CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
VITAMIN B-12 CONTENT OF BEERS
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Home Economics
by
Mark Stuart Meskin
May, 1983
The Thesis of Mark Stuart Meskin is approved;
Tung-Shan Chen, Ph.D.
Christine Hamilton Smith, Ph.D.
Chairperson
California State University, Northridge
ii
DEDICATION
For my mother, whom I often miss,
DIANE BANGHART MESKIN
1933-1981
She taught me the joy of learning
and was my greatest teacher
iii
ACKNOWLEDGMENTS
The completion of this thesis represents only a small
part of a much longer process of growth and maturation.
I have not only acquired a competent working knowledge of
basic nutrition, I have also developed the skills and selfconfidence to teach nutrition and to exchange ideas with
experts in my field.
The completion of this degree could
not have happened without excellent teachers and without
the care, love, cooperation and encouragement of my friends
and family.
I must begin by thanking my wife, Ilana.
The thesis
itself could not have been completed without her excellent
typing and editorial skills but her influence was much more
profound than the physical product she helped to produce.
She gave me the love, freedom and time to complete my degree in my own way and at my own pace.
She was my friend,
confidante, cheerleader and therapist.
I plan to colla-
borate with her for many years to come.
I am deeply
~ndebted
and grateful to Dr. Christine H.
Smith, my committee chairperson, my teacher and my friend.
She has been involved with my education from my first day
at CSUN through the completion of this thesis.
She is re-
sponsiblefor much of what I know about nutrition and I hope
iv
to continue to learn from her in the future.
I thank her
for her wisdom, keen insight and the many precious hours
of time she has given me.
I would like to thank Dr. Tung-Shan Chen for his advice and participation in this study.
My laboratory
skills were developed in his chemistry courses and the
environment he created in the food chemistry laboratory
made graduate study enjoyable.
The combination of his
keen critical abilities and his knowledge of microbiological
vitamin assays proved invaluable in discovering
flaws and solving problems in this study.
Special thanks go to my friend Arlene Kirsch who
constantly shared her time and knowledge with me in the
laboratory.
She helped solve many of the critical day to
day problems that emerged during the hours of laboratory
work involved in this study.
I am grateful to Dr. Margaret Holzer for reading this
thesis and for serving as a member of my committee.
Finally, I would like to thank my father, Mort Meskin,
and my brother, Joel Meskin, for their love, encouragement
and unconditional support throughout my education.
v
TABLE OF CONTENTS
APPROVAL PAGE • • . .. • . • . • . . • . . • . • . • • . . • . • . • • • • • • • • • .
ii
DEDICATION • • •.. • • • • • . • . • . • . • • • . . . • . . . • . . . • .. • . • • • • •
iii
ACKNOWLEDGMENTS . • • • . • • • . . . • . • . • • • . • • . . • . • • • . • . • • •
iv
LIST OF TABLES • . • • • • • • . • . • . • • • . • . • • • . • . • . • • • • • . • •
ix
LIST OF FIGURES ••••.•.•••••.•..•.••.•.•••••.•..••
xi
ABSTRACT
•• ·- • • • • • . • • • • • .• • • • • • • • . • . • • • • • • • • • • • • • • • •
xi i
CHAPTER 1.
INTRODUCTION .••..•.•••..••.••.•••..•••
1
CHAPTER 2.
REVIEW OF LITERATURE ••••...••.••.••••
7
Vitamin B-12 (Cyanocobalamin)
. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
Structure and Terminology •.••.•.•••••.•
8
. .. . . . . . . . . .. . . . . . . . . . . . . .. ...
11
Metabolism . . . . . . . . . . . . . . . . . . ... .. . . . . . . . .
12
Vitamin B-12 Deficiency: Causes
and Occurrence • . . • . • • . • • . • . • • • • • . • • •
14
Recommended Dietary Intakes •.•••••.••..
16
. . . . . . . . . . . . . . . . . . . . . .. . .. .
18
History
Chemistry
~
Food Sources
Vitamin B-12 Nutriture of Vegans
Beer
7
20
Non-meat Product Sources of
Vitamin B-12....................
22
Human Ability to Store Vitamin B-12
26
High Folacin Intake in Vegans
26
Serum Vitamin B-12 Levels in
Vegetarians ••..••.•••••••.•••••
27
Assay Methods • . • • • • • • • • . • • • • • . • . • • • . • • •
28
. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
32
vi
Historical Background of Beer..........
32
The Brewing Process •...•••••.•.•••.•••.
33
Types of Beer
. . • . . . . • . • . • . • • • . • . ..• . . • . •
36
Lager Beer •.•.••.••••.•.•.•.•.•.••
36
Dark Beer •.•.••..••••.••.•.•.•••••
36
Low-Calorie (Light) Beer..........
37
Ale . . • .. . . . . . . . . • . . . • . . . . . . . . . .. . . . .
37
Malt Liquor ••.. ~..................
37
Porter . . . . . . . . . . . . . . . . . . . . . . . . . . . .
38
Stout . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
38
Consumption of Beer....................
38
Nutritive Value of Beer
39
MATERIALS AND METHODS
44
CHAPTER 3.
. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .
44
Beer Samples . . . . . . . . . . . . . . . . . . . . . . . . . . .
44
Assay Microorganism....................
54
Chemical Reagents and
Microbiological Media
54
Glassware Maintenance ••..••.•.•••••.•••
54
. . . .. . . . . . . . . . . . . . . . . . . . . . . . . .
55
••••••••. ••. •••••. ••••••. ••••••. •••••
57
Sample Preparation ••.•••••.•••.•••.••••
57
Microbiological Assay for Vitamin B-12 •
59
Maintenance of Oahromonas
malhamensis ••....•••.•..••.••••
59
Assay Medium . • . • • • . • . . . • . • . • . • . • • •
60
Inocul urn . . . . . . . . . . . . . . • . . . . . . . . • . .
61
Materials
Equipment
~~tlloCls
vii
Standard Curve Preparation .•••••••
61
Sample Flask Preparation..........
62
Sterilization, Inoculation,
and Incubation •..•••••.•.••••••
62
Absorbance Measurement ••.•••.•••••
66
Treatment of Data •.••••••••..•••.•.••.•
67
Standard Curve Construction
67
Calculation of Vitamin B-12
Concentration in Beer Samples
67
Statistical Analysis ••..••••••.•••
67
RESULTS AND DISCUSSION...............
70
Vitamin B-12 Content of Different Brands
of Beer Within Each Beer Type ••..••••.•••
70
CHAPTER 4.
Domestic Lager Beer
70
Imported Lager Beer
75
Dark Beer .•...•.•....•. ·-·..............
77
Low-Calorie (Light) Beer ..••.•••••••.••
77
Ale . . . . . . . . . . . . . • . • . . . . . • . . . . . . . . . • . . . .
80
Malt Liquor . . • . . . . . . . . . . . . . . . . . . . . . . . . .
82
Porter . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . .
82
Stout . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . .
82
Vitamin B-12 Content of Eight Types of Beer •
85
CHAPTER 5.
CONCLUSION AND RECOMMENDATIONS •••••••
91
LITERATURE CITED ..•••...•.•••.•.•.•••.•••.•••••••
95
. . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . .
102
APPENDICES
viii
LIST OF TABLES
Table
Page
1.
Recommended Daily Intake of Vitamin B-12 ••.•
17
2.
Some Microorganisms Known to Be
Able to Synthesize Vitamin B-12
19
.........•.
3.
Common Food Sources of Vitamin B-12
21
4.
Non-Meat Product Sources of Vitamin B-12
23
5.
Growth Response of Various Assay Microorganisms to Clinically Active Molecules
("True B-12") vs. Clinically Inactive
Molecules (Analogs With Little or No
Activity) ••••••••••.•.••••..•••••..••••••
31
Selected Nutrients in Lager Beer
Composition per 355 ml (12 oz.)
41
7.
Vitamin B-12 and Alcohol Content of Beers •••
43
8.
Domestic Lager Beers
46
9.
Imported Lager Beers
6.
.. . .. .. .. . . .... . . .. .. . . .
47
10.
Dark Beers..................................
48
11.
Low-Calorie (Light) Beer •••••.•••.••.••.••••
49
12 .
Ale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
50
13.
Malt Liquor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
51
14.
Porter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
52
15.
Stout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
53
16.
Protocol of Vitamin B-12 Standard Curve
63
17.
Protocol of Vitamin B-12 Assay Flasks
for Beer Samples •.•••••.•••••••.•••.•••••
64
Vitamin B-12 Content of Domestic
Lager Beers •.•••••••.•.•.•.•.••••..•.••••
71
Vitamin B-12 Content of Imported
Lager Beers ••..•••.••.••.•••••••.•••.•..•
76
18.
19.
ix
Table
Page
20.
Vitamin B-12 Content of Dark Beers .•..••••••
78
21.
Vitamin B-12 Content of Low-Calorie
(Light) Beers •••••.••.••••.••.•.•••••••••
79
. . . . . . . . .. . . ....
81
22.
Vitamin B-12 Content of Ales
23.
Vitamin B-12 Contents of Malt Liquors
83
24.
