
Robyn Gaines-Moss
January, 1984
The ~hesis of Robync:=>nes-Moss is approved:
Chr1stine Hamilton Smith, Ph.D.
Tung-Shan Chen, Ph.D., Chairman
Califo~nia
State University, Northridge
ii
DEDICATION
To my beloved father who instilled in
me his thirst for knowledge and desire
to strive for excellence.
iii
ACKNOWLEDGEMENTS
I wish to express my gratitude and sincere appreciation to the members of my graduate committee, Dr. Robert
Lamb, Dr. Christine H. Smith and Dr. Tung-Shan Chen.
I
appreciate the time, effort and advice Dr. Robert
Lamb and Dr. Christine H.
I
~mith
have given to this study.
am particularly grateful to Dr. Tung-Shan Chen, my
committee chairman for his continuing advice, encouragement
and concern he has given me in the preparation and writing
of this thesis.
I
truly appreciate not only his patience,
time and accessibility but his difficult, yet rewarding
lesson of learning to think for yourself.
I
especially thank my co-worker in the laboratory,
Andree Armand who has given me tremendous help, encouragement and support.
I
am also thankful and grateful to Arlene Kirsch for
her expertise, help and guidance in the laboratory.
I
am grateful to Ahern Winery for providing the
samples used in this thesis.
I
am especially thankful to
Steve Hagata for his help in attaining the samples and information necessary for this research.
iv
Warm personal acknowledgements go to my husband
Gregg, my mother, brother, sisters and to Josie for their
tolerance, encouragement and pride in my achievements.
v
TABLE OF CONTENTS
Page
ii
APPROVAL PAGE • •
. . . . . . . . . .. .
ACKNOWLEDGEMENTS
. . .
LIST OF TABLES
. . .
...
LIST OF FIGURES . . . . . . .
ABSTRACT . . . . . . . .
. ..
iii
DEDICATION
iv
ix
X
xii
Chapter
1
1. INTRODUCTION
Statement of the Problem
3
Limitations •
4
Definitions
4
2. REVIEW OF LITERATURE
6
History and Terminology of Folacin
6
Structure and Chemistry
7
Importance of Folacin in Human Nutrition. •
10
Folacin Requirements and Deficiency •
11
Metabolism of Folacin
14
Recommended Dietary Allowances
Sources of Folacin
(RDA)
. . . . .. . . . . . .
Determination of Folacin in Foods •
vi
16
17
17
Chapter
Page
History of Grapes, Must and Wine
20
History of the Wine Industry
21
Technology of Wine Making
22
Wine Production
23
pH Variations in Wine Production
27
Nutritional Value of Wine
27
Vitamin Content of Grape, Must and Wine.
•
Economics of Wine.
3. MATERIALS AND METHODS
Materials
29
33
36
36
Grape, Must and Wine •
36
Chemical Reagents and Microbiological
Media
. • • . • . • •
42
Equipment
42
Glassware Maintenance
44
Methods
44
Variations of Sample Preparation Methods
Studied
• • • • • • • • •
44
Methodology of Folacin Determination in
Grape, Must, Juice and Wine . • •
49
Microbiological Assay for Folacin
54
Data Analysis
61
4. RESULTS AND DISCUSSION
64
Variations of Sample Preparation Methods
Studied
• • • • • • • . •
• • •
64
Methodology of Folacin Determination in
Grape, Must, Juice and Wine . . • • • •
74
Changes in Total Folacin Activity During
Wine Production . • • • . • • • • • • •
77
vii
I
Chapter
Page
5. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
99
LITERATURE CITED
102
APPENDICES
113
viii
'
LIST OF TABLES
Table
Page
1.
Protocol of folacin standard curve •
57
2.
Protocol of folacin assay tubes for
Paragon samples • • • • • • • • •
59
Protocol of folacin assay tubes for
MacGregor samples • • • • • • • •
60
Comparison of addition of ascorbate
containing phosphate buffer to direct
addition of ascorbate on the total
folacin cencentration of MacGregor
Chardonnay wine • . • • • • • . • • • • •
66
Effect of various levels of ascorbic
acid and autoclaving on the pH and
folacin content of wine sample • •
68
Comparison of heat treatment methods
on releasing folacin in wine samples
71
3.
4.
5.
6.
7.
8.
9.
10.
Comparison of two conjugase treatments
on their ability to release total
folacin content from wine samples • • .
•
73
Comparison of the folacin concentration
in the grape, must and hand-peeled grape •
76
Sugar content ( 0 Brix), pH, total acid,
alcohol percent and free sulfur dioxide
(FS02) values of must and wine
• • •
78
Comparison of total folacin activity of
unfiltered and filtered Paragon
Chardonnay grape, crushed grape and
must samples • • • • • • • • . • . •
80
ix
LIST OF TABLES
Page
Table
11. Comparison of total folacin activity
of unfiltered and filtered MacGregor
Chardonnay grape, crushed grape and
must samples • • • • • • • • • • • • •
. . .
81
12. Comparison of total folacin activity of
unfiltered and filtered Paragon Chardonnay juice and wine samples during
commercial vinification . • • • .
87
13. Comparison of total folacin activity of
unfiltered and filtered MacGregor
Chardonnay juice. and wine samples
during commercial vinification • .
89
14. Comparison of total folacin concentration
of white wine found from previous
research to this current research • • •
98
X
LIST OF FIGURES
Figure
Page
1.
Chemical structure of folacin . • • • • •
2.
Schematic flow chart illustrates sampling
day of wine production and significant
changes in the composition of Paragon
Chardonnay grape, must, juice and wine samples 39
3.
Schematic flow chart illustrates sampling
day of wine production and significant
changes in the composition of MacGregor Chardonnay grape, must, juice
and wine samp~es • • • • • • • • • • • • •
41
Schematic diagram of different stages in
the folacin assay, illustrating the
variations of sample preparation methods
studied with the sample used and day of
vinification
• • • • • • • • • • . • •
46
Flow chart of Ahern Winery's operations
in the production of Chardonnay wine
53
Total folacin activity of unfiltered
and filtered Paragon Chardonnay
samples during the vinification
process
• • • . • • • • • . • • . • •
84
Total folacin activity of unfiltered and
filtered MacGregor Chardonnay samples
during the vinification process • • • •
86
4.
5.
6.
7.
xi
9
ABSTRACT
CHANGES IN FOLACIN LEVELS DURING COMMERCIAL
PRODUCTION OF TWO CALIFORNIA CHARDONNAY WINES
by
Robyn Gaines-Moss
Master of Science in Home Economics
Changes in folacin activities were followed in the
commercial fermentation and aging of two Chardonnay wines
produced from grapes obtained from Paragon and MacGregor
Vineyards, California.
Ascorbic acid was added directly to all sample extracts to a final concentration of 0.15 percent.
Samples
were diluted with sodium phosphate buffer (pH 6.1) containing 0.15 percent asborbate.
Total folic acid activity
was assayed after treatment with conjugase prepared from
Difco chicken pancreas by a microbiological method using
Lactobacillus casei.
The folacin content of the Paragon and MacGregor
Chardonnay grapes was 5.6 and 3.5pg/100 gm respectively.
The folacin levels increased significantly in the must for
Paragon and l1acGregor to 8.6 and 9.6 pg/100 gm, respectively.
A 93-97 percent decrease in folate content was obX
ii
xiii
served in the free-run pressed juices when compared to the
musts.
The folacin content of the juice was 0.63 and 0.31
pg/100 ml for Paragon and MacGregor, respectively.
Following yeast inoculation, a 95-98 percent increase
in folacin activity was observed in the unfiltered and
filtered samples.
The folacin content of the Paragon un-
filtered and filtered sample was 14.9 pg/100 ml and 5.81
pg/100 ml respectively.
The folacin content of the Mac-
Gregor unfiltered and filtered samples was 4.lpg/100 ml
and 6.33 pg/100 ml, respectively.
Upon completion of fermentat.ion, a significant decrease in folacin activity occurred in all samples.
There
were, however, little further changes in folacin activities
during aging of the wines.
At the time of bottling, fola-
cin values for the unfiltered and filtered wines were not
significantly different.
The folacin content for the un-
filtered Paragon Chardonnay wine was 2.3 pg/100 ml and for
the MacGregor Chardonnay, 4.9 pg/100 ml.
Chapter 1
INTRODUCTION
Folacin is a water soluble B vitamin.
was first discovered in the 1940s
This vitamin
after most of the
vitamins had already been identified (Food and Nutrition
Board, 1968).
The vitamin is composed of a group of com-
pounds (pteroylglutamates) differing in the number of
glutamic acid residues attached to a pteridine nucleus.
Folacin exists primarily as polyglutamates in most natural
foods (Herbert and Bertino, 1967; Streiff and Rosenberg,
1967).
Rich dietary sources for folacin include liver,
yeast, green leafy vegetables, legumes, wheat germ, and
egg yolk (Butterworth, et al., 1963; Herbert, 1963;
Heppner, et al., 1972; Hurdle, et al., 1968; Perloff and
Butrum, 1977; Rodriquez, 1978; Santini, et al., 1964;
Tamura and Stokstad, 1973).
Folacin is essential in human nutrition and hence
must be included in the diet (Krause and Mahan, 1979).
The Recommended Dietary Allowances (RDA) for folacin were
first established in 1968 by the Food and Nutrition Board
of the National Academy of Science.
The recent inclusion
of folacin in the RDA exemplifies the recent recognition
1
2
Q '
of folacin as an important human nutrient {Food and Nutrition Board, 1968).
Since that time, there has been an in-
creased need for information regarding the folate content
in foods to improve and supplement the existing data.
present, this information is inadequate.
At
The most current
compilation of the folacin content of foods is limited to
299 items and excludes the beverage wine {Perloff and
Butrum, 1977).
Generating folacin data are difficult due to the lack
of a standardized method for determination.
Generally, the
folacin content of food is determined microbiologically.
The microbiological assay is based on the principle that
certain species of microorganisms, such as Lactobacillus
casei
{~.
casei), require the presence of folic acid for
its growth.
Presently, the microbiological assay is the
most accepted method for the determination of food folate
{Bell, 1974).
Day and Gregory {1983) however, state that
a lack of reproducibility exists with the use of the microbiological assay.
The variability of the microbiological
assay is exemplified when reviewing the various folacin
values stated for grape, must and wine.
From the first
study on the folacin content of grapes and wine {Castor,
1953) to the most recent study of California wines (Voigt,
et al., 1978) tremendous discrepancies in the folacin
values exist.
The differences in folacin values observed
may be due to variations in the microbiological assay
method and to technological changes of the wine industry.
3
Wine consumption in the United States has grown
steadily despite high inflation and a recession (Standard
and Poor, 1982}.
Consumption increased from 267 million
gallons in 1970 to 480 million gallons in 1980, representing an 80 percent increase.
This growth has witnessed a
steady increase in the per capita consumption jumping from
1.7 gallons per person in 1975 to 2.2 gallons per person in
1981 (Wine Institute, 1982}.
In 1980, wine consumption
topped liquor consumption for the first time which clearly
indicates changing patterns in social drinking (Mintz,
1981; Standard and Poor, 1982).
California's white wine production predominates the
United States wine market.
White wine accounts for 68
percent of the total market while other states represent
10 percent and European imports, 21 percent, based on the
latest available data in 1979 (Standard and Poor, 1982}.
In response to the increase in white wine production large
investments in California vineyards are anticipated at the
international level (Standard and Poor, 1983; Summar,
1980}.
This interest in wine production and wine con-
sumption does represent a major contribution to the economy
of this state.
Statement of the Problem
Castor (1953} was the first to follow changes in the
B-complex vitamins during must fermentation.
Hallet al.,
(1956} also studied the B-complex vitamin content including
4
folic acid in grapes, must and wine.
However, the micro-
biological assay method for folacin has improved since the
1950s
(Bell, 1974).
Modifications in viticultural prac-
tices and cellar technologies have also occurred in the
past 30 years.
However, folacin research in wine produc-
tion has not kept pace with these advancements.
Hence, a
current understanding of the folacin content of wine and
changes occurring during wine production is needed.
The
purpose of this research was to examine systematically the
changes and stability of folacin during grape-must fermentation, aging, storage and bottling of the wine.
Limitations
This study was limited to the use of two 100 percent
premium Chardonnay wines.
The two wines were produced
from grapes grown in different vineyards in the same geographical area.
winery.
The wines were produced by one California
Therefore, values and trends of folacin activity
found in these wines may not be applicable for all wines.
Definitions
Folacin, folates.
Comprehensive terms, used inter-
changeably, to describe the group of related compounds
which are derivatives of folic acid (pteroylglutamic acid).
Folacin activity.
It refers to the ability of a sub-
stance to support the growth of L. casei as measured
against a folic acid standard.
5
Conjugase.
The common name for enzymes which hydro-
lyze the gamma peptide bonds that link glutamyl residues
to folic acid.
Free folic acid (FFA) •
Folacin present in food-
stuffs that can be utilized by L. casei without prior
treatment with conjugase.
Total folic acid (TFA).
Folacin present in food-
stuffs that is utilized by L. casei after conjugase treatment of the sample.
Fermentation. A metabolic process bringing about
chemical changes in organic substrates through the action
of enzymes of microorganisms.
Must.
A product of crushed, destemmed grapes.
Lees.
The sediment present in inoculated white
grape juice/wine which is composed primarily of yeast
cells.
Vinification.
The conversion of grape juiceinto wine
by fermentation.
Brix. The soluble solids content or sugar content of
the grapes.
Racking.
A process of siphoning or pumping the fer-
mented grape juice from the lees in the fermentation vat
to a clean tank or barrel.
Fining.
A process which removes yeast proteins,
pectins, gums, unstable grape pigments, tannins and other
compounds with the use of fining agents.
Chapter 2
REVIEW OF LITERATURE
History and Terminology of Folacin
Folacin was first discovered by Wills (1931) when
liver and yeast extracts were used to cure tropical macrocytic anemia in India and was known as the Wills factor.
Day et al.
(1938) disclosed a deficiency in monkeys that
was relieved with yeast and liver extracts.
This unknown
factor contained in yeast and liver extracts was termed
Vitamin M.
In the years following, this factor became
known also, as Factor U, yeast Norite eluate factor,
Vitamin Be, and Lactobacillus casei factor (Burkholder et
al., 1945; Laskowski et al., 1945; Pfiffner, 1945) and was
found in all green leafy plants (Mitchell et al., 1941).
Mitchell et al.
(1941) coined the term folic acid from
follium, the Latin word for leaf.
The most commonly used terms for this vitamin are
folic acid, folacin and pteroylglutamic acid (PteGlu).
term folic acid has two meanings:
The
1) generically, it in-
cludes the monoglutamate and polyglutamate forms of the
pteroic acid compounds termed folacin and 2) chemically, it
is known as the non-reduced monoglutamic acid, pteroylmono6
7
glutamic acid (PGA)
(Food and Nutrition Board, 1980; Her-
bert and Bertino, 1967; Malin, 1975).
Structure and Chemistry
Rodriquez (1978) stated that there are more derivatives of folic acid than any other vitamin.
Folic acid,
the parent compound of the folacin group, is formed by the
linkage of three components:
a pteridine nucleus, a para-
aminobenzoic acid (PABA) conjugated with one to six Lglutamic acids (Nystrom and Nystrom, 1967; Stokstad and
Koch, 1967)
(Figure 1).
Additions to and reductions of
folic acid result in the many varieties of this vitamin.
The empirical formula of folic acid is
c19 H19 N7 o6
and its
molecular weight is 441 (Eigen and Shockman, 1963).
The pyrazine portion of the pteridine ring can be
reduced, chemically and enzymatically to give dihydro- or
tetrahydro-derivatives which are metabolically active
(Stokstad and Koch, 1967).
The functional forms of folic
acid are derivatives of 5,6,7,8-tetrahydrofolic acid
(THFA) (Malin, 1975; Nystrom and Nystrom, 1967).
The
tetrahydrofolic acid derivatives are responsible for the
coenzyme action in one-carbon unit transfers (Stokstad and
Koch, 1967).
Folic acid is a yellowish-orange crystalline powder
(Eigen and Shockman, 1963).
It is sensitive to heat,
light, oxygen, pH and endogenous conjugase (Rodriquez,
1978).
Folic acid is insoluble in alcohol, acetone, ben-
8
Figure 1.
Chemical Structure of Folic
Acid.
OH
N3t.x•
I~ I
u N~
~r~H~-~-o-~
•
..,•
16
.
-
.
H~OOH
00-N-CH-C u.-cu.-coou
1
2
N
N
Pleridine
p -Aminobenzoic
Glutamic acid
acid
~
IL
Pteroic acid
Pteroylglutamlc acid
Folic acid
\.0
""'
10
zene, chloroform, and ether.
It is soluble in dilute
solutions of alkali hydroxides, carbonates and hot diluted
HCl and H2S04 (Eigen and Shockman, 1963).
