CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
CHANGES IN FOLACIN LEVELS DURING COMMERCIAL
PRODUCTION OF A CALIFORNIA CABERNET SAUVIGNON WINE
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Home Economics
by
Andree Kathryn Armand
August 1984
The Thesis of Andree Kathryn Armand is approved:
Ro ert B. Lamb, Ph.D.
California State University, Northridge
ii
DEDICATION
A mon grand-pere maternel
et
a rna grand-mere paternelle
Ainsi qu'a tous ceux qui auraient voulu
etudier mais n'en aient eu la chance •••
iii
ACKNOWLEDGEMENTS
I wish to express my appreciation and gratitude to my
advisors, Dr. Robert Lamb, Arlene Kirsch and Dr. Tung-Shan
Chen.
Thank you Arlene for all your help, advice and fun
times shared in the laboratory.
I would like to give a special thank you to Dr. TungShan Chen whose guidance led me to discover new horizons
and whose presence and support made some of the rough
times not so rough •••
I must mention Robyn Gaines-Moss my laboratory
partner but above all good friend without whom this
project would not have been all that it was •••
I am grateful to Ahern Winery and especially thankful
to Steve Hagata without whose contributions this research
would not have been undertaken.
Thank you Richard Orkand, a friend more than an
employer, for your patience and understanding.
I am most grateful to my friends, parents, brother
and sisters for their continuous support and
encouragement.
iV
TABLE OF CONTENTS
Page
• • • • • iii
Dedication. • • • • • • • • •
• • •
Acknowledgments • • • • • •
• • • • • • • • • .iv
• • • • • • • • • .viii
List of Tables •• • • • •
List of Figures • • • • • • • • • • • • • • • • • •
Abstract. • • • • • •
• • • •
• • • • • • •
X
.xi
CHAPTER
1.
INTRODUCTION • • • • • • • •
Definitions • • • • • •
2.
• • • • • • • 1
• • • • • • • • 2
REVIEW OF LITERATURE • • • • • • • • • • • • 5
Folacin • • • • • • • • • • • • • • • • • 5
Structure and Chemistry of Folacin. • • 5
Folacin in Human Nutrition. • • • • • • 8
Recommended Dietary Allowances. • •
• 9
Sources of Folacin. • • • • • • • • • • 9
Folacin Determination in Foods. • • • .10
Wine. • • • • • • • • • • • • • • • • • .13
History of Winemaking • • • •
Winemaking. •
Grapes. •
• • .13
• • • • • • • • • • • .14
• • •
• •
Yeast • • • • • • • •
• • • • • .15
• • • • • •
.17
Fermentation. • • • • • • • • • • • • .19
v
CHAPTER
2. continued
Wine Production Technology. • • • • • .21
Folacin in Red Grapes, Must and Wine •• 26
Wine Consumption and Market Trends • • • 29
3.
MATERIALS AND METHODS. • • • • • • • • • • .32
Materials • • • • • • • • • • • • • • • • 32
Crushed Grapes, Must and Wine • • • • • 32
Assay Microorganism • • • • • • • • • .35
Chemical Reagents and Microbiological
Media • • • • • • • • • • • • • • • • 35
Glassware Maintenance • •
. . . • • • • 35
. . . • • • • • • • 37
Methods • • •
• • • • • . . . . . . • • 39
Sample Preparation Methodology
Studies. • • • • • • • • •• . . . .39
Equipment • • • • •
•
Methodology for Folacin Determination
in Crushed Grapes, Must and Wine •• 43
Sample Preparation. • • •
. . . • • • • 46
Wine and Must Analysis. • • •
• •• 47
Microbiological Assay for Folacin • • .47
Data Analysis •
4.
..•
• • • •
RESULTS AND DISCUSSION • • • • • • •
• • .55
..•
.57
Studies of Wine Sample Preparation
Methodology • • • • • • • • • • • • • • 57
Comparison of the Addition of
Ascorbate Phosphate Buffer to the
Direct Addition of Ascorbate. • • • .57
Ascorbic Acid Levels • • • • • • • • • • 60
vi
CHAPTER
4. continued
Heat Treatment. •
....•
• • • • • .62
Conjugase • • • • • • • • • • • • • • • 62
Filtration Treatment • • • • • • • • • .65
Overall Evaluation. • • • • • • • • • .67
Changes in Folacin Activity during the
Vinification Process • • • • • • • • • • 68
Grapes and Must • • • • • • • • • • • .68
Day 1 of Vinification • • • • • • • • .75
Day 3 of Vinification • • • • • • • •
Day 10 of Vinification.
.
.so
• • • • • • .81
Day 17 of Vinification. • • • • • • • .84
Day 31 of Vinification. • • • •
Day 49 of Vinification. •
.
.
• • .87
• • • •
Day 64 of Vinification. • • • • • •
. .89
. .89
Days 90, 190, 211 and 293 of
Vinification.
• • • • • • .90
• • •
.
5.
.
SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS •• 95
LITERATURE CITED • • • • • • • • • • • • • • • • • • 99
APPENDICES
A.
Preparation of Buffer Solutions. • • • • • 108
B.
Preparation of Chemical Solutions and
Culture Media • • • • • • • • • • • • • 110
vii
LIST OF TABLES
Table
1.
Page
Sampling Dates and Sample Types Collected during
the Vinification Process of Cabernet Sauvignon
Wine • • • • • •
• • • • • • • • • • 36
......
2.
The Variables in the Folacin Assay and the
Samples Used • • • • • • • • • • • • • • • • • • 40
3.
Protocol of Standard Curve Tube Preparation • • • 52
4.
Protocol of Folacin Assay Tubes for Ahern
Cabernet Sauvignon Samples • • • • • • • • • • • 54
5.
Comparison of Direct Addition of Ascorbate to
the Addition of Ascorbate Containing Phosphate
Buffer on the Folacin Activity of a Cabernet
Sauvignon Wine Sample. • • • • • • • • • • • • • 59
6.
Effect of Levels of Ascorbate Added during
Sample Preparation on Assayable Folacin in
Cabernet Sauvignon Wine. • • • • • • • • • • • • 61
7.
Comparison of Heat Treatment Methods of Folacin
Activity of Cabernet Sauvignon Wine • • • • • • • 63
8.
Comparison of Two Sources of Conjugase on
Assayable Folacin in Cabernet Sauvignon Wine • • 64
9.
Effect of Filtration Treatment on Assayable
Folacin in Samples Drawn from Various Stages of
Winemaking • • • • • • • • • • • • • • • • • • • 66
10.
Folacin Activity in Unfiltered and Filtered
Cabernet Sauvignon Samples during Wine
Production • • • • • • • • • • • • • • • • • • • 69
11.
Sugar Content ( Brix), Tartaric Acid, pH,
Alcohol and Free Sulfur Dioxide (FSO ) Values
for Cabernet Sauvignon Grapes and Wine • • • • • 70
viii
Table
Page
12.
Comparison of Folacin Activity of Red Grapes
and Must Found from Previous Research to the
Present Study • • • • • • • • • • • • • • • • • • 72
13.
Temperatures Recorded on Days of Sampling
during the Winemaking Process. • •
• • • • • • 79
14.
15.
.
Folate Contributions from Yeast Cells after
Inoculation of the Must. • • • • • . • • • •
• • 82
Folacin Activity in Several Varieties of Dry
Red Wines 4-6 Months after Fermentation. • • • • 92
ix
LIST OF FIGURES
Figure
Page
1.
Chemical Structure of Folic Acid • • • • • • • • • 7
2.
Wine Making Process of 1982 Cabernet Sauvignon
as Produced at Ahern Winery • • • • • • • • • • • 34
3.
Sample Preparation Method. • • • • • • • • • • • 45
4.
Flow Chart of Steps in the Folacin Assay • • • • 50
5.
Changes in Total Folacin Activity of Unfiltered
and Filtered Cabernet Sauvignon Samples during
Commercial Production. • • • • • • • • • • • • • 77
6.
Changes in Folacin Activity Extracted from
Yeast Contrasted to Changes in Folacin Activity
in the Filtered Cabernet Sauvignon Samples
during Commercial Production • • • • • • • • • • 86
X
ABSTRACT
CHANGES IN FOLACIN LEVELS DURING COMMERCIAL
PRODUCTION OF A CALIFORNIA CABERNET SAUVIGNON WINE
by
Andree Kathryn Armand
Master of Science in Horne Economics
Various conditions for preparing samples for the
microbiological assay of folacin in wine were studied.
The optimal conditions were established and applied in
monitoring the changes in folacin activity in a Cabernet
Sauvignon wine during commercial fermentation and aging
processes.
The optimal conditions in sample preparation methods
included the direct addition of ascorbic acid to samples
to a final concentration of 0.15 percent prior to autoclaving at 121°C, 15 psi for 10 minutes.
Conjugase
prepared from commercial dried chicken pancreas (Difco)
was used to hydrolyze the folacin polyglutarnates.
Folacin
activity was determined in filtered and unfiltered samples
in order to determine the source of folacin and to follow
folacin activity during the course of vinification.
The folacin content of the Cabernet Sauvignon grapes
was 9.0 ~g/100 g.
Folacin levels increased, although
insignificantly, in the must to 9.8
~g/100
g.
A 95
percent decrease resulted from must extraction in the
unfiltered and filtered samples.
Following yeast inoculation, a drastic increase in
folacin activity was observed in both unfiltered and
filtered samples due to folacin synthesis that accompanied
the growth of yeast.
The folacin content of the
unfiltered and filtered samples was 12.0
1.8
~g/100
~g/100
ml and
ml, respectively.
Upon completion of fermentation and throughout the
aging process, an initial decrease followed by little
further change in folacin activity was observed in the
wine.
The folacin content of the wine approximately nine
months after fermentation was 3.8 ~g/100 ml and 3.6 ~g/
100 ml in the unfiltered and filtered samples,
respectively.
rii
CHAPTER 1
INTRODUCTION
Folacin is the generic term for a group of compounds
having nutritional properties and chemical structure
similar to folic acid.
Folic acid, the parent compound of
the folacin group, was first isolated in 1941 from spinach
leaves by
Mitchell~
AI. (1941), after most of the
vitamins had already been identified {Food and Nutrition
Board, 1968).
This vitamin has since been found in a wide
variety of foodstuffs.
Foods which have been found to be
rich sources of folacin include liver, yeast, green leafy
vegetables, legumes and egg yolk (Perloff and Butrum,
1977).
The inclusion of folacin in the Recommended Dietary
Allowances in 1968 exemplifies the recognition of folacin
as an important human nutrient {Food and Nutrition Board,
1968).
Since that time, there has been an increased need
for information regarding the folate content in foods and
beverages to improve and supplement the existing limited
data.
However, generating folacin data has been difficult
due to the lack of a standardized method for analysis.
Generally, the folacin content of food is determined
microbiologically, with Lactobacillus casei (L. casei)
1
2
being the most widely used assaying organism (Bell, 1974).
The wide variations in sample preparations and conjugase
treatment procedures, however, have resulted in
inconsistent folacin data reported in the literature.
Castor (1953) was the first to follow changes in the
B complex vitamins during must fermentation.
Hall
~
gl.
(1956) also studied the B-complex vitamin content,
including folic acid, in grapes, must and wine.
However,
modifications in viticultural practices and cellar
technologies have occurred in the past 30 years and
folacin research in wine production 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 objectives of this research
were to determine optimal conditions for the
microbiological assay procedures for folacin in wine and
to investigate the changes in folacin activity during
commercial fermentation and aging of wine made from
Cabernet Sauvignon grapes.
Definitions
Folacin activity
The term refers to the ability of a substance to
support the growth of L. casei as measured against a
folic acid standard.
3
Conjugase
The common name for enzymes which hydrolyze the gamma
peptide bonds that link glutamyl residues to folic acid.
Free folic acid (FFA)
Folacin present in foodstuffs that can be utilized
by L. casei without prior treatment with conjugase.
Total folic acid (TFA)
Folacin present in foodstuffs 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 or
microorganisms.
Vinification
The conversion of grape juice into wine by
fermentation.
A mixture of crushed grapes, grape juice, skins and
seeds.
The sediment which forms after fermentation and which
is composed primarily of yeast cells and other debris such
as grape pulp and seeds.
4
Racking
A process of siphoning off the wine from the
lees and pumping it into clean tanks or barrels.
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
Folacin
Structure and Chemistry of Folacin
Folic acid or pteroylglutamic acid (PteGlu), the
parent compound of the folacin group, consists of three
parts:
the pteridine ring, para-amino-benzoic acid and L-
glutamic acid (Figure 1).
Other forms of folacin are
derived from reductions or substitutions at the 5-nitrogen
or 10-nitrogen position of the pteridine moiety (Eigen and
Shockman, 1963; Herbert and Bertino, 1967; Nystrom and
Nystrom, 1967) •
Bell (1972) has stated that there are more
biologically active forms of folic acid than any other
vitamin.
Natural active forms of folates exist largely in
the form of conjugates called polyglutamates (Stokstad and
Koch, 1967).
The glutamyl moiety of folic acid is
commonly linked through a gamma peptide linkage to a
polyglutamyl side chain which may consist of one to six
glutamyl residues (Bauch and Krumdieck, 1971).
The gamma
peptide linkage can be split by enzymes commonly called
conjugase (gamma glutamyl carboxypeptidase) (Rodriguez,
1978) •
5
6
Figure 1.
Chemical Structure of Folic Acid
NO:,•~rfiH.-~-o-~ H~OOH
OH
H,NAN I ;,••
2
Plerldine
.•
-
CO-N-CH-C
p-Amlnobenzolc
acid
u.-cu.-coou
Glutamic acid
Pterolc acid
Pteroylglulamlc acid
Folic acid
'-..]
8
In the literature, folacin activity is reported as
free and/or total folic acid.
Free folic acid (FFA)
refers to the folacin that can be utilized by L. casei
without treating the food sample with conjugase.
Total
folic acid (TFA) refers to the folacin that is utilized by
L. casei after conjugase treatment of the food sample.
Before polyglutamates can be absorbed and utilized by man,
they must be broken down to monoglutamates (Coleman and
Herbert, 1979).
Human intestinal juice contains conjugase
capable of hydrolyzing the polyglutamate forms of folacin
to simpler forms (Butterworth, 1968; Perloff and Butrum,
.1977).
TFA may thus be considered an approximation of
folacin available to man.
Folacin in Human Nutrition
Folates play an important role in human nutrition.
Folic acid is converted to its coenzyme form by a pyrimidine nucleotide-dependent enzyme.
The folacin coenzymes
are involved in the transfer of one carbon units
throughout the body and are required for the oxidation and
reduction of these single carbon units.
Folate coenzymes
play an essential part in the synthesis of purine and
pyrimidine bases which are the precursors to DNA and RNA.
Folate coenzymes also participate in the metabolism of
certain amino acids (Food and Nutrition Board, 1980;
Malin, 1975; Rodriguez, 1978).
9
Deficiency of folacin can result in impaired cell
division, alterations of protein synthesis and
megaloblastic anemia (Malin, 1975; Martin, 1981).
Recommended Dietary Allowances
Folacin deficiency is probably the most common
vitamin deficiency found in the United States (Martin,
1981).
To prevent folacin deficiency and to maintain
folate tissue reserves, the Food and Nutrition Board of
the National Research Council of the National Academy of
Science set the Recommended Dietary Allowance (RDA) for
folacin at 400 pg for normal non-pregnant, non-lactating
adults and adolescents.
The RDA for infants has been
estimated at 5 pg/kg of body weight.
For healthy children
between the ages of 1 and 10 years the RDA has been set to
supply approximately 8-10 pg/kg of body weight, providing
amply for growth and allowing for varying availability of
folacin from diets.
As a result of the increased
nutritional needs imposed by pregnancy, the RDA for
folacin is set at 800 pg per day during pregnancy.
During
lactation, the RDA for folacin is set at 500 Pg per day
(Food and Nutrition Board, 1980).
Sources of Folacin
Folacin is present in a wide variety of foods.
Rich
sources of folacin include yeast, liver, egg yolks, green
leafy vegetables, legumes and wheat germ (Herbert and
Bertino, 1967; Perloff and Butrum, 1977; Streiff, 1971).
10
Folacin Determination in Foods
The most widely used method of folacin determination
is the microbiological assay8
The microbiological assay
measures the ability of food samples to support the growth
of folate requiring bacteria.
Lactobacillus casei (L.
casei) is the most extensively used folate dependent
organism for quantitating food folacin because it can
utilize the widest spectrum of folacin derivatives
(Rodriguez, 19787
Tamura~
gl., 1972).
The high
sensitivity of the microbiological assay has favored its
use over the spectrophotometrical and chemical methods for
determining the folacin content of most natural materials
(Blakley, 19697 Herbert and Bertino, 19677 Rodriguez,
1978).
More recently, however, the microbiological assay
has been criticized for its lack of reproducibility (Day
and Gregory, 1983).
In nature, folacin exists primarily in the conjugated
forms of hexa- and hepta-glutamates (Broquist, 19557
Butterworth, 19687
Stokstad and Koch, 1967).
The
polyglutamate forms of folacin cannot be utilized by
~.
casei until they are hydrolyzed to mono-, di- or triglutamate forms of folic acid.
Hydrolysis of the
polyglutamates can be achieved by the use of conjugases
which are widely distributed in nature.
Conjugase
activity has been identified in numerous animal and human
tissues including hog kidney, rat liver, chicken pancreas,
ll
chicken liver and chicken intestine
Laskowski~
gl., 1946i
(Bird~
A!.,l945; Rao and Noronha, 1977).
The two types of conjugases extensively used in
folacin determination of foods are:
1) a carboxypeptidase
which is widely distributed in animal tissues, especially
liver and kidney, and whose optimum pH is 4.5; and 2) a
gamma glutamic acid carboxypeptidase present in chicken
pancreas and whose optimum pH range is 7-8 (Broquist,
1955; Eigen and Shockman, 1963).