Vitamin B-12 Content
84
25.
Vitamin B-12 Content
26.
Average Vitamin B-12
.......
of Porters . . . . . . . . . . . . .
of Stouts . . . . . . . . . . . . . .
Content of Beers . . . . . . .
X
86
88
LIST OF FIGURES
Page
Figure
1.
2.
3.
Structure of Vitamin B-12
(Cyanocobalamin) • . • . • • • . • • • • • • • . • . • . • • . • . •
9
Schematic Outline of Vitamin B-12
Assay Procedure . . . . . . . . . . . . . . . . . . . . . . . . . .
58
Typical Vitamin B-12 Standard Curve.........
68
.;
xi
ABSTRACT
VITAMIN B-12 CONTENT OF: ',BEERS
by
Mark Stuart Meskin
Master of Science in Home Economics
The vitamin B-12 content of beers regularly available
in local retail stores was determined.
brands of beer were assayed.
Eight types and 58
A microbiological assay em-
ploying the protozoan Ochromonas malhamensis was utilized
because it is the most sensitive and specific way to measure the metabolically and clinically active forms of vitamin B-12 in foods.
Vegetable products are not likely
sources of vitamin B-12 and researchers have largely ignored the assay of these foods for vitamin B-12.
The re-
sults of this study demonstrate that at least one vegetable
product, beer, contains vitamin B-12.
amounts of vitamin
B~l2
Small, measurable
were found in all beers assayed.
The average values for vitamin B-12 content of beers ranged from a low of 38.3 ng per 355 ml (one 12-ounce bottle)
in low-calorie beers to a high of 94.0 ng per 355 ml in
xii
dark beers.
The mean value for all beers assayed was 67.8
ng per 355 ml.
A number of individual brands of beer con-
tained at least 100 ng of vitamin B-12 per 355 ml.
The
data showed that there were significant differences among
the various brands of beer in each category but there were
not significant differences, in general, among the categories.
These data indicate that the most important fac-
tor in determining the vitamin B-12 content of a beer in
this study is the manufacturer and not the actual category
in which the beer is found.
Animal products are the best
sources of vitamin B-12 but for people who do not regularly consume these products, beer could contribute small but
significant amounts of vitamin B-12 to their diets as part
of a well-balanced diet.
xiii
Chapter 1
INTRODUCTION
Vitamin B-12 or cyanocobalamin is a water soluble B
complex vitamin essential for the adequate functioning of
all mammalian cells.
The dominant forms of vitamin B-12 in
mammalian tissues are coenzyme B-12
~min)
(5'-deoxyadenosylcobal-
and methylcobalamin (methyl-B-12)
and Jacob, 1980).
(Herbert, Colman
These are the two vitamin B-12 coenzymes
known to be active in humans.
As a component of these en-
zymes, vitamin B-12 plays a vital role in nucleic acid formation and in fat and carbohydrate metabolism.
It is also
involved in the synthesis of methionine and possibly other
aspects of protein metabolism.
quired for the synthesis of DNA.
With folacin, it is reThe neurological damage
that occurs with vitamin B-12 deficiency appears to indicate that vitamin B-12 participates in the synthesis of
myelin.
Microorganisms synthesize all of the vitamin B-12
found in natural foods (Smith; 1965).
1
Vitamin B-12 pro-
2
ducing micro9rganisms are highly active in the rumen of
ruminant animals making the rumen and other organ meats of
these animals the best sources of this vitamin for humans
(Orr, 1969).
Fruits, vegetables, nuts, seeds and grains
do not typically provide a suitable environment for vitamin B-12 producing microorganisms and vitamin B-12 is
only found in these products when they are contaminated by
soil or fecal matter.
Individuals or groups of people who
regularly exclude animals and animal by-products from their
diets have the potential problem of being unable to obtain
enough vitamin B-12 from dietary sources.
The majority of the world's population subsists on a
vegetarian or semi-vegeta-rian diet (American Dietetic
Association, 1980).
In recent years the number of Ameri-
cans choosing to eat a vegetarian diet for economic, ecologic~
philosophic, religious, health, and/or other rea-
sons has grown rapidly.
A significant number of these
vegetarians are vegans or total vegetarians.
Logically,
vegans would be at high risk of developing vitamin B-12
deficiency disease and most medical and nutrition textbooks present this risk as an inevitable outcome of choosing such a diet if no supplemental vitamin B-12 accompanies the diet.
In a review of the most often quoted case
studies of vitamin B-12 deficiency disease which attribute
the disease to a lack of vitamin B-12 in the diet, Meskin
(1981) concluded that the evidence cited in almost all
cases did not support such an attribution.
In fact,
it
3
seems that in relationship to the large numbers of vegetarians and vegans in the world, the incidence of vitamin
B-12 deficiency disease is quite low.
A number of possible answers to this paradoxical situation include: the ability to store large quantities of
vitamin B-12 in the liver (Herbert, Colman and Jacob,
1980); an almost perfect reabsorption mechanism for recovering vitamin B-12 excreted in the
b~le
(Herbert, Col-
man and Jacob, 1980); the presence of vitamin B-12 producing microorganisms in the small intestine (Albert,
Mathan and Baker, 1980); and, the possibility of oral,
tonsillar and pharyngeal vitamin B-12 producing bacteria
(Thrash, 1979).
It might also be possible that people do
obtain some vitamin B-12 from vegetable foods.
As previously noted, vegetable foods are not likely
sources of vitamin B-12 and researchers have largely ignored the assay of these foods for vitamin B-12.
Orr
(1969) has done some work in this area but the Nutrient
Data Research Group of the United States Department of
Agriculture recently reported that almost no data are
available for vitamin B-12 content of any foods
(Stewart,
1981).
The Recommended Dietary Allowances (RDA) (Food and
Nutrition Board, 1980) and the Food and Agriculture Organization/World Health Organization (FAO/WHO)
(l970) recom-
mend a daily intake of 3 and 2 pg of vitamin B-12, respectively, but Herbert (1968) suggests that 0.1 pg/day
4
will sustain normality in a normal subject.
There are
indications that although vegetable foods may be poor
sources of vitamin B-12, they may provide quantities
large enough to meet daily requirements Of 0.1 pg/day.
Abdulla et aZ.
(1981) reported a daily intake of 0.3
to 0.4 pg/day of vitamin B-12 from a strict vegan diet.
Fermented vegetable foods are more likely to provide a
suitable environment for vitamin B-12 producing
organisms than non-fermented vegetable foods.
micro~
Vitamin
B-12 has been found in tempeh (Liem, Steinkraus, and
Cronk, 1977), a fermented soybean product; kimchi
(Ro,
Woodburn and Sandine, 1979), a fermented cabbage product;
and California wines
(Voigt et aZ., 1978).
The first
three foods are of interest but are not widely consumed
in the United States.
The amounts of vitamin B-12 in
California red wines ranged from 20-50 ng/250 ml with an
average of 35 ng/250 ml.
White wines had significantly
less vitamin B-12 than the red wines.
The most popular fermented vegetable product in the
United States is beer.
Darby (1979) reports that there is
very little information available on the nutrient composition of beers and that tables of nutrient composition for
different varieties of beers are needed.
Darby (1979) did
report a vitamin B-12 value of 5 pg/1 or 1.77 pg/355 ml
(12 oz.} bottle of beer.
This value was based on a single
determination carried out by members of the U.S. Brewers
Association for the Food Data Bank of the Consumer and
5
Food Economics Institute of the U.S.D.A.
A single 12-
ounce bottle of beer containing vitamin B-12 at this level
could provide 59 percent of the RDA and 88 percent of the
FAO/WHO daily recommendations.
Paul and Southgate (1978) .
list values for ten types of beer ranging in vitamin B-12
content from 0.11 pg per 100 ml to 0.37 pg per 100 ml with
an average of 0.166 pg per 100 ml.
These values corres-
pond to a range of 0.385 pg per 355 ml (12 oz.) bottle of
beer to 1.295 pg per 355 ml (12 oz.) with an average of
0.581 pg per 355 ml (12 oz.) bottle of beer.
At these
levels, a single 12-ounce bottle of beer could provide
from 13 to 43 percent of the RDA and 19 to 65 percent of
the FAO/WHO daily recommendations.
Vitamin B-12 found at
these levels in beer could be very significant because a$
Roe (1979) indicates
11
beer has been the alcoholic beverage
which contributed most in the way of nutrients as well as
energy to the diet in the United Kingdom, and most probably in the United States 11
•
The assay technique used by the Brewers Association
was not indicated.
Paul and Southgate (1978) reported that
their values were obtained by microbiological assay with
Lactobacillus leichmanni (ATCC 7830).
As will be discussed
in Chapter 2, a number of the available assay techniques,
including assay with Lactobacillus leichmanni, can overestimate the amount of clinically active vitamin
foods.
B-12 in
It is important to verify reported values such as
these using a sensitive and specific assay.
The use of a
6
protozoan assay method. utilizing Ochromonas malhamensis in
this study attempts to provide such verification.
Throughout history and continuing today, beer has
been an important source of nutrients in the diets of peoples around the world (Steinkraus, 1979).