Folic acid activity is reported as free and total
folic acid.
An enzyme known as conjugase releases folic
acid from the glutamate acid molecule(s)
man, 1963).
(Eigen and Shock-
Free folic acid (FFA) represents that amount
of folic acid present in foodstuffs without prior conjugase
treatment.
Total folic acid (TFA) refers to the total
amount of folic acid in foodstuffs after conjugase treatment.
The polyglutamate forms found in foods are to some
extent hydrolyzed by man's intestinal
forms
conjug~se
to simpler
(Butterworth, 1968; Dong and Oace, 1973; Halsted et
al., 1977).
Thus, TFA may be considered an approximation
of folacin available to man.
Importance of Folacin in Human Nutrition
The principle function of folacin is transportation,
oxidation and reduction of single carbon units.
These
steps are essential in the biosynthesis of purine and pyrimidine bases which influence the synthesis of nucleic
acids.
Folic acid is also responsible for normal meta-
bolism of certain amino acids and chain initiation of RNA
and DNA synthesis (Food and Nutrition Board, 1980; Goodhard
and Shils, 1980; Malin, 1975; Rodriquez, 1978).
The single
carbon unit transfer function of folacin also occurs in
the formation of heme.
tein in hemoglobin.
Heme is the iron containing pro-
Therefore, if folic acid is absent,
11
the red blood cell will increase in size (macrocytosis)
and the concentration of hemoglobin in the erythrocytes
will decrease causing megaloblasts (abnormal cells) in the
blood.
This process is known as megaloblastic anemia and
is characterized by lethargy (Williams, 1981).
Infertility,
sterility and growth may be stunted due to the impaired
cell division and alterations of protein synthesis (Malin,
1975; Marks, 1975).
Folacin Requirements and Deficiency
Folacin deficiency is probably the most common vitamin
deficiency found in the U.S.A. today (Martin, 1981).
Folacin deficiency results because man's folate requirements depend on factors such as age, state of health, drug
use and abuse, dietary intake, and absorption.
In
addition, requirements depend on the total number of cells
in the body and/or the rate of cell synthesis.
As these
entities increase so does the folacin requirement.
Con-
sequently, folate deficiency may result from insufficient
dietary intake, altered requirement, impaired absorption
in the small intestine or from abnormalities in nutrient
demand and/or metabolism (Rosenberg and Dyer, 1979).
populations at an increased risk include:
The
low socio-
economic groups; chronic alcoholics; drug users of anticonvulsants, barbiturates, methotrexate, aminopterin, and
sulfasalazine; patients after gastric operations; patients
suffering from malabsorption syndromes, such as tropical
12
sprue; premature infants; pregnant and lactating women and
women taking oral contraceptives (Chanarin et al. 1968;
Butterworth, 1968; Iyengar and Rajalakshmi, 1975; Malin,
1975; Rosenberg and Dyer, 1979; Shaw and Hoffbrand, 1970;
Streiff, 1970}.
Chronic folic acid deficiency will result
in megaloblastic anemia (Wu et al., 1975).
Approximately
50 percent of all patients admitted to the hospitals from
low socioeconomic communities shows evidence of this
anemia (Shaw and Hoffbrand, 1970).
Megaloblastic anemia is
characterized by glossitis, anorexia, irritability, forgetfulness, depression, and often hostility and paranoid
behavior (Goodhard and Shils, 1980).
A deficiency of folic
acid may also lead to a reduction of host resistance and a
decrease in the immune system (Beisel, 1982).
This anemia
is indistinguishable from the Vitamin B12 deficiency anemia
(Herbert, 1968).
Folate deficiency may result from an inability to hydrolyze polyglutamate folate to monoglutamate folate at the
absorption stage.
Inhibitors, which have been identified
in beans, peas, and yeast (Colman and Herbert, 1979) may
suppress intestinal conjugase and thereby decrease absorption of ingested folate as much as 75 percent.
Folacin deficiency is prevalent in the chronic alcoholic.
Forty percent of all alcoholics has a folic acid
deficiency (Eichner and Hillman, 1971).
This folic acid
deficiency may be a result of a decreased nutrient density
intake (Windham et al., 1983), malabsorption or an im-
13
paired utilization or hyperexcretion of folacin (Roe,l979;
Visocan, 1983).
Malin (1975) and Lindenbaum (1980) sug-
gested that alcohol is a folate antimetabolite.
However,
it is not clear whether the folate deficiency is a result
of a poor dietary intake of folacin or if alcohol itself
alters dietary folacin absorption, storage and requirements
(Rodriquez, 1978).
Sullivan and Herbert (1964) suggested a
direct role of alcohol causing folate deficiency independently of dietary deficiency which is further supported by
Lindenbaum (1980).
On the other hand, Wu et al.,
(1975)
determined that the amount of alcohol consumption did not
itself significantly affect the folate status.
Eichner and
Hillman (1973) indicated that ethanol accelerated the
development of megaloblastic anemia when folate stores were
depleted.
Halsted et al.
(1971) suggested that malabsorp-
tion and thus, folate deficiency were caused by poor
nutrition rather than a toxic effect of ethanol on the
jejunum.
Shaw and Hoffbrand (1970) and Roe (1979) stated
that the folate deficiency was a result of a decreased
dietary intake, malabsorption, liver damage with reduced
pools of activated vitamin and excess excretion.
There may
also possibly be a direct effect of alcohol on enzymes involved in folate metabolism (Shaw and Hoffbrand, 1970; Roe,
1979).
Eichner and Hillman (1971) supported the assumption
that alcohol interfered with the formation of N5-methyltetrahydrofolic acid within the liver.
In order to handle alcohol with a minimum of damage,
14
the body must be well-nourished.
A well-nourished body re-
quires the consumption of a nutritionally adequate diet.
The B-complex vitamins are especially important in handling
alcohol consumption.
In order to prevent folate deficiency
of alcoholism, fortification of the beverage has been
recommended (Kaunitz and Lindenbaum, 1977; Perlman and
Morgan, 1945).
ages.
Folic acid is soluble in alcoholic bever-
The vitamin does not alter the taste of the bever-
age and is well-absorbed (Lindenbaum, 1980).
Therefore,
folacin fortification may be a worthwhile prophylactic
measure in the malnourished alcoholic.
A well-nourished
body can aid in handling alcohol, yet cannot protect
against the damage of chronic heavy drinking (Houtkooper,
1983) .
Metabolism of Folacin
Food folate exists primarily in conjugated forms
(gamma-linked polypeptide chain of 1 to 6 glutamic acid
residues).
Food folate is resistant to hydrolysis by the
usual proteolytic enzymes normally present in the intestine.
In order for man to absorb folic acid, the food
folate must be cleaved by a specific group if intestinal
enzymes, pteroyl-polyglutamate hydrolases (Halsted et al.,
1977; Martin, 1981).
jugases.
These enzymes are simply termed con-
Conjugase activity hydrolyzes pterolyheptagluta-
mate to pteroylmonoglutamate in the small intestine (Malin,
1975; Nystrom and Nystrom, 1967).
The hydrolysis of fola-
cin is a rapid and non-rate limiting step.
However, the
15
extent of deconjugation occurring in man is not known.
The monoglutamyl form is absorbed into the mesenteric
circulation and reduced by the enzyme, dihydrofolate reductase, to tetrahydrofolate (THFA) then methylated to N5 methyl-THFA (Martin, 1981; Streiff and Rosenberg, 1967).
The N5-methyl-THFA enters the blood to be carried to the
liver and to the tissues via systemic circulation (Martin,
1981).
Folate is found in all organs, especially the
liver, as well as the kidneys, spleen, bone marrow cells,
erythrocytes, leukocytes, intestinal mucosal cells, cerebral spinal fluid and the brain (Goodhard and Shils, 1980;
Rodriquez, 1978).
from 5 to 10 mg.
Normal total body folate stores range
At least half of this amount is found in
the liver.
Folate is excreted in the feces, urine and bile.
The
feces contains 5 to 15 times the amount of folacin than ingested (Rodriquez, 1978).
This increase may be a result of
unabsorbed dietary folacin, folacin synthesized by oral and
intestinal microflora (Herbert, 1963) or that which is
secreted in the bile and saliva and reabsorbed or from the
degradation of gastrointestinal cells (Rodriquez, 1978).
Folacin has a half life of 24 hours (Rodriquez, 1978).
The principle breakdown products of folacin in the urine
and bile are both in the metabolically active and inactive
forms.
Seventy-five percent of ingested folic acid is ex-
creted in the urine (Rodriquez, 1978).
The principle cata-
bolic pathway is the oxidative cleavage of the folate
16
molecule at the 9-10 bond forming acetamidobenzoylglutamate (Goodhard and Shils, 1980; Stokstad, 1979).
Bile
contains about lOOpg of metabolically active folate per
day (Goodhard and Shils, 1980).
However, Rodriquez (1978)
stated that the folate contained in the bile may be reabsorbed.
Folate absorption is impaired in those patients
suffering from malabsorption syndromes such as idiopathic
steatorrhea and tropical sprue and in those lacking a
small intestine.
Alcohol may also interfere with entero-
hepatic circulation of the vitamin (Goodhard and Shils,
1980).
Recommended Dietary Allowances
Recommended Dietary Allowances (RDA) was first established by the Food and Nutrition Board of the National
Research Council of the National Academy of Science
1968 and has since been revised in 1980.
in
The RDA are set
at 400 pg per day of total folacin activity for normal
non-pregnant, non-lactating adults and adolescents.
The
RDA is based on the assumption that 100 to 200 pg are required to maintain tissue reserves.
The RDA for infants
are estimated at 5 pg/kg body weight per day.
For healthy
preadolescent children the RDA is 8 to 10 pg/kg body
weight per day allowing for growth and variability in
diet.
Due to the added burden of pregnancy, the RDA are
increased to 800 pg/day.
In response to the demand of
17
lactation, the RDA are set at 500 pg/day (Food and Nutrition Board, National Research Council, 1980).
Sources of Folacin
Folacin is found in most plant and animal tissues.
The rich sources of folacin are yeast, liver, and green
leafy vegetables such as, asparagus, endive, broccoli,
lettuce and spinach (Butterworth, 1968; Herbert and Bertino, 1967; Streiff and Rosenberg, 1967).
Determination of Folacin in Foods
The microbiological assay method is a common method in
determining the folacin activity of food.
However, assay
of the food folate is difficult due to deviations in the
method and the ultrasensitivity of the vitamin.
Folic acid exists predominately (75 percent) in the
conjugated form of hexa- and heptaglutamates (Butterworth
et al., 1963; Nystrom and Nystrom, 1967; Stokstad and Koch,
1967).
Since~-
casei can only utilize mono-, di- and tri-
glutamates, enzymatic treatment should be employed in the
assay to determine the total folate activity of the foodstuff available to man.
The two sources of conjugase used extensively in the
determination of food folacin are:
1) carboxylpeptidase
found in animal tissue (hog kidney), optimum pH 4.5 which
hydrolyzes polyglutamic acids to monoglutamic acid forms
and 2) a gamma-glutamic acid carboxylendopeptidase isolated
from chicken pancreas, optimum pH 7 to 8 which hydrolyzes
18
polyglutamic acids to diglutamic acid forms (Buehring et
al., 1974; Eigen and Shockman, 1963).
The foundation for the microbiological assay is dependent on certain species of microorganisms lacking the
ability to synthesize folic acid yet requiring its presence
for growth {Buehring et al., 1974; Rodriquez, 1978).
these microorganisms are Lactobacillus casei
{~.
Among
casei),
Streptococcus faecalis {§_. faecalis) , and Pediococcus
cerevisiae {P. cerevisiae).
organism because
~-
L. casei is the favored
casei can utilize mono- di- and tri-
glutamate and the oxidized and reduced forms of folic acid.
Furthermore, L. casei responds well to low potency folate
materials {Eigen and Shockman, 1963) and its assay range
of folic acid is 0 to 1 ng/tube (Bell, 1974).
Moreover, L.
casei more closely resembles man's response to the vitamin
than
s.
faecalis, the organism of choice in earlier studies
{Butrum and Perloff, 1975; Toepfer et al., 1951).
The growth of L. casei is stimulated by both the use
of ascorbic acid and phosphate buffer {Colman and Herbert,
1979; Herbert, 1961).
The growth
of~-
casei results
from the protection afforded the heat-labile reduced forms
of folic acid by the ascorbic acid and buffer solution
{Butrurn and Perloff, 1975; Hurdle et al., 1968; Toennies
et al., 1956).
Hurdle et al.
{1968) determined that the
optimum ascorbate level in the buffer solution used was
0.15 percent, yet 1.5 percent inhibited the growth of
casei.
~­
19
The inclusion of ascorbic acid in the food sample
during autoclaving is essential.
Autoclaving is used to
release the bound folacin and to sterilize the sample
(Bird et al., 1945).
It is recommended that the sample be
autoclaved at 15 psi for 15 minutes.
A boiling waterbath
may also be used (Hurdle et al., 1968).
During the heat
treatment a reducing agent, such as ascorbic acid, is
necessary to prevent oxidation of folacin (Chen and Cooper,
1979).
The absence of a reducing agent would cause the
oxidized folic acid to decompose into a biologically inactive product which would not function as a growth factor
for the microorganism during assay (Stokstad et al., 1947).
Toepfer et al.
(1951) published the Agriculture Handbook
No. 29 prior to the need for ascorbate in the assay procedure was recognized.
Thus, the values for folacin in
the Agriculture Handbook No. 29 are significantly lower
than those values found with the use of ascorbate during
assay (Perloff and Butrum, 1977; Rodriquez, 1978).
Day
and Gregory (1983) refute the use of enhancing impractical
experimental conditions, such as buffer.
However, they
do support the use of ascorbic acid to improve the thermal
stability of folacin.
In addition, pH may affect the total folate activity
assayed.
PGA is most stable under neutral and alkaline
conditions and less stable under acidic conditions (Stokstad and Thenen, 1972).
Tamura et al.
(1976) noted that
the pH of orange juice inhibits conjugase activity.
20
Orange juice, with acidic pH of 3.7 decreased the availability of pteroylheptaglutamate acid 54 percent (Tamura,
1976).
Paine-Wilson and Chen (1979) determined that the
rate of destruction of PGA and its variations increased
rapidly with decreasing pH below 4.0.
Hence, pH of the
food sample and standard solution used in the microbiological assay may influence the total folate activity.
There-
fore, the recommended ascorbic acid level is 0.15 percent
(Hurdle et al., 1968) and sodium phosphate buffer pH 6.1
(Chen, 1979).
History of Grapes, Must and Wine
One definition of wine as defined by the Office International de la Vigne et de Vin (OIV, 1975) in Paris
follows:
Wine is exclusively the drink that results
from complete or partial alcoholic fermentation
of grape, freshly pressed or not, or of must
of grape •••
The exact origin and date of the vine (Vitis vinifera)
and its product wine are not clear.
It is believed that by
3200 B.C. wine and vine were already dispersed throughout
the middle east and the eastern Mediterranean areas (Darby
et al., 1977).
There are records of wine production during
the first Egyptian dynasty.
Evidence of wine production
was found in tombs of the first two dynasties.
In
addi~
tion, hieroglyphics of the ancient wine press indicates the
production of wine (Darby et al., 1977).
Wine was des-
cribed as "A Gift from God", a "Gift from Heaven", the
21
"Dispenser of Joy"
(Gastineau et al., 1979).
Wine was used
as a ritual during festivities, religious gatherings,
sacrifices, and funerals.
The medicinal uses of wine were many.
The lees of the
wine were rubbed on the body and then allowed to dry in the
sun to heal the skin.
Wine was used orally to:
1) aid
swallowing a dry medicine, 2) act as a dispensing agent,
3) kill tapeworms, and 4) act as a soothing agent.
Wine
was prescribed to alleviate anorexia, protect against disease characterized by a cough and to ease childbirth
(Darby et al., 1977).
History of the Wine Industry
United States wine production first began with the
European settlers in 1562 (Amerine, 1981).
Wine production
came to California in 1769 with the missionaries from Baja,
California.
By 1806, the wine industry had spread through-
out the mission land holdings of California.
The gold rush
era further enhanced the demand for grapes and wine in
California.
The growing California wine industry en-
couraged immigrants from Europe to bring their vines to
California.
The increase in the wine industry led to the scientific study of wine--Viticulture and Enology.
Eugene
Waldemar Hilgard, Professor of Agriculture at the University of California, assisted in creating the Board of
State Viticultural Commissioners in 1880.
However, the
economics of the wine industry were unstable.
Thus, in
22
1894, the California Wine Association (CWA) was established
to aid in stabilizing wine prices.
By 1918, the CWA con-
trolled 84 percent of the wine production.
Even though the
wine industry increased, the prices decreased sharply in
the mid 1920s.