Hydrolysis of the poly-
glutamates by chicken pancreas conjugase results in
diglutamates while monoglutamates are the end products of
hog kidney conjugase hydrolysis
Eigen and Shockman, 1963).
(Buehring~
Al., 1974;
Inhibition by different
compounds and buffer type of the reaction mixture further
differentiate the two sources of conjugase.
The addition of ascorbic acid to the extraction
solvent to prevent the destruction of heat labile reduced
forms of folic acid was first recognized by Toennies
Al. (1956).
~
Since then, the use of ascorbate during
extraction of folate as well as assay to ensure that heat
labile folates are protected throughout the determination
has been confirmed time and again (Chen and Cooper, 1979;
Herbert, 1963;
Streiff, 1971).
Hurdle~
al., 1968; O'Broin
~AI.,
1975;
However, discrepancies exist as to the
optimal usage of ascorbate.
While O'Broin
~
al. (1975)
found that 0.2 percent ascorbate was required and Chen and
Cooper (1979) determined that 0.1 percent ascorbate was
12
sufficient to protect folacin, Hurdle
~ gl
(1968)
found
no significant difference between the effects of ascorbic
acid concentrations of 0.15 percent and 10 percent.
Ascorbic acid is usually added during the extraction
procedure and the assay to ensure that the heat labile
folate forms are protected (Chen and Cooper, 1979; Hurdle
~
gl., 1968).
The use of heat treatment is required to
release bound forms of folacin and to inactivate
interfering compounds
1984).
(Bird~
gl., 1945; Kirsch and Chen,
The boiling waterbath method has been used
extensively as a substitute for autoclaving
1945; Dong and Oace, 1973;
Keagy~
(Bird~
al., 1975).
£l.,
The
absence of a reducing agent such as ascorbic acid would
cause the oxidized folic acid to decompose into a
biologically inactive compound which would not function as
a growth factor for the microorganism during assay
(Stokstad
~
O'Broin
gl., 1947).
~
gl.
(1975) demonstrated that pH affected
the stability of folacin.
Pteroylglutamic acid has been
found to be most stable in neutral or slightly alkaline
solution and less stable under acidic conditions (Blakley,
1969; Stokstad and Thenen, 1972).
Paine-Wilson and Chen
(1979} found buffer pH to have a profound effect on the
thermal stability of folacin.
The rate of destruction of
folic acid increased rapidly with decreasing pH below 4.0
while most naturally occurring folates were more stable in
~3
neutral or alkaline pH.
History of Winemaking
The exact origin of wine is unclear although
paleontology has uncovered evidence of masses of grape
seeds, skins and stems which had apparently been crushed
by prehistoric man {Grossman, 1964).
The earliest records
show that the Fertile Crescent was the site of an active
wine industry as early as 3200 B.C.
1979).
{Gastineau~
gl.,
From the Tigris-Euphrates basin in Egypt the grape
vine was introduced to the Mediterranean coastal trading
posts by Phoenician traders and later it was introduced
into France, Germany and across the Channel into England
by the Greeks and Romans.
The Romans advanced the art of
winemaking, however, as a result of spoilage, winemaking
remained an industry of high risks until the nineteenth
century.
Louis Pasteur revolutionized the wine industry
with conclusive proof that the spoilage of wines was due
to aerobic microorganisms of the Acetoba&ter type
{Grossman, 1964) •
Since the early Pasteur experiments and discoveries,
winemaking has developed into a well defined scientific
discipline.
The wine industry has made immense progress
in the application of pure yeast culture, the use of
antiseptics and the clarification of wines.
In addition,
14
the Industrial Revolution introduced new types of
machinery for cultivating vineyards, for handling or
crushing grapes, for pumping crushed grapes to fermenting
tanks, and for filtration or priming wines from one tank
to another (Amerine and Singleton, 1965).
Today, the
application of scientific principles to fermentation and
the care of wines continues with the introduction of new
technology in variety selection, location of vineyards,
crop level, process control, aging and blending.
Winemaking
The principle of winemaking is characterized by the
complex metabolism of the ingredients, beginning with the
grapes and ending with wine.
The chemistry of winemaking
is a biological-technological sequence of events.
The
sequence which originates with the grapes, proceeds to
grape destemming, crushing and pressing technology and to
fermentation is greatly influenced by vinification
practices (Drawert, 1974).
The steps of winemaking
include harvesting, yeast selection, sulfur dioxide
application, fermentation conditions, aging and others.
Although certain principles have been established within
each step of winemaking, the quality of wine is quite
variable and is often related to the winemaker's art in
the execution of many winemaking steps.
l5
Grapes
The chemical composition of grapes is influenced by
numerous factors including variety, maturity, soil, root
stock, climatic conditions, crop yield and post harvest
handling (Amerine gt
AI., 1980; Gallander, 1974). The
composition of the grape plays a major role in the
character and quality of the finished wine.
Dextrose and levulose are the predominant sugars in
grapes and occur in approximately equal amounts (Amerine
gt
Al., 1980).
Sugar content, together with titratable
acidity (as tartaric acid), is one of the most important
measures of maturity thereby allowing for harvest at the
proper maturity, a critical factor in the production of
maximum quality wines (Amerine gt AI., 1972).
Sugars are
of paramount importance as they are the source of ethyl
alcohol production in wine.
In addition to ethyl alcohol,
other important wine components which contribute to the
sensory properties of wines are derived from sugars during
fermentation.
These components whose amounts in wine are
generally dependent on numerous vinification practices
include glycerol, acetic and lactic acids, higher alcohols
and acetaldehydes (Gallander, 1974).
Organic acids are important constituents which affect
the sensory properties of wine.
Organic acids have a
marked effect on pH and buffer wines at a relatively low
pH (3.0 to 4.0) thereby providing a biologically stable
environment (Gallander, 1974).
Tartaric and malic acids
l6
are the two major organic acids of grapes while citric
acid is present in very small amounts (Fong
~
Al., 1974).
Total acidity measurements taken during the course of
winemaking are calculated as tartaric acid.
Nitrogen compounds and inorganic constituents greatly
influence wine quality as they are essential for the
growth and development of yeast in the fermentation
process (Kunkee and Amerine, 1970).
Complete utilization
of nitrogen compounds is critical to the keeping quality
of wines since incomplete utilization of these compounds
may result in refermentation and bacterial spoilage of the
finished wines
(Amerine~
al., 1980).
Ammonia, ammonium
salts and inorganic constituents are particularly critical
in the development and reproduction of yeast during
fermentation.
Grape musts usually contain ample
quantities of nitrogenous substances and inorganic
compounds to support an active fermentation (Kunkee and
Amerine, 1970).
The vitamins of grapes are primarily important as
essential microbial growth factors although some may be
present in large enough amounts to be of nutritional value
in man's diet.
The significance of vitamins in winemaking
is attributed to their influence on the fermentation
process as several vitamins are considered major growth
factors for yeast multiplication and development
(Gallander, 1974).
l7
Yeast
Wines were made for thousands of years before it was
recognized that yeasts were responsible for the
fermentation.
Wine yeasts are present as part of the
natural grape microflora, however, they cannot be trusted
to produce good fermentations (Kunkee and Amerine, 1970).
Today, modern winemaking makes use of pure yeast cultures
as they offer the winemaker many advantages.
The original
attraction of pure yeast cultures was that_the growth of
undesirable microorganisms could be inhibited by
conducting the fermentations under strictly anaerobic
conditions (Kunkee and Amerine, 1970).
Inoculation of the
must with pure yeast culture ensures a clean fermentation
in a reasonable time with efficient conversion of the
grape sugars (Thoukis, 1974).
Pure yeast cultures are of
great value to the wine industry.
Combined with the
inhibitory effects of sulfur dioxide, they have made wine
spoilage rare (Kunkee and Amerine, 1970).
The use of pure
cultures is now common in many parts of the world.
In
California, nearly all wineries use pure yeast cultures
(Rankine, 1972) •
The selection of pure yeast strains may be based
primarily on fermentation technology, depending on
temperature and duration of fermentation, and the yield of
alcohol (Kunkee and Amerine, 1970).
An important reason
for the use of pure cultures is to produce wines with most
desirable flavors and aromas.
The selection of pure yeast
18
strains may also be influenced by their ability to produce
hydrogen sulfide from sulfate, sulfur dioxide, sulfur
containing amino acids and other sulfur compounds
(Rankine, 1972).
The presence of hydrogen sulfide in more
than trace amounts in wine is most objectionable.
Rankine
(1972) believes that the by-products formed by yeast
during fermentation are quantitative rather than
qualitative and the selection of pure strains can
ultimately eliminate wine disorders caused by the presence
of large amounts of undesirable by-products such as
hydrogen sulfide, acetic acid, ethyl acetate and higher
alcohols.
The genus Saccharomyces is characterized by its
efficient capacity to convert sugar to ethanol.
Wine
yeasts are those yeasts which function well at the
relatively high acidity of grape juice, which are
resistant enough to allow the formation of greater than
10% ethanol, and which can adapt to low concentrations of
sulfur dioxide added as an antiseptic (Kunkee and Amerine,
1970).
It was established early that the primary agent of
alcoholic fermentation in wine was
cerevisiae var. ellipsoideus.
Saccharomyc~s
The shape of the cells is
typically short ellipsoidal and cell size is approximately
6 x 12
~m
(Amerine~
Al., 1980).
Good wine yeasts are
characterized by their high alcohol tolerance, their
flocculation (agglutination) capability and their steady
persistent fermentation capacity (Austin, 1968).
~}
'
l9
Yeast autolysis is of importance in winemaking as it
influences the development of odors and flavors in wines.
Yeast autolysis is defined as •an enzymatic selfdestruction of the yeast cell and essentially involves
hydrolysis of the protoplasmic constituents and their
excretion into the medium• (Joslyn, 1955).
Inasmuch as
yeast autolysis contributes to the quality of wines, it
can also be detrimental to its value (Kunkee and Amerine,
1970).
When yeast cells begin to autolyze under the
highly anaerobic conditions of the new wine, undesirable
off flavors from hydrogen sulfide, mercaptans can develop
(Thoukis, 1974).
In addition, the release of amino acids,
nucleotides and vitamins from yeast can serve as nutrients
for spoilage bacteria such as lactic acid bacteria.
However, a possible exception for the desired release of
amino acids and other nutrients is to stimulate the growth
of the bacteria responsible for the malolactic
fermentation (Phaff and Amerine, 1979).
Fermentation
Amerine
~
Al. (1980) describe fermentation as "the
metabolic processes bringing about chemical changes in
organic substrates through the action of enzymes of
microorganisms or other cells.•
Alcoholic fermentation of
grape juice takes place in the yeast cell by a series of
reactions, usually referred to as the Embden-MeyerhofParnas pathway (Phaff and Amerine, 1979).
Under anaerobic,
20
conditions, each molecule of sugar is converted to two
molecules each of ethanol and carbon dioxide according to
the Gay-Lussac equation:
Yeast
~
2C 2H5 0H + 2C02
ethanol carbon dioxide
The overall reaction theoretically yields 51.1% ethanol
and 48.9% carbon dioxide on a weight basis.
This
theoretical yield cannot be achieved due to the formation
of by-products, the entrainment of ethanol, and the
utilization of approximately 1% of the sugar for yeast
growth and metabolism
(Amerine~
A!., 1980J Kunkee and
Amerine, 1970J Phaff and Amerine, 1979).
By-products of
alcoholic fermentation of grape must include glycerol,
acetic acid, acetaldehyde, 2,3-butanediol, succinic acid,
and higher alcohols as well as hydrogen· sulfide, and ethyl
and methyl mercaptans (Phaff and Amerine, 1979).
In
practice, actual yields amount to 90-95% of the
theoretical value.
The yield varies with yeast strain,
must composition, fermentation temperature, and pumping
over.
The rate of fermentation is affected by sugar
concentration.
The optimum fermentation rate for grape
juice into red table wine occurs in the range 15-20 °Brix
(Ough, 1966a).
Above 25 °Brix the must fermentation is
retarded and the maximum amount of alcohol produced
decreases.
In addition, at higher sugar concentration
21
there is an increase in acetic acid production possibly
resulting from the inhibited fermentation rate (Kunkee and
Amerine, 1970).
Alcohol yield and fermentation rate are affected by
temperature.
Temperature is also an essential factor in
flavor formation (Phaff and Amerine, 1979).
Slow
fermentations at low temperatures are preferred because
more bouquet is formed in the wines.
Temperature is the
one factor to have the greatest influence on fermentation
rate, however, other important variables include sugar
content (measured as
the juice
(Amerine~
0
Brix), pH, and ammonia content of
Al., 1980).
Wine Production Technology
The term wine refers to the natural beverage produced
by normal alcoholic fermentation of the juice of sound and
ripe grapes, in strict accordance with state and federal
regulations (Thoukis, 1974).
However, the ultimate
character and quality of wine results from the winemaker's
use of old and new technology coupled with artistic
creativity.
The winemaking process begins with the grape harvest.
The time when grapes must be picked depends on the
measurement of their pH and their sugar and acid contents.
Recommended ranges for making red table wine are 20.5-23.5
0
Brix, titratable acidity (TA) higher than 0.65 g/100 ml
and pH lower than 3.4
(Amerine~
A!., 1980).
22
The grapes are harvested by hand-picking or
mechanical harvester.
Rapid processing of the grapes and
the crushed grapes is important to inhibit the growth of
undesirable microorganisms and to prevent wild yeast
fermentation, and enzymatic and oxidative browning
{Thoukis, 1974).
The grapes are first fed through a crusher-stemmer to
separate the berries from their stem, and then pumped into
vats.
The berries are crushed to yield the must which
contains grape juice and pulp, skins and seeds.
dioxide
{so2 )
Sulfur
is added at the time of crushing to protect
the must from premature or wild fermentation.
In addition
to its antifungal and antimicrobial properties,
so2
gives
lasting protection against enzymatic oxidation (Amerine
Al., 1980).
A desired reaction is the binding of
so2
~
with
acetaldehyde which is formed during fermentation and which
has undesirable organoleptic properties {Benda, 1982).
The effectiveness of
so 2
as a bacteriostatic agent or an
antioxidant is, however, dependent on the amount of free
so 2
present in the medium (Burroughs and Sparks, 1973).
In the production of red wine, fermentation is
induced as soon as possible after crushing and destemming
by inoculation of the must with 2-3% selected pure yeast
culture (Thoukis, 1974).
Red wines are fermented in
contact with the skins to extract the anthocyanin
pigments.
Usually the must is allowed to ferment on the
skins until the desired amount of color has been
23
extracted.
During fermentation, the skins, pulp and seeds
are buoyed by carbon dioxide to the surface to form a cap.
The cap must be pumped over several times a day for
efficient color and tannin extraction from the skins and
seeds
(Amerine~
A!., 1980).
The solvent action of
ethanol produced during fermentation enhances extraction
of anthocyanin pigments and tannins from the skins.
Pumping over is also necessary to prevent thermophilic
bacteria from infecting the cap (Thoukis, 1974).
When sufficient color has been extracted, the freerun wine is drawn off the grape skins (pomace) to finish
its fermentation.
the pomace
Press wine is recovered from pressing
(Amerine~
£1., 1972).
In the making of high
quality wines the smoother, less astringent free-run
should be kept separate from the press
~ine
(Amerine
~
al., 1980).
Warm temperatures (65-75°F or 18.3-23.9°C) are
desirable for red wine fermentation partly because color
extraction is facilitated.
Warmer temperatures increasing
up to 80-85°F (26.7-29.4°C) give better flavor and color
for most red grape fermentations
Ough and Amerine, 1966).
(Amerine~
AI., 1980;
Above ideal temperatures will
result in the loss of varietal flavors by evaporation, the
development of cooked off flavors, and the death of yeast
cells causing a stuck fermentation and leaving a wine with
residual sugar and low alcohol content (Thoukis, 1974).
24
By the time alcohol fermentation is complete or
nearly complete, winemakers may consider the initiation of
malo-lactic fermentation, an acid reducing fermentation
which involves the decarboxylation of malic acid to lactic
acid and
co2 •
A convenient time to add the malo-lactic
bacteria (Leuconostoc oenos) is at pressing where the red
wine must is pressed before the end of alcoholic
fermentation (Kunkee, 1974).
In cold climate regions
where high acid wines are produced or in warm areas where
the pH of the wine is so high that it is difficult to
prevent the malo-lactic fermentation, induction of malolactic fermentation is recommended (Kunkee, 1974).
Under
California conditions, however, the desirability of
promoting malo-lactic fermentation has not been clearly
established since the wines are not generally acidic
(Thoukis, 1974) •
It should be noted that to the
California winemaker, malo-lactic fermentation may be
desirable in that it brings about bacteriological
stability and flavor complexity to the wine (Rankine,
1972).
The increase in flavor complexity is generally
considered to result from the end-products of the malolactic bacterial metabolism and not merely from the
deacidification
(Amerine~
gl., 19801 Kunkee, 1974).
Rankine (1972) suggests, however, that except for flavors
from diacetyl formation, one cannot reliably detect malolactic fermentation by taste.
Inconsistencies have also
25
been found among experienced tasters in their capabilities
of detecting the fermentation.
Once alcoholic fermentation is complete, the
fermentation solids {lees) must be separated from the wine
(racked).
In the case of red wines where a malo-lactic
fermentation is desired, the initial racking may be
delayed.
The delay will encourage yeast autolysis and the
release of micronutrients necessary for bacterial growth
(Kunkee, 1974) •
Upon prolonged contact, the lees which
include yeast, seeds, finely divided grape pulp and grape
skin particles could be detrimental to the quality of the
wine {Thoukis, 1974).