This phenomenon
is especially true of rural populations in developing
countries.
Beer consumption in developed countries is al-
so high (Katz, 1979) but the available nutrients, other
than kilocalories, are often restricted due to the filtration and clarification processes used in the production of
beer.
There is still reason to believe that beers
m~y
contain small but significant amounts of vitamin B-12 and
that this information would be a valuable addition to food
composition tables.
The purpose of this study was to determine the vitamin B-12 content of beers regularly available in local retail stores.
Eight types and 58 brands of beer were as-
sayed for their vitamin B-12 content.
A microbiological
assay employing the protozoan Ochromonas malhamensis was
utilized because it is the most sensitive and specific way
to measure metabolically and clinically active forms of
vitamin B-12 in foods
(Baker and Frank, 1968).
Chapter 2
REVIEW OF LITERATURE
Vitamin B-12 (Cyanocobalamin)
History
The first description of the anemia caused by a deficiency of vitamin B-12 was given by Combe in 1824 (Herbert, 1976).
He attributed the anemia to a malfunction
of the digestive system which interfered with digestion
and absorption of the vitamin and eventually led to death.
The imminence of death after the onset of this anemia led
to its common name, "pernicious anemia".
Over 100 years
later, Minot and Murphy (1926) discovered that the intake
of large amounts of liver could cure pernicious anemia.
It then took more than twenty years to isolate the actual
vitamin.
Researchers at two independent laboratories,
Rickes et al.
(1948) in the United States and Smith and
Parker (1948) in England, simultaneously isolated crystallized B-12.
In an effort that eventually won her the 1964
Nobel Prize for Chemistry, Dorothy Hodgkin used X-ray
7
8
crystallographic analyses to delineate and define the
structure of vitamin B-12 in 1957 (Herbert, 1976).
The
first full chemical synthesis of vitamin B-12 was achieved
in 1973 but with 70 steps involved it was not a practical
industrial tool (Florent and Ninet,
1979) •
Today vitamin
B-12 is produced by bacterial fermentation processes.
Structure and Terminology
The structure of vitamin B-12 (cyanocobalamin) is
shown in Figure 1.
The major distinguishing features of
the vitamin B-12 molecule are the presence of cobalt and
the corrin ring system which surrounds the cobalt.
The
corrin ring system consists of four reduced and highly
substituted pyrrole rings encircling a single cobalt atom
(Harper, 1979).
The corrin ring is similar to the por-
phyrin ring of chlorophyll.
All compounds which contain
the corrin nucleus are referred to as corrinoids (Fasman,
1976).
All corrinoids are chemically related to vitamin
B-12 because of the corrin nucleus but only those with a
biological activity qualitatively equivalent to cyanocobalamin should be termed vitamin B-12.
The remaining
major part of the molecule is a nucleotide, 5,6 dimethylbenzimidazole, which is attached to ribose by an alphaglycoside linkage (Harper, 1979).
These two parts of the
molecule are attached in two places.
One of the nitrogen
atoms of the nucleotide is joined to the central cobalt
atom.
The other nitrogen atom is attached to ring D (See
9
CH
~H
c
Corrin
ring
system
3
CH 3
CH:l.
I
CH:l
I
CH 3
C=O
I
I
CH-CH -NH
'
o...'
4
oe .--- ------------,
,j.
/
p'\.
.
N
I
lp~
:I:'
?" ~
"' CH3 I:
0 : . HO!.r-: _____ 1__ ~--:~gj
5,6-Dimethylbenzimidazole
H H
Figure 1.
Structure of Vitamin B-12 (Cyanocobalamin)
10
Figure 1) of the tetrapyrrole nucleus through phosphate
and D-1-amino-2-propanol.
Finally, cyanide is coordi-
nately bound to the central cobalt atom (Harper, 1979).
Cyanide is present in the molecule due to contamination
from reagents used in the isolation procedure (Stadtman,
1971).
Without the cyanide, the molecule is called cobal-
amin.
Cyanocobalamin is not the form of the vitamin that
occurs in nature but it is far more stable than other
natural cobalamins and it does have clinical activity
against pernicious anemia.
The Commission on Biochemical
Nomenclature of the International Union of Pure and Applied Chemistry (IUPAC)
(1966) states that the permissive
name for vitamin B-12 is cyanocobalamin and that the words
vitamin B-12 without qualification mean cyanocobalamin
exclusively.
Unfortunately, vitamin B-12 is usually used
in reference to all cobalamins active in humans.
The
permissive term for vitamin B-12 without the cyanide group
is cobalamin.
The cyanide group can be replaced by other
ions or groups to form other cobalamins.
These cobalamins
are named by using the name of the replacement group as a
prefix to the word cobalamin.
Substitution of the cyanide
group by a hydroxy group, nitro group or methyl group
yields hydroxocobalamin (vitamin B-12a), nitritocobalamin
(vitamin B-12c), or methylcobalamin, respectively.
All
three of these derivatives have clinical activity similar
to cyanocobalamin.
11
The cyanide group is not the only group that can
vary.
The nucleotide or 5,6 dimethylbenzimidazole can be
replaced by other nitrogenous bases (Baker and Frank,
1968).
Cobalamins containing adenine {pseudovitamin
B-12~
2-methyladenine (Factor A), and guanine (Factor C) are
found in natural sources.
Factor B is a cobalamin that
contains no nucleotide base.
~ty
These cobalamins have activ-
for some microorganisms that utilize vitamin B-12
but they have no clinical activity against pernicious
anemia in humans.
Two additional terms need definition, coenzyme B-12
and vitamin B-12 coenzyme.
Coenzyme B-12 only refers to
5'-deoxyadenosylcobalamin.
Vitamin B-12 coenzyme can be
applied to any coenzyme form of vitamin B-12.
The major
vitamin B-12 coenzymes in mammalian tissues, and the only
ones known to act as specific coenzymes in humans are coenzyme B-12 and methylcobalamin (methyl-B-12).
Chemistry
The empirical formula for cyanocobalamin is c 63 H co88
N14o14P and its molecular weight is 1355.42.
dark red, odorless crystals.
The crystals are hygroscopic
and can absorb up to 12 percent water.
tals are stable to air.
water.
It forms
The hydrated crys-
One gram dissolves in 80 ml of
It is also soluble in alcohol but is insoluble in
organic solvents such as chloroform, actone and ether.
is relatively stable in solution at pH values between 4
It
12
and 7.
Its maximum stability in solution is at pH 4.5 to
5 and within this range it can be autoclaved for 20 minutes at 121°C.
It decomposes quickly at pH 2 or pH 9.
It is also inactivated and rapidly decomposed by heavy
metals, strong oxidizing agents and reducing agents.
It
is moderately sensitive to ultraviolet or strong visible
light (Windholz, 1976; Fasman, 1976; Baker and Frank,
1968).
Metabolism
There is an active (physiologic) and a passive
(pharmacologic) mechanism for vitamin B-12 absorption in
the body (Harper, 1979; Herbert, Colman and Jacob, 1980).
Gastric acid and gastric and intestinal enzymes release
vitamin B-12 from foods.
Vitamin B-12 then attaches it-
self to a receptor site on a glycoprotein given the name
intrinsic factor (IF) by Castle.
The vitamin B-12-IF
complex travels to the ileum where it attaches to the intestinal microvilli if there is a neutral pH and ionic
calcium is present.
Vitamin B-12 is released from the
complex by an unknown mechanism and passes through the
intestinal mucosal cell into the portal venous blood.
The alternative absorptive mechanism is a passive absorption of approximately one percent of megadose amounts of
vitamin B-12 by diffusion along the entire length of the
small intestine.
Transport, distribution and storage of vitamin B-12
13
in the body are carried out by three vitamin B-12-binding
proteins, transcobalamin I, II and III (Herbert, Colman
and Jacob, 1980; Harper, 1979).
Transcobalamin I and III
seem to be mainly involved in storage of vitamin B-12.
Transcobalamin III may also play a role in the redelivery
of vitamin B-12 to the liver.
Transcobalamin II is the
carrier responsible for delivering vitamin B-12 to the
tissues.
The mechanism is similar to the method by which
IF delivers vitamin B-12 to intestinal mucosal cells.
Transcobalamin II and vitamin B-12 form a complex and when
this complex reaches tissues such as bone marrow cells or
reticulocytes, the vitamin B-12 is released if the pH is
neutral and ionic calcium is present.
Vitamin B-12 coenzymes appear to play roles in a
variety of reactions in animal tissues but the absolute
necessity for vitamin B-12 has been demonstrated in only
two reactions
1980).
(Harper, 1979; Herbert, Colman and Jacob,
As previously noted, methylcobalamin and 5'-deox-
yadenosylcobalamin are the only two forms of vitamin B-12
that are known to act as coenzymes in mammalian tissues.
Coenzyme B-12 is involved in fat and carbohydrate metabolism through its role in hydrogen transfer and isomerization in the conversion of methylmalonyl-CoA to succinylCoA.
Vitamin B-12 coenzymes, as well as folate coenzymes,
are involved in protein synthesis through their roles in
the methylation of homocysteine to methionine.