The prices of wine remained depressed
through prohibition and World War II.
After World War II,
the Wine Institute, a non-profit trade organization, was
established in San Francisco in 1934, and still remains the
authoritative voice of the California wine industry.
American Society of Enologists was founded in 1950.
The
This
organization functions as an avenue for presentation and
publication of research papers.
The publication is known
as the American Journal of Enology and Viticulture.
The
Wine Institute and the American Society of Enologists both
function today (Amerine, 1981).
Technology of Wine Making
The principle of wine production is based on the fermentation process of grapes and its juice.
Fermentation is
defined as the "metabolic process bringing about chemical
changes in organic substrates through the action of enzymes
of microorganisms"
(Amerine et al., 1980).
Grapes contain a considerable amount of sugar.
The
sugars are important as a source of ethyl alcohol production.
Dextrose and levulose are the primary sugars found
in the grapes in a ratio of 1:1 (Amerine et al., 1972).
The yeast, Saccharomyces cerevisiae possesses the ability
to convert sugar to ethanol.
Thus, Gay-Lussac's equation
23
of 1815 notes the overall reaction of alcoholic fermentation (Amerine et al., 1980).
Sugar+ Saccharomyces yeast= Alcohol+co 2 (wine)
or
c 6 H12 o 6 + yeast --~
2C 2 H5 0H + 2co 2 (wine)
The grape juice in white wine production is fermented to a
low sugar content of 0.2 percent or less which is termed
dry.
White wine, namely, Chardonnay, has a low total acidity of less than 0.6 percent (Amerine et al., 1980).
The
two principle acids present are D-tartaric and L-malic
acid.
The D-tartaric acid is a relatively strong acid and
accounts for the total acidity of the wine (Amerine et al.,
1980).
There is very little
L~citric
acid (0.01 to 0.03
percent) present (Fong et al., 1974).
Wine Production
The production of wine is an art.
The manner in which
winemakers create their wine is subject to their taste.
However, there is a basic scientific process underlying
each winemaker•s practice.
Wine production
begi~s
September through October.
with harvesting the grapes in
The decision to harvest
depends on grape maturity based on the balance of sugar,
acid and flavor.
As the grape ripens, the sugar content
increases, color changes, malic acid is metabolized, phenolic synthesis occurs and the berries soften.
The solu-
ble solids content or sugar content of the juice,
24
measured in °Brix, is the most common indicator of maturity
(Amerine et al., 1980).
to 23 °Brix.
Optimum
0
Brix for maturity is 21
Titratable acidity, pH of the grape and the
year's climatic conditions also determine harvest time.
Temperature, rainfall, humidity, wind, and soil have a
tremendous effect on the grapes growth and the quality of
the wine produced.
The grapes are harvested by hand-picking or machineharvesting.
Machine-harvesting is advantageous in that
night harvesting is possible allowing delivery to occur
during the cool night temperature.
It is also a more
economical and quicker process, allowing the grapes to come
closer to optimum maturity.
The grapes are then crushed using a machine that removes the stems and breaks the berry's skin.
Sulfur di-
oxide (S02) is added at this point as an antioxidant, reducing agent to prevent enzymatic browning (caused by polyphenoloxidase) and as an antimicrobial agent against
spoilage microorganisms and wild yeasts (Burroughs, 1981).
These functions require the presence of "free so 2 ".
Free
so2 is the portion which does not become bound to the
carbonyl compounds of the must (Burroughs and Sparks, 1973).
The exact amount of so 2 added depends on whether the grape
is bruised, broken, moldy, high in pH or low in titratable acidity (Amerine, 1981).
additional S02 is required.
If these conditions exist
Nevertheless, it is important
to control the quantity of so 2 present in wine.
The added
25
sulfur can combine with hydrogen gas to form hydrogen sulfide gas (H2S)
(Amerine, 1967; Eschenbruch, 1974).
In
addition, S02 can be very irritating to the mucous membranes of the nasal passages, eyes and lungs (Nelson,
1982).
In the production of white wine, the must travels
from the crusher either directly to the presser to obtain
the "free-run" yield or it is allowed to remain with the
skin from 2 to 36 hours (Singleton et al., 1980).
The in-
creased skin contact time will provide the wine with a
richer or heavier characteristic.
Arnold and Noble (1979)
noted an increase in total aroma and fruity aroma of
Chardonnay grapes with a pomace contact time of 16 hours.
As the length of pomace content time increases, acid content decreases, pH increases, phenolic extraction increases and S02 content decreases
(Liu and Gallander,
1982; Ough, 1969; Singleton et al., 1980).
The skin-
contacted must is then pressed to yield the free-run juice
(Amerine, 1980).
The juice is allowed to clarify.
The pressed juice
settles in a tank at a cool temperature to prevent fermentation.
The lees (grape solids) will settle on the
bottom of the tank.
The clarified juice is then trans-
ferred to a clean tank and yeast is added to commence fermentation.
The yeast added is Saccha·romyces cerevisiae.
Yeast is 8x7 urn in size (Amerine, 1972).
This strain of
yeast in particular can ferment sugar more completely
26
than any other strain (Lichine, 1974).
However, it is more
sensitive to large additions (300 ppm) of so2 than Schizosaccharomyces pombe (Nakano et al., 1981; Yang, 1975).
Fermentation of white wine does occur at a cool controlled temperature (7 to 15°C.)
Cool temperature fer-
mentation provides a fruity flavor to the wine.
tion takes 2 to 8 weeks to complete.
Fermenta-
Once fermentation is
complete, the yeast and remaining lees are separated from
the wine.
There are three basic methods to separate or
remove the yeast and lees from the wine:
centrifugation,
filtration or settling and racking the wine.
The wine is aged to provide time for it to soften,
balance, mellow and develop its flavor and bouquet.
White
wine may be aged in barrels (cooperage) or stainless steel
tanks.
The barrels allow the wine to achieve its charac-
teristic, rich, intense, complex style.
The age of the
cooperage influences the characteristic of the wine.
New
oak barrels contribute more oak to the wine than older
barrels.
also.
The type of oak of the cooperage is important
European oak contributes more extract and tannin but
less flavor than American oak (Amerine et al., 1972 and
1980).
The wine is then fined to enhance its sensory and
clarity properties.
Common fining agents are bentonite
(fine clay) , casein, and gelatin.
These agents remove
excess protein and phenol compounds (Amerine et al., 1972
and 1980).
27
pH Variations in Wine Production
The pH increases during the ripening of the grapes.
It also increases during the pressing of the grapes, where
the first press is lower in pH than the second.
pH is important in the fermentation process.
If the
pH is too low, sulfur dioxide should not be added in order
to avoid the pH from becoming too low as to delay fermentation (Amerine et al., 1980).
The pH range of 3.3 to 4.0
favors the rate of fermentation.
However, the best utili-
zation of sugar occurs at pH 2.8.
Moreover, pH values
above 3.3 are to be avoided as they favor lactic acid formation (Amerine et al., 1980).
During alcoholic fermentation, pH values generally
decrease as the buffer capacity of the wine increases.
The
buffering capacity is in response to the buffer substances
of yeast, tannins, coloring materials, and other polyphenolic compounds (Amerine, 1954).
The higher the
original pH in the grape, the greater the decrease observed.
taste.
The pH of the wine has a direct influence on
The lower the pH, the more acid the taste (Amerine
et al., 1972 and 1980).
Nutritional Value of Wine
Wine is distinguishable from other alcoholic beverages in that it possesses a beneficial effect on man's
metabolic processes.
Wine has been used as:
relief of
emotional tension, treatment of obesity and anorexia,
28
aid in increasing high density lipoproteins, aid in postgastrectomy malabsorption, aid in iron deficiency anemia
and as a source of energy (Lucia, 1954; McDonald and
Margen, 1976).
The sedative effect of wine is due to the interaction
bebween the aldehydes in balance with the alcohol (Lucia,
1972).
Wine is metabolized differently than fat, carbo-
hydrate and protein.
The body can oxidize approximately
half a gram of alcohol per pound of body-weight over a 24
hour period.
Wine in small amounts will also serve to
stimulate the appetite, thus decreasing anorexia (Lichne,
1974).
Wine is an alcoholic beverage which contains nutrients.
Pasteur claimed that it is the "most healthful of (dietary)
beverages"
(Lucia, 1972).
Wine is beneficial in the diet
as a source of calories {7 kilocalories/gram), nitrogen,
minerals, and vitamins.
One liter of wine may contain
600 to 1000 kilocalories and more than one hundred
chemical ingredients.
Nitrogen is composed of amino acids.
Some of the wine containing amino acids disappear during
fermentation or used by yeasts to form higher alcohols.
Other amino acids are formed by the yeast (Lichne, 1974).
The most common amino acids found in wine are:
alanine,
arginine, aspartic acid, cystine, glutamic acid, glycine,
histidine, isoleucine, lysine, methionine, proline, serine,
threonine, tryptophan, tyrosine, valine, and phenylalanine
(Lichne, 1974).
Many minerals necessary to human life are
29
found in wine such as:
calcium, phosphorus, magnesium,
sodium, potassium, chlorine, sulfur, iron, copper, manganese, zinc, iodine, and cobalt (Lichne, 1974; Spring et al.1
1979).
Some vitamins are present in sufficient amounts to
be of importance of human nutrition.
Those vitamins in-
clude vitamin A, ascorbic acid, thiamin, riboflavin, pyridoxine, folacin, nicotinic acid, pantothenic acid, and
biotin (Lichne, 1974; Spring et al., 1979}.
Vitamin Content of Grape, Must and Wine
Morgan et al.
(1939) conducted one of the first studies
on the vitamin (thiamin, riboflavin, pyridoxine, and pantothenic acid) content of American grape juices and wines.
Later, Hall et al.
(1956) studied the vitamins thiamin,
riboflavin, niacin, pantothenic acid, pyridoxine, vitamin
B12, and folic acid in grape must and their wines.
The red
grapes and musts were richer in all of the above vitamins
than the white grapes and musts.
Specifically, folic
acid activity was lost during the extraction of the must.
However, there was little further loss and even a small increase of folic acid activity during fermentation of the
wine (Hallet al., 1956).
The skins and seeds of the
grapes contribute folic acid and other B complex vitamins
to the finished wine.
The presence of skins and seeds
accounts for the higher values of folic acid and B complex
vitamins in the red wines.
The average folic acid content, using S. faecalis as
the assaying organism, in white grapes, musts and wines
30
was 4.9 pg/100 gm, 1.8 pg/100 gm, and 1.8 pg/100 gm, respectively (Hallet al, 1956).
the research by Hall et al.
In the past 27 years since
(1956), the wine industry and
vitamin determination methods have changed greatly.
Therefore, Voigt et al.
(1978) conducted research to
determine the B complex vitamin content of California
wines.
Folic acid was assayed according to the Baker and
Frank (1968) method utilizing L. casei as the assaying
organism.
The reported folacin value for white wine by
Voigt et al.
(1978) was lower than Hall et al.
(1956) where
3.1 ng/ml is compared to 14 to 20 ng/ml, respectively.
The
difference between the folacin values may be attributed to
the different microorganisms used.
used by Hall et al.
s.
faecalis which was
(1956) as the assaying organism is not
recommended to assay food folate activity because it may
respond to clinically inactive forms of folic acid (Voigt
et al., 1978).
The variations found in the folacin activity as reported by Hallet al.
(1956), and Voigt et al.
(1978) con-
tinue to be noted as each researcher attempts to correlate
folic acid values.
Hardinge and Crooks (1961) stated that
the folic acid content of 100 gms of grapes was 5.2 pg and
120 gms or
~
cup of grape juice contained 3.6 pg.
Dong
and Oace (1973), utilized L. casei as the assaying organism, determined the free and total folic acid content of
grape juice as 1.4 pg/100 ml and 2.3 pg/100 ml, respectively.
Paul and Southgate (1978), utilized L. casei as the
31
assaying organism and conjugase treatment to determine
the folacin content of white dry wine in the absence of
ascorbic acid.
Both the free and total folic acid content
of white dry wine was 0.2 pg/100 gm.
Most recently, Per-
loff and Butrum (1977} have cited free and total folic
acid values of grapes and grape juice.
White raw grapes
contained 4 pg free folic acid and 7 pg total folic acid
per 100 gm edible portion.
Canned or frozen reconstituted
grape juice yielded 2 pg/100 gm free and total folic acid.
Streiff (1971} used L. casei as the assaying organism and
indicated that canned grape juice contained mean folate
level of 0.1 pg/100 ml of the monoglutamate form.
The total folate content of grape juice was generally
greater than the free folate amount because a portion of
the folate is in the polyglutamyl form (Dong and Oace,
1973}.
Therefore, conjugase treatment released additional
folate.
Different methods were utilized to determine the folic
acid activity in all of the aforementioned studies.
The
variations in the methodology of each experiment were
significant enough to accurately estimate the folic acid
content of grape, must, and wine.
In contrast to the aforementioned studies, Herbert
(1963} reported that wine folacin values were larger than
that for grapes and musts.
Grape juice (Westfield brand)
contained 0.95 pg/100 gm folate activity which was less
than 1.2 pg/100 gm, 1.0 pg/100 gm, and 1.3 pg/100 gm folate
32
activity for homemade Italian Red Wine, Blanchard Muscatel
and Guild Muscatel, respectively.
The increased folacin
values found in wine may be attributed to the fact that
yeast is added to the must to encourage fermentation.
Yeast is a well-known source of folacin (Rodriquez, 1978).
Brewers yeast, for example, contains approximately 4,000
pg of total folate activity in 100 gm (Perloff and Butrum,
1977).
The consequence of the addition of yeast was exemplified in yeast-leavened bread and doughnuts
and Calloway, 1972; Keagy et al., 1975).
(Butterfield
These researchers
measured the folacin content in bread and doughnut dough
before and after fermentation.
after fermentation.
Folate values were higher
The yeast's production of folacin
counteracted the destruction of folacin by the cooking temperature.
Thus, yeast is not only a source of folacin but
also produces folacin during fermentation (Butterfield and
Calloway, 1972; Keagy et al., 1975; Perloff and Butrum,
1977).
Brewers and active dry yeast contain 95 percent of
its folate in the conjugated form (Butterfield and Calloway, 1972).
In order to get an accurate measure of the
clinically active form, conjugase treatment must be employed.
However, only 10 percent of the folate contained
in yeast was available (Babu and Srikantia, 1976).
Rosen-
berg and Godwin (1971) demonstrated the presence of conjugase inhibitors in yeast.
Thus, the possible conjugase
33
inhibitor in yeast would explain the poor folate availability (Babu and Srikantia, 1976; Butterfield and Calloway,
1972}.
Economics of Wine
Wine consumption has been in an uptrend for more than
15 years (Standard and Poor, 1982}.
The market demand for
wines in the United States increased more than 10 percent
during the years 1970 to 1972 (Folwell et al., 1974}.
The
estimated United States-produced table wine market in the
United States was 321 million gallons in 1980.
The United
States market for all wine was estimated to be 467 million
gallons in 1980 with an estimate greater than 735 million
for 1990.
The increase in demand and usage of wine is in
relation to the higher education and affluence of society.
An increase in education and income results in an increase
in wine consumption.
California possesses one of
th~
highest market potentials in the United States due to its
relatively high per capita income and population density
(Folwell, 1971}.
Associated with the increasing affluence
of society has been the change in tastes and preferences
of its consumers.
In addition, state regulations, such as the twentyfirst amendment (repeal of prohibition}, gave each state
the authority to regulate the sale, price, distribution
and taxation of wines (Folwell, 1974 a and b).
These
regulations may affect the market size and potential.
For
34
example, as the level of taxation increased, the per
capita consumption decreased (Folwell, 1971).
Therefore,
the greatest market potential for United States table
wines would be in regions of high population density with
low degrees of regulation (Folwell, 1971).
Despite recession and high inflation, the United
States wine consumption has grown 7 to 8 percent in 1980
compared to 1979 (Standard and Poor, 1982) and it slowed
to 5.2 percent during 1981.
However, wine consumption is
expected to rise a further 4 to 6 percent in 1982 (Standard
and Poor, 1983).
White wine accounts for more than 57
percent of the California table wine market (Standard and
Poor, 1982).
For the first time, wine consumption has
topped liquor consumption in 1980.
White wine is less
expensive than hard liquor and has become a popular
beverage at cocktail hour (Standard and Poor, 1983).
Wine consumption is influenced by age, sex, education,
profession, family status, and income (Gastineau et al.,
1979).
The highest consumption rate is among 30-39 year
old men, who consume more wine than women.
A greater
proportion of those individuals with a higher education
drink wine rather than other alcoholic beverages.
In
addition, first time drinkers have an initial increased
consumption (Folwell, 1974b).
However, marital status and
employment have little influence.
Most important, is the
influence of income on wine consumption (Gastineau et al.,
1979).