After the first racking, the new wine is clarified by
the use of fining agents which adsorb suspended materials
such as tannin, acid and protein.
Common fining agents
include bentonite, gelatin, casein, tannin, and egg
albumen
(Amerine~
Al., 1980).
Red wines are aged from 1 to 10 years or more in
stainless steel tanks or wood barrels (cooperage) to
develop bouquet and flavor
(Amerine~
Al., 1972).
A
general shift from wood to stainless steel cooperages has
been a factor in changing production practices around the
world.
This shift has led to the production of young
wines with a fresh and fruity character resulting from the
shortened vinification time.
However, many of the world's
finest red wines owe part of their character, complexity
and quality to maturation in wood (Singleton, 1974).
26
Prior to bottling, the wines are again fined, followed by
a finishing filtration
(Amerine~
£1., 1980).
Folacin in Reg Grapes, Myst and Wine
The significance of vitamins in winemaking is
attributed to their influence on the fermentation process.
Some of the vitamins are essential in the media for growth
of yeasts, and others are synthesized by yeasts during
their growth.
Although numerous studies have investigated
the vitamin B-complex content of grapes, must and wine,
few have examined folacin.
Hall
~
A!. (1956) were among the first to have
reported on the folacin content of grapes, must and wine.
The folacin content of red grapes ranged between 5.8 and
10.2
~g
per 100 g.
were published.
More recently, similar folacin values
Hardinge and Crooks (1961) listed the
folacin content of red grapes as 5.2 vg per 100 g, Perloff
and Butrum (1977) as 7.0
Southgate (1978) as 5.0
~g
~g
per 100 g and Paul and
per 100 g.
While the
aforementioned authors listed folacin values of red grapes
in the same range as that reported by
Hall~
Al. (1956),
Lafon-Lafourcade and Peynaud (1958) and Peynaud and
Lafourcade (1957) reported considerably lower folacin
values of 0.14 vg per 100 ml and 0.1 to 0.2
respectively, for red grapes.
~g
per 100 ml,
The large discrepancy may,
however, be explained by the difference in the sample
analyzed.
The higher folacin contents were listed for
D ,
27
whole red grapes while Lafon-Lafourcade and Peynaud {1958)
and Peynaud and Lafourcade {1957) determined the folacin
content of the juice expressed from the grapes.
The
higher concentration of folacin found in the whole grapes
may be attributed to the presence of skins and seeds which
have been found to be sources of folacin {Gaines-Moss,
19847
Hall~
A!., 1956).
Mathews {1958) similarly found
folacin in Swiss commercial grape juices to occur in small
amounts of 0.19 to 0.34
Hall
~
per 100 ml.
~g
Al. (1956) reported the changes in B vitamin
content during the fermentation of wines.
They noted a
loss of folacin during extraction of the must and reported
the folic acid content of musts from California red grapes
to range from 1.2 to 3.9
~g
per 100 ml.
Bordeaux grapes, Lafon-Lafourcade and
For musts of
P~ynaud
(1958)
showed a slight increase in pteroylglutamic acid during
maturation.
Musts of Bordeaux grapes contained 0.12
~g
folacin per 100 ml.
Hall
~
Al. (1956) observed that folic acid suffered
the main losses in juice extraction, however, there was
little further loss or some increase in fermentation to
wine.
The levels of folacin found in dry red table wines
ranged from 1.3 to 2.1
~g
per 100 ml.
Red wines were
higher in folate content than whites as a result of folate
contributions from skins and seeds.
Similar folacin
concentrations were observed by Herbert (1963) with
homemade Italian red wine containing 1.2
~g
per 100 g.
0
28
Lafon-Lafourcade and Peynaud (1958) and Paul and Southgate
(1978) reported much lower folacin values of 0.1
100 ml and 0.2
~g
~g
per
per 100 ml, respectively, for red wine.
More recently, Voigt
~
AI. (1978) investigated the
folacin content of California wines and reported an
average value of 0.7
~g
per 100 ml for red wine.
The
difference in folacin values for red wines revealed in the
published data indicates that the differences may be the
result of differing folacin determination methods inasmuch
as they reflect the original nutrient content of the
grapes from which the wines were made.
Chemical analysis and nutritional studies of
unfiltered wines reveal that they reflect the nutrients of
the yeasts and of the grapes from which they are prepared
(Gastineau, 1979).
The yeast cell is outstanding in its
ability to produce vitamins.
MacKenzie
~
Al. (1954)
reported that yeast (Saccharomyces cerevisiae) synthesized
34
~g
folacin per 100 g dry cells.
Calloway (1972) and Keagy
~
gl.
Butterfield and
(1975) showed that yeast
not only contains folacin but also produces folacin during
fermentation.
As a result of nutrient synthesis,
fermented beverages, including wines, can often make a
significant contribution to the food value of the diet.
It should be mentioned, however, that the folacin content
of unfined wines and other fermented beverages may be more
significant than in their fined, filtered and clarified
counterparts because unfined beverages contain yeast cells
'
29
which are excellent nutrient sources (Butterfield and
Calloway, 1972; Gastineau, 1979; Perloff and Butrum,
1977).
Wine Consumption and
The
u.s.
Mark~t
Trends
is one of the fastest growing wine markets
in the world.
During the past decade, the total volume of
wine sold in the nation rose more than 50 percent.
In
1983, total wine sales are estimated at 535 million
gallons and with
u.s.
wine sales projected to increase at
a 6% average annual rate during the next seven years, they
are expected to reach 800 million gallons by 1990 (Cannon,
1983).
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 512 million gallons in 1982.
Nontheless, the
u.s.
wine market is in a very early stage
of development as per capita intake increased from 1.7
gallons in 1975 to 2.2 gallons in 1982, still an
insignificantly low consumption level compared to most
European countries such as Italy, France and Portugal
whose annual per capita consumption easily exceeds 20
gallons (Cannon, 1983; Standard and Poor, 1982).
Growth in wine consumption is almost assured to
continue steadily through the next two decades.
Historically, the consumption of wine rises as more people
30
are introduced to wine (Cannon, 1983J
1974a).
Folwell~
Al.,
In addition, per capita consumption also grows as
wine sales regulations are relaxed, as the consumer
disposable income rises and as the population in the 25 to
45 year of age group increases (Folwell and Dailey, 1971J
Folwell~
£1., 1974b).
The uptrend in wine consumption during the past 15
years is clearly indicative of changing patterns in social
drinking since, in 1980, wine consumption topped liquor
consumption for the first time (Standard and Poor, 1982).
While the drinking of white wine as a cocktail increases
in popularity there should also be an increased
consumption of varietal wines as the drinking of table
wines with meals increases.
Changing consumer tastes have
dictated that wineries produce higher quality table and
sparkling wines with an emphasis on whites (Cannon, 1983}.
White wine's share of the table wine market showed an
exceptional growth with 63% in 1982 compared to 32% in
1974.
The share held by red wine declined to 17% from 41%
(Cannon, 1983).
The marked increase in white wine
consumption is partially related to its acceptance as a
cocktail (Standard and Poor, 1982).
Sales growth of white
wine has been greater than that of red since 1960.
This
trend is attributed to the American consumers' devotion to
cold drinks as white wines are properly served chilled
(Cannon, 1983).
However, growth in the more expensive
premium wines is expected to be more balanced between reds
31
and whites because the premium segment of the market
reflects wine drinkers' taste for the more complex styles
found only in reds.
California's wine production dominates by far the
u.s.
wine market although its share is projected to drop
from its current 68% to 65% by 1990 as a result of rapid
gains of imports and additional competition from increased
wine production in other states.
California's wine
industry will, however, continue to grow during the
remainder of the 1980's but in an environment of
competition (Cannon, 1983).
CHAPTER 3
MATERIALS AND METHODS
Materials
Crushed Grapes, Must and Wine
Cabernet Sauvignon grapes (Vitis vinifera L.) were
grown and harvested on Priest Ranch Vineyard located in
eastern Napa Valley, California.
Twenty tons of nitrogen
packed grapes were transported to Ahern Winery, San
Fernando, California, within 24 hours of harvest.
Crushed grapes, must and wine samples used in this
study were obtained from Ahern Winery •. The winemaking
procedures followed by Ahern Winery in the production of
the Cabernet Sauvignon wine are outlined in Figure 2.
Crushed grape and must samples were placed in Ziploc
plastic bags, wrapped in aluminum foil and immediately
packed in dry ice.
for sampling.
Whole grapes had not been available
Free-run, press and young wine samples
taken at various time intervals during the winemaking
process were placed in brown glass or aluminum wrapped
clear glass reagent bottled and ice packed until frozen.
All samples were transported directly from the winery
to the California State University, Northridge Food
32
33
Figure 2.
Wine Making Process of 1982 Cabernet Sauvignon
as Produced at Ahern Winery.
34
Addition of
potassium
Grapes
Crusher
meta-bisulfite
1
pumped to
Must in temperature
Crusher-stemmer - - - - - - - . controlled staintanks
less steel tanks
FERMENTATION
pumping
over of
must
Pressing of must with
hydraulic basket
press
./Inoculation with
' malo-lactic bacteria
Free-run and press
returned to tanks
End of alcoholic and
malo-lactic fermentation
3 rackings each followed
by subsequent addition of
Young wine
----------------i~young
CuS04 to clear H2S
racked to
wine
------!~aging
barrels
wine
35
Science Laboratory.
minutes.
The estimated transit time was 20
All samples were stored in a freezer at -22°C
until analysis.
A flowchart of sampling dates and sample
descriptions is shown in Table 1.
Assay Microorganism
LactQbacillus casei (ATCC 7469) (lot no. 362612)
culture was purchased from Difco Laboratories, Detroit,
Michigan.
Chemical Reagents ang MicrQbiolQgical Media
All chemicals were reagent grade.
Crystalline
pteroylglutamate (folic acid) (lot no. 7924) was purchased
from ICN Pharmaceuticals, Inc., Life Sciences Group,
Cleveland, Ohio.
Bacto folic acid casei medium (lot no.
685522), Bacto chicken pancreas (lot
no~
681226), and
Bacto agar (lot no. 566771) were obtained from Difco
Laboratories, Detroit, Michigan.
The composition and preparation of sodium phosphate
ascorbate buffer,
~
casei maintenance medium, inoculum
broth, sterile saline solution, basal medium, chicken
pancreas and hog kidney conjugase solutions, and standard
folic acid assay solutions are described in Appendices A
and B.
Glassware Maintenance
Thorough cleaning of glassware is essential in
folacin assay as extraneous folacin contamination would
36
TABLE 1
Sampling Dates and Sample Types Colle~ted during the
Process of Cabernet Sauvignon Wine
Date of
Sampling
Day of
Vinification
10-28-82
0
Grapes
No sample
10-28-82
0
Crushed
grapes
Crushed grapes dosed with S02
10-28-82
0
Must
11-2-82
0
Inoculation with Saccharomyces
cerevisiae (Red Star Brand French Red
Yeast)
11-3-82
1
Free-run
11-5-82
3
Free-run
H2S production by yeast detected
11-12-82
10
Press +
free-run
Heavy fermentation stage
Pressing; sample is combination of press
and free-run. Recombined in tank to
resume fermentation. Addition of malolactic bacteria.
11-19-82
17
Press +
free-run
Still in fermentation stages
Samples
Comments/Events
Alcoholic fermentation completed alcohol 13.8%
12-3-82
31
Young wine
12-21-82
49
Young wine
1-5-83
64
Young wine
Malo-lactic fermentation completed
1st racking followed by addition of CuS04
2nd racking
1-31-83
90
Young wine
Young wine
Mid March/83
fol~owed
by addition of CuS04
3rd racking followed by final addition
of CuS04
No sample, wine racked into barrels
5-11-83
190
Wine
Aging
6-1-83
211
Wine
Aging
6-27-83
238
Wine
Aging; sample used in preliminary
experiment
8-22-83
1
293
Wine
2
Aging
20 tons of grapes were harvested at Priest Ranch Vineyards,
Napa Valley, California.
2
Wine was produced by Ahern Winery, San Fernando, California.
37
interfere with results.
Therefore, all glassware used in
the folacin assay was cleaned by the following procedure:
1.
After use, all glassware was rinsed with hot tap water
and scrubbed with a brush, if necessary.
2.
The glassware was washed in an Ultrasonic Cleaner with
two percent Micro detergent (International Products
Corporation, Trenton, New Jersey) for 45 minutes.
3.
Each piece of glassware was rinsed ten times with hot
tap water.
4.
Each piece of glassware was rinsed three times with
deionized water.
5.
All glassware was air dried in an inverted position.
6.
All glassware was wrapped in aluminum foil and
sterilized in an autoclave for 15 minutes at 121°C, 15
psi.
Eguipment
The following equipment were used for this research:
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.
Mettler Electronic Analytical Balance, Model AC
100, Mettler Instruments Corporation, Princeton, New
Jersey.
38
Sartorius Balance, Model 2255, Brinkman
Instruments, Westbury, New York.
5.
Autoclave, Model STM-E, Type
c,
No. 45018, Market
Forge, Everett, Massachusetts.
6.
Ultrasonic Cleaner, Model B52, Bransonic Co.,
Stamford, Connecticut.
7.
Eppendorf Digital Pipette, 100-1000
~1,
No.
22
33 660-7, Brinkman Instruments, Inc., Westbury, New York
Micropipette, 50-200
~1,
No. 53517-820, Wheaton
Instruments, Millville, New York
Micropipette, 200-1000
~1,
No. 851268, Wheaton
Instruments, Millville, New York.
8.
pH Meter, Model pH 102, Brinkman Instruments,
Inc., Westbury, New York.
9.
Freezer, Model FV 188 R, Westinghouse Electric
Corporation, Pittsburgh, Pennsylvania.
10. Touch Mixer, Model 12-810, Fisherbrand,
Highstown, New Jersey.
11. Osterizer, Model - PulseMatic 10, Oster
Corporation, Milwaukee, Wisconsin.
12. Corning Hot Plate - Stirrer, Model PC 351,
Corning Glass Works, New Jersey.
13. Additional equipment:
Culture test tubes
(various sizes)1 test tube racks; sterile disposable
pipettes, miscellaneous laboratory glassware and utensils.
39
Methode
Sample preparation Methodology Studies
Optimum conditions for folacin determination in wine
were established from experimental results from variations
in sample preparation methods.
The variations in sample
preparation are illustrated in Table 2.
Ahern Winery
samples of crushed grapes and wine drawn on days 17, 211
and 238 of vinification were used in the preliminary
experiments.
Direct addition of ascorbic acid versus addition of
ascorb9te sodium phosphate
b~ffer.
The addition of sodium
phosphate buffer containing 0.15 percent ascorbate to wine
samples drawn on day 211 of vinification prior to
autoclaving was compared to the direct addition of 0.15%
ascorbate to wine samples (also from day 211).
The
samples were then autoclaved, diluted with ascorbate
sodium phosphate buffer, treated with conjugase and
assayed for folacin.
Ascorbic akig levels.
The effect of ascorbate on
folacin extraction was studied with different ascorbate
levels added to wine samples.
The percents of ascorbic
acid added directly to wine samples taken from day 238 of
vinification included O, 0.15, 0.3, 0.5, 0.7 and 1.0
percents.
After the direct addition of each ascorbate
level to separate wine samples, the samples were
autoclaved, diluted with buffer, conjugase treated and
TABLE 2
The Variables in the Folacin Assay and the Samples Used
Steps in Folacin Assay
Variables Studied
Sample Preparation
Use of Ascorbate
Direct addition of ascorbate
vs. addition of ascorbate
buffer
· Levels of ascorbate
Sample Used
Wine from day 211 of vinification
Wine from day 239 of vinification
(0-1. 0%)
Filtration
Filtration vs. no filtration
Crushed grapes
Wine from day 17 of vinification
Wine from day 211 of vinification
Heat Treatment
· Autoclaving
Wine from day 211 of vinification
Boiling water bath
No heat treatment
Conjugase Treatment
Hog kidney vs. chicken pancreas
Wine from day 211 of vinification
Microbiological Assay
Determination of folacin
concentration
~·
0
4l
assayed for folacin.
The buffer used in all the assay and
standard curve tubes contained the standard 0.15 percent
ascorbic acid.
Heat treatment.
The effects of two heating methods
and no heat treatment on folacin extraction were
investigated.
The heating methods compared were
autoclaving and heating in a boiling water bath.
A sample
of wine obtained on day 211 of vinification and to which
was added 0.15% ascorbate was divided into three 20 ml
aliquots for the comparison.
The first aliquot received
no heat treatment, the second was autoclaved at 121°C,
15 psi for 10 minutes, and the third was heated in a
boiling waterbath for 5 minutes.
Samples were then cooled
to room temperature, diluted, conjugase treated and
assayed.
Conjugase treatment.
Two types of conjugase
treatments were compared: fresh hog kidney and commercial
dried chicken pancreas conjugases.
The sample used was
from day 211 of vinification.
Fresh hog kidneys were obtained from Farmer John
slaughterhouse (Vernon, California) and utilized within a
few hours of slaughter.
The hog kidney conjugase extract
preparation recommended by Eigen and Shockman (1963) and
modified by Chen (1979) is described in Appendix B.2.
Exactly 0.5 ml of the conjugase solution prepared from hog
kidney, 0.5 ml of sample, and 4.0 ml of sodium acetate
buffer (pH 4.7) (Appendix A) containing 0.15% ascorbate
42
were mixed together in a test tube.
The reaction mixture
was incubated at 37°C for two hours in an air incubator.
Chicken pancreas conjugase was prepared from Difco
dried chicken pancreas (lot no. 681226) as described in
Appendix B.l.
Exactly 0.1 ml of the chicken pancreas
conjugase solution was added to a test tube containing
0.5 ml sample and 4.4 ml sodium phosphate buffer (pH 6.1)
containing 0.15% ascorbate.