This reac-
tion is vital in the synthesis of thymidylate which is
14
essential for DNA synthesis.
The reaction regenerates
tetrahydrofolate which is needed to form the 5,10-methylene tetrahydrofolate necessary for thymidylate synthesis.
The major route of excretion of vitamin B-12 is
the bile.
in
Most of the vitamin B-12 excreted in this way
is reabsorbed in the ileum.
Any vitamin B-12 which is not
reabsorbed would be lost in the feces.
The reabsorption
mechanism, which will be discussed later in this paper, is
highly efficient and little vitamin B-12 is lost by this
route.
Small amounts of vitamin B-12 can be lost in the
urine but the amounts are not significant.
There is no
significant catabolism of vitamin B-12 by humans (Harper,
1979; Herbert, Colman and JacobJ 1980).
Vitamin B-12 Deficiency: Causes and Occurrence
Herbert, Colman and Jacob (1980) suggest six possible
reason~
for nutritional deficiency of vitamin B-12: inade-
quate ingestion; inadequate absorption; inadequate utilization; increased requirement; increased excretion; and
increased destruction.
Nutritional deficiency could re-
sult from any single cause or combinat·ion of these causes.
The two major causes of vitamin B-12 deficiency are inadequate absorption and inadequate ingestion.
Inadequate
absorption is usually due to a defect in IF metabolism,
lesions of the stomach or lesions of the ileum.
The de-
ficiency is reversed by parenteral administration of vitamin B-12 on a temporary or permanent basis depending on
15
the extent of the problem.
Inadequate ingestion of vita-
min B-12 is corrected by adding vitamin B-12 containing
foods to the diet or by taking oral vitamin B-12 supplements.
Both pernicious anemia, due to a lack of IF, and
macrocytic megaloblastic anemia, due to dietary deficiency
of vitamin B-12 have the same clinical picture (Herbert,
Colman and Jacob, 1980).
Symptoms include weakness,
tiredness, dyspnea, sore tongue, paresthesia, constipatio~
anorexia, syncope, headache and palpitation.
Clini-
cal signs include megaloblastic bone marrow, glossitis,
postural hypotension and neurologic damage due to demyelination.
As the disease progresses the neurologic damage
becomes evident with a deereased sense of vibration and
position, poor muscular coordination, poor memory,and
hallucinations.
The incidence of vitamin B-12 deficiency is rare in
the United States (Food and Nutrition Board, 1980) •
prevalence of vitamin B-12
uncertain (FAO/WHO, 1970).
defic~ency
The
around the world is
The deficiency does appear
more often in developing countries than in developed countries.
India and South-East Asia seem to have an increas-
ed incidence of the deficiency disease but the causes of
the disease are difficult to ascertain.
Malabsorption
syndromes, tropical sprue, fish tapeworm and dietary deficiency are all possible causes in that area of the wond.
16
Studies of the incidence and causes of vitamin B-12
deficiency are needed.
Recommended Dietary Intakes
The Food and Nutrition Board of the National Research Council, National Academy of Sciences (FNB/NRC)
included RDA for vitamin B-12 for the first time in 1968
(Food and Nutrition Board, 1968).
Table l compares the
current vitamin B-12 recommendations of the FNB/NRC
(1980), the Department of National Health and Welfare of
Canada (1975) and the FAO/WHO Expert Group (1970).
The
Canadians and FAO/WHO recommend 0.3 pg per day during the
first year while the FNB/NRC recommends 0.5 pg per day
for the first six months and 1.5 fg per day for the last
six months of the first year of life.
For children one
to three years old, the Canadians and FAO/WHO recommend
0.9 pg per day while the FNB/NRC recommends 2.0 pg per
day.
For children four to six years old the Canadians
and FAO/WHO recommend 1.5 pg per day while the FNB/NRC
recommends 2.5 pg per day.
For children seven to ten
years old the Canadians and FAO/WHO recommend 1.5 pg per
day while the FNB/NRC recommends 3.0 pg per day.
For ages
eleven and older FAO/WHO recommends 2.0 pg per day the
FNB/NRC and the Canadians both recommend 3.0 pg per day.
All three groups recommend an additional 1.0 pg per day
during pregnancy.
During lactation the Canadians and
FAO/WHO recommend an additional 0.5 pg per day while the
17
Table 1
Recommended Daily Intake of Vitamin B-12
Age (years)
or Status
United States
(pg)
a
Canada b
(pg)
FAO/WHOc
(pg)
0.0-0.5
0.5
0.3
0.3
0.5-1.0
1.5
0.3
0.3
1-3
2.0
0.9
0.9
4-6
2.5
1.5
1.5
7-10
3.0
1.5
1.5
11-14
3.0
3.0
2.0
15-18
3.0
3.0
2.0
19-22
3.0
3.0
2.0
23-50
3.0
3.0
2.0
51+
3.0
3.0
2.0
Pregnant
+1.0
+1.0
+1.0
Lactating
+1.0
+0.5
+0.5
asource: Food and Nutrition Board,
(1980).
bsource: Canada, Department of National Health and
Welfare, (1975).
csource: FAO/WHO Expert Group,
(1970).
18
FNB/NRC recommends an. additional 1.0 pg per day.
The FNB/NRC (1980) recommendations are based solely
on studies that measure the body turnover of radioactive
viatmin B-12.
This turnover rate is not related to the
actual minimum daily requirement which is estimated at
0.1 pg per day of exogenous vitamin B-12 (Herbert, 1968).
The turnover rate of radioactive vitamin B-12 tends to be
a fixed percentage of approximately 0.1 percent of total
body stores per day.
This fixed percentage means the ac-
tual range of B-12 loss per day in normal individuals is
quite variable and the amount of vitamin B-12 needed daily
to maintain equilibrium would vary widely among individuals.
The FNB/NRC sets its recommendation at the upper
limits of this turnover rate.
The FAO/WHO (1970) looks at
the radioactive vitamin B-12 turnover rate but their recommendations also take into account studies of minimum
amounts of vitamin B-12 needed to prevent or cure megaloblastic anemia and studies of tissue levels of vitamin B-12
in healthy and deficient individuals.
The FAO/WHO values
are more likely to approximate actual needs than the FNB/
NRC values.
The FNB/NRC (1980) recommendations have been
revised down since 1968 when the adult recommendation was
as high as 5.0 ?g per day (Food and Nutrition Board, 1968).
Food Sources
Microorganisms are responsible for the production of
vitamin B-12 in nature.
' &
Table 2 lists a number of micro-
19
Table 2
Some Microorganisms Known to Be Able
To Synthesize Vitamin B-12
Nocardia rugosa
Butyribacterium rettgeri
Streptomyces aureofaciensb
Rhodopseudomonas spheroides
Streptomyces griseusb
Streptomyces olivaceus
Pseudomonas denitrificansb
Clostridium tetanomorphum
Protaminobacter ruber
Clostridium sticklandii
Clostridium thermoaceticum
Rhizobium meliloti
Propionibact£rium
shermanii
Crithidia fasciculata
Propionibacteriu~
freudenreichi
Strigomonas oncopelti
Propionibacterium
technicum
Propionibacterium
arabi no sum
Propionibacterium
pentosaceum
Propionibacterium
peterssoni
asource: Beck (1982).
bMicroorganisms used for industrial vitamin B-12 produc~
tion.
20
organisms that are known to have the ability to synthesize vitamin B-12.
The number of microorganisms capable
of producing vitamin B-12 is unknown.
Studies of micro-
organisms isolated from animals, foods, soil and water
revealed that a substantial number of the microorganisms
produced measurable amounts of vitamin B-12 (Beck, 1982).
The microorganisms used for industrial vitamin B-12 production include Propionibacterium freudenreichii,Propionibaaterium shermanii, Pseudomonas denitrificans, Streptomyces grisues and Streptomyces aureofaciens · . (Florent and
Ninet, 1979; Beck, 1982).
The typical food sources of vitamin B-12 are meat and
meat products and milk and milk products
bert,Colman and Jacob, 1980).
sources.
(Orr, 1969; Her-
Table 3 summarizes these
The best sources are liver, heart, and kidney,
closely followed by muscle meats, milk, seafood (especially clams), fish, fermented cheese such as Camembert or
Limburger, and eggs.
A significant portion of the world's population does
not regularly consume any of these foods.
In spite of
this, vitamin B-12 deficiency disease is not widespread.
The next section details some possible explanations for
this paradoxical situation.
Vitamin B-12 Nutriture of Vegans
For those people who either cannot or choose not to
consume meat products, a number of factors acting singly
21
Table 3
Common Food Sources of Vitamin B-12a
Food
pg Vitamin B-12 per
100 gm Wet Weight
Greater than or
Orgaz:t:1 meats:
equal to 10
la.!!lb & beef liver
lamb & beef kidney
lamb & beef heart
Rating
High
Bivalves:
clams
oysters
Nonfat dry milk
3 to 10
Moderately
High
to 3
Moderate
Some Seafood:
crabs
rockfish
salmon
sardines
Egg yolk
Muscle meats
l
Some Seafood:
lobster
scallops
flounder
haddock
swordfish
tuna
Fermenting cheeses:
Camembert
Limburger
Fluid1milk products
Less than 1
Some cheeses:
cream
cheddar
cottage
asource: Herbert, Colman and Jacob (1980).