Although grape production and wine consumption are
35
I
on the rise, strict traffic legislation, such as blood
alcohol limits and penalty for drunk driving, may deter
consumption of wine as well as other liquor in the near
future
(Gastineau et al., 1979).
'
36
~1
Chapter 3
MATERIALS AND METHODS
Materials
Grape, r1ust and Wine
The Chardonnay grapes used in this study were grown at
Paragon and MacGregor vineyards located in San Luis Obispo
County, California.
Paragon and MacGregor vineyards pos-
sess a cool climate and relatively heavy soil containing
calcareous material such as lime.
The unique feature of
1982's growing condition was a heat spell which occurred
prior to harvest.
The intense heat caused the grapes to
immediately ripen. The premature ripening may have caused
the grapes to become dehydrated resulting in a higher acid
content of the grape.
on hillsides.
Both vineyards' grapes were grown
MacGregor vineyard's grapes were grown at
a lower elevation than Paragon vineyard's grapes.
The
lower elevation and slightly cooler climate of the Mac
Gregor vineyard's grapes resulted in a later harvest than
the Paragon vineyard's grapes, October 17 and October 14,
respectively (Efirv, 1983).
The freshly harvested Paragon and MacGregor vineyards'
'
37
grapes were packed in nitrogen bags and transported within
24 hours to Ahern Winery, San Fernando, California.
Upon
arrival samples of grapes were taken and placed in ziplock
baggies, wrapped in aluminum foil and immediately placed on
dry ice in a portable cooler.
Sugar content ( 0 Brix), pH
and tartaric acid (TA) were measured.
Samples of must and wine were taken at various time
intervals during the wine making process.
The schematic
flow chart (Figures 2 and 3) depicts the sampling day of
wine production.
Must samples were placed in ziplock
baggies, wrapped in aluminum foil and immediately frozen.
Juice and wine samples were stored in brown reagent bottles
and immediately frozen.
All samples were transported from Ahern ltV'inery to the
California State University, Northridge Food Science
Laboratory.
minutes.
The transit time was estimated to be twenty
The samples were stored in the freezer at -22°C.
until analysis.
In the preliminary experiments a 100 percent Cabernet
Sauvignon wine and a 100 percent MacGregor Chardonnay wine,
both produced by Ahern Winery were assayed.
The Cabernet
Sauvignon sample used was taken from the 220th day of wine
production.
The MacGregor Chardonnay samples used were
taken from the 80th and 106th days of wine production.
The wine samples were stored in the freezer at -22°C,
until use.
38
Figure 2.
Schematic flow chart illustrates
sampling day of wine production
and significant changes in the
composition of Paragon Chardonnay
grape, must, juice and wine samples.
39
Paragon Samples!
t~-----41~G~ra~p~e~s~da~y~o~l------1t
~rush II day
jcrush I day Qj
I
I
T~mk
Tank
~
!Must I day
J
~
Ol
(Must II day
~
!Pressed Juice I day 11
!Juice 1 day
I
01
Ol
!Pressed Juice II day
91
jJuice II ;day 9]
I
Day 10 Inoculated with
Pasteur Champagne Red Star Brand Active Dry Wine Yeast
.J;
!Inoculated Juice day llJ
I
.
Heavy Fermentation Stage
J,
jwine day 151
{
jwine day 2 21
"'
!wine day 291
*
!~vine day 36
.
I
I
.
Ma 1 olact1c Fermentat1on
I
Racked to:
I
!Racked Wine day 1411
Fined
"'
!Bottled Wine day 163 2 1
1 25 tons of grapes were harvested at Paragon Vineyards,
San Luis Obispo, California.
2 wine was produced by Ahern Winery, San Fernando, California.
11
40
Figure 3.
Schematic flow chart illustrates
sampling day of wine production
and significant changes in the
composition of MacGregor Chardonnay
grape, must, juice and wine samples.
41
MacGregor Samplesl
jGrapes day oj
Pres ling
!Pressed Jtice day
11
!Post-Pressed Juice day 1]
Inoc~lated
Day 6
with
Pasteur Champagne Red StarWBrand Active Dry Wine Yeast
61
(Inoculated Juice day
Heavy FermeAtation Stage
/wine Jay 8/
jwine day 121
~
jwine day 191
,...,
jwine day
'l'
261
jWine day 3 3 Racked to Barrelsl--aging
.J.,
jwine day 4
71
I
Malolactic Fermentation
Jwine iay sol
jwine day 106j
I
Racked to Stainless Steel Tanks
{.
jRacked Wine day 13 sj
.I d
FJ.ne
.J..
_!Bottled Wine day 16o
1
2
j
25 tons of grapes were harvested at MacGregor Vineyards,
San Luis Obispo, California.
2 wine was produced by Ahern Winery, San Fernando, California.
42
Chemical Reagents and Microbiological Media
All chemicals were reagent grade.
Sterile deionized
water was used in the preparation of all solutions.
Crys-
,talline pteroylglutamate (folic acid) was purchased April 6
1982 from ICN Pharmaceuticals, Inc., Life Sciences Group,
Cleveland, Ohio (Lot No. 7924).
Lactobacillus casei cul-
ture (ATCC 7469), received August 1983 (Control No.
362616), Bactofolic Acid Casei Medium (Lot No. 685522) and
dried chicken pancreas (Lot No. 7924) were all purchased
from Difco Laboratories, Detroit, Michigan.
The composition and preparation of L. casei maintenance medium, standard folic acid solution, Difco chicken
pancreas conjugase, inoculum broth, folic acid assay basal
medium, sodium phosphate buffer and sterile saline solution
are described in Appendices A-G.
Equipment
The following equipment was used for sample preparation and measurement purposes.
1. UV-Visible spectrophotometer, Model 24, Beckman
Instruments, Inc., Fullerton, California
2. Gravity air incubator, Model 3211, National
Appliance Co., Portland, Oregon
3. Refrigerated centrifuge, Model J-21B, Beckman
Instruments, Inc., Fullerton, California
4. pH meter, Model pH 102, Brinkman Instruments, Inc.,
Westbury, New York
43
5. Sartorius balance, Model 2255, Brinkman Instruments
Westbury, New York;
Mettler electronic analytical balance, Model AClOO,
Mettler Instrument Corporation, Princeton, New
Jersey
6. Autoclave, Model STM-E, Type C, No. 45018, Market
Forge, Everett, Massachusetts
7. Freezer, Model FU 188R, Westinghouse Electric Corp.i
Pittsburgh, Pennsylvania
8. Ultrasonic cleaner, Model B52, Bransonic Co.,
Stanford, Connecticut
9. Filters:
Nalgene membrane syringe filters, poros-
ity 0.2pm (Lot No. 545897 C3), Nalgene, Division
of Sybron Corp., Rochester, New York; Autoclavable
and grided GA-6 Millipore filters, porosity 0.45
pm (Lot No. 152307), Millipore Corp., Bedford,
Massachusetts; Whatman filter paper No. 42, porosity 2.5 pm, Whatman Ltd., England
10. Syringe, Luer-lok tip disposable 10 cc (Lot No.
3B516), Becton-Dickinson and Co., Rutherford, New
Jersey
11. Additional equipment:
Eppendorf and vfueaton micro-
pipettes, Brinkman dispensettes, Vortex mixer,
Waring blender, Heater with stirring magnet, Corning Model PC 351, Gelman Filter 250 ml, and miscellaneous laboratory glassware.
44
Glassware Maintenance
In order to keep glassware free of folic acid, washing
procedures were carefully maintained.
All glassware was
soaked overnight in hot tap water with Micro detergent
(International Products, Corp., Trenton, New Jersey).
The
next day the glassware was immersed, without air bubbles
inside the glassware and washed with hot tap water and
fresh Micro detergent (20 ml/1 water) in an Ultrasonic
cleaner for 45 minutes.
The glassware was then thoroughly
rinsed in hot tap water ten times and rerinsed three times
with deionized water.
allowed to air dry.
The glassware was inverted and
After drying, the glassware was
covered with alumi:num foil.
Prior to use the glassware
was sterilized in the autoclave with a fast exhaust at
121°C and 15 psi for 15 minutes.
Methods
Variations of Sample Preparation Methods Studied
The optimum conditions for folacin determination were
established from preliminary experiments on the variations
in sample preparation methods.
Ahern Winery's Cabernet
Sauvignon and MacGregor Chardonnay wines were used in the
following three preliminary studies (see also Figure 4):
1. Use of ascorbate in sample preparation.
A. Addition of ascorbate phosphate buffer vs.
direct addition of ascorbate.
Butrum and Perloff (1975),
45
Figure 4.
Schematic diagram of different
stages in the folacin assay,
illustrating the variations of
sample preparation methods
studied with the sample used
and day of vinification.
Steps in Folacin Assay
Variables Studied
1. Sample f'reparation -------Ascorbate:
2. Heat Treatment
---------Method:
Sample Used
Addition of ascorbate
phosphate buffer
Direct addition of
ascorbate
MacGregor day 106
Varying percent
ascorbate levels
MacGregor day 80
Not-Autoclaving
Autoclaving
Cabernet
Sauvignon day 220
Boiling Waterbath
Filtratfon Treatment
Dilutiof
3. Conjugase Treatment
Microb~logical
- - -Conjugase:
Fresh Hog Kidney
Cabernet
Sauvignon day 220
Dried Chicken Pancreas
Assay
Folate Concentration
Determined
~
0'1
47
Herbert (1961), Hurdle et al.
(1968) and Lui (1980) stated
that ascorbic acid should be added to the buffer solution
in sample preparations during the extraction process and/or
autoclaving to protect the labile folate forms.
The use of
ascorbate in the extracting phosphate buffer solution was
compared to the direct addition of ascorbic acid in the
sample preparation procedure.
One portion of the sample
was diluted with (0.15 percent) ascorbate buffer, autoclaved, conjugase treated and microbiologically assayed
for total folic acid activity.
The other portion of the
sample received ascorbic acid directly to a final concentration of 0.15 percent ascorbate, autoclaved, conjugase
treated, diluted with (0.15 percent) ascorbate phosphate
buffer and microbiologically assayed for total folic acid
activity.
B. Varying levels of ascorbic acid.
The effects
of various levels of ascorbic acid added directly to
samples were examined.
The percentages chosen included:
0 percent, 0.15 percent, 0.3 percent, 0.5 percent, 0.7 percent, and 1.0 percent.
The various ascorbic acid levels
were added directly to each sample, autoclaved, conjugase
treated and microbiologically assayed for total folic acid
activity.
The buffer in the assay and standard-curve
tubes contained ascorbic acid with a final concentration
of 0.15 percent for all samples.
2. Heat treatment.
A heat treatment is recommended
for use in sample preparation to enhance the release of
48
bound folacin and to eliminate bacterial contamination
(Bird, et al., 1945).
Non-heat treatment and two heat
treatment methods were examined.
Twenty ml aliquots of
Cabernet Sauvignon wine containing 30 ml ascorbic acid
were used.
One sample received no heat treatment.
The
second sample was autoclaved at 121°C and 15 psi for 10
minutes.
The third sample was heated in a boiling water-
bath for 5 minutes.
All samples were then diluted and
microbiologically assayed for total folic acid activity.
3. Conjugase treatment.
Conjugase treatment is used
to hydrolyze the polyglutamate folate forms making them
available to the assay organism.
were compared:
Two sources of conjugases
fresh hog kidney, and commercial dried
chicken pancreas.
The two conjugases were chosen to
examine their ability to hydrolyze polyglutamate folate
forms in the presence of citric acid and yeast which are
found in grapes and wine.
Fresh hog kidneys were obtained at Farmer John's
slaughter house, Los Angeles, California.
Hog kidney con-
jugase was prepared according to the method of Eigen and
Shockman (1963) as described in Appendix H.
Exactly 0.5
ml of the hog kidney supernatant was added to 0.5 ml sample
containing 4.0 ml of ascorbate (0.2 percent) in a sodium
acetate buffer (pH 4.7)
(Appendix I).
The mixture was
then incubated at 37°C for 2 hours in an air incubator.
The chicken pancreas conjugase was prepared from
dried Difco chicken pancreas (Lot No. 7924) purchased from
49
Difco Laboratories, Detroit, Michigan.
The method of
preparation is outlined in Appendix C.
Exactly 0.1 ml of
the chicken pancreas supernatant were added to 0.5 ml
sample containing 4.4 ml of ascorbate (0.15 percent) in a
sodium phosphate buffer (pH 6.1).
The mixture was then
incubated at 37°C for 2 hours in an air incubator.
To determine the amount of folacin present in the hog
kidney and chicken pancreas conjugase, a blank sample of
each conjugase preparation was made.
The blanks contained
0.5 ml of hog kidney or chicken pancreas and 4.5 ml
sodium acetate buffer or sodium phosphate buffer, respectively.
The blanks were incubated at 37°C for 2 hours in
an air incubator along with the samples.
Methodology of Folacin Determination in Grape, Must, Juice
and Wine
From the experiments previously described, conditions
for the folacin assay were identified.
Maximum folacin
yields were obtain-d using the following procedures.
All
samples received the direct addition of ascorbic acid to
a final concentration of 0.15 percent.
The samples were
0
then autoclaved at 121 C and 15 psi for 10 minutes.
The
total folic acid content was determined after conjugase
treatment utilizing the chicken pancreas.
This methodology
was incorporated into the standard procedure to obtain the
maximum extractable folacin in the grape, must, juice and
wine samples.
50
~1
Grapes, Must and Hand-peeled Grapes
The folacin content of grapes, must and hand-peeled
grapes was examined.
Hall et al.
(1956) stated that a
significant portion of the folacin found in grapes was
contained in the skin.
Frozen samples of Paragon grapes and must were defrosted slightly at room temperature.
Forty gm of grapes
were hand-picked from the vine, 40 gm of must was taken
from a frozen block and 25 gm of hand-peeled grapes were
obtained by hand-peeling the berries.
The samples were
homogenized in a Waring blender for 1 minute and 10
seconds.
Ascorbic acid (0.15 percent) was added directly
to the homogenized samples and then autoclaved at 121°C
and 15 psi for 10 minutes.
at 3,000 x g and -5
0
c
The samples were centrifuged
for 20 minutes.
The supernatant was
aseptically decanted, diluted, conjugase treated and
microbiologically assayed for total folic acid activity.
Sample Preparation
Grape and Must.
Grape and must samples were removed
from the freezer and allowed to defrost for 30 minutes at
room temperature.
Fifty gm of each sample were homage-
nized in a Waring blender for one minute and transferred
to a sterile beaker.
Ascorbic acid (0.15 percent) was
then mixed into the slurry with a sterile glass rod.
The
homogenates were then autoclaved at 121°C and 15 psi for
10 minutes.
The samples were removed from the autoclave
'
51
and allowed to cool to room temperature.
The liquid por-
tion of the sample was aseptically decanted into sterile
centrifuge tubes and centrifuged at 3,000 x g and -5°C for
20 minutes.
The supernatant was then ready for the fil-
tration procedure.
Juice and Wine.
The liquid samples were removed from
the freezer and allowed to thaw in a waterbath in the
refrigerator for 30 minutes.
Once defrosted, the samples
were ready for the filtration procedure.
Filtration Procedure.
Grape, must, juice and wine
samples were divided into two sets, those which were filtered and those which were not filtered.
procedure is described below.
The filtration
The unfiltered samples were
set aside in a dark, cool place during this procedure.
Approximately 5 ml of the supernatant from the centrifuged grape and must samples were filtered into sterile
test tubes with a 250 ml Gelman filter apparatus attached
to a vacuum pump using Whatman filter paper No. 42, porosity 2.5 jllll·
In between samples the filter apparatus was
rinsed with sterile deionized water and then air dried.
A
new filter was used for each sample.
Approximately 5 ml of the Paragon juice and wine
samples were filtered into sterile test tubes with a Gelman
filter apparatus attached to a vacuum pump using sterile
Millipore filter paper, porosity 0.45
pm.
The filter
apparatus was rinsed with sterile deionized water and air
52
dried between samples.
A new sterile filter was used for
each sample.
Approximately 3 ml of the MacGregor juice and wine
samples were filtered into sterile test tubes with a luerlok tip disposable syringe attached to a pre-sterilized
membrane syringe disposable filter, porosity 0.2
Dilution.
pm.
In preparation for dilution, precisely 1 ml
of the Paragon and MacGregor unfiltered and filtered juice
and wine sample was transferred to a sterile test tube and
ascorbic acid was added directly to provide a final concentration of 0.15 percent ascorbate.
The samples were
autoclaved at 121°C and 15 psi for 10 minutes.
The samples
were removed and allowed to cool in a dark, cool place to
room temperature.
The unfiltered and filtered grape, must, juice and
wine samples were then diluted with 0.05 M sodium phosphate
buffer with pH 6.1 containing (0.15 percent) ascorbic acid.