The reaction mixture was
incubated at 37°C for 2 hours in an air incubator.
To determine the amount of folacin present in the
conjugases, the preparation of blanks for each conjugase
was necessary.
The blanks were composed of 0.5 ml of hog
kidney or chicken pancreas conjugase and 4.5 ml sodium
acetate and sodium phosphate buffers, respectively, both
containing 0.15% ascorbate.
The blanks were incubated at
37°C for 2 hours in an air incubator along with the
samples undergoing conjugase treatment.
The conjugase treated samples and blanks were then
assayed microbiologically.
Filtration of sample.
The effects of sample
filtration were studied 6n three different samples:
crushed grapes (day 0 of vinification), free-run sample
obtained during vigorous fermentation (day 17 of
vinification) and wine in the aging stage (day 211 of
vinification).
Crushed grapes were homogenized and then autoclaved
at 121°C for 10 minutes with 0.15% ascorbic acid.
The
43
resulting juice was decanted into centrifuge tubes and
centrifuged at 1000 x g for 20 minutes.
One portion of
the supernatant was filtered with a No. 42 Whatman filter
paper (porosity 2.5 vm) using a Gelman filter apparatus
attached to a vacuum pump.
The filtrate and the
unfiltered sample were diluted with buffer, conjugase
treated and assayed.
Portions of the samples obtained on days 17 and 211
of vinification were filtered using a pre-sterilized
disposable membrane filter (Nalgene syringe filter,
porosity 0.2 vm) attached to a disposable syringe.
The
filtrates and unfiltered samples to which 0.15% ascorbate
was added, were autoclaved, diluted with buffer, conjugase
treated and assayed for folacin.
Methodology fo;r fQlagj.n Determination fn Crushed Grape"
Myst ang Wj.ne
The experimental design was set forth by several
conditions determined from the previously described
preliminary experiments.
A standard procedure for sample
preparation was established whereby maximum folacin yields
were obtained.
The various steps involved in the sample
preparation methodology are outlined in Figure 3.
procedures are described as follows.
The
Ascorbic acid was
added directly to all samples to a final concentration of
0.15 percent.
The samples were then autoclaved at 121°C,
15 psi for 10 minutes.
Total folacin activity was
44
Figure 3.
Sample Preparation Method.
45
Crushed grapes
Must
Free-run, Press
Young wine
I
Jr
Thawing
~
Homogenization
J
Addition of 0.15% Ascorbate
~
Autoclaving
l
l
Centrifuging
!
. r-~
Supernatant
ij_i
Filtered
F~ltered
Not Filtered
t
I
Not Filtered
l
l
l
Addition of 0.15% Ascorbate
J
AUTOCLAVING
Dilution
l
l
All
with
l
Samples
Sodium
Ready
Phosphate
For
L
Ascorbate
l
Conjugase
Buffer
i
l
Treatment
46
determined in unfiltered and filtered samples after
conjugase treatment using chicken pancreas conjugase.
Sample Preparation
Crushed
grap~p
and must.
Crushed grape and must
samples were removed from the freezer and allowed to thaw
for 30 minutes.
When thawed, 100 g samples of crushed
grapes and must were homogenized for one minute in an
Osterizer blender.
The homogenates were then transferred
to beakers and re-weighed.
Ascorbic acid was added to the
homogenates to a final concentration of 0.15% just prior
to autoclaving at 12l°C, 15 psi for 10 minutes.
After
autoclaving, samples were cooled to room temperature in a
water bath.
The liquid portion of the samples was
aseptically decanted into centrifuge tubes and centrifuged
at 1000 x g for 20 minutes.
The supernatants were divided
into two sets, filtered and not filtered.
The filtered
samples were further prepared by filtering approximately
5 ml aliquots of the supernatant through a no. 42 Whatman
filter paper (porosity 2.5 vm) using a Gelman filter
apparatus attached to a vacuum pump.
Both filtered and
unfiltered samples were diluted with 0.05 M sodium
phosphate buffer containing 0.15 percent ascorbic acid.
The sample extracts were kept in a dark cool place for
subsequent TFA determination.
Free-run, press and wine.
The frozen samples were
removed from the freezer and allowed to thaw in a
47
0
waterbath in the refrigerator for 30 minutes.
When
defrosted the samples were divided into two sets, those
which were filtered and those which were not.
The
filtered samples were filtered using a pre-sterilized
disposable membrane filter (Nalgene syringe filter,
porosity 0.2
~m)
attached to a disposable syringe.
Both
sets of samples received the addition of ascorbate to a
final concentration of 0.15%.
The samples were then
autoclaved at 121°C, 15 psi for 10 minutes, cooled to room
temperature in a waterbath, diluted with 0.05 M sodium
phosphate buffer containing 0.15% ascorbate, and stored in
a dark cool place for subsequent analysis.
Wine ang Must Analysis
The
0
Brix, pH, total acidity (TA), alcohol and free
sulfur dioxide (Fso 2 ) measurements were taken by the
enologist at the Ahern Winery's laboratory (Hagata, 1983).
The
0
Brix was measured with a hydrometer and the pH with a
pH meter.
The TA was measured by titration with NaOH to
an endpoint of pH 8.2.
Alcohol content of the wine was
determined with a hydrometer following distillation of the
wine.
FS0 2 was measured by the Ripper method as described
by Amerine and Ough (1974).
Microbiological Assay for Folacin
The microbiological assay measures the growth of
folate requiring bacteria in food sample extracts.
Lactob9cillus casei is one organism which is extensively
.
48
used in folacin analysis because it can utilize the widest
spectrum of folacin derivatives {Tamura, et al., 1972).
The major steps in the folacin microbiological assay
method utilizing L. casei are summarized in Figure 4.
Maintenance of Lactobacillus casei (ATCC 7469).
L.
casei was obtained from Difco Laboratories, Detroit,
Michigan.
The culture was transferred to and maintained
in a maintenance medium {Appendix B.l) by stab
inoculation, followed by incubation at 37°C for 20 hours.
The stabs were stored in the refrigerator until use.
The
culture was transferred monthly to new maintenance medium.
Inoculum.
One day before microbiological assay was
to take place, L. casei was subcultured from a stab into a
culture tube containing 5 ml of inoculum broth {Appendix
B.2).
The subculture was incubated at 37°C for 20 hours
in an air incubator.
After incubation, the suspension was
centrifuged at 1000 x g for 10 minutes at -5°C.
were harvested by decanting out the supernatant.
The cells
The
cells were then washed by suspending the cells in 5 to
8 ml of sterile 0.9 percent saline solution {Appendix
B.3), centrifuging the suspension, and discarding the
supernatant.
This washing procedure was repeated three
times to deplete the cells of folacin because any carried
over folate would have induced excessive growth in the new
medium and erroneous results.
A final suspension of the
cells into an additional 10 ml of sterile 0.9% saline was
49
Figure 4.
Flow Chart of Steps in the Folacin Assay
(Reprinted from Chen, 1983a).
50
Standard
curve
Medium
L.
casei
culture
t.: buffer
:Folic acid:
: stc•cK sol n:
Basal
I medium :
' ____ .;.. ___ _ '
·----------·
·------1
0
Cul tur·e
stab
I
'
'
·---------·
·---------1
t-Ja:
phosphe.te:
buffer
I
Sample
ext r· act i c•n :
'
'
•-----------·
·------------·
I
I
---"'+'-----
: C.c·nju9~·:<?:
: t r- e-~. tmer. t :
---""--: __________.:Inoculum:
: F o 1 i c acid:
I
: __________________ .,.:
'
'
·---------·
__ ....' __ _
br-oth
----------.•·--------·
__________ ·------·-------,
I
,
'
:
I
...,
I
I
Tr·aros.fer·
to
mai ntenar1ce:
stab
____ ____ _
a~:say
_____ ..,' _____
·------------·
I
'
IA~:.corb.
Si.mpl e
pr·epar· e.. t i c•n:
:
·-------·
_____
,... _____ _
I
: F o 1 i c acid:
: wor·~~ing
lsolutic•n
:scolutic•ro
Food
sample
I
I
I
____., ___ _
:;:ompl::
~-lashed
'------·
I
f
:
TF;.;;
I
cell
:SUE-pen: ion:
'
----·,' ________ ,:.'
I
't
I
--------
'
I
------·-------~.
I
I
---¥------------~--------V------V-
Star1dard
curve
tubes
I
I
-W---W--------------------~--
tube:.
Sample
'
'
·---------------------------------·
·-----------------------------'
Incubation
, _____________________
,
I
I
I
0
--~--------------~---
I
I
'
'
--~--------------~--Opt i c:a 1 dero~: i ty
de t errroi nation
,
I
_____________________ ,
0
'
'
---------~--Standard : . ________ : --~---------Sample O.D.
·-------------'
:
TFA
:
I
curve
I
: ___________ :1--------~
I
-------------er, t r·
i
, ____________
,
: cern c
I
at
c,r, :
0
51
made and stored in the refrigerator 1 to 3 hours until
used to inoculate each assay tube.
Preparation fQr standard curve assay tubes.
A
standard curve plotting the growth of L. casei against
known folic acid concentrations was prepared for each
assay.
The details of the preparation of assay tubes for
folic acid standard curve are given in Table 3.
The standard curve tubes were prepared in triplicate
for each folic acid concentration.
The test tubes were
first filled with basal medium and sodium phosphate buffer
containing 0.15% ascorbic acid, covered with aluminum foil
and autoclaved at 121°C, 15 psi for 5 minutes.
After
autoclaving the tubes were cooled in a water bath.
Standard folic acid assay solution (Appendix B.5.3) was
pipetted precisely into each test tube to obtain a final
concentration range of 0.05 ng per tube through 1.2 ng per
tube.
The tubes were stored in the dark until inoculated.
PreparatiQn Qf sample tubes.
To each sample tube was
added a 2.5 ml aliquot of basal medium.
Diluted sample
extract was added to these tubes and then diluted to a
final volume of 5 ml with ascorbate sodium phosphate
buffer.
Each sample extract was assayed at two different
concentrations.
From preliminary experiments, the
dilutions necessary to obtain folacin activity within the
range of the standard curve were established.
The
52
TABLE 3
1
Protocol of Standard Curve Tube Preparation
Vol. FA
Assay
solution
Tub~
no.
(ml)
Vol. of
buffer
(ml)
Vol. of
basal
medium
(ml)
Final
vol.
(ml)
Final
FA cone.
per tube
(ng/5ml)
Inoculum
added,
1 drop of
L. casei
3
1
0
2.5
2.5
5.0
0
2,3
0
2.5
2.5
5.0
0
+
4,5,6
0.1
2.4
2.5
5.0
0.05
+
7,8,9
0.2
2.3
2.5
5.0
0.1
+
10,11,12
0.4
2.1
2.5
5.0
0.2
+
13,14,15
0.6
1.9
2.5
5.0
0.3
+
16,17,18
0.8
1.7
2.5
5.0
0.4
+
19,20,21
1.0
1.5
2.5
5.0
0.5
+
22,23,24
1.2
1.3
2.5
5.0
0.6
+
25,26,27
1.6
0.9
2.5
5.0
0.8
+
28,29,30
2.0
0.5
2.5
5.0
1.0
+
31,32,33
2.4
0.1
2.5
5.0
1.2
+
4
1
Source:
Chen, 1983a.
2
Tubes were in triplicates for each concentration.
3
Tube no. 1 was the blank, used to zero the spectrophotometer.
4
Tube nos. 2 & 3 were inoculated blanks to establish the
baseline of growth caused by folic acid carried over by the inoculum cells
and any potential contamination of folic acid.
53
preparations and the dilutions used for the sample extract
are listed in Table 4.
Prepa{ation of conjugase blanks.
The folacin
concentration of the conjugase solutions was determined by
assaying blank tubes.
Blank tubes were prepared for all
experiments at the same time sample extracts were treated
with conjugase.
The blanks were made with 0.5 ml
conjugase preparation and 4.5 ml sodium phosphate buffer
containing 0.15% ascorbate.
The buffer solution and
incubation conditions were identical for the blank tubes
and the sample extracts.
After incubation, 0.5 ml
aliquots of the prepared blanks were assayed.
Inoculatipn and incubation.
A sterile 1 ml
disposable pipette was used to deliver one drop of the L.
casei inoculum into each standard
tube.
curv~
and sample assay
The tubes were capped, mixed with a Vortex mixer
and incubated 20 hours at 37°C in an air incubator.
Measurement of L. casei growth.
After 20 hours of
incubation, further growth of L. casei was stopped by
autoclaving the tubes for 5 minutes at 12l°C, 15 psi.
The
microbial growth of the tubes was determined turbidimetrically by measuring the optical density (O.D.) at
640 nm using a Beckman spectrophotometer model 24 equipped
with a sipper system.
Each tube was thoroughly mixed
using a Vortex mixer prior to each
o.o. reading.
il
'
54
TABLE 4
Protocol of Folacin Assay Tubes for Ahern Cabernet
Sauvignon Samples 1
Dilution prior to
conjugase treatment
Sample
Dilution from
conjugase treatment
Final
Dilution
Facto:r2
Unfiltered/
Filtered
Crushed Grapes
1:4
1:10
1:40
Unfiltered
Must
1:5
1:10
1:50
17-31
1:5
1:6
1:10
1:10
1:50
1:60
Filtered
Must
1:4
1:10
1:40
1:4
1:5
1:10
1:10
1:40
1:50
Free-run/
Press/Wine
Day 1-10; 49-293
Free-run/
Press/Wine
Day 1-10; 49-293
17-31
1
Samples were diluted with ascorbate (0.15%) sodium phosphate
buffer (pH 6.1) to the established dilution level prior to conjugase
treatment.
2
Each sample was assayed in triplicate at two concentrations
55
Data Analysis
The optical densities were used to construct a
standard curve and to determine the folacin concentration
of the samples.
The results were then evaluated by
statistical analysis.
Standard curve.
The means of the optical densities
for each triplicated folic acid concentration were plotted
on millimeter graph paper against known concentrations of
folic acid.
A smooth curve was fitted through the data
points using a French curve.
The standard curve was also constructed using a least
square polynomial method.
Using the optical densities,
the computer program •Least-Squares Polynomials• at CSUN
(California State University, Northridge, 1983a)
determined the best-fit equation for the standard curve.
Determination of folacin concentration in crushed
grape, must and wing samples.
The folate concentration in
each sample assay tube was determined by comparing its
O.D. reading to the standard curve.
When the •Least-
Squares Polynomials• program was used to construct the
standard curve, folacin concentrations were determined
with the program •Folacin Calculations• (Chen, 1983b).
The volume of sample in the tube and the final dilution
factor were used to calculate the folacin content as
micrograms per 100 grams or 100 milliliters of crushed
grapes, must and wine.
The portion of the total folic
acid (TFA) value that was contributed by folates present
56
in the conjugase preparation was determined from the
blanks and subtracted from the TFA values of the crushed
grapes, must and wine.
Stati~tical
analysis.
Each sample was assayed in
triplicate at two different concentrations.
The mean and
standard deviations were calculated from the six
measurements.
The significant differences among samples
were determined using CSUN's •one-Way Analysis of
Variance• computer program (California State University,
Northridge, 1983b).
When the F statistic showed a
significant difference, the program further computed t and
Z statistics to compare pairs of means.
Significant
differences between the means of two variables or
treatments were tested using student t test (Ferguson,
1966) •
CHAPTER 4
RESULTS AND DISCUSSION
Studies of Wine Sample Preparation Methodology
The stability of folacin in wine appears to be
affected by numerous variables during the extraction
process.
To establish optimal conditions of wine sample
preparation for folacin assay, crushed grapes and young
wine samples obtained on days 17, 211 and 238 of
vinification were used to determine the effects of sample
preparation on folacin activity.
ComparisQn of the Addition Qf hscorpate PhQsphate Buffer
tQ the Direct AdditiQn
of_A9~rbate
Ascorbic acid is routinely added to the extracting
buffer to ensure that the heat labile forms of folacin are
protected from degradation during extraction (Hurdle
~.,
1968).
~
Chitwood (1983), however, showed that
significantly higher folacin activities were detected in
beer when ascorbate was added directly to beer prior to
autoclaving as compared to adding phosphate buffer
containing ascorbate to beers.
Since wine samples are
similar in nature, the addition of sodium buffer that
contained ascorbate was compared to the direct addition of
57
58
ascorbate to the wine samples.
As shown in Table 5, the
direct addition of ascorbate to samples prior to
autoclaving resulted in significantly higher folacin
values (p<O.OOl).
The direct addition of ascorbate to
unfiltered and filtered wine samples yielded 11.387
9.996
~g
~g
and
TFA per 100 ml, respectively, while the addition
of sodium phosphate ascorbate buffer to the unfiltered and
filtered samples resulted in 5.802
100 ml, respectively.
~g
and 4.539
~g
TFA per
In a similar study conducted on
Chardonnay wine, Gaines-Moss (1984) reported findings of
the same nature where 20% higher folacin activity was
found in the wine sample to which ascorbic acid had been
directly added prior to autoclaving.
Paine-Wilson and
Chen (1979) found that the buffer pH had a profound effect
on the thermal stability of folacin
an~
that most
naturally occurring folates are more stable in neutral pH
than under acidic conditions.
Thus, the addition of
sodium phosphate buffer (pH 6.1) which increased the pH of
the wines was expected to enhance the stability of
folacin.
However, the direct addition of ascorbate to the
wine samples resulting in lowered pH values, appeared to
provide better protection to the labile folates than the
diluting buffer.