Low
22
or in various combinations can either provide sufficient
vitamin B-12, delay the onset of vitamin B-12 deficiency
disease or mask the symptoms of vitamin B-12 deficiency
disease.
Non-meat Product Sources of Vitamin B-12.
Table 4
presents the possible and potential sources of vitamin
B-12 for vegans.
The natural habitats of vitamin B-12
producing microorganisms range from the soil to ponds,
lakes and seas.
When these natural habitats are left un-
treated, as is the case in many underdeveloped countries
lacking modern sanitation facilities, they can serve as
significant sources of vitamin B-12 (Smith, 1965). Vitamin B-12 has been found in rain (Parker, 1968).
Human
feces contain substantial amounts of vitamin B-12 and
people who do not regularly wash their hands, which is
often the situation with small children, can take in vitamin B-12 either directly through hand to mouth contact or
indirectly by contaminating food (Alfin-Slater and Kritchevsky, 1980; Herbert, 1976).
There are certain legumes which have root nodules
containing vitamin B-12 producing microorganisms (Herbert,
Colman and Jacob, 1980).
When consumed with their
these legumes can provide vitamin B-12.
nodule~
Wokes, Badenoch
and Sinclair (1955) reported that ground nuts had detectable quantities of vitamin B-12.
Ericson and Banhidi
(1953) tested seaweeds for various nutrients while looking for new sources of animal feeds and found vitamin B-12
23
Table 4
Non-Meat Product Sources of Vitamin .B-12
Source
Beer
Reference
Darby, 1979; Paul and Southgate,
1978
Fermented soybean oroducts
Tempeh
Natto, Mise, Shoyu
Liem, Steinkraus, and Cronk,l977
Douolass and Fleiss, 1980;
Shurtleff, 1979
Fortified breakfast cereals
Food labels
Ground nuts
Wokes, Badenoch and Sinclair,l955
Human small intestinal
bacteria
Albert, Mathan, and Baker, 1980;
Nutrition Reviews, 1980
Kimchi
Ro, Woodburn and Sandine, 1979
Legume root nodules
Herbert, Colman and Jacob, 1980
Nutritional yeasts
Food labels
Oral, tonsillar, and
pharyngeal bacteria
Thrash, 1979
Rain
Parker, 1968
Sea Vegetables
Ericson and Banhidi, 1953
Brown and red alqae
Combs, 1952
Chlorella algae
Shurtleff, 1979
Kombu, wakame, nori,
hijiki, dulse, arame,
kelp, alaria, green nori
flakes, unprocessed agar
Soil and water contaminated
by feces
Smith, 1965
Unsanitary personal habits
Alfin-Slater and Kritchevsky,l980;
Herbert, 1976
Wine
Voigt ,et aZ., 1978
24
in one brown and two red species of algae.
In a number
of places around the world seaweed has become a common
human food.
In a similar study, Combs (1952) found vita-
min B-12 in Chlorella algae.
The previously mentioned foods are not usually consumed by large numbers of people but there are a number
of foods regularly eaten in Eastern cultures which do
contain vitamin B-12 in significant amounts.
are also becoming available in the West.
These foods
One group of
vitamin B-12 containing foods are fermented soybean products.
Tempeh, cultured soy cakes, are a popular Indo-
nesian food.
A typical serving (100 gm) contains :3.:9
pg
of vitamin B-12 which ii greater than the suggested RDA
(Liem, Steinkraus, and Cronk, 1977).
When the tempeh
culture is inoculated with a high yield vitamin B-12 producing microorganism, a 100 gm serving can provide 14.8 pg
of vitamin B-12.
Natto (sticky fermented whole soybeans),
miso ( a seasoning made from soybeans, grain, salt, water
and Aspergillus oryzae culture) and shoyu (soy sauce) all
contain vitamin B-12 but in lesser quantities.than in
tempeh (Douglass and Fleiss, 1980; Shurtleff, 1979).
Kimchi, a staple food in the diet of Koreans, is a
fermented vegetable dish which includes cabbage.
Kimchi
contains considerable amounts of vitamin B-12 (Ro, Woodburn, and Sandine, 1979).
As with tempeh, the vitamin
B-12 content can be increased by inoculation with highyield vitamin B-12 producing microorganisms.
If kimchi
25
contains vitamin B-12, then sauerkraut, a similar food
popular in the West, might contain vitamin B-12.
This
possibility has not yet been studied.
Shurtleff (1979) lists the following sea vegetables
as sources of vitamin B-12: kombu, wakame, nori, hijiki,
dulse, arame, kelp, alaria, green nori flakes, and unprocessed agar.
The vitamin B-12 in these foods is pro-
duced by bacteria in the sea.
These same bacteria pro-
duce the vitamin B-12 found in seafood.
Finally, fortified breakfast cereals and nutritional
yeast are sources of vitamin B-12.
Neither of these would
be natural sources but they are sources that are commonly
eaten and do not compromise the values of vegans.
The
yeast get their high vitamin B-12 content through growth
in a vitamin B-12 rich medium.
Yeast cannot synthesize
vitamin B-12.
Vitamin B-12 is produced by bacteria in the colon in
humans.
This vitamin B-12 is unavailable for absorption
because vitamin B-12 is absorbed in the ileum (Herbert,
Colman and Jacob, 1980).
Albert, Mathan and Baker,
(1980)
recently found vitamin B-12 producing microorganisms in
the small intestine of healthy Indian lactovegetarians.
This study is only the first of its kind but if future
research duplicates its results, vegans may have a reliable internal source of vitamin B-12.
Thrash (1979)
suggests that "it is very likely that oral, tonsillar, and
pharyngeal bacteria produce more than adequate quantities
26
of vitamin B-12 to supply the requirements of a vegetarian".
The possibility of internal sources of vitamin B-12
requires further study.
Human Ability to Store Vitamin B-12. Vitamin B-12 is
unique among the water soluble vitamins.
It is the only
water soluble vitamin which can be stored in the human
body.
The stores range from l to 10 mg and are found pre-
dominantly in the liver (Herbert, Colman and Jacob, 1980).
The RDA is set at 3 pg (Food and Nutrition Board, 1980).
If vitamin B-12 is used up at a rate of 3 fg per day,
stores of l to 10 mg could last from l to 10 years.
There
is an enterohepatic circulation of vitamin B-12 in which
the vitamin is excreted in the bile and reabsorbed in the
ileum (Harper, 1979; Herbert, Colman and Jacob, 1980).
This reabsorption mechanism conserves virtually all the
vitamin B-12 excreted.
If a person becomes a vegan after
having eaten animal products for a number of years, a
substantial amount of vitamin B-12 will have been stored
in the body.
With the liklihood of such a person having
at least minimal vitamin B-12 intake from the sources
mentioned in the previous section in addition to stored
vitamin B-12 and an almost perfect reabsorption mechanism,
it could take many years or even decades before a deficiency would appear.
High Folacin Intake in Vegans. Folacin does not pose
the same problem for vegetarians as does vitamin B-12.
Folacin can be found in many fruits and vegetables, espe-
27
cially fresh green vegetables (Food and Nutrition Board,
1980).
A typical vegan would probably have a high intake
of folacin.
High levels of folacin will mask the symptoms
of megaloblastic anemia which usually appear in persons
with vitamin B-12 deficiency (Ellis, 1974).
Neurological
damage from vitamin B-12 deficiency can progress inthese
persons while their blood continues to appear normal.
Without testing for neurological damage, a case of vitamin
B-12 deficiency could go unnoticed.
Serum Vitamin B-12 Levels in Vegetarians. Studies
carried out to determine average serum B-12 levels in
humans were done using non-vegetarian populations.
The
averages for these populations may not be accurate for
vegetarians.
If clinical symptoms of vitamin B-12 disease
do not appear, it is difficult to claim a person has the
disease based solely upon serum vitamin B-12 levels.
Mehta, Rege and Satoskar (1964) studied serum vitamin B-12
levels in lactovegetarians compared to non-vegetarians.
The lactovegetarians had a mean serum vitamin B-12 level
of 121 pg per ml while the non-vegetarians had levels of
366 pg per ml.
''Despite considerably low serum vitamin
B-12 values, the lactovegetarians had no apparent signs or
symptoms of vitamin B-12 deficiency state".
al.
Armstrong et
(1974) studied 431 vegetarians and found that low
serum levels did not necessarily indicate vitamin B-12
ficiency disease.
Other studies also report low serum
levels did not necessarily indicate vitamin B-12 but
de~
28
again the low levels
~o
not indicate vitamin B-12 defi-
ciency disease (Ellis and Montegriffo, 1970; Sanders,
Ellis and Dickerson, 1978).
It is possible that non-
vegetarians have higher levels than actually needed.
In
conclusion, serum B-12 levels are poor indicators of
megaloblastic anemia.
Assay Methods
Large quantities of vitamin B-12 can be assayed
spectroscopically, colorimetrically, fluorometrically or
chemically (Florent and Ninet, 1979).