This buffer was used as the standard buffer solution
throughout the folacin
determinat~on.
Chardonnay Wine Production
The samples of grape, must, juice and wine used in
this research were obtained from Ahern Winery.
Ahern
Winery's operations of Chardonnay wine production are outlined in Figure 5.
Samples of Paragon and MacGregor grape, juice and
wine were analyzed for
0
Brix, pH, tartaric acid (TA),
53
ADDITIONS
BY- PRODUCTS
HARVEST
mecltoniCIII
so.
(or}
~
CRUSH
h•nd
stema
·~
l
!
PRESS
sklnaonds-
juiceii'IICiion
-lltion
j
JUICE CLAAIFICAnON
--~
. ,_..
( - 1 . juice solicb}
-Of-1
pecllc--
!
FERMENTATION
CO•
!
M•l
linioh dry
AGING
lanka
lining
and/or
~
-~··----------.... 1
lor unsory or c l l l r i l y -
+
CLARIFICATION FOR B O T T L I N G - - - - - - - - - " 1 - "
l
,_
l
BOTTLE
Figure 5.
Flow chart of Ahern Winery's operations
in the production of Chardonnay wine.
Source: Amerine (1981).
54
alcohol and free sulfur dioxide (FS0 2 ) by the enologist
(Hagata, 1983) at Ahern Winery's laboratory.
was measured by a hydrometer.
pH meter.
The
0
Brix
The pH was determined by a
The TA was analyzed by a pH meter and titration
using NaOH to endpoint of pH 8.2.
The percent alcohol of
the wine was measured with a hydrometer following distillation of the wine.
The FS02 content was measured by the
Ripper method as described by
Am~rine
and Ough (1974).
Microbiological Assay for Folacin
The principle of the microbiological assay is based on
the fact that certain species of microorganisms can grow
only in the presence of folic acid.
L. casei is the micro-
organism of choice because it responds to the widest
spectrum of folate forms.
Maintenance of Lactobacillus casei (ATCC 7469).
L.
casei was obtained from Difco Laboratories, Detroit,
Michigan.
The culture was transferred upon receipt to
maintenance culture stabs which were then incubated at
37°C for 20 hours.
The stabs were kept refrigerated at
4°C until needed for inoculum preparation.
The culture
was then transferred monthly to new maintenance medium
(Appendix A).
Inoculum.
The day prior to each assay, inoculum was
prepared by subculturing L. casei from a stab into inoculum
broth (Appendix D).
The subculture was incubated at 37°C
55
for 20 hours.
After incubation, the cells were harvested
by centrifuging the cells at 3,000 x g and
minutes.
-s 0 c
for 10
The supernatant was discarded and the sedimented
cells were washed with 0.9 percent sterile saline {Appendix
G).
The cells were washed three times to deplete the cells
of folacin.
The harvested cells were suspended in 10 ml of
0.9 percent sterile saline.
to inoculate the assay tubes.
This suspension was then used
One drop of the cell sus-
pension from a 1 ml disposable pipette was used to inoculate each assay tube.
Assay medium.
The commercial, dehydrated folic acid
casei medium {Lot No. 685522) was purchased from Difco
Laboratories, Detroit, Michigan.
The double strength basal
medium was prepared following the instructions provided by
Difco Laboratories {Appendix E).
A 2.5 ml aliquot of
prepared medium was dispensed into each assay or standard
tube.
Diluted sample extracts or standard folic acid
solution were added to these tubes and then further diluted
with sodium phosphate ascorbate (0.15 percent) buffer to a
final volume of 5 ml.
Thus, the initial double strength
medium had been diluted to single strength.
Preparation of standard curve assay tubes.
A standard
curve plotting bacterial growth against known folic acid
concentrations was prepared for each assay.
A standard
curve was prepared for each experiment to compensate for
the variability of the microorganism.
The standard curve
56
tubes were prepared in triplicate for each folic acid concentration.
The following steps outline the preparation of
a standard curve.
1. 2.5 ml basal medium (Appendix E) was dispensed into
each culture rube.
This growth medium contained all the
nutrients for the growth of L. casei except folacin.
2. Sodium phosphate (0.15 percent} ascorbate buffer,
pH 6.1 (Appendix F) was added in amounts sufficient to give
a final volume of 5 ml.
The buffer provided and maintained
the proper pH to ensure similar growth response of
to several forms of folic acid.
~-
casei
The ascorbate protected
the folacin from oxidation during the assay.
3. The culture tubes were capped with plastic caps
and autoclaved at 121°C and 15 psi for 5 minutes as a
final sterilization technique before the aseptic addition
of folic acid.
4. Standard folic acid solution (folic acid solution
C, Appendix B.3} was pipetted into the cooled culture
tubes in calculated volumes to obtain a final concentration
range of 0.05 ng/tube to 1.2 ng/tube.
The test tubes were
stored in the dark until inoculated (Table 1}.
Preparation of sample assay tubes.
The sample tubes
were prepared by following the procedures outlined above
in steps 1 through 3 for the standard curve preparation.
Rather than adding a known amount of folic acid, diluted
grape, must, juice or wine extract was added to the tubes
Table 1
Protocol of Folacin Standard Curvel
Tube
No2
Folic acid
cone.
(ng/tube)
1
2,3
4,5,6
7,8,9
10,11,12
13,14,15
16,17,18
19,20,21
22,23,24
25,26,27
28,29,30
31,32,33
1 source:
Folic acid
ass a~
solution (ml)
0
0
0.05
0.1
0.2
0.3
0.4
0.5
0.6
0.8
1.0
1.2
Phosphate
ascorbate
buffer (ml)
0
0
0.1
0.2
0.4
0.6
0.8
1.0
1.2
1.6
2.0
2.4
2.5
2.5
2.4
2.3
2.1
1.0
1.7
1.5
1.3
0.9
0.5
0.1
Basal
Medium4
(ml)
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.-5
Total
volume (ml)
per tube
L. casei
Inoculum
added (1 drop)
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
_5
+6
+
+
+
+
+
+
+
+
+
+
Chen, 1979
2Each concentration was assayed in triplicate, except tubes Nos. 1, 2, and 3
3 Appendix B.
Solution C
4Appendix E
5Tube No. 1 was used as reference blank
6Tubes Nos. 2, 3 were used as zero concentration
U1
-...]
58
at step 4.
Each test sample was assayed at two different
strengths (0.1 ml and 0.2 ml) in triplicate.
for the samples varied.
The dilutions
Details of sample dilutions are
shown in Tables 2 and 3.
Preparation of conjugase blanks.
The folate content
of the conjugase preparations was determined by assaying a
conjugase blank.
A 0.5 ml aliquot of the conjugase blank
was mixed with 2.5 ml basal medium and 2.0 ml buffer and
assayed.
Inoculation and incubation.
Assay tubes for samples
and standard growth curve were inoculated with one drop of
L. casei inoculum.
The inoculum was delivered into each
test tube via a sterile 1 ml disposable pipette.
The tubes
were capped, mixed with a vortex mixer and incubated at
37°C in an air incubator for 20 hours.
Measurement of growth of L. casei.
After 20 hours of
incubation, the assay tubes were autoclaved at 121°C and
15 psi for 5 minutes to halt any further growth of
casei.
bath.
~­
The tubes were then allowed to cool in a waterEach tube was thoroughly mixed by a vortex mixer
and the turbidity of the sample was measured at 640 nm with
a Beckman UV-Visible Spectrophotometer model 24 equipped
with a sipper system.
The turbidity was recorded in op-
tical density units and determined quantitatively the
microbial growth.
Table 2
Protocol of Folacin Assay Tubes for Paragon Samples 1
Sample
Dilution prior
to conjugase
treatment
Dilution of
conjugase
treatment
Final
dilution
factor2
Unfiltered/Filtered:
Grape
1:2
1:10
1:20
Crush and Must
1:2.5
1:10
1:25
Press3
1:3
1:10
1:30
Juice and Wine
1:5
1:10
1:50
1:4
1:10
1:40
Unfiltered:
Filtered:
Juice and Wine
1 samples were diluted with ascorbate (0.15%) sodium phosphate buffer (pH 6.1) to the
established dilution level prior to conjugase treatment.
2Each sample was assayed in triplicate at two dilutions (0.1 ml, 0.2 ml).
3only an unfiltered press sample could be obtained.
U1
1.0
Table 3
Protocol of Folacin Assay Tubes· for MacGregor Samples
Sample
Dilution prior
to conjugase
. treatment
1
Dilution of
conjugase
treatment
Final
dilution
factor2
Unfiltered/Filtered:
Grape
1:2
1:10
1:20
Crush and Must
1:2.5
1:10
1:25
Day 1-12; 47-160
1:4
1:10
1:40
Day 19-33
1:5
1:10
1;50
Day 1-12; 47-160
1:3
1:10
1:30
Day 19-33
1:4
1:10
1:40
Unfiltered Juice/Wine
Filtered Juice/Wine
1 samples were diluted with ascorbate (0.15%) sodium phosphate buffer (pH 6.1) to the
established dulution level prior to conjugase treatment.
2Each sample was assayed in triplicate at two dilutions (0.1 ml, 0.2 ml)
0'1
0
61
Data Analysis
Construction of Standard Curve.
The means of the op-
tical densities for each triplicated folic acid concentration were plotted against known concentrations of folic
acid.
A standard curve was obtained by the computer pro-
gram "LIB POLFIT" and "LIB XYPLOT".
The computer program "POLFIT" fits least-square polynomials to bivariate data, using an orthogonal polynomial
method.
The program's lowest degree polynomial to fit was
set at the second degree, where Y
optical density; X
=
=c
+ BX + AX2 (Y
folic acid concentration) •
=
The index
of determination was computed which indicated the curve's
degree to fit.
Coefficients were determined and were sub-
sequently used in other programs.
The computer program "XYPLOT" performs a plot of
single-value functions.
B{X) - AX2.
The function used was Y
=C
+
The coefficients A, B, and C were obtained
from "POLFIT".
The limits of the plot were set by the
user and a plot was constructed.
A standard curve was also obtained by hand-fitting
the data points.
The mean data points were plotted
against the known concentrations of folic acid and a
standard curve was drawn using a French Curve.
Determination of Folacin Concentration in the Test
Sample.
A computer program designed by Chen (1983) cal-
culated the folacin values of the sample tube from the
62
optical density.
The second degree equation and resulting
coefficients obtained from the computer program "POLFIT"
were used.
The folacin activity was also calculated by
hand using the handfitted standard curve.
A correction value was calculated by determining the
folacin content per milliliter of conjugase preparation.
The amount of conjugase and thus, the correction value for
the sample assay tube was calculated.
This factor was sub-
tracted from the folacin concentration in the tube.
The
amount of sample in the tube and the dilution factor were
also taken into account to calculate the extractable folacin concentration expressed aspg/100 gm (ml) of grapes,
must, juice or wine.
Appendix J diagrams the work sheets
used for this research.
Statistical Analysis.
Each sample was assayed at two
concentrations in triplicate.
The mean and standard devi-
ation were calculated from those six values.
In order to determine if statistical differences
existed, the computer program "LIB DAM" was used.
The
program "DAM" performs a one-way analysis of variance
(ANOVA) to test for differences among means.
The program
utilized the raw data given by the user and calculated the
mean, standard deviation and variance.
tic showed significant differences (P
When the F statis~0.05)
existed, the
program "DAM" further computed T and Z statistics which
compared pairs of means.
The T statistic and correspond-
63
ing degrees of freedom were compared to the distribution of
a t statistical chart (Joseph and Joseph, 1979; Larmond,
1977).
The significance was determined at a minimum of 5
percent level.
Chapter 4
RESULTS AND DISCUSSION
A comparison of variations in sample preparation
methods was first studied in preliminary experiments.
From
these studies, the optimal conditions for folacin determination in grape and wine were established and utilized to
determine the changes in folic acid activity during wine
production from Paragon and MacGregor Chardonnay grapes.
Variations of Sample Preparation Methods Studied
1. Use of ascorbate in sample preparation.
A. Addition of ascorbate phosphate buffer vs.
direct addition of ascorbate.
The addition of ascorbate
to the extracting buffer or directly to the sample during
the extraction was studied.
The presence of ascorbate
has a protective effect on the folates.
Ascorbic acid's
reducing properties inhibit oxidative destruction of the
labile forms of folacin.
(Herbert, 1963; Hurdle et al.,
1968).
During the sample extraction process, the samples are
usually autoclaved at 121°C and 15 psi for 10 minutes.
In
order to protect the folacin against thermal destruction,
64
65
sodium phosphate buffer containing ascorbate is added
prior to autoclaving.
Paine-Wilson and Chen (1979) found
that the pH of the buffer had a profound effect on the
thermal stability of folacin.
Folacin is most stable in
neutral or slightly alkaline solutions and less stable
under acidic conditions (Blakeley, 1969; Stokstad and
Thenen, 1972).
Wine has acidic pH.
Thus, the addition of
sodium phosphate buffer at pH 6.1 would increase the pH
of the wine thereby providing a suitable environment for
folacin stability.
Ahern's MacGregor Chardonnay wine samples treated with
either phosphate buffer containing ascorbate or ascorbate
only prior to autoclaving were compared.
As shown in
Table 4, the wine initially had a pH of 2.91 and was reduced to 2.65 with the direct addition of ascorbic acid
and increased to 3.82 with the addition of buffer.
The
sodium phosphate ascorbate buffer sample yielded 5.23pg/
100 ml of total folic acid which was significantly less
(p
~0.001)
than 6.63 pg/100 ml found in the sample treated
only with ascorbic acid.
The direct addition of ascorbic acid caused the pH to
become more acidic.
However, a greater amount of folacin
was extracted when autoclaved in the absence of sodium
phosphate buffer.
Chitwood (1983) also found that the
direct addition of ascorbic acid prior to autoclaving
yielded a greater amount of folacin.
Therefore, a decision
was made to autoclave all samples using the direct ascor-
66
Table 4
Comparison of addition of ascorbate containing phosphate
buffer to direct addition of ascorbate on the total
folacin concentration of MacGregor Chardonnay winel
Treatment
pH2
Buffer 4
3.82
5.23 + 0.65a
Ascorbate5
2.65
6.63
Total
Folacin Concentration
. (pg/100 ml) 3
±
0.45b
1 The sample used was MacGregor Chardonnay wine derived
from the 106th day of wine production.
2pH of the wine sample was 2.91.
3values represent the mean ± S.D. of the mean from six
replicates. Means followed by different letters are
significantly different at 0.1% level.
4sodium phosphate buffer with pH 6.1 containing 0.15%
ascorbate was added to the sample.
5Ascorbate was added directly to the sample to a final
concentration of 0.15 percent.
67
bate method to extract the greatest amount of folacin
available.
B. Varying levels of ascorbic acid.
Various levels
of ascorbate were examined during the extraction process to
determine the optimal amount required to protect the labile
folacin forms.
The ascorbic acid was added directly to a
MacGregor Chardonnay wine sample in the following percents:
0, 0.15, 0.3, 0.5, 0.7, and 1.0.
The pH of the sample was
measured prior to autoclaving and again after autoclaving
to examine the effect of the heat on the pH.
The results
of the change in pH with the addition of ascorbic acid and
autoclaving are shown in Table 5.
The addition of ascor-
bate caused the original pH of the wine sample to decrease
proportionately to the amount of ascorbate added.
After
the sample was autoclaved, the pH rose above that of the
original pH of the wine for all levels of ascorbate.
Per-
haps, the heat caused the pH of the wine to become less
acidic providing a suitable environment for folacin stability.
There was no significant difference
(p~
0.05) in the
folacin concentration assayed among the various percent
levels of ascorbic acid added when analyzed by ANOVA with
the exception of 0 percent ascorbic acid (Table 5).
bert ( 1961) , Hurdle et al.
(196 8) , 0' Brain et al.
Her-
( 197 5) ,
Chen and Cooper (1979), and Lui (1980) found that the
presence of ascorbic acid was necessary to protect the
labile folate forms during assay.
However, the exact per-
Table 5
Effect of various levels of ascorbic acid and autoclaving on
the pH and folacin concentration of wine samplel
Percent
Ascorbic Acid
pH before
Autoclaving 2
pH after
Autoclaving 3
0.0
3.25
3.75
0.15
3.20
3.53
11.49 ± 1.94b
0.3
3.19
3.48
11.56 ± 1.05b
0.5
3.16
3.40
10.68 +
- 0.8 4b
0.7
3.13
3.34
10.67
±
0.47b
1.0
3.11
3.24
10.66
±
0.64b
Total
Folacin Concentration
(pg/100 ml) 4
5.07
±
0.48a
1 The sample used was MacGregor Chardonnay wine from the 80th day of fermentation. The
sample received the direct addition of various percent ascorbate levels and autoclaved
at 121°C and 15 psi for 10 minutes.