The direct addition of ascorbic acid to
the wine samples resulted in 50% higher folacin activity
in both unfiltered and filtered Cabernet Sauvignon wine
samples than the samples diluted with buffer, and thus
confirmed the works of Gaines-Moss (1984) and Chitwood
59
TABLE 5
Comparison of Direct Addition of Ascorbate to the Addition
of Ascorbate Containing Phosphate Buffer on the Folacin Activity
of a Cabernet Sauvignon Wine Sample!
Treatment
pH2
Folacin Activity
{f.lg/100 ml23
Unfiltered
Filtered
3.71
11.387 ± 1.189
5.24
5.802 ± 0.457
4
Ascorbate
a
5
Buffer
a
9.996 ± 2.30
b
b
4.539 ± 0.239
1
The Cabernet Sauvignon wine sample was drawn on day 211 of
vinification.
2
pH of the wine measured after autoclaving.
sample had pH of 3.78.
The original
3
Values represent the mean ± S.D. of the mean from six
replicates. Means followed by different superscripts within the same
column are significantly different (p<0.01).
4
Ascorbate was added directly to the sample to a final
concentration of 0.15 percent.
5
Sodium phosphate buffer (pH 6.1) containing 0.15% ascorbate
was added to the sample prior to autoclave treatment.
60
(1983).
As a result, all samples were treated with the
direct addition of ascorbate.
The mechanism for this
unusual observation needs to be studied in the future.
Ascorbic Acig Levels
The effect of five different levels of ascorbic acid
added directly to wine samples was studied.
No
significant difference (p>0.05) in folacin values was
found among the various levels of ascorbate added to the
Cabernet Sauvignon wine samples (Table 6).
The absence of
ascorbate in the samples resulted in the lowest, although
not statistically significant, folacin value.
Numerous
research have shown the inclusion of ascorbate necessary
during extraction for the protection of folates.
However,
discrepancies exist as to the optimal usage of ascorbate.
Herbert
~
Al. (1961) recommended the use of 0.15%
ascorbate while Hurdle
~
gl.
(1968) reported no
significant difference between 0.15 and 10.0% ascorbate.
O'Broin
~
gl.
(1975) found that 0.2% ascorbate was
required yet Chen and Cooper (1979) determined that a
level of 0.1% ascorbate was sufficient to protect folacin.
Based on the results of past works, the appropriate level
of ascorbate appears to be dependent on the food sample
and the inclusion of preliminary experiments to establish
optimum ascorbate concentrations during extraction should
be an integral part of folacin analysis for foodstuffs.
As L. casei is most active in the presence of 0.15%
6l
Q
TABLE 6
Effect of Ascorbate Added during Sample Preparation
on Assayable Folacin in Cabernet Sauvignon Winel
Folacin Activity
(llg/100ml) 3
Percent Ascorbate
pH2
0
3.77
a
8.28 ± 1.50
0.15
3.69
a
9. 78 ± 1.00
0.3
3.68
9.34 ± 0.74
0.5
3.63
a
9.41 ± 1.42
0.7
3.60
a
9.16 ± 1.63
1.0
3.58
9.10 ± 1.47
a
a
1
The wine sample used was drawn on day 238 of vinification.
2
pH of the wine was measured after autoclaving.
3
Values represent the mean ± S.D. of the mean from six
replicates. Means followed same superscripts are not significantly
different (p>0.05).
'
62
ascorbic acid
(Herbert~
gl., 1961), this level of
ascorbate was used for the subsequent preparation of
crushed grape, must and wine samples.
Heat Treatment
The results from three heat treatment methods are
shown in Table 7.
The samples receiving heat treatment
resulted in significantly (p<0.05) greater folacin
activity than the sample not heat-treated.
The use of
heat treatment is required to release bound forms of
folacin and to inactivate interfering compounds (Bird
al., 1945; Kirsch and Chen, 1984).
~
The boiling waterbath
method has been used extensively as a substitute for
autoclaving
~
(Bird~
£l., 1975).
Al., 1945; Dong and Oace, 1973; Keagy
Satisfactory results were obtained using
either the boiling waterbath or the autoclave treatment.
However, since young wine samples that may have contained
contaminating microorganisms were used in this research,
the autoclave method was chosen.
Conjugase
Two sources of conjugase were compared to determine
which was more suitable to deconjugate the polyglutamyl
forms of folacin present in wine.
As shown in Table 8,
significantly higher (p<O.Ol) folacin activity was found
in the wine sample treated with chicken pancreas
conjugase.
Kirsch and Chen (1984) and Tamura and Stokstad
(1973) studied variations in conjugase treatment of food
63
TABLE 7
Comparison of Heat Treatment Methods on Folacin
Activity of Cabernet Sauvignon Wine 1
Folacin Activity
(1-lg/lOOml)
Treatment
3
No heat treatment
b
4.71 + 0.61
a
4
Boiling waterbath
7.18 + 1.91
a
5
Autoclaving
8.84 + 2.32
1
The sample used was drawn on day 211 of vinification and had 0.15% ascorbate added.
2
Values represent the mean ± S.D. of the mean from
six replicates. Means followed by different superscripts
are significantly different (p<0.05).
3
Wine sample was set aside in a cool dark place
until analysis.
4
Wine sample was heated in a boiling waterbath for 5
minutes.
5
Wine sample was autoclaved at 121°C,
minutes.
1~
psi for 10
64
TABLE 8
Comparison of Two Sources of Conjugase on
Assayable Folacin in Cabernet Sauvignon Wine 1
Folacin
Acti~ity
(~g/lOOml)
Conjugase Source
3
b
Fresh hog kidney
5.87 + 0.52
4
a
9.54 + 0.56
Dried chicken pancreas
(Difco)
1
The sample used was drawn on day 211 of vinification.
2
*
Values represent the mean
S.D. of the mean from three
replicates. Means followed by different superscripts are
significantly different (p<O.Ol).
3
Fresh hog kidney was prepared as described in Appendix B.6.2.
4
Commercial dried chicken pancreas (Difco) was prepared as
described in Appendix B.6.1.
65
samples and found the conjugase source to have no
significant effect on the total folacin activity of the
samples.
However, Kirsch and Chen (1984) found the buffer
species to influence conjugase activity.
Chicken pancreas
conjugase was found to be inhibited by citrate phosphate
buffer while fresh hog kidney conjugase was not affected.
Small amounts of citric acid are produced as a by-product
of normal fermentation and thus citric acid is present in
wines in small amounts {0.01 to 0.05%) (Fong
1974).
~
gl.,
In this experiment, however, the wine sample was
diluted 40 times before treatment with conjugase and the
low concentration of citrate did not appear to affect
chicken pancreas conjugase activity.
In contrast, Babu
and Srikantia (1976) and Butterfield and Calloway (1972)
have found hog kidney conjugase to be inhibited by the
presence of yeast.
The low folacin activity observed
using hog kidney conjugase in this study may be attributed
to the presence of yeast or yeast cell components released
into the medium during autolysis.
Filtration Treatment
Samples drawn from 3 different stages of winemaking
were divided into two sets:
those which received
filtration treatment and those which did not.
As shown in
Table 9, a significant difference in folacin activity
(p<O.OS) was found between unfiltered and filtered samples
drawn from the period of vigorous fermentation whereas no
66
TABLE 9
Effect of Filtration Treatment on Assayable Folacin in
Samples Drawn from Various Stages of Winemaking
Day of
Vinification
Sample
Folacin Activity
(ug/100ml) 1
Unfiltered
0
a
9.006 + 0.36
a
7.949 + 1.27
17
a
3.913 + 0.77
0.860 + 0.56
211
a
5.816 + 0.84
2
Crushed grapes
Sample drawn from
vigor~us fermentation
stage
Young wine in aging
stage3
1
Filtered
b
a
6.041 + 0.47
Values represent the mean ± S.D. of the mean from six
replicates. Means followed by different letters within the same row
are significantly different (p<0.05).
2
Filtered crushed grape samples were filtered using a cellulose
type filter.
3
Filtered samples drawn from the vigorous fermentation stage and
the aging stage were filtered using a membrane type filter.
67
9
difference was found between unfiltered and filtered
crushed grapes or young wine samples.
The significantly
higher (p<O.OS) folacin activity noted in the unfiltered
sample drawn on day 17 was attributed to yeast
characteristically present in large amounts during the
fermentation stage.
Kirsch (1983) studied the effect of
filtration of spinach homogenate through filter paper and
found decreased total folacin values.
The significantly
lowered TFA values were attributed to the adsorption of
the long chain polyglutamates onto the filter paper.
As a
result, Kirsch (1983) did not recommend filtration of
samples through filter paper as filtration might result in
decreased total folacin activity of the sample.
In the
present study, however, filtration treatment was found
necessary to determine the sources of folacin in the
samples.
Therefore, all the samples drawn during the
commercial production of the Cabernet Sauvignon wine were
divided into two sets, those receiving and those not
receiving filtration, in order to monitor folacin
activity.
Overall Evaluation
Based on the results of the variations in sample
preparation studies, optimal conditions for folacin assay
of crushed grapes, must and wine samples included the
following:
'
68
0
1)
The direct addition of ascorbic acid to samples
to a final concentration of 0.15 percent.
2)
Autoclaving at 12l°C, 15 psi for 10 minutes.
3)
Deconjugation of the polyglutamyl folate forms
with commercial dried chicken pancreas conjugase (Difco).
4)
Monitoring folacin activity in samples receiving
and not receiving filtration treatment.
Changes in
Fola~in
Activity during the Vinification
Process
The folacin activity in Cabernet Sauvignon samples
collected during the vinification process were determined.
Table 10 lists the folacin values of the unfiltered and
filtered samples at each sampling date during wine
production.
Grapes and Must
As described earlier, samples of crushed grapes and
must were obtained on the first day of winemaking.
Circumstances were such that grapes with whole berries
were not available for sampling, however,
0
Brix, tartaric
acid and pH values of the grapes are given in Table 11.
The difference between the whole and crushed grape
clusters lie in the sulfiting and slight crushing of the
berries prior to the next treatment, destemming.
During
sample preparation for folacin assay of the crushed
grapes, care was taken not to include visible extraneous
'
69
TABLE 10
Folacin Activity in Unfiltered and Filtered Cabernet Sauvignon
Samples during Wine Production
Date of
Sampling
Significant
Day of
Folacin Activity in Folacin Activity
Difference b/w
ViniUnfiltered Samp~ in Filtered Samples 1 Unfiltered & 2
fication (iJg/lOOg or JJg/1
(JJg/lOOg or JJg/lOOmlJ Filtered sample
10-28-82
(crushed
grapes)
9.006 :t 0.363b
7.949 :t 1.266a
NS
9.830 :t 0.528
b
10-28
(must)
a
9.314 :t 0.515
NS
g
11-3
1
0.481 ± 0.179
g
11-5
3
10
11-12
0.647 :t 0.058
11.966
± 0.891
a
c
11-19
17
7.604 ± 0.816
12-3
31
9.794 ± 0.389
12-21
49
4.582 ± 0.385
1-5-83
64
7.838 ± 0.369
1-31-83
90
3.691 ± 0.246
b
d,e
c
f
190
5-11
6-1
211
8-22
293
4.180
4.662
± 0.306
± 0.329
d,e,f
d
e,f
3.796 ± 0.402
1
0.517 :t 0.083
f
NS
f
0.504 ± 0.106
e
1.801 ± 0.234
d
2.690 ± 0.503
b
5.639 ± 0.112
c,d
3.444 ± 0.573
b,c
4.678 ± 0.788
c,d
3.281 ± 0.414
c,d
3.611 ± 0.514
c
4.239 ± 0.307
c,d
3.656 ± 0.750
Values are the mean ± S.D. of the mean from six replicates.
Values followed by different superscripts within the same column are
significantly different (p<0.05).
2
NS - Not Significant;
**
p<O.Ol.
NS
**
**
**
**
**
NS
NS
NS
NS
TABLE 11
Sugar Content ( 0 Brix), Tartaric Acid, pH, Alcohol
and Free Sulfur Dioxide (FS0 2 ) values for Cabernet Sauvignon Grapes and Wine 1 ' 2
0
Sample
Crushed grapes
Brix 3
24.2
Tartaric Acid
(g/100 ml)
pH
Alcohol
% by Volume
0.86
3.79
Day 30 of vinification
0.80
3.67
13.8
Day 105 of vinification
0.59
3.62
13.8
Day 210 of vinification
0. 59
3.42
13.8
1
2
FS02
(ppm)
20
Cabernet Sauvignon grapes grown and harvested at Priest Ranch Vineyard, Napa Valley, California.
Values for the analyses were determined by Ahern Winery's laboratory.
3 0 Brix indicates grams of sugar per 100 g of grape juice.
-....]
0
7l
materials such as stems and leaves.
Gaines-Moss (1984)
analyzed Chardonnay grapes with whole berries as they
arrived from the vineyard and sulfited crushed grapes as
they came out of the crusher.
She found no significant
difference (p>0.05) in folacin activity between the
samples of whole and crushed berries.
The folacin activity in the unfiltered crushed grapes
was 9.01
~g
yielded 7.95
per 100 grams while the filtered sample
~g
per 100 grams (Table 10).
In the initial
stages of sample preparation for folacin determination, it
had been decided to autoclave grape and must samples prior
to filtration because the presence of wild yeast was
assumed to be negligible.
Statistical analysis showed no
difference in folacin activity between unfiltered and
filtered crushed grape samples.
This
r~sult
was expected
since folate contributions by wild yeast would have been
accounted for in both unfiltered and filtered samples.
The release of folates from yeast would have occurred
during autoclaving prior to the filtration process.
Table 12 lists literature values of folacin activity
in grapes and must.
Lafon-Lafourcade and Peynaud (1958)
and Peynaud and Lafourcade (1957) in similar studies using
Streptococcue f9ecalis and St{eptococcus lactis,
respectively, assayed the juice expressed from grapes for
folacin.
0.2
~g
They reported grapes to contain 0.14
~g
and 0.1-
folacin per 100 ml juice respectively, expressed
from grapes.
The values reported by Lafon-Lafourcade and
72
TABLE 12
Comparison of Folacin Activity of Red Grapes and Must
Found from Previous Research to the Present Study
Folacin Activity
Cvg/lOOg or ml)
2
Reference
Assaying Organism
Grapes
Must
Hall £!_ al. ( 1956)
S. faecalis
7.9
2.0
Lafon-Lafourcade &
Peynaud (1958)
S. lactis
0.14
0.12
Peynaud &
Lafourcade (1957)
S. faecalis
Hardinge and Crooks
(1961)
L. casei
5.2
Perloff and Butrum
(1977)
L. casei
7.0
Paul and Southgate
(1978)
L. casei
5.0
This study
L. casei
0.1 - 0.2
Unfiltered
9.01 3
Filtered
7.95 3
Unfiltered
9.83 3
Filtered
9.31 3
1
Current research on total folacin activity of Cabernet
Sauvignon grapes from Priest Ranch Vineyard, Napa Valley, California.
2
Microorganism used in the microbiological assay for total
folacin activity.
3
Values represent the mean from six replicates.
73
~1
Peynaud (1958) and Peynaud and Lafourcade (1957) do not
agree with the folacin value for grapes obtained in the
present study.
The large discrepancy observed may be due
to the absence of ascorbate and the use of
s.
s.
faecalis and
lactis in the microbiological assay for folacin by the
previous researchers.
Furthermore the folacin activity in
the juice expressed from grapes does not accurately
represent the folacin content of grapes.
Gaines-Moss
(1984) determined the folacin content of whole grapes,
hand-peeled grapes and juice expressed from grapes.
From
her study, it appeared that folacin is concentrated in the
skins of the berry and that the folacin found in grapes is
primarily attributed to the skins and a few seeds.
The
folacin values of the Cabernet Sauvignon grapes are
similar to those reported by Hall
~ ~.
(1956) and those
listed by Perloff and Butrum (1977) while values listed by
Hardinge and Crooks (1961) and by Paul and Southgate
(1978) were slightly lower.
Discrepancies in the
aforementioned values may be a result of the grape variety
and the growing conditions of the grape.
Soil,
temperature, rainfall and various other environmental
conditions affect the growth of the grape and hence its
vitamin content
(Amerine~
gl., 1980).
In addition,
variations in the microbiological assay methodology would
also have affected the folacin values for grapes.
The folate yields of the must were 9.83
9.31
~g
~g
and
per 100 ml in the unfiltered and filtered samples,
'
74
0
respectively (Table 10).
No significant difference
(p>0.05) was found between the two samples.
This result
was expected since no apparent characteristic should
differentiate the two samples at this stage of the
winemaking process.
In addition, as was discussed with
the grapes, autoclaving had also been performed prior to
the filtration process.
The literature values for folacin in must listed in
Table 12 do not agree with the values obtained in the
current study.
reported 0.12
Lafori-Lafourcade and Peynaud (1958)
~g
folacin per 100 ml while Hall
(1956) reported 2.0
~g
folacin per 100 ml.
~
al.
The large
discrepancy observed may have stemmed from the
interpretation of the term must and thus in the method of
sampling.
It appears from the literature that the authors
sampled the liquid portion of the must which would tend to
yield results similar to those obtained for juice
expressed from grapes as was reported by Gaines-Moss
(1984), Lafon-Lafourcade and Peynaud (1958) and Peynaud
and Lafourcade (1957).
In the present study, the must
sample was comprised of the slurry of grapes, skins,
seeds, free run juice and possibly stems and leaves, and
was analyzed as such.
The folacin values of the must were
therefore similar to the values obtained for the crushed
grapes (Table 12).
Comparison of the folacin activity in unfiltered and
filtered crushed grapes with the unfiltered and filtered
'
75
must, respectively, showed no significant difference
(p<0.05) in folacin activity among the unfiltered and
filtered sample sets (Table 12).
The crushed grapes and
must samples were of similar composition and sampled
within several hours of each other.
As a result, no
difference in folacin activity was expected.