These methods are
usually appropriate for industrial scale assay of vitamin
B-12 only.
The most suitable assay methods for vitamin
B-12 in foods are the microbiological and radiosotope
dilution assays
(Butner, Cury and Baker, 1958; Baker and
Frank, 1968; Voigt and Eitenmiller, 1978).
It is likely
that all the reported vitamin B-12 values for foods were
determined by microbiological methods
(V-oigt and Eiten-
miller, 1978).
Bacteria were the first microorganisms used to assay
for vitamin B-12.
Lactobacillus lactis was used to iden-
tify the factor in refined liver extracts that cured pernicious anemia.
Lactobacillus lactic Dorner (ATCC 8000),
Lactobacillus leichmannii
(ATCC 7830),
Es~herichia coli
mutants and certain thermophilic bacteria have all been
used to assay for vitamin B-12.
There are potential
problems when these bacteria are used.
Assay results can
29
be invalidated with lactic acid bacteria because deoxyribosides can replace vitamin B-12 in the assay.
Methi-
onine can replace vitamin B-12 in the assays utilizing
Escherichia coli mutants of thermophilic bacteria (Baker
and Frank, 1968).
In spite of these problems, the Offi-
cial Method of Analysis (Association of Official Analytical Chemists, 1975) is the assay utilizing Lactobacillus
leichmannii
(ATCC 7830).
Radioisotope dilution assays are available but they
regularly produce inappropriately high results due to the
fact that the proteins used in the assay have an affinity
for both vitamin B-12 and biologically inactive vitamin
B-12 analogs (Kolhouse et al., 1978).
This method re-
quires expensive radioisotopes and counters and many lab.oratories would find the cost prohibitive.
Biological assays using mice, rats and chicks are
reliable but also expensive.
The protozoan method of
assay is the assay of choice due to its sensitivity and
specificity. This assay can measure as little as 1 pg of
vitamin B-12 per ml and will not respond to
methionine or vitamin B-12 analogs
deoxyriboside~
(Baker and Frank, 1968).
Two protozoan microorganisms, Euglena gracilis and Ochromonas malhamensis are used to assay for vitamin B-12.
Of
the two, Ochromonas malhamensis is the preferred assay
microorganism because its vitamin B-12 requirement is
exactly like human requirements while Euglena gracilis
does respond to some pseudo-forms of vitamin B-12.
Table
30
5 summarizes tqe response of Escherichia coli
Lactobacillus
leichmannii~
113-3~
Euglena gracilis and Ochromonas
malhamensis to vitamin B-12 and other substances.
The
table clearly shows that 0. malhamensis is the most specific of the assay microorganisms responding exclusively to
clinically active cobalamins.
England has recognized the
advantages of this method and has made the 0. malhamensis
assay its official method of analysis (Analytical Methods
Committee, 1956).
Comparative studies have shown that the 0. malhamensis assay is the superior method.
The results of the 0.
malhamensis assay agree with results from chick assays
(Ford and Butner, 1955).
Assays of blood, serum and urine
show that 0. malhamensis is more accurate than Escherichia
coli~
Lactobacillus leichmannii ahd Euglena gracilis for
the detection of clinically active forms of vitamin B-12
(Baker and Frank, 1968).
Finally, in assays of fruits
and vegetables, O. malhamensis has been found to be more
specific than Lactobacillus leichmannii
(Voigt and Eiten-
miller, 1978).
In natural materials vitamin B-12 is found in unstable coenzyme forms bound to cellular protein.
The extrac-
tion medium needs a reducing agent such as metabisulfite,
and sodium cyanide to convert labile hydroxocobalamin to
stable cyanocobalamin.
Heating in an autoclave libertes
protein-bound cobalamins (Voigt and Eitenmiller, 1978).
31
Table 5
Growth Responae of Variou• Assay HicroorQanisrns to Clinically Active Molecules
("True .B-12")
Vs. Clinically Inactive Molecules
Growth
E. coZl
Name
(Analoqs With Little or No Activity)•
respon~e
taotobaoiU-u•
of microoroaniam
Eughna
Ochro111oruu
Base of nucleotide
Clinical Activity
in Animals
cyanocobalamin
s. 6-Dimethylbenzimidazole
PseuC.ovitamin B-12
Adenine
Inactive
'F~c:tor
2-Hethy !adenine
Little or no
Activity
A
Active aQainst
pernicious anemia
Factor B
No nucleotide
Inactive
Fec:tor C
Guanine (guanosine
Unknown
Fa:tor D
Unknown
Fa:tor E
Unknown
diphosphate)
:ra:tor
r
Unknown
+++
Unknown
-or+
2-Hethylmercapto-
Unknown
adenine
Unknown
Fa:tor G
Hypoxanthine
Fe:tor H
2-Hethylhypoxanthine
Fe:tor I
5-Hydroxybenziminazole
+++
Benziminazole
+++
Unknown
Active
:B-12-Factor III)
Nc name
S .. Hethylbenziminazole
No name
Naphthiminazole
De:)xyriboaides
Thymine, adenine,
hypoxanthine,
cytosine
aSources Baker and Frank (1968).
Active
Active
Active
Inactive
Ir::.act DNA
He:hionine
+++
Inactive
+++
Inactive
32
Beer
Historical Backround of Beer
The origin of the word beer is probably from the
Latin word bibere, to drink.
Most agree that beer origi-
nated about 6000 years ago in Egypt and Mesopotamia.
Throughout history descriptions about of societies developing an alcohblic beverage from local sources of sugar or
starch such as barley, corn, millet, oats, potatoes, rice,
rye, fruits or vegetables.
It is difficult to look back
and determine why alcoholic beverages became such important parts of religious rituals and ceremonial occasions.
The preservative qualities of alcohol, the physiological
effects, or both of these could explain the interest and
wide acceptance of this beverage.
Beer moved across Europe with the various migrations
of the Celts, Teutons and Saxons.
Eventually brewing
centers were set up in Czechoslovakia, Germany, England
and Ireland.
Throughout the Middle Ages the church was involved
in the brewing industry and the involvement continues today.
Much of the original science of brewing was devel-
oped under the watchful eye of the church.
American Indians made a beer-type beverage out of
maize but it was not similar to the British varieties.
Beer was one of the staples brought along on the Mayflower.
The British brought their ale brewing traditions to America~
with them, mainly a top fermentation at fairly warm
33
temperatures.
In the mid-nineteenth century German lager
beer became popular and eventually became the most popular
type of beer in America.
fermented.
The German lagers were bottom
In this process the lager was fermented at
cool temperatures and the yeast settled to the bottom. The
fermentation is slower but it produces a stable beer.
To-
day, German type lagers dominate the American beer market
(Westermann and Huige, 1979; Robertson, 1978; Katz, 1979).
The Brewing Process
U.S. Federal Regulations define beer as a malt beverage produced from an alcoholic fermentation of the aqueous
extract of malted barley with hops.
Additional carbohy-
drate materials, called adjuncts, can also be added.
To
malt the barley, the kernel is separated from the stalk
and chaff and then germinated under controlled temperature
and humidity conditions.
Germination continues until high
levels of diastatic and proteolytic enzymes are produced
and some degradation of the barley starch granules has
occurred.
Drying or kilning stops the growth of the grain
and reduces the moisture level to approximately four percent.
Enzymatic activity is suspended but not destroyed.
The first step in brewing is the mashing process.
The malt is carefully crushed with rollers to release the
kernel without releasing excessive polyphenolic compounds.
A mash is made from the crushed grain and water.
If
adjuncts are desired, they are added at this point.
These
34
adjuncts are commonly added in the United States
to o- 1e
.
the beer a lighter flavor and are usually made from r
or corn.
~e
The mash is subjected to controlled time an
temperature cycles.
The dormant malt enzymes begin t
break down the carbohydrate material in the mash.
Ei 1ty
percent of the starch is converted to maltose with so
~
glucose and trisaccharides also produced.
When mashing is complete, the insoluble portions
removed in a lauter tub or filter press.
extract is called wort.
1re
The clear l
[Uid
The insoluble grain residue
spent brewers grain can be used as an excellent anima
feed.
The wort is boiled in a brew kettle to which hop
hop extracts have been added.
or
Hops are the blossoms .
clones of the hop plant, Humulus ZupuZus.
Hops conta
essential oils and bittering substances which contrib·
e
to the characteristic flavor and aroma of beer.
rt
The '
is boiled for approximately an hour during which the
of the beer develops.
lor
Spent hops are strained from tl
wort and then the wort is allowed to stand until the
ulated proteins settle out.
<
ag-
OJ
so-
The wort is aerated and
cooled which causes additional proteins to fall out
lution.
The primary fermentation begins when the cooled,
aerated wort is inoculated with a pure yeast culture c d
allowed to ferment.
un~que
yeast strain.
Each brewery prides itself on
it~
There are only two species of YE st
35
important in brewing, Saccharomyces cerevisiae and
Saccharomyces carlsbergensis. For 48 hours the yeast
actively grow or bud and then the budding stops.