2pH of wine sample was taken after the addition of ascorbic acid prior to autoclaving.
3pH of wine sample was taken after it was autoclaved and cooled.
4values represent the mean ± S.D. of the mean from six replicates.
different letters are significantly different at 5% level.
Means followed by
0'\
(X)
69
cent level chosen varied.
Herbert (1961) recommended the
use of 0.15 percent ascorbic acid, yet Hurdle et al.
(1968) reported no significant difference between 0.15
percent and 10.0 percent ascorbate.
O'Broin et al.
(1975)
found that 0.2 percent ascorbate was required during autoclaving to protect the folacin.
Yet, Chen and Cooper
(1979) determined that a level of 0.1 percent ascorbate
was sufficient, and Lui (1980) found buffers that contained 0.1 to 0.2 percent ascorbic acid were best.
The
fact that there was no significant difference between the
levels of ascorbate in this research was supported by the
discrepancie~
found in previous research.
Perhaps, the
optimum level of ascorbate to be used depends on the pH
and the foodstuff being analyzed.
L. casei is most active in the presence of 0.15 percent ascorbic acid (Herbert, 1961) .
The percent ascorbate
recommended for use in the sodium phosphate buffer in the
microbiological assay for spinach was 0.15 percent (Song,
1982).
In addition, there was no statistical difference
(p=- 0.05) among the various percents of ascorbic acid
added.
Thus, for simplicity, a final concentration of
0.15 percent ascorbic acid was chosen as the level to be
added directly to the sample prior to autoclave treatment
and utilized in the buffer throughout the entire microbiological assay.
2. Heat treatment.
The effect of heat treatment on the folacin extrac-
70
tion was studied employing three different conditions:
non-heat treatment, autoclaving at 121°C and 15 psi for 10
minutes and heating in boiling waterbath for 5 minutes.
Ahern's Cabernet Sauvignon was utilized to determine the
optimum method to enhance the release of bound folacin and
to eliminate bacterial contamination (Bird, et al. 1945).
The non-heat treatment method resulted in 4.88 pg/
100 ml, the autoclave sample yielded 8.46 pg/100 ml and the
boiling waterbath sample contained 7.42 pg/100 ml of folic
acid activity (Table 6).
The statistical analysis (ANOVA)
indicated there was no significant difference
(P~
0.05)
between the autoclave and boiling waterbath treatment.
There was a statistical difference
(P~
0.05) in the non-
heat treatment method compared to the two heat treatments.
Since autoclaving is a method of sterilization, the
high folacin yield in the autoclave sample would have to be
attributed to the heat effect on releasing the bound folacin.
Furthermore, the insignificant results found between
the boiling waterbath treatment and the autoclave method
support the assumption that the effect of heat releases
bound folacin in the sample.
The boiling waterbath method
has also been used extensively as a substitute for autoclaving (Bird, et al. 1945; Dong and Oace, 1973; Keagy, et
al. 1975).
However, the autoclaving treatment at 121°C
and 15 psi for 10 minutes was chosen for this research.
3. Conjugase treatment.
All grape, must, juice and wine samples were treated
,, .
71
Table 6
Comparison of heat treatment methods on releasing
total folacin in wine samplesl
Heat Treatment
Total
Folacin Concentration
.. (pg/100 ml) 2
Autoclave3
8.46 ± 2.2Sa
Waterbath 4
7.42 ± 2.22a
Non-Heat Treatments
4.88 ± 0.73b
lThe sample used was Ahern's Cabernet Sauvignon from the
220th day of fermentation.
2values represent the mean ± S.D. of the mean from three
replicates. Means followed by different letters are
significantly different at S% level.
3wine sample was autoclaved at 121°C, lS psi for 10
minutes.
4wine sample was heated in the boiling waterbath for S
minutes.
Swine sample was set aside in cool, dark place.
72
with conjugase in order to hydrolyze the polyglutamate
forms of folacin thereby providing the total folic acid
activity of the sample.
Grape and wine contain yeast
which are present on the grape prior to the addition of
sulfur dioxide and thatwhich are added to grape juice prior
to fermentation.
Grape and wine also contain small amounts
of citric acid (0.01 to 0.03 percent)
(Fong, et al. 1974).
Hog kidney conjugase is inhibited by the presence of yeast
(Babu and Srikantia, 1976; Butterfield and Calloway,
1972).
Chicken liver conjugase is inhibited by the pres-
ence of citric acid (Rao and Noranha, 1977).
In addition,
Kirsch (1983) noticed that the chicken pancreas conjugase
was affected by citrate phosphate buffer and thus concluded
that citrate inhibited the chicken pancreas conjugase.
Therefore, fresh hog kidney conjugase was compared to
commercial dried Difco chicken pancreas conjugase to
determine which enzyme had the greater ability to hydrolyze polyglutamate folate forms in the presence of yeast
and minute amounts of citric acid.
The Cabernet Sauvignon sample treated with hog kidney
conjugase resulted in 5.51 pg folacin per 100 ml.
The
chicken pancreas treated sample yielded 8.77 pg folacin
per 100 ml.
Table 7 illustrates the results.
The chicken
pancreas conjugase treatment resulted in significantly
(p <
0. 01) greater detectable folacin than the hog kidney
conjugase treatment.
Thus, it can be concluded that the
minute amount of citric acid present
in the wine did not
73
Table 7
Comparison of two conjugase treatments on their ability
to release total folacin content from wine samplesl
Conjugase
Total
Folacin Concentr~tion
. . ... (pg/100 ml)
Hog Kidney 3
5.51 ± 0.40a
Chicken Pancreas 4
8.77 ± 0.63b
1 The sample used was Ahern's Cabernet Sauvignon from the
220th day of fermentation.
2values represent the mean ± S.D. of the mean from one
replicate. Means followed by different letters are
significantly different at 0.1% level.
3Fresh hog kidney was utilized to hydrolyze the polyglutamate folate forms (Appendix Hand I).
4 commercial dried Difco chicken pancreas was utilized
to hydrolyze the polyglutamate forms of folacin
(Appendix C) •
74
inhibit the chicken pancreas conjugase in comparison to
the effect of yeast used during fermentation on the hog
kidney conjugase.
Methodology of Folacin Determination in Grape, Must, Juice
and Wine
The results of the above studies on the variations in
sample preparation methods led to the selection of the
optimal conditions for the assaying of total folac acid
activity in grape, must, juice and wine.
The optimal
conditions included adding ascorbic acid to a final concentratioh of 0.15 percent directly to the sample prior to
autoclaving at 121°C and 15 psi for 10 minutes, and hydrolyzing the folacin polyglutamates with commercial dried
Difco chicken pancreas.
Half of Paragon and MacGregor
Chardonnay samples were subjected to filtration treatment.
The filtration procedure employed in the methodology was
extremely important in order to determine the source or
sources of folacin in the samples.
Yeast as well as
leaves, skins of the grape and seeds which are excellent
sources of folacin were present in the juice and wine
samples.
The porosity of the filters were small enough
(0.2 pro to 2.5 pm) to eliminate yeast cells and other extrinsic folacin containing material from the samples.
These procedures were utilized to monitor the folic acid
activity during the commercial production of Paragon and
MacGregor Chardonnay wines.
75
Grape, Must,· and Hand-peeled Grape
Paragon grapes, must and hand-peeled grapes were
analyzed to examine which portion of the berry contained
the greatest proportion of folacin.
The must contained
the greatest source of folacin of 5.1 pg, the whole grape
yielded 3.9 pg, and the hand-peeled grape resulted in 1.9
pg folacin per 100 gm (Table 8).
The results of ANOVA show
that the must contained significantly
(p~
0.05) greater
proportion of folacin activity than the hand-peeled grape.
The must consisted of grapes, seeds, skin, leaves,
stems, and other debris.
The skins, seeds, leaves and
stems are excellent sources of folacin (Hall et al. 1956;
Amerine, 1980).
Thus, the presence of these extraneous
materials explained the increased folacin content.
Simi-
larly the grape sample contained skins and a few seeds.
However, the grape samples lacked leaves and other extraneous material which may have contributed a substantial
amount of folacin.
On the other hand, the hand-peeled
grapes consisted of grape pulp and a few seeds.
Apparent-
ly, a substantial source of folacin was absent in the handpeeled grapes.
It can be concluded that a significant
portion of the folacin was contained in the skins of the
grape.
The presence of folacin in the skins of the grape
is supported by comparing the folacin values of red wine
to white wine.
Red wine is generally a greater source of
folacin than white wine (Hallet al., 1956).
Certain red
sweet wines contain 45 percent more folic acid than the
76
Table 8
Comparison of the folacin concentration in the
grape, must and hand-peeled grape 1
Sample
Total
Folacin Concentration
(pg/100. gm} 2
Grape
3.99 ± 0.16a
Must
5.13 ± 0.42b
Hand-peeled Grape
1.96
±
0.21c
1 Grape, must and hand-peeled grape samples were obtained
from freshly harvested Paragon grapes.
2values represent the mean ± S.D. of the mean from six
replicates. Means followed by different letters are
significantly different at 5% level.
77
grape it originated from (Lucia, 1954).
Grape juice for
red wine remains with its skins intact much longer than
for white wine (Amerine, 1980) thus providing more folic
acid to the final end product.
Changes in Total Folacin Activity During Wine Production
The total folic acid activity of wine during vinification beginning with freshly harvested Chardonnay grapes to
the final bottling of wine was monitored by microbiological
assay.
The grapes were harvested at Paragon and MacGregor
Vineyard in San Luis Obispo, California and fermented into
wine at Ahern Winery in San Fernando, California.
Samples
were taken from Ahern Winery at various intervals throughout the wine making process and assayed for total folic
acid content.
Degree Brix, tartaric acid, pH, alcohol and
free sulfur dioxide values were determined by Ahern
Winery as shown in Table 9.
The grape extracts were randomly sampled from Paragon
and MacGregor lots.
The folacin content of the unfiltered
Paragon grape extract was 5.6 pg/100 gm and 4.68 pg/100 gm
in the filtered sample.
The folacin content of the un-
filtered MacGregor grape extract was 3.49 pg/100 gm and
2.93 pg/100 gm in the filtered sample.
The differences
between the folacin content of the unfiltered and filtered
grape extract samples were insignificant (p < 0. 05) as
analyzed by ANOVA (Tables 10 and 11).
The folacin grape
values found in the unfiltered samples were similar to
Table 9
0
Sugar content ( Brix), pH, total acid, alcohol percent and free
· sulfur dioxide (FS0 2 ) values of must and winel
Day of
Vinification
Day 5
Sample
Paragon
Brix 2
(gm/100 gm)
0
Tartaric Acid
(TA)
· (gm/100 ml)
Alcohol
% by volume
3.37
1.0
Day 49
3.39
0.9
13.4
Day 124
3.40
0.87
13.4
Day 164
3.34
0.81
13.4
3.29
0.93
Day 46
3.39
0.91
13.6
Day 121
3.32
0.91
13.6
Day 161
3.32
0.86
13.6
Day 4
MacGregor
23.8
pH
24.0
FSO
(ppm)
35
36
1values for these analyses were obtained from Ahern Winery's laboratory.
2°Brix indicates grams of sucrose per 100 gm of grape juice.
-.....!
co
79
those reported by Hall et al.
(1956) and Hardinge and
Crooks (1961) which were 4.9 pg/100 gm and 5.2 pg/100 gm,
respectively.
Perloff and Butrum (1977) listed higher
value of 7 pg folacin per 100 gm of grapes.
The discrep-
ancy in these values may be a result of the growing conditions of the grape.
Soil, temperature, rainfall and
various other environmental conditions affect the growth
and maturity of the grape.
Soil, temperature, rainfall and
various other environmental conditions affect the growth
and maturity of the grape (Amerine, 1980) and hence its
vitamin content.
In addition, variations in the micro-
biological assay methodology would also affect the folacin
content found in the sample.
The grapes were crushed to break the skin of the
berry.
Sulfur dioxide was added at this stage.
The sulfur
may cause the folic acid content to decrease as noted by
Hallet al.
(1956}.
The folacin content of the crushed
grape extracts were similar to that of the grape extracts.
The folacin content of the crushed Paragon grape extract
was 4.24 pg/100 gm in the unfiltered sample and 3.87
100 gm in the filtered sample (Table 10}.
~g/
The MacGregor
crushed grape extract contained 4.54 pg of folacin per
100 gm in the unfiltered and 3.78 pg of folacin per 100 gm
in the filtered samples (Table 11).
The crushed grapes were then destemmed, macerated and
transported to refrigerated stainless steel tanks.
The
must samples consisted of skins, seeds, and leaves and
80
Table 10
Comparison of total folic acid activity of unfiltered
and filtered Paragon Chardonnay grape,
crushed grape and must samples
Sample
Total
Folacin Concentration
(pg/100 gm) 2
Unfiltered
1.07b
Filtered 3
0.67b
5.63
Crushed Grape
4.24 + 2.8lb
-
3.87 + 2.47b
Must
8.61 ± o.o8a
7.94 + 1. 56a
Prepressed Must 1
7.88 + 1. 06a
--- -
±
-
4.68
±
Grape
1 The prepressed must sample was obtained 18 hours prior to
the pressing of the must.
2values represent the mean ± S.D. of the mean from six
replicates. Means followed by different letters within
the same column are significantly different at 5% level.
3samples were filtered with a Gelman Apparatus attached to
a vacuum. Whatman filter papers (porosity, 2.5 pro> were
used.
81
Table 11
Comparison of total folic acid activity of unfiltered
and filtered MacGregor Chardonnay grape,
crushed grape and must samples
Sample
Total
Foiacin Concentration
(pg/100 gm) l
Unfiltered
Filtered 2
Grape
3.49
±
0.67b,x
3.10
Crushed grape
4.54
±
0.65b,x
3.78 + 0.17b,y
Must
9.61 ± 1.17a,x
7.96
±
±
0.44b,x
0.82a,y
1values represent the mean ± S.D. of the mean from six
replicates. Means followed by different letter (a or b)
within the same column are significantly different at 5%
level. Means followed by different letter (x or y) in
the same row are significantly different at 5% level.
2sarnples were filtered with a Gelman filter apparatus
attached to a vacuum. Whatman filter papers (porosity,
2.5 pm) were used.
82
remained in the tanks about 24 hours.
The folacin content
for both unfiltered and filtered Paragon and MacGregor must
increased significantly (p c:::::. 0. 05) when compared to the
crushed grape extracts (Tables 10 and 11).
The folacin
content of the unfiltered and filtered Paragon must samples
was 8.61 pg/100 gm and 7.94 pg/100 gm, respectively.
The
folacin content of the MacGregor unfiltered and filtered
must yielded 9.61 pg/100 gm and 7.96 pg/100 gm, respectively.
The increase in the total folic acid content in the
must was a result of the lapsed time between the crushing
and the pressing of the grape.
The time lapse permitted
the folacin which is contained in the skins, seeds, and
leaves (Hallet al., 1956) to be absorbed into the liquid
portion of the must.
A sample of the Paragon must 18 hours prior to pressing was taken.
The prepressed must folic acid content
decreased slightly to 7.88 pg/100 gm in the unfiltered
sample, a filtered sample could not be obtained.
The musts were pressed at 2 atmospheres and again at
4 atmospheres to obtain the free-run juice.
The absence
of skin, seeds, and leaves as well as other debris in the
free-run juice explains the tremendous decrease (pc::::: 0. 05)
of folacin in the grape juice on day 1 (Figures 6 and 7).
Paragon contained 1.29 pg folacin per 100 ml in the unfiltered sample and 0.55 pg folacin per 100 ml in the
filtered sample (Table 12).
MacGregor resulted in 0.49 pg
folacin per 100 ml in the unfiltered sample and 0.26 pg
83
Figure 6.
Total folacin activity of
unfiltered and filtered
Paragon Chardonnay samples
during the vinification
process.
84
"C
cu
~
cu
0
---· ...
c
::»
"C
U)
cu
~
cu
u...
0
!:
bI
0
-
N
c
0
0
0
.....
co
0
c
0
CIQ
>
0
>.
0
U)
0
..
N
Ill
:I
E
!
~
2
CIQ
0
N
ca
c
i1
'
85
Figure 7.
Total folic acid activity of
unfiltered and filtered MacGregor Chardonnay samples
during the vinification process.
86
"'0
...
cv
cv
0
--·--· -...
"'0
!2
Ql
Q)
c
~
t
u...
0
3:
%
0
N
c::
0
0
0
-
ro
-·(.)
c:
0
CD
>
0
>.
0
CD
.
0
0
N
...
II
:I
.-
E
-
N
CD
CD
•
0
ro
0
87
Table 12
Comparison of total folacin activity of unfiltered
and filtered Paragon Chardonnay juice and wine
samples during commercial vinification.