Day 1 of Yinification
Figure 5 describes the changes in folacin activity
during the commercial production of the Cabernet Sauvignon
wine at Ahern Winery.
On day 1 a liquid sample was drawn from the must
containing tanks.
The day prior, the must had been
inoculated with SacchgiQrnyces cerevisiae var. ellipRQideus
(French Red wine yeast, Red Star brand, active dry wine
yeasts).
The amounts of folacin found·in both the
filtered and unfiltered samples were extremely low (Figure
5).
The filtered sample had slightly higher TFA with
0.52
~g
per 100 ml while the unfiltered sample showed
0.48
~g
per 100 ml (Table 10).
This difference, however,
was statistically insignificant (p>0.05).
The sudden drop in TFA from crushed grape and must
samples may be explained in part because the tank's
sampling spout allowed only free run juice to be drawn,
excluding any skins or pulp.
The low folacin
concentrations found in both the unfiltered and filtered
samples would thus confirm findings by Gaines-Moss (1984)
76
Figure 5.
Changes in Total Folacin Activity of Unfiltered
and Filtered Cabernet Sauvignon Samples during
Commercial Production.
77
z
z
0
"C
&J
-·--- ..·-1I
~
&J
-
(!;)
"C
&J
>
::l
<
UJ
~
CD
c
:::;)
~
w
z
a:
w
£D
<
()
c
-0
ca
u
c
>
0
>.
ca
c
ID
N
CIw OOfl! n')
uoneJ~ue~uoJ
upeJO.:f
1 elOl
78
whereby folacin in grapes was found to be concentrated in
the skins.
Higher folacin was expected than was actually found
in the unfiltered sample for two reasons:
One, several
researchers have found yeast to be a rich source of
folacin (Butterfield and Calloway, 1972; Perloff and
Butrum, 1977;
Singh~
£1., 1948) and two, autoclaving
would have release folates from yeast cells present as a
result of inoculation the day before.
The insignificant
difference found between the unfiltered and filtered
samples suggested that yeast cells were not present in
abundant amounts in the unfiltered sample.
It thus
appears that 24 hours after inoculation the yeast activity
was still low at this early stage in fermentation and that
the cells may not have fully adapted to their surrounding
medium.
In the commercial making of the Cabernet Sauvignon
wine under study, the low temperatures used in the early
stages of fermentation (Table 13) may have affected yeast
growth and in turn, folate levels.
Ough (1966a,b) and
Ough and Amerine (1966) studied the effect of temperature
on yeast growth and fermentation rates.
They found that
lower fermentation temperatures (50-55°F) resulted in
slower yeast cell growth and grape juice fermentation
rates.
The low temperature used in the making of the
Cabernet Sauvignon may account for the low yeast activity
witnessed on day 1 of vinification.
Folacin activity in
79
v .
TABLE 13
Temperatures Recorded on Days of Samfling during
the Wine Making Process
Date of Sampling
Day of Vinification
Temperature OF
10-28-82
0
53
11-3-82
1
55
11-5-82
3
58
11-12-82
10
70
11-19-82
17
62
12-3-82
31
53
12-21-82
49
52
1-5-83
64
50
1-31-83
2
3-4-83
90
56
122
60
5-11-83
190
60-65
6-1-83
211
60-65
8-22-83
293
60-65
3
3
3
1
Between days 0 and 122 temperatures were recorded from
thermostat of temperature-controlled fermentation vessel.
2
No sample obtained on day 122.
3
Wine aging in barrels under usual cellar temperatures
maintained between 60 and 65°F.
80
the samples appears to have been contributed by the
freshly inoculated must itself rather than by the yeast.
Day 3 of VinificatiQn
Three days after the addition of the yeast culture,
another liquid portion sample of the must was drawn for
folacin analysis.
The folate levels in both the
unfiltered and filtered samples increased slightly, though
insignificantly, and were again quite low {Figure 5).
filtered sample contained 0.65
filtered 0.50
~g
per 100 ml.
~g
The
TFA per 100 ml and the
Folacin activity in the
unfiltered sample was slightly higher than in the filtered
{Table 10), though insignificantly so {p>0.05).
Three days after inoculation, higher folacin levels
were expected in both samples, particularly in the
unfiltered sample.
After the addition of the yeast
culture, the must had been pumped over to ensure even
distribution of the cells and to encourage yeast growth
and multiplication
Singh~
{Amerine~
gl., 1948).
Singh~
sl., 1980; Joslyn, 1955;
gl.
(1948) studied the
influence of aeration and agitation on the yield and
vitamin content of several food yeast.
s.
Higher yields of
cerevisiae were obtained as a result of both aeration
and agitation.
Although changes in folic acid content of
the yeast were irregular under various conditions of
aeration and agitation,
rich source of folacin.
~.
cerevisiae was found to be a
It was therefore anticipated that
Bl
conditions of aeration and agitation from pumping over and
the time lapse since inoculation would have led to higher
folate levels in the unfiltered sample.
Comparison of the
folacin yields from days 1 and 3 of vinification (Table
10) showed no significant difference in the amounts of
measured folacin in either unfiltered or filtered samples.
Table 14, however, indicates the folacin contributed by
yeast to have risen slightly since the last sampling date
(day 1 of vinification).
This small rise may be a sign of
increased or increasing yeast activity although actual
events cannot be ascertained since no cell counts had been
made.
Pay 10 of
Vinific9~
Shortly after the last sampling (day 3 of vinification), the formation of hydrogen sulfide had been
detected.
After 14 days on the skins and 10 days of
inoculation, the fermenting must was pressed and a sample
of pressed must was drawn.
Analysis of the unfiltered and
filtered pressed must samples showed folacin activity to
have increased significantly (p<0.05) since the last
sampling (Figure 5).
The folacin contents were 11.97
per 100 ml of the unfiltered sample and 1.80
~g
~g
per 100 ml
of the filtered sample.
Several events may explain the marked increases in
folate levels observed in the samples.
Skin contact time
and the pressing of the must which allowed the folacin to
82
TABLE 14
Folate Contributions from Yeast Cells after
Inoculation of the Must
1
Day of Vinification
Folacin from yeast cells
(llg/100 ml)
1
0 (-0.036)
3
0.14
10
10.17
17
4.91
31
4.15
49
1.14.
64
3.15
90
0.41
190
0.57
211
0.42
293
0.14
1
Folacin from yeast was determined by subtracting folacin in
unfiltered samples from folacin in filtered samples.
83
be extracted from the skins, seeds and leaves into the
liquid medium caused the increase of folacin in the
filtered sample.
Furthermore, the must was warmed (Table
13) in preparation for the inoculation with LeuconostQC
Qenos ML-34 because recommended temperatures for malolactic bacterial growth should be no lower than l8-22°C
{64.4-71.6°F) {Kunkee, 1974).
The warming of the must
also necessary for color extraction encourages yeast
growth, fermentation rate and malo-lactic fermentation
(Amerine~
gl., 1980; Kunkee, 1974; Ough, 1966a,b; Ough
and Amerine, 1966).
Analysis of the filtered sample demonstrated a 74%
increase in folacin activity since last sampling.
The
chief cause of the increase was the production and
liberation of folacin by the multiplication and ongoing
autolysis of yeast cells during fermentation {Castor,
1953).
Burkholder~
AI. {1945) reported on the folacin
production by bacteria and yeast and observed that certain
yeast strains including three fermenting strains of
SaccharQmyc~
capacity.
had considerable folacin synthesizing
The increase in folacin activity witnessed in
the filtered sample was, however, not entirely the result
of folacin synthesis by yeast.
As was mentioned earlier,
contributions from skin contact time and pressing are of
consideration in the filtered sample as well.
The high folacin activity found in the unfiltered
sample largely results from yeast present in large
84
proportion which is an indication of a vigorous
fermentation period.
Figure 6 illustrates the changes in
folacin activity extracted from yeast (folacin activity in
unfiltered sample minus folacin activity in filtered
sample) and the changes in folacin activity in the
filtered Cabernet Sauvignon samples during the
vinification process.
As shown in Figure 6, a large
amount of folacin was derived from yeast cells on day 10,
thereby accounting for a major portion of the folacin
activity measured in.the unfiltered sample.
In addition, the largest difference in folacin
activity between unfiltered and filtered pressed must
samples represented by the highest peak in Figure 6, was
observed on the lOth day of vinification (Table 14).
The
large amount of yeast cells that had been removed in the
filtered sample caused this difference.
Although sulfur may cause the folic acid content to
decrease
(Hall~
A!., 1956) the effects of hydrogen
sulfide formation by yeast on folacin activity are
unknown.
Day 17 of Vinification
One or two days following pressing, the free-run was
inoculated with Leuconostoc
oenQ~
ML-34 to induce malo-
lactic fermentation (exact inoculation date unknown).
day 17, a decrease in folacin activity was noted in the
On
85
Figure 6.
Changes in Folacin Activity Extracted from
Yeast Contrasted to Changes in Folacin Activity
in the Filtered Cabernet Sauvignon Samples
during Commercial Production.
86
-
1
CP
c::
"i
Cl)
"C
CP
~
>. CP
CQ
CP
:::
- .s:
-II
E
0
~
c::
.(.)
E (.)
CQ
0
CQ
0
c::
g
--
C)
>
0
C)
CQ
(.)
c::
0
•
0
>.
C)
•
...
C)
0
C)
""
•
•
CQ
0
87
~1
unfiltered sample while continued increase was observed in
the filtered sample (Figure 5).
Folacin activity in the unfiltered and filtered
samples was 7.60 vg and 2.69 vg TFA per 100 ml,
respectively (Table 10).
The significant (p<0.05)
increase in folacin activity in the filtered sample from
day 10 is indicative of further synthesis or release of
the vitamin by yeast.
From Figure 6, a decrease in the
amount of folacin activity contributed by yeast is
apparent.
Correspondingly, on day 17 a decrease in
folacin activity in the unfiltered sample is obvious from
Figure 5.
The decrease in folacin activity in the
unfiltered sample thus appears to correspond to a relative
decrease in the number of yeast cells.
Day 31 oi Vinification
Alcoholic fermentation was completed by day 31 as
indicated by the presence of 13.8% alcohol (Table 11).
The folacin activity in unfiltered and filtered samples
was 9.79 vg and 5.64 vg TFA per 100 ml, respectively. The
significant increase (p<0.05) in extracted folacin in the
unfiltered sample from day 17 (Table 10) was unexpected at
first since the fermentation process was complete and
correspondingly yeast activity would be low.
A possible
cause for the higher folate content found may be the
result of settling of the folacin containing yeast cells.
In the end stages of fermentation, yeast tend to
•
88
agglomerate and form floes which settle to the bottom of
the fermentation vessel
{Amerine~
gl., 1980).
This
phenomenon of flocculation is desirable as it greatly
assists in the removal of yeasts from wine {Rose, 1980).
It may therefore be, that in the drawing of the sample, a
higher concentration of cells was obtained, resulting in
the observed higher folacin activity.
The continued folacin increase witnessed in the
filtered sample {Figure 5) may more easily be interpreted.
Following fermentation dead yeast cells begin to autolyse
under anaerobic conditions in the new wine, thereby
releasing more folacin into the medium (Thoukis, 1974).
Figure 6 shows a decline in the amount of folacin
extracted from the yeast cells.
It appears that the
proportion of cells has decreased steadily since day 10
where the highest apparent yeast activity was noted {Table
14).
The gradual decrease in folacin extracted from the
cells may be reflective of ongoing autolysis which is more
pronounced toward the end and after the completion of
fermentation {Eschenbrucb, 1974).
In addition, with the
completion of fermentation, yeast inhibition by alcohol
compounded with the depletion of vital carbon and energy
sources have a direct effect on the reduction in the
number of viable cells (Castor, 1953).
Compounding the tedious interpretation of these
events was the malo-lactic fermentation, which by this
stage was not yet completed.
The effects and extent of the
89
interrelationship of this fermentation on yeast and
folacin activity are essentially unknown.
Day 49 pf Vinification
Shortly after completion of malo-lactic fermentation,
the wine was racked to stainless steel tanks.
The
addition of cupric sulfate (Cuso 4 ) was required to
counteract the hydrogen sulfide (H 2 S) formed in the early
stages of yeast fermentation (exact date of Cuso 4 addition
unknown). Upon completion of both fermentations, the new
Cabernet Sauvignon wine was in its aging process.
Alcohol, tartaric acid, pH and free sulfur dioxide values
determined by Ahern Winery are listed in Table 11.
In both the unfiltered and filtered samples, sudden
drops in folacin activities were witnessed (Figure 5)
resulting in 4.58 yg and 3.44 yg TFA per 100 ml wine,
respectively (Table 10).
Figure 6 shows the amount of
folacin extracted from yeast to have decreased since last
sampling.
This was not unexpected because the wine had
been cleared of a major portion of yeast lees during the
first racking.
Suspected causes for the observed folacin
decreases in both unfiltered and filtered samples include
racking of the wine, addition of cuso 4 and unknown
contributions from post malo-lactic fermentation.
Day 64 of VinifjQation
An
unexpected increase in folacin activity occurred
on this date of sampling (Figure 5).
Folacin activity
90
increased noticeably from 4.58 vg to 7.84
~g
TFA per
100 ml in the unfiltered sample (p<0.05) and from 3.44
to 4.68
~g
~g
TFA per 100 ml in the filtered wine sample
(p>0.05) (Table 10).
Since the last sampling on day 49,
the wine had been racked and cuso 4 treatment had been
repeated. It would appear unlikely that the outcome of
this treatment be manifested by an increase in folacin
activity as previously (prior to day 49), this same
treatment resulted in a decrease in folacin activity.
Reasons for the observed increase are unknown and cannot
be explained.
Days 90,
1~0,
211 9Pd 293 9!-Yinific9tion
After day 64 of vinification, an initial decrease and
subsequent stabilization in folacin activity was observed
(Figure 5).
In addition, there were no significant
changes (p>0.05) in folacin activity in both filtered and
unfiltered samples between days 90 and 293 of vinification
(Table 10).
A few days prior to sampling on day 90, the wine had
been racked once again followed by a final addition of
cuso 4 •
The resulting folate content of the unfiltered and
filtered wine samples was lower than that of the previous
sampling.
The wine yielded 3.69 vg and 3.28 vg TFA per
100 ml in the unfiltered and filtered aliquots,
respectively (Table 10).
As was mentioned earlier, due to
the unexpected results obtained on day 64, the effects of
91
the racking and the Cuso 4 treatment on the vitamin at this
stage of winemaking could not be determined.
In March, approximately between days 130 and 140 of
vinification, the wine was racked to barrels and stored
under usual cellar conditions with temperatures maintained
between 60 and 65°F (Table 13).
However, no new samples
were drawn until day 190 of vinification.
On day 190,
approximately five and a half months after fermentation,
the wine was examined (Table 10).
resulted in 4.66
~g
and 3.61
~g
Analysis of the samples
TFA per 100 ml in the
unfiltered and filtered samples, respectively (Table 10).
The racking of the wine from tanks to barrels did not
appear to have caused any vitamin loss since an
insignificant increase (p>0.05) in folacin activity was
noted (Table 10).
Hall~
AI. examined the folacin
content of two varieties of dry red wine 4 to 6 months
after fermentation.
The folacin values for those wines
along with the values obtained in the present study are
listed in Table 15.
Folacin activity in the Cabernet
Sauvignon wine in both unfiltered and filtered samples was
considerably higher than the folacin activity found in the
wines examined by
(1956) use of
Hall~
gl.
(1956).
Strepto~o~cgs f~ecalis,
Hall~
gl.'s
the microorganism's
growth response measurement by titration and the absence
of ascorbic acid in the sample preparation and the
microbiological assay are factors which may have partaken
in
Hall~
gl.'s (1956) observed folacin values in dry red
92
TABLE 15
Folacin Activity in Several Varieties of Dry Red Wines
4-6 Months after Fermentation
Year and Variety
Folacin Activity
(llg/100 ml)
1
Tinta Madeira
1952
1.3
1954
2.1
1
Zinfandel
1952
1.7
1953
1.3
Average vitamin content
of all dry red wines 2
1.5
3
Cabernet Sauvignon
1982
4
4.184 (Unfiltered)
3.61 (Filtered)
1
Wine variety examined by Hallet al. (1956).
2
Represents averages of all folacin values obtained from all
the varieties and every lot studied (year), reported by Hallet al.
(1956).
3
Results from this study.
4
Values represent the mean from six replicates •
.
'
93
wines.
In addition to the use of different methodologies,
other factors which may explain the observed differences
include laboratory versus commercial wine production,
grape variety, and soil and weather conditions, to name a
few.
The last samples examined in this study were
collected on days 211 and 293 of vinification.
folacin activity on day 211 was 4.66
~g
The
and 4.24
~g
TFA
per 100 ml wine in the unfiltered and filtered samples,
respectively (Table 10}.
On day 293, folacin activity in
the unfiltered and filtered wine samples was 3.80
~g
3.66
A
~g
TFA per 100 ml, respectively (Table 10).
and
significant decrease was noted (p<O.OS} in the unfiltered
sample from day 211 to day 293.
Overall examination of
the folacin activity between days 90 and 293 does not
indicate that a significant change was in order since
there was no significant difference in the filtered sample
between days 211 and 293.
In addition, statistical
analysis showed that there was no difference (p>O.OS)
between unfiltered and filtered samples from day 90 to day
293 thereby indicating that the yeast had been removed
from the wine (Table 14).
Therefore, the observed
difference in the unfiltered samples would appear to have
been the result of the microbiological assay rather than
be an indication of a significant change occurring at this
point of the winemaking process.
94
v '
The folacin levels determined in the last samples
showed that the amount of folacin was higher in the young
wine than in the freshly inoculated liquid portion of the
must (day 1).