Fer-
mentable sugars are converted to ethyl alcohol and trace
amounts of flavor ingredients.
Fermentation continues
actively after the budding has stopped.
This primary
fermentation usually lasts from four to seven days and by
the end of this period the yeast will have settled to the
bottom of the fermenting tank.
The beer is pumped into
aging tanks and the yeast are recovered to be reused or
sold as food.
The length of time the beer is aged, or
lagered, depends completely on the brewer.
The lagering
varies from days to a month or more and is done to improve
the flavor and stability of the beer.
The aging usually
takes place in a cold environment, about 0-10 0 C.
When the aging process is complete, the beer still
contains yeast cells, proteinaceous materials, microorganisms and other colloidal substances.
The beer is clari-
fied by filtering,.to a greater or lesser extent, these
materials out.
Modern processing methods are efficient at
removing most of the unwanted material but with the current trend towards "natural" and "old-style" brewing techniques, at least some beers will retain quite a bit of
extraneous matter.
After filtration, the beer is carbonated.
Sometimes
the natural carbonation is retained but usually the carbon
dioxide is recovered from the fermentation, purified and
36
then returned to the finished product.
Most beer is
. packaged in bottles or cans and are usually pasteurized
(Hardwick, 1979; Westermann and Huige, 1979).
Types of Beer
Lager Beer.
The word lager means to store or stock
and it refers to the period of storage when the beer
undergoes a slow second fermentation.
It is bottom fer-
mented and aged for as short a time as a week or for premium products as long as several months.
In the United
States the fermentation takes place at 4.4°C to 7.2°C (and
possibly higher) and goes on for five to eight days while
in Europe the fermentation usually is done near 0°C and
can take up to fourteen days.
The flavor, the body and
the shelf life of the lager all improve with increased
aging.
The alcohol content is 3.0 to 3.8 percent by weight
or 3.4 to 4.2 percent by volume.
Lagers are usually a pale
gold color, light in body, flavored with a medium to light
hop taste and are fairly high in carbonation (Robertson,
1978; Hardwick, 1979).
Dark Beer.
This is a lager beer to which roasted bar-
ley, caramel or roasted barley malt has been added.
Dark
beers are full-bodied, have a sweet malt flavor and a
slight hop taste.
These beers tend to have a higher alco-
hol content than lighter lagers and can approach five percent alcohol by weight (Robertson, 1978).
37
Low-Calorie (Light) Beer.
lager beers.
Low-calorie beers are also
They range in kilocalorie content from 70 to
108 kilocalories and 2.8 to 8.8 grams of carbohydrate per
355 ml (12 oz.) bottle.
Their kilocalorie content compares
to 140 kilocalories and 11 grams of carbohydrate per bottle
of regular lager beer (Darby, 1979).
exception among the low-calorie beers.
(Michelob Light is an
Labeling informa-
tion on the bottle indicates 134 kilocalories and 12.4
grams of carbohydrate per 355 ml bottle.)
Low-calorie
beers have a lower alcohol content than regular lager beer&
The alcohol content is in the range of 3 percent by weight
or lower (Darby, 1979).
The low-kilocalorie content is
most likely produced by the addition of water.
Ale.
Ale is produced by a special yeast.
Ale yeast
carries out a top fermentation in the presence of air.
Ale
is predominantly a product of the British Commonwealth.
As
opposed to European lager which is fermented near 0°C, ale
is fermented at 4.4°C to 7.2°C.
It is fermented for a
shorter period of time than European lagers but is aged in
the bottle for an additional length of time to develop both
flavor and strength.
by weight.
Ale is four to five percent alcohol
Compared to lager, ale has a winelike characte4
is more aromatic, more full-bodied and has a stronger hop
flavor and tartness (Robertson, 1978; Hardwick, 1979).
Malt Liguor.
Malt liquor is brewed as a lager beer.
Its distinctive qualities are achieved through the use of
38
less malt, less hops and more fermentable carbohydrate than
in lager beer (Hardwick, 1979).
Malt liquor is 4.5 percent
or more alcohol by weight (Darby, 1979). Ahy beer with five
percent alcohol by weight or more can be labeled malt liquor in the United States.
Typically, malt liquor is
darker in color and more fruity in flavor than lager (Robertson, 1978).
Porter.
Porter is fermented in a manner much the same
as lager beer.
Charcoal or dark colored malts and less
hop bitter are used than in lager beer.
This procedure
produces a dark brown, malty flavored, sweet tasting beer.
It is usually approximately five percent alcohol by weight
(Robertson, 1978; Hardwick, 1979).
Stout.
Stout is similar to porter.
dient is roasted barley.
Its major ingre-
It has a dark, almost black colo4
a strong malt flavor and a much stronger hop character than
porter.
Stout tends to have 5 to 6.5 percent alcohol by
weight (Robertson, 1978; Hardwick, 1979).
Consumption of Beer
Between the time of the repeal of Prohibition in 1933
and 1974, total malt beverage consumption more than doubled
in the United States.
In 1974, American consumed 144,196,
705 barrels (31 gallons/barrel) of malt beverages.
Based
on the total population residing in the United States, per
capita consumption equalled 21.1 gallons.
Based on the 21
39
year old and over population
only~
per· capita consumption
was 33.9 gallons or 542 eight ounce glasses.
For the 21
year old and over population, per capita beer consumption
increased every year from 1960 (24.4 gallons) until 1974
(33.9 gallons)
(United States Brewer's Association, 1975).
In 1981 total domestic and imported malt beverage
sales equalled 182,225,100 barrels (Clark Gavin Associates,
1983).
The 1981
u.s.
population estimate for the number
of persons 21 years of age or older was 153,664,000 (U.S.
Department of Commerce, 1981).
Utilizing these sales and
population statistics, per capita consumption equalled 36.7
gallons per person, an increase over 1974 figures.
Pennington (1983) developed representative
u.s.
diets
based on the data from the 1977-78 U.S.D.A. Nationwide Food
Consumption Survey and the Second National Health and Nutrition Examination Survey which was carried out from 1976
to 1980.
The representative diet.contained canned beer.
The average daily intake of canned beer for a 25 to 30 year
old female was 34.6 gm.
The average daily intake of canned
beer for a 25 to 30 year old male was 299.2 gm.
Nutritive Value of Beer
Throughout history fermented beverages, served in
their fresh state, have contributed significant amounts of
nutrients to the diets of many.
Mead (from honey) , palm
wine (from palm sap), Kaffir beer (from sorghum) and maize
beer from Africa as well as pulque
(from the juice of the
40
Century plant) from Mexico are all highly nutritious
(Steinkraus, 1979).
As these beverages become refined
through filtration, clarification or distillation there is
a significant drop in their kilocalorie content as well as
in their nutrient density (Darby, 1979). To illustrate this
drop in nutrient density with vitamin B-12, home brewed
palm wine can be compared to California red wines.
Vitamin
B-12 has been found in palm wine at levels of 190 to 280 ng
per ml (Steinkraus, 1979).
prepared home brew.
Voigt et al.
per ml.
The palm wine was a freshly
In processed California red wines,
(1978) found 0.09 to 0.20 ng of vitamin B-12
This comparison demonstrates one of the possible
effects of modern processing on the nutrient content of
fermented beverages.
Table 6 lists reported values for selected nutrients
in beer.
The major nutritional contribution of these bev-
erages is in the form of kilocalories.
However, small
amounts of riboflavin and niacin are also present.
The
other listed vitamins and minerals are found in insignificant amounts.
With respect to vitamin B-12, Darby (1979) reports a
value of 1.77 pg of vitamin B-12 per 355 ml (12 oz.) for an
assay of a single beer sample.
Paul and Southgate (1978)
report vitamin B-12 values for a number of beers assayed
with Lactobacillus leichmannii
(ATCC 7830) by the Labora-
tory of the Government Chemist, the British equivalent of
41
Table 6
Selected Nutrients In Lager Beer
Composition per 355 ml (12 oz.)
Source
u.s. Food
u.s.
Composition
Brewers b
Tablea
Association
Nutrient
British Food
Composition
Tablec
Kilocalories
151
140
103
Protein (gm)
1.1
1
.71
Carbohydrate
(gm)
13.7
11
5.3
18
18.8
14
108
27
43
Ca (mg)
P (mg)
Fe (mg)
Tr
Na (mg)
25
18.5
14
(mg)
90
108
121
K
Vitamin A (IU)
Thiamin (mg)
0
Tr
No value
0
.01
.013
Tr
Riboflavin (mg).11
.114
.07
Niacin (mg)
2. 2
2.34
1.17
0
0
0
Ascorbic acid
(mg)
asource:
Adams (1975).
bsource:
Darby (1979).
c
Source
Paul and Southgate (1978).
42
the U.S.D.A. Nutrient Composition Laboratory.
are listed in Table 7.
These values
The amount of vitamin B-12 found in
beers ranges from a low of 0.385 pg per 355 ml in brown ale
to 1.295 pg per 355 ml in strong ale.
0.49 ?g per 355 ml.
Pale ale contained
The major difference between these
three beers was the alcohol content with brown, pale and
strong ales having 7.81 g, 11.7 g and 23.43 g of alcohol
respectively.