Total
Folacin Concentration
yug/100 ml) 1
Day of
Vinification
Unfiltered
Filtered 2
1
1. 29 ± 0.38f,x
0.55 ± O.l3e,y
9
0.63
± o.o9h,x
0.25 + 0.02g,y
11
0.87
± 0.14g,x
0.39
15
3.57 + 1.03d,x
2.08 + O.l9d,y
22
0.99c,x
5.33 + 0.38b,y
29
10.73 ± 0.33b,x
4.18 ± 0.82c,y
36
14.95 + 0_._79a,x
5.81 + 0.65a,y
50
4.12 ± 1.19d,x
2.20
141
5.16 ± 0.33d,x
5.36 ± o.s2b,x
163
2.27 + O.l4e,x
2.03 + 0.16d,y
-
9.68
±
± o.o6f,y
-
± 0.23d,y
1 values represent the mean ± S.D. of the mean from six
replicates. Means followed by different letter (a-h)
within the same column are significantly different at
5% level. Means followed by different letter (x or y)
in the same row are singificantly different at 5% level.
2samples were filtered with a Gelman filter apparatus
attached to a vacuum. Millipore filter papers
(porosity, 0.45 prn) were used.
88
folacin per 100 ml in the filtered sample (Table 13).
The
difference between the unfiltered and filtered samples
within each group was significant (p < 0. 05) owing to the
presence of debris containing folacin in the unfiltered
samples.
A sample of MacGregor was taken 6 hours after the
must was pressed.
There was a significant (p c:::: 0. 05) in-
crease in folacin content (Table 13).
Exactly 0.71 pg of
folacin per 100 ml was contained in the unfiltered and 0.49
pg of folacin per 100 ml was present in the filtered sample.
The increase was a result of folacin-containing mate-
rial present in the sample.
The lapsed time permitted in-
creased amounts of folacin to be extracted.
The increased
folacin extraction was exemplified by the significant
difference (pc::::::.0.05) between the unfiltered and filtered
sample owing to the presence of folacin-containing
material.
The 9th day sample of Paragon and the 6th day sample
of MacGregor showed a significant
the folacin content from day 1.
(p~
0.05) decline in
The folacin content of
the unfiltered Paragon sample was 0.63 pg/100 ml and 0.25
pg/100 ml in the filtered sample.
Both the unfiltered and
filtered MacGregor samples resulted in 0.31 pg folacin per
100 ml.
The decrease in folacin activity may be attri-
buted to the settling of the sample leaving the body of
the juice free from excess sediment.
However, there was a
significant difference (p <:::: 0. 05) in the Paragon sample
89
Table 13
Comparison of total folacin activity of unfiltered
and filtered MacGregor Chardonnay juice and
wine samples during commercial vinification
Total
Folacin Concentration
(pg/100 ml)
Day of
Vinification
Unfiltered
Filtered 2
1
0.48 ± O.l5h,x
0.26 ± O.l8h,y
1 + 6 3 hours
0.70 ± 0.15g,x
0.49 + O.lOf,y
6
0.31 ± o.o5i,x
0.31 + o.62g,x
8
2.61 ± 0.99f,x
2.06 ± 0.19e,x
12
4.30 ± 0.4le,x
3.37 + 0.29d,y
±
±
0.19a,x
6.33 + 0.29c,y
0.66d,x
3.63 ± 0.19d,y
19
14.05
26
7.56
33
5.92 ± 2.34d,x
±
±
4.30
±
0.45d,y
0.80b,x
11.14 + 0.77a,x
0.75c,x
10.21 + 2.17a,x
106
8.29 + 0.46d,x
8.05 + 0.57b,x
138
10.03 ± 0.22c,x
160
4.99 ± 0.32d,x
47
11.58
80
10.67
9.71
±
0.22a,y
4.94 ± 0.17d,x
]Values represent the mean ± S.D. of the mean from six
replicates. Means followed by different letter (a-h)
within the same column are significantly different at
5% level. Means followed by different letter {x or y)
in the same row are significantly different at 5% level.
2 samples were filtered with Nalgene membrane syringe filters, {porosity, 0.2 pm) attached to a Luer-lok disposable syringe.
3 A sample was taken 6 hours after the must was pressed on
day 1 of vinification.
90
between the unfiltered and filtered sample owing to the
presence of sediment and debris as a source of folacin
(Table 12).
There was no significant difference between
the unfiltered and filtered MacGregor samples on day 6 of
vinification (Table 13).
The Paragon and MacGregor juice was inoculated with
Red Star Brand, Pasteur Champagne Active Dry Wine Yeast on
day 10 and 6, respectively.
Immediately after inoculation
a steep (93 to 97 percent) increase in folic acid activity
was observed (Figures 6and 7).
Sixteen days after inocula-
tion the folacin content of the unfiltered and filtered
Paragon Chardonnay samples were 14.95 pg/100 ml and 5.81
pg/100 ml, respectively (Table 12).
Thirteen days post
inoculation, the unfiltered and filtered MacGregor
Chardonnay samples contained 14.05 pg folacin per 100 ml
and 6.33 pg folacin per 100 ml, respectively (Table 13).
Yeast fermentation of sugar to alcohol and carbon
dioxide was proceeding during the period when an increase
in folic acid activity was observed.
On the onset of fer-
mentation when aerobic conditions existed there was a
period of rapid multiplication of yeast cells.
As fer-
mentation progressed anaerobic conditions resulted and
the yeasts needed to utilize the sugar present in the
grape juice to obtain carbon necessary for energy
(Guilliermond and Tanner, 1920).
The yeast's utilization
of the sugar resulted in the production of carbon dioxide
and alcohol.
The production of carbon dioxide caused the
" .
91
yeast to become dispersed throughout the juice.
The
tremendous increase in folic acid content of the unfiltered
sample during fermentation supported the fact that yeast is
an excellent source of folacin as noted by Castor (1953);
Rodriquez
(1978).
Folic acid is also an essential factor
for the multiplication of yeast (Castor, 1953).
Folic acid in the filtered samples were significantly
(pc:::::0.05} lower than the unfiltered samples.
However,
the folacin content of the filtered samples were significantly
(p~
samples.
0.05} greater than the uninoculated juice
MacKenzie, et al.
(1954} reported that yeast
(Saccharomyces cerevisiae} synthesized 34 pg folacin per
100 gm dry cells.
In addition, Butterfield and Calloway
(1972} determined that yeast synthesized folacin into its
medium in the baked product, bread.
Thus, the synthesis
of folacin by the yeast explains the increase in folacin
content observed in the inoculated filtered samples.
In the filtered Paragon sample there was a significant
drop (p-= 0.05) in folic acid activity from day 22 to day
29.
There was also a slight lag in activity compared to
the steep rise seen before and after this day in the unfiltered sample.
On day 22 the folacin concentration of
the unfiltered sample was 9.68 pg/100 ml and 10.73 pg/
100 ml on day 29.
The folacin content of the filtered
sample on day 22 was 5.33 pg/100 ml and 4.18 pg/100 ml on
day 29.
An explanation for this decrease in folacin
activity is not known.
Nevertheless, the folic acid
92
activity resumed its increase as noted on day 36.
On day
36 of vinification, Paragon Chardonnay reached maximum
folic acid activity.
The folacin content of the unfiltered
and filtered Paragon samples were 14.95 pg/100 ml and 5.81
pg/100 ml, respectively.
The MacGregor Chardonnay achieved
maximum folic acid activity on day 19 of vinification.
The folacin content of the unfiltered and filtered MacGregor samples were 14.05 pg/100 ml and 6.33 pg/100 ml,
respectively.
Prior to completion of fermentation the
folic acid activity began to decline in both the Paragon
and MacGregor Chardonnay wines.
The para-aminobenzoic acid increase and decrease
during fermentation has been described by Castor (l953) .
Para-aminobenzoic acid is a constituent of the vitamin
folacin.
However, in the 1950s, para-aminobenzoic acid
was considered to be the vitamin folacin.
The para-
aminobenzoic acid content as noted by Castor (1953) increased during fermentation, at the period of rapid multiplication of yeast cells and then decreased to its original
must level prior to the end of fermentation.
Alcoholic fermentation was essentially completed by
day 49 in Paragon and day 46 in MacGregor samples as the
alcoholic content was determined at the winery to be 13.4
percent and 13.6 percent for Paragon and MacGregor, respectively (Table 9).
The presence of 12 percent alcohol
content or so indicates the completion of fermentation
(Webb, 1974).
The decrease in total folic acid observed
93
for MacGregor on day 26 which was perhaps prior to the completion of fermentation was supported by Castor's observation (1953} as described previously.
Castor's observation
(1953} may also support the decrease in folic acid activity
observed prior to day 50 for the Paragon sample.
However,
the alcoholic content was first taken on day 49 but the
folacin activity dropped below that of the must prior to
that day.
After fermentation is completed it is essential that
the wine by separated from the fermentation solids (lees}.
The lees include yeast, seeds, finely divided grape pulp
and grape skin particles which may impair the wines
quality.
In addition, dead yeast cells will begin to
autolyse in anaerobic conditions of the new wine (Webb,
1974).
Thus, the new wine was racked to separate the wine
from the lees.
Paragon wine was racked to barrels and
stainless steel tanks on day 50.
MacGregor wine was
racked to barrels and stainless steel tanks on day 26 and
again on day 33.
Thus, the decrease in folic acid
activity was a result of racking the samples thereby
eliminating lees which included yeast.
However, not all
the yeast lees were removed by racking as there was a
significant difference
and filtered samples.
(p~
0.05) between the unfiltered
The unfiltered and filtered
Paragon sample on day 50 contained 4.12 pg folacin per
100 ml and 2.2 pg folacin per 100 mk respectively.
folacin content of MacGregor unfiltered and filtered
The
94
samples on day 26 resulted in 7.56 pg/100 ml and 3.63 pg/
100 ml, respectively.
MacGregor sampling day 33 yielded
5.92 pg folacin per 100 ml and 4.3 pg folacin per 100 ml
for the unfiltered and filtered samples, respectively.
Malolactic fermentation occurred on day 50 for the
Paragon Chardonnay and on day 47 for the MacGregor Chardonnay (Hagata, 1983).
Malolactic fermentation carried out
by lactic acid bacteria occurs often in California wines to
deacidify the wines.
The fermentation process will cause
an increase in pH, although changes are not conclusive indicators (Webb, 1974) (Table 9).
The exact effect of malo-
lactic fermentation on nutrients is unknown (Webb, 1974).
Perhaps the slight decrease in acidity provided a stable
environment for folacin, thus accounting for the subsequent
increase in folic acid activity.
An increase in the folic acid activity from day 50 to
day 141 was observed in the Paragon sample.
On day 50 the
unfiltered sample yielded 4.12 pg folacin per 100 rnl and
the filtered sample contained 2.2 pg folacin per 100 ml.
On day 141 the folacin content of the unfiltered and filtered samples increased to 5.16 pg/100 ml and 5.36 pg/100
ml, respectively.
The increase in the unfiltered sample
was not significant (p;- 0.05), but the increase in the
filtered sample was significant (p <
0. 05) •
The folacin content of the MacGregor sample significantly (p<::::" 0. 05) increased on day 47.
The folacin content
of the unfiltered and filtered samples were 11.58 pg/100
95
ml and 11.14 pg/100 ml, respectively.
From day 47 to day
80, a gradual yet significant decrease (p oc:::::::: 0. 05) in folic
acid activity was observed.
On day 80, the wine was racked
to barrels and a significant decrease
acid activity was noted.
(p~
0.05) in folic
The loss of yeast lees resulting
from the racking supported the insignificant difference
(p~
0.05) found between the unfiltered and filtered wine
samples.
This continual loss of folic acid activity was
noted again on day 106 when the wine was again racked to
stainless steel tanks.
However, the folacin activity in-
creased significantly (p-=::::. 0.05) on day 138.
There was
also a significant difference (p-c:::. 0.05) between the unfiltered and filtered samples, 10.03 and 9.71 pg folacin
per 100 ml, respectively.
Thus, yeast may still be present
in the wine.
The increases in total folic acid observed in Paragon
and MacGregor wines on day 141 and 138, respectively, may
reflect the nutrient content of the yeast and grape musts
from which they were prepared (Gastineau, et al. 1979).
Furthermore, the autolysis of yeast still present could
cause a release of micronutrients into its environment
(Webb, 1974).
Thus, the similarity in folic acid activity
in the unfiltered and filtered wine samples on day 50
through 141 for Paragon and on day 47 through 138 for MacGregor could be explained.
There was overall no change or an increase in the
folic acid activity of the wine compared to the grape for
96
Paragon and MacGregor, respectively, prior to bottling.
Hall et al.
(1956) noted a loss of folacin in the extrac-
tion of the musts but little further loss or some increase
in total folic acid activity during fermentation to wine.
In this study, a loss of folic acid activity (63 percent}
occurred in the extraction of musts but there was little
further loss or some increase in folic acid activity in
fermentation to wine.
Hall et al.
(1956} concluded that
the skin and seeds of the grape contributed folic acid to
the wine.
Paragon and MacGregor wines were bottled on day 163
and 160, respectively.
Prior to bottling the wines were
racked and clarified with bentonite clay to remove protein,
remaining yeast cells and other debris.
Beverages that are
filtered, or clarified show a decrease in the nutrient
content (Gastineau, et al. 1979}, thus, explaining the
decrease in folic acid activity observed.
In addition,
fining and bottling of the wine exposed the folacin to
environmental conditions in which it is labile, such as,
light, oxygen and agitation (Rodriquez, 1978}.
Hence, the
fining and bottling procedures may also attribute to the
remaining folic acid activity.
Overall, in the Paragon wine, a significant
(p~
0.05}
decrease occurred from the freshly harvested grape on day 0
to the wine at the time of bottling on day 163.
The
freshly harvested grape contained 5.63 pg folacin per 100
gm in the unfiltered sample and 4.68 pg/100 gm in the fil-
97
tered sample.
The folacin content of the wine at the time
of bottling decreased to 2.27 pg/100 ml in the unfiltered
sample and 2.03 pg/100 ml in the filtered sample.
In con-
trast, the MacGregor wine illustrated a significant
(p
<
0. 0 5) increase in the folacin activity from the
freshly harvested grape on day 0 to the wine at bottling
on day 160.
The folacin activity of the unfiltered and
filtered grape was 3.49 pg/100 gm and 3.1 pg/100 gm, respectively.
The wine at the time of bottling contained 4.99
pg folacin per 100 ml in the unfiltered sample and 4.94pg
folacin per 100 ml in the filtered sample.
The folic acid values determined in the bottled
Paragon and MacGregor wines were 95 times greater than
literature values (Table 14).
All the researchers des-
cribed in Table 14, except Hall et al.
(1956) and possibly
Leake and Silverman (1966) utilized the recommended
assaying organism, L.
~asei.
Hall et al.
(1956) used S.
faecalis as the assaying organism and it is not known
which organism Leake and Silverman (1966) used.
A large
variation in folacin values exists within the literature
as well as in comparison with this research.
The high
values found in the two 100 percent Chardonnay wines
studied probably resulted from the use of improved extraction and assay conditions.
98
Table 14
Comparison of total folacin concentration of white winy
found from previous research to this current research
Folacin
Concentration
(pg/100 ml)
Researcher
Assaying
Organism 2
Hall et al., 1956
s.
Leake
Unknown
1.8
L. casei
0.2
Voigt et al., 1978
L. casei
0.3
Paragon, 1983
L. casei
2.3 3
MacGregor, 1983
L. casei
4.9
Paul
1
&
&
Silverman, 1966
Southgate, 1978
faecalis
1.8
3
current research on total folic acid concentration of two
100% Chardonnay wines: Paragon and MacGregor from Ahern
Winery, San Fernando, California are compared to the
literature values.
2 The microorganism used in the microbiological assay of
total folic acid by each researcher is stated.
3values represent the mean from six replicates.
Q
•
Chapter 5
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
There has been little advancement in the knowledge of
folacin content of wines during fermentation since Hall et
al.
(1956}.
Moreover, modifications in the microbiological
assay method for folacin determination and viticultural
practices have occurred in recent years.
This research
investigated the changes in folacin activity during commercial fermentation and aging of two California Chardonnay
wines.
The folacin content of the Paragon and MacGregor Chardonnay grapes was 5.6 and 3.5 pg/100 gm, respectively.
The folacin levels increased significantly in the must for
Paragon and MacGregor to 8.6 and 9.6 pg/100 gm, respectively.
A 93 to 97 percent decrease in folate content was
observed in the free-run pressed juices as compared to the
musts.
The folacin content of the juice was 0.63 and 0.31
pg/100 ml for Paragon and MacGregor, respectively.
These
results indicate that the folacin compounds are concentrated in the skin, seeds, stems and leaves of the grape.
After inoculation with pure yeast cultures, rapid increases of 95 to 98 percent in folacin activity were ob-
99
100
served in both the unfiltered and filtered samples.