The fate of the vitamin, however, is not
completely known since an additional 1 to 2 years of aging
was foreseen for this Cabernet Sauvignon.
Additional
handling of the wine including fining and bottling may
further affect its folacin levels.
/
/
CHAPTER 5
SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS
The microbiological assay for the determination of
'
folacin in foods has often produced
widely divergent data.
The variability of the microbiological assay is exemplified when reviewing the various folacin values given for
grapes, must and wine.
From the first study on the
folacin content of grapes, must and wine
(Hall~
Al.,
1956) to the most recent study of California wines (Voigt
~
gl., 1978) tremendous discrepancies in the folacin
values exist.
In this study, variations in sample
preparation methods for the microbiological assay for
folacin in wine were investigated.
These variations
included the comparison of the direct addition of
ascorbate to the addition of ascorbate buffer, the
determination of an optimal ascorbic acid level, the
comparison of three heat treatments, the comparison of two
sources of conjugase and the evaluation of filtration
treatment.
Also, the changes in folacin activity were
followed during the commercial fermentation and aging of a
California Cabernet Sauvignon wine.
The direct addition of ascorbate resulted in significantly higher folacin values in wine than the addition
95
96
of ascorbate buffer.
Although most naturally occurring
folates are more stable in neutral or slightly alkaline
pH, the direct addition of ascorbate to the wine samples
resulting in lowered pH values provided better protection
to the labile folates than the diluting buffer.
As much
as 50% higher folacin activity was obtained in the samples
receiving the direct addition of ascorbate.
The effect of five different levels of ascorbic acid
added directly to wines was studied.
No significant
difference in folacin values was found among the various
levels of ascorbate.
As L. casei is most active in the
presence of 0.15% ascorbic acid (Herbert, 1968), this
level of ascorbate was used for the subsequent preparation
of grape, must and wine samples.
The results from three heat treatment methods showed
that the use of heat treatment was necessary to release
bound forms of folacin.
Satisfactory results, however,
were obtained using either the boiling waterbath or
autoclave method.
Treatment with chicken pancreas conjugase yielded
significantly higher results than with hog kidney
conjugase.
The lower folacin values found for wine using
hog kidney conjugase was attributed to the presence of
yeast, a known inhibitor to hog kidney conjugase.
Upon evaluation, filtration treatment was found
necessary in the determination of the source of folacin
97
and in the monitoring of folacin activity during
vinification.
The'folacin content of the Cabernet Sauvignon grapes
was 9.0
~g/100
g.
Folacin levels increased, although
insignificantly, in the must to 9.8
~g/100
g.
A 95
percent decrease in folacin content was observed in the
extraction of the must.
After yeast inoculation rapid increases in folacin
activity were witnessed in both the unfiltered and
filtered samples.
sample was 12.0
1.8
~g/100
ml.
The folacin content of the unfiltered
~g/100
ml and the filtered sample was
The increase in folacin activity is
attributed to folacin synthesis that accompanied the
growth of yeast during fermentation because folacin
activity had significantly increased in. unfiltered and
filtered samples since must extraction.
When fermentation was complete, there was a large
initial decrease and then little further changes during
aging.
The folacin content of the wine approximately 9
months after fermentation was 3.8
unfiltered sample and 3.6
~g/100
~g/100
ml in the
ml in the filtered
sample, not statistically different.
The folacin levels
in these last samples showed that the amount of folacin
was higher in the young wine than in the extracted must.
The fate of the vitamin, however, is not completely known
since an additional 1 to 2 years of aging in barrels was
98
foreseen for this wine.
Also, further handling of the
wine may affect its folacin levels.
The levels of folacin found in this study were
markedly higher than those reported by
Hall~
Lafon-Lafourcade and Peynaud (1958) and Voigt
(1978).
Hall
100 ml and
~
gl.
Voigt~
(1956) reported 1.5
gl.
(1978) 0.7
~g
~g
gl.
~
(1956),
gl.
folacin per
folacin per 100 ml.
The use of an optimized sample preparation technique and
microbiological assay may be the reason for the higher
results found in this study.
In view of the increased
interest in wines and nutrition, it is recommended that
future research generates folacin data for a wider
spectrum of wines and investigates the mechanisms and
conditions leading to folacin synthesis by yeast in
fermented beverages.
Further research.on the application
of the optimal procedures determined in this investigation
to other foodstuffs is needed to help establish the
standardization of folacin methodology.
A specific matter
which needs further clarification is the direct addition
of ascorbate to foodstuffs.
Increased folacin values
obtained in wine with the direct addition of ascorbate has
not previously been reported and its action mechanism
needs elucidating.
/
LITERATURE CITED
Amerine, M.A., Berg, H.w., and Cruess, w.v. 1972. ~
,X_e_c..b.D.Ql.9.g,Y_.Q.f_Fine Making. Third edition. Westport,
Connecticut: The AVI Publishing Co., Inc.
Amerine, M.A., Berg, H.w., Kunkee, R.E., Ough, c.s.,
Singleton, V.L., and Webb, A.D. 1980. ~
Technology Qf Win~ Making. Fourth edition.
Westport, Connecticut: The AVI Publishing Co., Inc.
Amerine, M.A. and Ough, c.s. 1974. Wine and Must
Analysis. New York: John Wiley and Sons.
Amerine, M.A. and Singleton, V.L. 1965. Wine: An
Introd~ction for Americans.
Berkeley and Los
Angeles: University of California Press. Pp. 9-25.
Austin, c. 1968. The Science of Wine. New York:
American Elsevier Publishing Company. Pp. 28-31.
Babu,
s. and Srikantia, S.G. 1965. "Availability of
Folates from some Foods." AID~ican Journ§l of
Clinic§l NutritiQn, 29: 376-379.
Baugh, C.M. and Krumdieck, c. 1971. "Naturally Occurring
Folates." Annals Qf th~ew York Acagemy of
Sciences, 186: 7-28.
Bell, G. 1972. The Vitamins. Stein (editor).
Churchill, Livingstone, Edinburgh: A University of
Nottingham Seminar. P. 171.
Bell, G. 1974. "Microbiological Assay of Vitamins of the
B Group in Foodstuffs." Laboratory Practice, 23:
235-252.
Benda, I. 1982. "Wine and Brandy." In: Prescott §nd
PYnn'a Indu~i§l Micr.Qbiology. Fourth edition.
Reed, G. (editor). Westport, Connecticut: The AVI
Publishing Co., Inc.
Bird, O.D., Blinkley, S.B., Bloom, E.S., Emmett, A.D. and
Pfiffner, J.J. 1945. "On the Enzymatic Formation of
Vitamin Be from Its Conjugase." Journ§l of
BiologicaJ Chemis~, 157: 413-414.
99
100
Bird, O.D., Robbins, M., Vandenbelt, J., and Pfiffner,
J.J. 1946. "Observations on Vitamin B Conjugase
from Hog Kidney." Journal of Biologica£ Chemistry,
163: 649-659.
Blakley, R.L. 1969. The Biochemistry of Folik Acid and
Belated Pterigines. Amsterdam, London: North
Holland Publishing Co.
Broquist, H. 1955. "Folic Acid Conjugase." In: Methogs
in Enzymology, vol. II. Colowick, s. and Kaplan, N.
(editors). New York: Academic Press. Pp. 629-631.
Buehring, K.U., Tamura, T. and Stokstad, E.L.R. 1974.
"Folate Coenzymes of Lactobacillus ~~ and
Streptococcus faecalis." Journal of Biological
Chemistry, 249: 1081-1089.
Burkholder, P.R., McVeigh, I. and Wilson, K. 1945.
"Studies on Vitamin 'B£' Produced by Microorganisms."
Archives of Biochemist y, 7: 287-303.
Burroughs, L.F. 1975. "Determining Free Sulfur Dioxide
in Red Wine." American Journal of Enology and
Viticulture, 26: 25-29.
Burroughs, L.F., and Sparks, A.H. 1973. "Sulfite-Binding
Power of Wines and Ciders." ~rnal of the Science
of Foog and Agriculture, 24: 187-198.
Butrum, R.R. and Perloff, B.P. 1975. "Folacin Content of
Foods--Compilation of Data Based on Modified Microb i ol og i cal Method. n &!Ab~s~t,..r..k!a~c:..!<t~i~n~Am~e,..r.J>i~c'-"'a.....,n.___,.C...,hu.e~m...i~cu.a.-.1
Society. Pp. 169-170.
Butterfield, s. and Calloway, D.H. 1972. "Folacin in
Wheat and Selected Foods." JQurnal of the American
Dietetic Association, 60: 310-314.
Butterworth, C.E. 1968. "The Availability of Food
Folate." British Journal of Haematology, 14: 339343.
California State University, Northridge, 1983a. "Least
Squares Polynomials." Application library for RSTS/E
computer system (unpublished).
California State University, Northridge, 1983b. "One-Way
Analysis of Variance." Application library for
RSTS/E computer system (unpublished).
Cannon, F.L. 1983. "Overview: A Competitive Growth
Environment." California Wine Report. Pp. 1-16.
10~
Castor, J.G.B. 1953. "The B-Complex Vitamins of Musts
and Wines as Microbial Growth Factors." ~~
MicrQbiology, 1: 97-102.
Chen, T.S. 1979. Folic Acid Microbiological Assay Used
in the Food Science Laboratory, California State
University, Northridge. Unpublished.
Chen, T.S. 1983a. Folic Acid Microbiological Assay Used
in the Food Science Laboratory, California State
University, Northridge. Third Revision.
Unpublished.
Chen, T.S. 1983b. "Folacin Calculations." Computer
program for RSTS/E computer system at California
State University, Northridge (unpublished).
Chen, T.S. and Cooper, R.G. 1979. "Thermal Destruction
of Folacin: Effect of Ascorbic Acid, Oxygen and
Temperature." JQurnal Qf FQQd Science, 44: 713-716.
Chitwood, J. 1983. "Folacin Content of Beer." Masters
Thesis, California State University, Northridge.
Coleman, N. and Herbert, V. 1979. "Dietary Assessments
with Special Emphasis on Prevention of Folate
Deficiency." In: Folic Acig in Neurplogy,
FPY9hiatry, and Internal Medicine. Botez, M.I. and
Reynolds, E.H. (editors). New York: Raven Press.
Pp. 23-33.
Day, B.P.F. and Gregory, J.F. 1983. "Thermal Stability
of Folic Acid and 5-Methyltetrahydrofolic Acid in
Liquid Model Food Systems." Journal of Food Science,
48: 581-587.
Dong, F.M. and Oace, S.M. 1973. "Folate Distribution in
Fruit Juices.• JQyrnal of the American Dietetic
AssociatiQn, 62: 162-165.
Drawert, F. 1974. "The Chemistry of Winemaking as a
Biological-Technological Sequence." In: ~hemistry
Qf Winemaking. Webb, A.D. (editor). Washington,
D.C.: American Chemical Society (Advances in
Chemistry Series 137). Pp. 1-10.
Eigen, E. and Shockman, G.D. 1963. "The Folic Acid
Group." In: Analytical MicrobiQlogy, I. Kavanaugh,
F.G. (editor). New York: Academic Press. Pp. 431488.
102
Eschenbruch, E. 1974. •sulfite and Sulfide Formation
during Winemaking--A Review.• AID~~Journal of
~Q9Y ang Yjticulture, 25: 157-161.
Ferguson, G.A. 1966. Statistical Analysis iiLP~yQhQlQ~
an9 EducatiQn. Second edition. New York: McGrawHill Book Company. P. 406.
Folwell, R.J. and Dailey, R.T. 1971. •u.s. Table Wine
Consumption, State Regulations and Market
Potentials.• Amerigan JQurnal of Enolo2Y- and
Vitigulture, 22: 210-214.
Folwell, R.J., Hodges, D.K. and Dailey, R.T. 1974a.
•price, Income and Expenditure Relationships in Wine
Purchasing.• Amerigan JQurnal Qf ~nQlogy and
Viticulture, 25: 108-110.
Folwell, R.J., Dailey, R.T. and Lee, P.S.T. 1974b.
Market Projections and Shares in the u.s. Wine
Market.• Americ~n JQurnal Qf_En9logy and
Viticulture, 25: 73-78.
•wine
Fong, D.C., Amerine, M.A. and Ough, c.s. 1974. •citric
Acid in Some California Wines.• AID~i&9~~rnal of
Enology and_yj~j~~~~, 25: 222-224.
Food and Nutrition Board. 1968. Regoromended Dietary
hlJQwanges. 7th edition. Washington, D.C.:
National Research Council, National Academy of
Science.
Food and Nutrition Board. 1980. Regoromended Dl~~
AllQwanges. 9th edition. Washington, D.C.:
National Research Council, National Academy of
Science.
Gaines-Moss, R.E. 1984. •changes in Folacin Levels
during Commercial Production of Two California
Chardonnay Wines.• Masters thesis. California State
University, Northridge.
Gallander, J.F. 1974. •chemistry of Grapes and Other
Fruits as the Raw Materials Involved in Winemaking.•
In: Chemistry of Winemaking. Webb, A.D. (editor).
Washington, D.C.: American Chemical Society
(Advances in Chemistry Series 137). Pp. 11-49.
Gastineau, C.F., Darby, W.J., Turner, T.B. (editors).
1979. Fermented FQod Be~..~:_g~..s__in NutritiQn. New
York: Academic Press. Pp. 84-857 110-111.
103
Gomori, G. 1955. •preparation of Buffers for Use in
Enzyme Studies.• In: Methods in Enzymology, vol. 1.
Colonick, s. and Kaplan, N. (editors). New York:
Academic Press. Pp. 138-146.
Grossman, H.J. 1964. Grossm~~uide tQ Wines, Spirits
and Beers. 4th edition. New York: Charles
Scribner's Sons. Pp. 15-22.
Hagata, s. 1983. Enologist at Ahern Winery.
Communication.
Personal
Hall, A.P., Brinner, L., Amerine, M.A. and Morgan, A.F.
1956. •The B Vitamin Content of Grapes, Musts and
Wines.• Food ResearQb, 21: 362-371.
Hardinge, M.G. and Crooks, H. 1961. •Lesser Known
Vitamins in Foods.• Journ~l_Qf_the Aroei.jQg~~~~
AssogigtiQn, 38: 240-245.
Herbert, V. 1963. nA Palatable Diet for Producing
Experimental Folate Deficiency in Man.• ~be American
Journal of ~linical Nutrition, 12: 17-20.
Herbert, V. and Bertino, J.R.
1967.
•Folic Acid.• In:
Pgthology,
Methods. Gyorgy, P. and Pearson, W.N. (editors).
2nd edition. New York: Academic Press. Pp. 243376.
j.'_b~_Y.i.t.Mli~.._Ol.emil3try_,_.£b,y_p_ipl.Qgy,
Herbert, v., Fisher, R. and Koontz, B. 1961. •The Assay
and Nature of Folic Acid Activity in Human Serum."
Journal of Clinical Investigation, 40: 81-91.
Hurdle, A.D.F., Barton, D., and Searles, I.H. 1968.
Method of Measuring Folate in Food and Its
Applications to a Hospital Diet." The AroeriQan
J.QYI.D9l_.Qi~j~j~l Nutrition, 21: 1202-1207.
"A
Joslyn, M.A. 1955. "Yeast Autolysis I." Wallerstein
Laboratoriel3 Cornmunicgtions, 18: 107-121.
Keagy, P.M., Stokstad, E.L.R. and Fellers, D.A. 1975.
"Folacin Stability during Bread Processing and Family
Flour Storage.• Cereal CbemiRtLY, 52: 348.
Kirsch, A.J. 1983. •Evaluation of the Microbiological
Method for Folacin Determination in Foods: Effect of
Variations in Conjugase Treatment on the Folacin
Content of Spinach." Masters thesis. California
State University, Northridge.
104
Kirsch, A.J. and Chen, T.s. 1984. "Comparison of
Conjugase Treatment Procedures 1n the Microbiological
Assay for Food Folacin." Journgl of Food Scienge,
49: 94-98.
Kunkee, R.E. 1974. "Malo-Lactic Fermentation and
Winemaking." In: ~hemistry of Winemaking. Webb,
A.D. (editor). Washington, D.C.: American Chemical
Society (Advances in Chemistry Series 137). Pp. 151170.
Kunkee, R.E. and Amerine, M.A. 1970. "Yeasts in
Winemaking." In: The Yeast. Rose, A.H. and
Harrison, J.S. (editors). Volume 3. London and New
York: Academic Press. Pp. 6-71.
Lafon-Lafourcade, s. and Peynaud, E. 1958. "L'Acide pArninobenzoique, l'Acide Pterolglutamique et la
Choline (Vitamines du Groupe B) dans les Vins."
Annales d~ Technologie Agricole, 7: 303-309.
Laskowski, M., Mims, v. and Day, P.L. 1945. "Studies on
the Enzyme which Produces the Streptococcus lactis RStimulating Factor from Inactive Precursor Substance
in Yeast." JQJ,lrnal of__lli.Ql..Q.gjcal Chemistry, 157:
731-739.
MacKenzie, F.B., Noble, W.M. and Peppler, H.J. 1954.
"Production of Vitamins Other than Riboflavin." In:
Indystrial Fermentation~, vol. 2. ·underkofler, L.A.
and Hickey, R.J. (editors). New York: Chemical
Publishing Co., Inc. P. 200.
Malin, J.D. 1975. "Folic Acid." HQLld Review of
NutritiQn and Dietetics, 21: 198-223.
Martin, D.W. 1981. "Water Soluble Vitamins." In:
Hgrper•s Review of Bioch~~. California: Lange
Medical Publications. Pp. 110-111.
Mathews, J. 1958. "The Vitamin B Complex Content of
Bottled Swiss Grape Juices." Vitis, 2: 57-64.
Mitchell, H.K., Snell, E.E. and Williams, R.J. 1941.