The
folacin content of the unfiltered Paragon sample was 14.9
pg/100 ml and the filtered was 5.81 pg/100 ml.
The un-
filtered MacGregor sample had 14.1 pg, and the filtered,
6.33 pg of folacin per 100 ml.
The increase in folacin
activity observed during vinification has never been reported before.
The increase in folacin activity is ex-
plained by the activity of yeast and its synthesis of folacin during fermentation since the folacin levels of the unfiltered samples were significantly greater than the filtered samples.
When the fermentation was complete, a large decrease
in folacin activity occurred.
There were, however, little
further changes during aging.
Prior to bottling, the wine
was fined which caused the folacin values to decrease
slightly.
The folacin content of the unfiltered Paragon
Chardonnay wine was 2.3 pg/100 ml and for the MacGregor
Chardonnay wine, 4.9 pg/100 ml.
There was no significant
difference in the folacin values for the unfiltered and
filtered wine samples.
The levels of folacin concentration of wine found in
this study were markedly higher than those reported by
Hall et al.
(1956) and Voigt et al.
(1978).
Hall et al.
(1956) reported folacin values for white wine of 1.4 to 2.0
pg/100 ml.
Voigt et al.
pg/100 ml for white wine.
(1978) found folate levels of 0.31
The high values obtained in the
present study were probably due to the use of an improved
101
microbiological assay.
From the results found in this study, a glass (240 ml)
of Chardonnay wine will provide 5.52 to 11.76 pg of folacin
or 1.42 to 2.9 percent of the RDA for a healthy adult.
The
amount of folacin present in wine may not be a substantial
contribution of the RDA.
However, in view of the increas-
ing popularity of white wine as a cocktail, it is important
for dietitians, nutritionists and food scientists to
possess accurate folacin values for wine in Food Tables and
Agriculture Handbooks.
Therefore, it is recommended that
further research includes a larger spectrum of grapes and
wines in order to provide accurate folacin values for Food
Tables and to confirm trends of folacin changes during
commercial vinification.
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10 8
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109
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110
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~~------~~~=--·
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11
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11
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11
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112
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APPENDICES
A. Culture Maintenance Medium
Lactobacillus casei was maintained in stabs of BactoLactobacilli Agar A.O.A.C., obtained from Difco Laboratories, Detroit, Michigan.
This commercially available
medium is composed of the following ingredients:
Difco yeast extract
D-glucose
Difco Bacto-Agar
Sodium acetate
Distilled water
2 g
1 g
3 g
1 g
(1.0%)
(0.5%)
(1.5%)
(0.5%)
200 ml
These ingredients were mixed in a 500 ml Erlenmeyer
flask and heated slowly with constant stirring until clear.
Ten ml aliquots of the medium was distributed into culture
tubes, capped and autoclaved at 121°C and 15 psi for 15
minutes.
The tubes were cooled in an upright position to
solidify and stored in the refrigerator at 4°C.
B. Standard Folic Acid Solution
Three different standard folic acid solutions, A, B,
and C, were prepared.
1.
Eg/ml).
Folic acid stock solution, solution A (25
Precisely weighed 25.0 mg of Folic Acid, crystal-
line (ICN Pharmaceuticals, Inc., Life Sciences Group,
114
Cleveland, Ohio) was dissolved in 100 ml of a sterile O.OlN
NaOH solution containing 20 percent ethanol in a one liter
volumetric flask.
(This NaOH solution was prepared by
combining 210 ml of 95 percent ethanol, 12.5 ml of 0.8 N
NaOH solution and sterile deionized water to 1 liter.
Additional 0.01 N NaOH solution containing 20 percent
ethanol brought the volume to one liter.
The solution was
aseptically distributed in 5 ml aliquots into sterile test
tubes.
Nitrogen gas was bubbled through the contents of
each tube to displace the oxygen, thereby minimizing oxidative degradation.
The tubes were immediately sealed with
parafilm, wrapped in foil and stored in a freezer at -22°C.
2. Folic acid working solution, solution B (25
ng/ml).
This solution was prepared by diluting precisely
1 ml of solution A to one liter with sterile O.OlN NaOH
solution containing 20 percent ethanol.
The solution was
aseptically distributed in 5 ml aliquots into sterile test
tubes.
The tubes were flushed with nitrogen gas, sealed
and stored in a freezer at -22°C.
3. Folic acid assay solution, solution C (0.5
ng/ml) .
Solution C was freshly prepared for each assay
just prior to use.
It was prepared by precisely pipetting
2 ml of solution B into a sterile 100 ml volumetric flask
and diluting to volume with sterile 0.05M sodium phosphate
buffer containing 0.15 percent ascorbic acid.
The unused
portion of solution C was discarded after each assay as it
was no longer stable.
115
c. Difco Chicken Pancreas Conjugase.
The commercial chicken pancreas was purchased from
Difco Laboratories, Detroit, Michigan.
A suspension of the
dehydrated chicken pancreas was prepared by dispersing 60
mg of the dried powder into 20 ml of sterile deionized
water with constant swirling for 10 minutes.
The suspen-
sion was centrifuged at 3000 x g for 10 minutes in a
refrigerated centrifuge at -5°C.
Approximately 5 ml of
the supernatant was aseptically decanted into sterile test
tubes, capped with parafilm and stored in a freezer at
-22°c.
One test tube was defrosted just before use by
placing it in a water bath in the refrigerator.
D. Inoculum Broth.
On the day prior to assay,
~·
casei was subcultured by
transferring the cells from the maintenance stab medium to
an inoculum broth.
The broth was composed of:
Folic acid assay solution {solution C)
Phosphate ascorbate buffer solution
Basal medium {double strength)
2.0 ml
0.5 ml
2.5 ml
The inoculum broth was prepared quantitatively and pipetted
5 ml into each sterile test tube.
The tubes were sealed
with aluminum foil, autoclaved at 121°C and 15 psi for 5
minutes.
use.
They were stored in the freezer at -22°C until
Just prior to use a test tube was removed from the
freezer and was defrosted in a waterbath in the refrigerator.
Each tube contained 1 ng of folic acid.
116
E. Basal Medium (Double Strength)
The commercial Dehydrated Folic Acid Casei Medium (No.
0822) was obtained from Difco Laboratories, Detroit,
Michigan, in 100 gm bottles.
The formula of the medium
consisted of:
Charcoal treated casitone
Bacto dextrose
Sodium acetate
Dipotassium phosphate
Monopotassium phosphate
DL-Tryptophane
L-Asparagine
L-Cysteine hydrochloride
Adenine sulfate
Guanine hydrochloride
Uracil
Xanthine
Tween 80
Glutathion (reduced)
Magnesium sulfate
Sodium chloride USP
Ferrous sulfate
Manganese sulfate
Riboflavin
Para-Aminobenzoic acid
Pyridoxine hydrochloride
Thiamine hydrochloride
Calcium pantothenate
Nicotinic acid
Biotin
10 g
40 g
40 g
1 g
1 g
0.2 g
0.6 g
0.5 g
10
10
10
20
0.1
mg
mg
mg
mg
g
5 mg
0.4 g
20 mg
20 mg
15 mg
1 mg
2 mg
4 mg
400
pg
800 pg
800 pg
20 p.g
The medium was rehydrated with sterile deionized water the
day of or day before assay.
The medium was rehydrated
following the instructions on the bottle.
A solution was
prepared by suspending 9.4 gm of the dehydrated powder for
every 100 ml of sterile deionized water.
The mixture was
heated to boiling for 1 minute on a magnetic stirrer/hot
plate.
The solution was cooled and stored in a refrigera-
tor at 4°C until use.
111
F. Sodium Phosphate Buffer
A 0.05M sodium phosphate buffer solution, pH 6.1 was
prepared as follows:
monobasic sodium phosphate
6.792g anhydrous NaH2Po 4
dibasic sodium phosphate
2.687g anhydrous Na2HP0 4
These two compounds were dissolved in one liter of sterile
deionized water in a one liter volumetric flask.
Ascorbic
acid was added to the buffer to obtain a final concentration of 0.15 percent ascorbate just prior to use.
The
ascorbic acid serves to protect the folate from destruction by oxidation during the assay.
Ascorbate buffer was
not made too soon in advance and any excess was discarded
as the buffer was no longer stable after the addition of
ascorbic acid.
G. Sterile Saline
Sterile saline was used to wash the L. casei cells
free of folacin and to suspend the cells for inoculation.
The solution was prepared by dissolving 0.9 gm sodium
chloride (NaCl) crystals with 100 ml sterile deionized
water to make a 0.9 percent sterile saline solution.
The
sterile saline was autoclaved at 121°C and 15 psi for 15
minutes prior to use.
H. Fresh Hog Kidney Conjugase
Fresh hog kidney was defatted, chopped and then homogenized in sterile 0.32 percent cysteine hydrochloride
solution that had been adjusted with NaOH to pH 5.4.
The
118
t<:l
suspension was poured into a one liter Erlenmeyer flask
and layered with toluene.
The flask was sealed with para-
film and the contents autolyzed by incubation for 2 hours.
Foam and fat floating on top of the autolysate were removed and discarded.
The suspension was then filtered
through glass wool and the filtrate centrifuged.
Fat
floating on top of the solution was removed and the
solution was centrifuged again.
The supernatant was de-
canted, adjusted to pH 4.5 with lN HCl and immediately set
in an ice bath.
Dowex was added to the supernatant and
the slurry was stirred occasionally for a period of 1 hour
to remove contaminating folate.
The slurry was centri-
fuged and the clear brownish red supernatant was collected.
The hog kidney conjugase preparation was further
purified using gel chromatography as suggested by Chen
(1979).
A pyrex chromatography column was packed to a
height of 42 em with Sephadex G-25 gel suspension.
The
solvent was allowed to drain out until the level of solvent
was just barely above the surface of the gel.
Approxi-
mately 20 ml of the crude conjugase solution were carefully poured into the column without disturbing the flat
surface of the gel.
The outlet was opened to obtain an
effluent rate of approximately 1 ml per minute.
As soon
as the conjugase solution level had dropped to 1 mm above
the gel surface, sterile sodium acetate buffer was carefully added to the column until the solvent level was 1
inch above the surface of the gel.
This 1 inch "head" was
•
119
maintained throughout the entire column chromatographic
process.
The enzyme fraction was brown in color and passed
quickly through the column.
The folic acid fraction was
yellow and moved slowly down the column.
The enzyme
fraction was collected and frozen in sealed test tubes at
-22°C until use {Bird, et al., 1945}.
The procedure is
illustrated in the diagram on page 121.
I. Sodium Acetate Buffer
A solution of 0.1 M acetate buffer, pH 4.7 was prepared by dissolving 8.2 gm sodium acetate {NaCH 3 COO} or
13.6 gm NaCH3C00,3H20 in 1 liter sterile deionized water.
The solution was titrated with the acetic acid solution
which contained 6 gm or 5.7 ml of glacial acetic acid in
1000 ml sterile deionized water.
The titration was stopped
when the pH of the solution was 4.7.
Approximately 700 ml
of acetic acid solution were used to adjust 1 liter of the
sodium acetate solution to the final pH of 4.7.
Then 1
gm ascorbic acid (0.2 percent} was added to 500 ml of the
above solution and used for hog kidney conjugase purification.
120
Diagram.
Schematic diagram of the steps
in the preparation and purification
of fresh hog kidney conjugase.
Reprinted from Kirsch (1983).
CRUDE ENmiE PREPARATION
Defat and
chop zoo g
fresh hog
kidney
llomogenlze In
1---t---600 ml 0.3Z%
cysteine hydrochloride
at pH 5.4
Autolyze
under toluene
for Z hours
at JJOC
Collect
supernatant
and store
Centrt fuge at
ZOOO x g for
30 minutes at
ooc (4500 rpm)
Add 30 g Dowex
1-XB (chloride form)
to supernatant.
Place In Ice water bath
and stir occasstonally
for 1 hour
r--
I--
- - - -
....._____ __
---
1--
:---
lllscard
foam and fat,
fIlter through
glasswool
Adjust
supernatant
to pH 4.5
with IN HCI
Centrifuge filtrate
at 1000 x g for
f---- ZO minutes at ooc
(approx. 3000 rpm
on a JS7.5 rotor;
~eckman JZI centrifuge
I--
'
Remove fat and
re-centrlfuge at
4000 x g for
30 minutes at ooc
(about 6000 rpm)
I
i
_I
------
PURIFICATION BY GEL CiiROMATOGRAPilY
Set up Pyrex
chromatography column
Collect brown enzyn~
fraction and store.
Discard yellow
folic acid eluate.
---
Suspend zo g
Sephadex G-Z5 powder
In 0. lH sodium acetate
buffer, pH 4. 1,
containing O.Z~ ascorbate
~
Just prior to when the crude
enzyme solution has completely
entered the gel, carefully add
enough sodium acetate buffer
f-- to create a 1 Inch head above I - the surface of the gel.
Maintain this head through
entire purification process.
Pour gel
Into column and
let gel settle-top should be flat
Adjust outlet to
obtain an effluent rate
of approximately
I ml per minute
----
let solvent drain out
until solvent level
Is just barel.v
above the gel
--
Carefully pour 20 ml
approximately 2 Inches height)
crude enzyme solution
Into column.
Do not disturb
flat surface of gel.
'
1-'
"'
1-'
122
Appendix J.
Work Sheets used in the
microbiological assay
for folacin.
::<lt!"l
.. Ill 0
~-t:
I. SANPLE PREPARATION
A
r==r F_~ -----
c
B
Extracting solution
1-T.·
Tube CIT
. no totl
Sample
Description
Vol. ~one.;
Wt used type
(g.) (ml.)
lst
Description:
lfilutn homgnztn,
Ascor- factor autoclvg,
bate
etc.
B+A/A
(%)
D ]_E
Con1ugase treatment
(for Total onlv) · lncubatn=
II
Addtnl
In d tlutn
factor
Vol.
Vol. Vol.
2nd
prior
sample conjgs type Total ~ilutn
to
ext ret preptn buffer vol. factor
assay
(ml.) (ml.) (ml.) (ml.) F/1>
'
s;
rn
n
'"cnH
. , :I' 'Z
-;-'= ~
........ 1-l
00 .-..
I "CC
tTJ
~
~~ ~
1-l,_.l-l
•
H
."'oo
..., z
§.~
~
I
I
i
I
.....~
"CC
t1
·----- -- -------------
------- --- --- -------
------ - --··· .. , --
---------
~
:I
z"
0
-
---
I I. CONJUGASE BLANK
Source of
conjugase
Tube
no
(e .g.Difco
C.P.)
--L--..
K
-- FA Jconcentration*
I
Method ·of preparation
Wt. of
Solvent
conjugase
used
(g.)
(ml.)
Vol.
assayed
(ml.)
0, D,
at
640nM
L-1~~
L-3
Correction values for TFA adjustmt
(Average K =
ng/ml)
per ml
per
conjugase
Ka~e~l0- 3
tube
J /I
(ng/O.OOlml
(ng/tube) (ng/ml)
KaveXlo-4.
(ng/10-~l
ng/
~
..
n
ml
------- ---------
r-1L
~
,..1L
~
38
r--I 39
-- --------------------··-·· - ---. --------- ----
-----
____.._______
----------
*Calculated from standard curve Or by using comput('r
1
TSCIIEI'I' 1 program
1-'
N
w
:<11::1"'1
111 1-> 0
III. RESULTS
M
N
Total
Vol
OD at
Free dilutn extrct
Tube or
F=CxH assyd 640nM
Sample no totl T=CxGxH (ml) X 103
0
p
I
~- ~
R
Q
Calc ltd
Corrctn for total only !FA in
ml conjgs Corrctn Adjstd FA
folacin
conctrn* pe:,V~ube value,L
0-P
(ng/tube) Ex ~FxH (ng/tube ) (ng/tube)
Folacin concn in
asyd extr
Original sample
(pg/100g)
F=O/N
T=Q/N
Std
R X M/10
He an
(ng/ml)
Dvtn
ll-::TZ
l1l
....,1111::1
lrtt"l
ex;,.... r;l
"tl :<f
0)"':::::
l,oJOQ
111 z
,....
;o.
I
H
.,_
>-'ll.ol>-'l
•
H
.ti>O,.., 0z
.,_
C"ll.ol
~
1-
z
.....
f--
l
>
'"
C"l
l1l In H
.....
.,_
.,_
I-~
I
I
..,!r
I--
111
11
....
f-;
~
I-I--
,..,
z0
;:l
f-I
1----
r
1--
l
f-I--
i
I
'
i
I
i
I
I
'
I
I
I
i
I.
~
~
I
f.--f.--f.--I-I--
I
I
,.,5'
111
I
I
I
1-1-1--
I
I
I
. !----=~
J
*Calculated from standard curve OR
by
j
using computer 'TSCllEN' program
1-'
I'V
o+:>.
<>