"The Concentration of Folic Acid." Journgl of the
American Chemical Society, 63: 2284.
Nystrom, B. and Nystrom, c. 1967. "On the Biochemistry
of Folic Acid." h&~DD~~~~~ica Gynecologia
Scandinayig, 46: supplement 7: 89-100.
105
O'Broin, J.D., Temperley, I.J., Brown, J.P. and Scott,
J.M. 1975. "Nutritional Stability of Various
Naturally Occurring Monoglutamate Derivatives of
Folic Acid." The Amezjcan JQyrngl_Qi_Clinica1
Nutrition, 28: 438-444.
Ough,
c.s. 1966a. •Fermentation Rate of Grape Juice II.
Effect of Initial Brix, pH and Fermentation
Temperature." American Joyrna1 of Enology and
Viticulture, 17: 20-26.
Ough,
c.s. 1966b. "Fermentation Rates of Grape Juice
III. Effects of Initial Ethyl Alcohol, pH and
Fermentation Temperature.• American Journal of
~n9l99Y and Viticulture, 17: 74-81.
Ough,
c.s. and Amerine, M.A. 1966. "Effects of
Temperature on Wine Making." California ExpeJjm~ntal
Station Bulletin, 827: 1-36.
Paine-Wilson, B. and Chen, T.S. 1979. "Thermal
Destruction of Folacin: Effect of pH and Buffer
Ions.• Journal Qf FQog Science, 44: 717-722.
Paul, A.A. and Southgate, D.A.T. 1978. "Vitamines in
Composition of Foods.• In: ~Cance ang WiddowsQn's
The CQmposijJ~f Foods. 4th edition. Ministry of
Agriculture, Fisheries and Food and Medical Research
Council. London: H.M. Stationery Office.
Amsterdam, New York: Elsevier/North Holland and
Biomedical Press.
Per1off, B.P. and Butrum, R.R. 1977. "Folacin in
Selected Foods.• JQurnal Qf the-American~tetic
~ciati~, 70: 161-172.
Peynaud, E. and Lafourcade, s. 1957. "Les Vitamines B
dans le Raisin et dans le Vin.• ~ongres
International pQYr l'etu~ scientifigue du ~j~et du
B.Ai.ai.D__,__lW~.YX.
Pp. 6 5-7 0 •
Phaff, H.J. and Amerine, M.A. 1979. "Wine.• In:
MicrQpigl TecbnQlogy. 2nd edition, vol. 2. Peppler,
H.J. and Perlman, D. (editors). New York: Academic
Press. Pp. 131-153.
Rankine, B.C. 1972. "Influence of Yeast Strain and MaloLactic Fermentation on Composition and Quality of
Table Wines.• Arnerican_J~~nal of Eno1Qgy ana
Viticulture, 23: 152-158.
//
106
Rao, N.K. and Noronha, J.M. 1977. "Studies on the
Enzymic Hydrolysis of Polyglutamyl Folates by Chicken
Liver Folyl Poly-gamma-Glutamyl Carboxypeptidase. I.
Intracellular Localization, Purification and Partial
Characterization of the Enzyme." Biochimica et
Biophysipa Acta, 481: 594-607.
Rodriguez, M.S.
1978.
"A Conspectus of Research on
Folacin Requirements of Man."
Xb~-- Jo~JLal
of
Nutriti9Dr 108: 1983-2103.
Rose, A.H. 1980. "Recent Research on Industrially
Important Strains of ~bsrQmyQe9 cereyisiae." In:
BiolQgy and ActiYities of Yeasta. Skinner, F.A.,
Passmore, S.M. and Davenport, R.R. (editors).
London: Academic Press. Pp. 103-121.
Singh, K., Agarwal, P.N. and Peterson, W.H. 1948. "The
Influence of Aeration and Agitation on the Yield,
Protein and Vitamin Content of Food Yeasts."
~jy~R-Qf BiQchemistry, 18: 181-200.
Singleton, V.L. 1974. "Some Aspects of the Wooden
Container as a Factor in Wine Maturation." In:
Chemistry of Win~msting. Webb, A.D. (editor).
Washington, D.C.: American Chemical Society
(Advances in Chemistry Series 137). Pp. 254-277.
Standard and Poor's Industry Surveys. ~984. "Wine:
Growth Prospects Very Bright." New York: Standard
and Poor's Corporation. Vol. 1, pp. F32-F33.
Standard and Poor's Industry Surveys. 1982. "Wines:
Wine Market Unscathed by Recession." New York:
Standard and Poor's Corporation." Vol. 1, pp. B76B78.
Stokstad, E.L.R., Fordham, D. and DeGrunigen, A. 1947.
"The Inactivation of Pteroylglutamic Acid (Liver
LactQbapillus caaei Factor) by Light." Journal of
Biological Chemistry, 167: 877-878.
Stokstad, E.L.R. and Koch, J. 1967. "Folic Acid
Metabolism." PhysiolQgipal Reyiews, 47: 83-116.
Stokstad, E.L.R. and Thenen, s.w. 1972. "Chemical and
Biochemical Reactions of Folic Acid." In:
Analytical Microbiology, II. Kavanaugh, F.J.
(editor). New York: Academic Press. Pp. 387-408.
Streiff, R.B. 1971. "Folate Levels in Citrus and Other
Juices." 1\I.n~.Ij.Qp.D_J.Qy.mgl Qf Clinicsl Nutrition, 24:
1390-1392.
107
Tamura, T., Buehring, K.U. and Stokstad, E.L.R. 1972.
"Enzymatic Hydrolysis of Pteroylpolyglutamates in
Cabbage." Proce~gjngs of the S9~et~-fQL
~~iiD~D~9.l_sn9 BiolQgical Megicine, 141: 1022-1025.
Tamura, T. and Stkstad, E.L.R. 1973. "The Availability
of Food Folate in Man." »~jtjsh Journgl_Qf
Haematology, 25: 513-532.
Thoukis, G. 1974. "Chemistry of Wine Stabilization: A
Review." In: ChemiRtxy of Win~~king. Webb, A.D.
(editor). Washington, D.C.: American Chemical
Society (Advances in Chemistry Series 137). Pp. 116133.
Toennies, G., Usdin, E., and Phillips, P.M. 1956.
"Precursors of the Folic Acid-Active Factors of
Blood." Journal of piological Chemistry, 221: 855863.
Voigt, M.N., Eitenmiller, R.R., Powers, J.J. and Ware,
G.O. 1978. "Water Soluble Vitamin Content of Some
California Wines." Journal-Qf Food Science, 43:
1071-1073.
APPENDIX A
PREPARATION OF BUFFER SOLUTIONS
A.l.
Sodium Phosphate Buffer
A 0.05 M sodium phosphate buffer (pH 6.1) was
prepared by dissolving and diluting:
6.792 g monobasic sodium phosphate, anhydrous
(NaH PO )
2
4
2.687 g dibasic sodium phosphate, anhydrous (Na HPO )
2
4
with sterile deionized water to a volume of 1 liter in a
1-1 volumetric flask.
A.2.
Ascorbate Sodium Phosphate Buffer·
Ascorbate sodium phosphate buffer solution was
prepared by adding ascorbic acid to the prepared 0.05 M
sodium phosphate buffer solution (Appendix A.l).
The
ascorbic acid was added to the buffer just prior to use to
obtain a final concentration of 0.15 percent.
A.3.
Sodium Acetate Buffer
Walpole's sodium acetate buffer solution was prepared
according to Gomori (1955).
The method of preparation was
as follows:
108
109
Stock solutions
1.
Solution A:
0.2 M solution of acetic acid
(11.55 ml of glacial acetic acid diluted to 1 liter with
sterile deionized water).
2.
Solution B:
0.2 M solution of sodium acetate
(27.2 g of NaC H 0 3H 0 dissolved in and diluted to 1 1
2 3 2
2
with sterile deionized water).
Buffer composition.
Stock solutions A and B were
combined to obtain a buffer of specific pH using the
formula x ml A + y ml B, diluted to 1 liter with deionized
water.
x
A.4.
For pH 4.7 buffer, the x andy values are
=
230 ml and y
=
270 ml
Cysteine Hydrochloride Buffer Solution
A 0.32% cysteine hydrochloride buffer solution was
prepared by dissolving 2.245 g of cysteine hydrochloride
into 700 ml sterile deionized water.
adjusted with 1.0 N NaOH to pH 5.4.
The solution was
0 '
APPENDIX B
PREPARATION OF CHEMICAL SOLUTIONS AND CULTURE MEDIA
B.l.
Culture Maintenance Medium
Lactobacillus gasei was maintained in stabs of agar
medium which was prepared by suspending the following
ingredients in 200 ml distilled water:
Difco yeast extract
2 g
D-glucose
1 g
Difco Bacto-agar
3 g
Sodium acetate
1 g
The medium was heated with constant
s~irring
until clear.
Ten milliliters of medium were distributed into culture
tubes, capped and autoclaved at 121°C, 15 p.s.i. for 15
minutes.
The tubes were cooled in an upright position
until solidified and were stored in the refrigerator at 4°
c.
B.2.
Inoculum Broth
On the day prior to assay, L. casei was subcultured
by transferring the cells from the maintenance stab medium
to inoculum broth.
The broth was prepared by pipetting
the following solutions into sterile culture tubes:
110
111
Standard folic acid solution C (see B.5.3)
2.0 ml
Sodium phosphate ascorbate buffer (see A.2)
0.5 ml
Basal medium (double strength) (see B.4)
2.5 ml
The inoculum broth was prepared quantitatively and
pipetted in 5 ml aliquots into sterile culture tubes.
The
tubes were sealed with aluminum foil, autoclaved at 121oc,
15 p.s.i. for 5 minutes and stored in a freezer at -22°C
until use.
B.3.
Each tube contained 1 ng of folic acid.
Sterile Saline Solution
Sterile saline solution was prepared by dissolving
0.9 grams sodium chloride in 100 ml sterile deionized
water and autoclaving for 15 minutes at 121°C, 15 p.s.i.
B.4.
Basal Medium {double strength)
The dehydrated Bacto Folic Acid medium was obtained
from Difco Laboratories, Detroit, Michigan.
The medium
was formulated as follows:
Charcoal treated casitone
10 g
Bacto-dextrose
40 g
Sodium acetate
40 g
Dipotassium phosphate
1 g
Monopotassium phosphate
1 g
D-L Tryptophane
0.2 g
L-Asparagine
0.6 g
L-Cysteine hydrochloride
0.5 g
Adenine sulfate
10 mg
112
Guanine hydrochloride
10 mg
Uracil
10 mg
Xanthine
20 mg
Tween 80
0.1 g
Glutathione (reduced)
5 mg
Magnesium sulfate
0.4 g
Sodium chloride, USP
20 mg
Ferrous sulfate
20 mg
Manganese sulfate
15 mg
Riboflavin
1 mg
p-Aminobenzoic acid
2 mg
Pyridoxine hydrochloride
4 mg
Thiamine hydrochloride
400 Jlg
Calcium pantothenate
800 Jlg
Nicotinic acid
800 Jlg
Biotin
20 Jlg
The medium was rehydrated with sterile deionized water the
day before or the day of assay.
The medium was prepared
by suspending 9.4 g of the rehydrated powder in 100 ml of
sterile deionized water.
The mixture was heated to
boiling for 1 minute on a magnetic stirrer/hot plate and
autoclaved for 5 minutes at 12l°C, 15 p.s.i.
The solution
was cooled and stored in the refrigerator until use.
B.S.
Standard Folic Acid Solution
Three different standard folic acid solutions A, B,
and C were prepared:
113
1.
Folic Acid stock solution, solution A (25
~g,
1 ml)
Twenty five milligrams of folic acid crystalline
(INC. Pharmaceuticals Inc., Life Sciences Group,
Cleveland, Ohio) were weighed out precisely on an
analytical balance, transferred to a 1-1 volumetric flask
and dissolved in 100 ml of sterile 0.01 N NaOH solution
containing 20% ethanol (To prepare NaOH (0.01 N) with 20%
ethanol solution, 210 ml of 95% ethanol were combined with
12.5 ml of 0.8 N NaOH and sterile deionized water to 1
liter.).
Additional 0.01 N NaOH with 20% ethanol was used
to bring up the volume to 1 liter.
The solution was
distributed in 5 ml aliquots into sterile test tubes.
Nitrogen gas was bubbled through the solution to chase out
oxygen in order to minimize oxidative degradation.
tubes were immediately sealed with
para~fin
The
film, wrapped
in foil and stored frozen at -22°C until use.
2.
Folic acid working solution, solution B (25 ng/ml)
This solution was prepared by diluting precisely 1.0
ml of solution A to 1 liter with sterile 0.01 N NaOH
solution containing 20% ethanol.
The solution was
distributed in 5 ml aliquots into sterile test tubes.
The
tubes were immediately flushed with nitrogen gas, sealed
with paraffin film, wrapped in aluminum foil and stored in
a freezer at -22°C until use.
114
3.
Folic acid assay solution, solution C (0.5 ng/ml)
The day of the assay solution C was freshly prepared
just prior to use.
It was prepared by precisely pipetting
2.0 ml of solution B into a sterile 100 ml volumetric
flask and diluting to volume with sterile 0.05 M sodium
phosphate buffer containing 0.15% ascorbic acid.
B.6.
1.
Conjugase Solutions
Chicken pancrease conjugase solution.
Commercial Difco chicken pancreas, purchased from
Difco Laboratories, Detroit, Michigan, was used in the
preparation of the conjugase solution.
A suspension was
prepared by dispersing 60 mg of chicken pancreas powder
into 20 ml of sterile deionized water with constant
swirling for 10 minutes.
The suspension was centrifuged
at 1000 x g for 10 minutes in a refrigerated centrifuge at
-5°C.
Approximately 5 ml of the supernatant were decanted
into sterile test tubes, capped with paraffin film and
stored in a freezer at -22°C (Kirsch, 1983).
Just before
use, one test tube was defrosted in a waterbath in the
refrigerator.
2.
Fresh hog kidney conjugase solution.
Hog kidney conjugase was prepared from fresh hog
kidneys according to the method of Eigen and Shockman
(1963).
A flow chart of the steps involved in the
procedure is shown on page 118.
llS
Fresh hog kidney was defatted, chopped and then
homogenized in sterile 0.32% cysteine hydrochloride
solution (Appendix A.4).
The suspension was poured into a
1-1 Erlenmeyer flask and layered with toluene.
The flask
was sealed with paraffin film and the contents were
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 again
centrifuged.
The supernatant was decanted, adjusted to
pH 4.5 with 1 N 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 in order to
remove contaminating folate.
The slurr¥ was centrifuged
and the clear brownish red supernatant was collected.
The conjugase preparation was further purified using
gel chromatography as suggested by Chen (1979).
A pyrex
chromatography column was packed to a height of 42 ern with
Sephadex G-25 gel suspension.
The solvent was allowed to
drain out until the level of the solvent was just barely
above the surface of the gel.
Approximately 20 rnl 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 rnl per minute.
As soon as the conjugase
116
solution level had dropped to 1 rom 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 maintained
throughout the entire column chromatographic process.
The
enzyme fraction was brown and passed quickly through the
column.
The folic acid fraction was yellow and moved
slowly down the column.
The enzyme fraction was collected
and stored frozen in sealed test tubes at -22°C until used
(Bird~
gl., 1945).
117
Diagram.
Schematic diagram of the steps
in the preparation and purification
of fresh hog kidney conjugase.
Reprinted from Kirsch (1983).
CRUOE ENZniE 11REPARATION
C~ntrlfuge flltrAt~
Defat a'1d
chop 200 g
fr~sh hog
f-
kldn~y
Co11ect
supernatant
and store
I--
IIOIIOCJl!n lze In
f----600 ~1 0. 321
cyst~ln~ hydrochlorld!
at pH 5.4
Centrifuge at
2000 x 9 for
JO 11lnutes at
OOC (4500 rpw)
f-.--
Auto lyre
und~r tolu~ne
f--.-
for 2 hours
at J70C
Add 30 9 Oowex
I-X8 (chlorld~ fonm)
to supernatant.
Place In Ice water bath f--and stir occasslonally
for I hour
lllscard
foa~ and fat,
rt 1t~r through
glasswool
Adjust
supernatant
to pit 4. 5
with IN HCI
~
r--
at 1000 x g for
20 ~lnut~s at one II
(approx. JOQO rpw I
on a JS7.5 rotor; j
hc1t1111n J21 c~ntrffuqe
'
RI!IIIOve rat and
at
4000 x q for
JO 11lnutes at OOC
(about 6000 rpM)
re-centrlfug~
PUR IF I CATION 8Y GEL CIIR(JfATOGRAPIIY
Set up Pyr~x
chromatography coltJnn
1---
Suspend 20 g
Sephade• G- 25 powder
In O. IH sodllllll ac~tate
buff~
pH 4 7
contalnlngrO.Z% a~c~rbate
t---
Pour gel
Into coiiJIII'I and
let g~l settl~-top should b~ flat
I---
Let solv~nt drain out
until solv~nt lev~ I
Is just bar~ly
abov~ th~ 9el
1
1
Collect brown entyll'll!
fract ton and Hore.
Discard yellow
folic acid eluate.
Just prior to when the crude
enzyme solution has completely
f"ntered the gel, carefully add
~ enough sodl1111 acetate buffer
to create a I Inch head above
the surface of the gel.
Maintain this head through
entln~ purification proct>ss.
1
1---
Adjust out let to
nbtaln an effluent rate
of aprro•llllltely
1 ml rer minute
f-.-
I
Carefully pour 20 ml
I
ilpproxllllltely 2 lnchi'!O helqht l1
1
crude enzyme ~olutlon
Into column.
Do not disturb
flat surfare of qel.
1-'
1-'
00