The increase in alcohol content reiulted
from an increase in fermentation times.
A possible expla-
nation for the higher vitamin B-12 content in beer fermented for additional time is that vitamin B-12 producing
microorganisms might have used this time to produce increased amounts of vitamin B-12. under the favorable conditions of the prolonged fermentation.
existed for stout.
The same situation
Bottled stout had 10.29 g of alcohol
and 0.385 pg of vitamin B-12 per 355 ml while extra stout
had 15.26 g of alcohol and 0.595 jlg of vitamin B-12 per
355 ml.
The average of the ten types listed was 0.581 pg
of vitamin B-12 and 12.17 g of alcohol per 355 ml.
This
quantity of vitamin B-12, the amount in one bottle, could
provide 19 percent of the RDA (Food and Nutrition Board,
1980) and 29 percent of the FAO/WHO (1970) daily recornrnended intakes.
research.
These significant values warrant further
The present study assayed a variety of beers for
their vitamin B-12 content utilizing Ochromonas malhamensis
which is more sensitive and specific than Lactobacillus
leichmannii.
43
Table 7
Vitamin B-12 and Alcohol Content of Beersa
Type of Beer
Vitamin B-12
)lg per
pg per
355 ml
100 ml
g Alcohol
per 355 ml
Brown ale :0bottled)
0.11
0.385
7.81
Canned beer (bitter)
0.15b
0.525
11.00
Draught beer (bitter)
0.17
0.595
11.00
Draught beer (mild)
0.15b
0.525
9.23
Keg beer (bitter)
0.15
0.525
10.65
Lager beer (bottled)
0.14
0.49
11.36
Pale ale (bottled)
0.14
0.49
11.7
Stout (bottled)
0.11
0.385
10.29
Stout (extra)
0.17
0.595
15.26
Strong ale
0.37
1. 295
23.43
Average
0.166
0.581
12.17
asource:
Paul and Southgate (1978).
bEstimated value.
Chapter 3
MATERIALS AND METHODS
Materials
Beer Samples
The sample consisted of 58 brands of beer assigned to
eight categories: domestic lager beer, imported lager beer,
dark beer, low-calorie beer, ale, malt liquor, porter and
stout.
The categories were based upon the definitions
given in Chapter 2.
Lager beer was divided into two sep-
arate categories, domestic and imported.
Information on
the formulation and fermentation times of the various domestic and imported lager beers was not readily available.
The decision to divide the lager beer category was based
on the fact that beer retailers treat domestic and imported lager beer qualitatively differently with respect to
advertising, store displays and pricing.
Beers were as-
signed to categories based upon information found on their
labels.
U.S. labeling laws do not guarantee that labels
are accurate with respect to type of beer (Robertson, 1978).
44
45
Label information, however, was the only information
available.
The sample was not randomly selected.
An attempt was
made to sample all beers that were regularly available &t
local markets or liquor warehouses.
The beer was purchased
at the local branches of three major supermarket chains
with large liquor departments (Alpha Beta Market, Northridge, California; Von•s Supermarket, Northridge, California; Ralph•s Supermarket, Northridge, California) or the
local branches of two major liquor warehouse chains (The
Liquor Barn, Granada Hills, California; MGM Liquor, Chatsworth, California) •
A beer was classified as "regularly
available,. if it met two criteria: 1) the beer had to be
available in at least two of the five retail outlets;
2) the manager of the store confirmed that the beer was a
normal stock item.
All samples purchased for the study were in 355 ml
(12 ounce) containers.
Fifty-four of the brands were pur-
chased in glass bottles while four brands were available
only in aluminum cans.
Fifty-one of the bottles were dark
brown or dark green in color.
The remaining three bottles
were made of clear glass.
The samples were purchased on the day before they were
to be assayed.
The actual time between production and pur-
chase was impossible to ascertain.
Tables 8 through 15 list the complete unabridged
name~
Table 8
Domestic Lager Beers
Name of Beer
Brewery
1.
Anchor Steam Beer
Anchor Brewing Company
2.
Augsburger Old World Bavarian Style Beer
Jos. Huber Brewing Company
3.
Budweiser Beer
Anheuser Busch, Inc.
4.
Coors Premium
Adolph Coors Co.
5.
Erlanger Classic 1893
Jos. Schlitz Brewing Co.
6.
Hamm•s Genuine Draft Beer
Olympia Brewing Company
7.
Henry Weinhard•s Private Reserve Beer
Blitz-Weinhard Company
8.
Lone Star Beer
Lone Star Brewing Company
9.
Lowenbrau Special Beer
Miller Brewing Company
10.
Michelob Beer
Anheuser Busch,
11.
Miller High Life
Miller Brewing Company
12.
Olympia Beer
Olympia Brewing Company
13.
Schlitz
Jos. Schlitz Brewing Co.
14.
Tuborg Beer Export Quality
Carling National Breweries
No.
Inc.
~
~
"'"
Table 9
Imported Lager Beers
No.
Name of Beer
Brewery
Country of
Origin
1.
Asahi Lager Beer
Asahi Breweries
Japan
2.
Carlsberg Beer
Carlsberg Breweries
Denmark
3.
Carta Blanca Beer
Cerveceria Cuauhtemoc, S.A.
Mexico
4.
Dos Equis Beer
Cerveceria Moctezuma, S.A.
Mexico
5.
Kirin Beer
Kirin Brewing Co., Ltd.
Japan
6.
Molson Golden
Mol~on
Canada
7.
St. Pauli Girl Beer
St. Pauli Brewery; Bremen
Germany
8.
San Miguel Beer
San Miguel Corp.
Philippines
Breweries of Canada Ltd.
.t:>.
-..]
Table 10
Dark Beers
No.
Name of Beer
Brewery
Country of
Origin
1.
Beck's Beer - Dark
Brauerci Beck & Co.
Germany
2.
Carta Blanca Dark
Special Beer
Cerveceria Cuauhtemoc, S.A.
Mexico
3.
Heineken Special Dark
Beer
Heineken Brouwerijen B.V.
Holland
4.
Kronenbourg Dark Beer
Kronenbourg Breweries
France
5.
Lowenbrau Dark Special
Miller Brewing Co.
U.S.A.
6.
St. Pauli Girl Dark Beer
St. Pauli Brewery
Germany
7.
San Miguel Dark Beer
San Miguel Corp.
Philippines
8.
Tuborg Deluxe Dark
Export Quality Beer
Carling National Breweries
U.S.A.
.~:>.
00
Table ll
Low Calorie (Light) Beers
Brewery
Country of
Origin
95
Amstel Breweries
Holland
Brisa Cerveza Ligena
90
Cerveceria Cuauhtemoc,S.A.
Mexico
3.
Budweiser Light Beer
108
Anheuser Busch, Inc.
U.S.A.
4.
Coors Light Beer
105
Adolph Coors Co.
U.S.A.
5.
Hamm's Special Light
99
Olympia Brewing Company
U.S.A.
6.
Kirin Light Beer
105
Kirin Brewing Co., Ltd.
Japan
7.
Lite Beer
Miller Brewing Company
U.S.A.
8.
Michelob Light Beer
Anheuser Busch, Inc.
U.S.A.
9.
Olympia Gold Light Beer
70
Olympia Brewing Company
U.S.A.
Schlitz Light
96
Jos. Schlitz Brewing Co.
U.S.A.
No.
Name of Beer
l.
Amstel Light
2.
10.
KiloCalor1es
per 355 mla
96
134
aKilocaloric content per 355 ml was taken directly from the label on each bottle.
,.J:>.
'-0
"'~
Table 12
Ale
No.
Name of Ale
Brewery
Country of
Origin
1.
Bass and Co.•s Pale Ale
Bass Brewing Ltd.
England
2.
McEwan's Scotch Ale
Scottish & Newcastle
Breweries PLC
Scotland
3.
Molson Ale
Molson Breweries of Canada, Ltd.
Canada
4.
Sierra Nevada Brewing Co.
Pale Ale
Sierra Nevada Brewing Company
U.S.A.
5.
Thos. Cooper & Sons Real
Ale
Cooper & Sons, Ltd.
Australia
U1
0
4\l
Table 13
Malt Liquor
No.
Name of Malt Liquor
Brewery
Country of
Origin
1.
Carlsberg Elephant Malt
Liquor
The Carlsberg Breweries
Denmark
2.
Colt 45 Malt Liquor
Carling National Breweries
U.S.A.
3.
Mickeys Fine Malt Liquor
Carling National Breweries
U.S.A.
4.
Olde English
Liquor
Blitz-Weinhard Company
U.S.A.
11
800 11 Malt
Ul
......
Table 14
Porter
No.
Name of Porter
Brewery
Country of
Origin
1.
Anchor Porter
Anchor Brewing Company
U.S.A.
2.
Taddy Porter
Samuel Smith Old Brewery
England
3.
Sierra Nevada Brewing Co.
Porter
Sierra Nevada Brewing Co.
U.S.A.
4.
Yuengling Dark Brew Porter
D.G. Yuengling & .Son, Inc.
U.S .A.
Vl
N
Table 15
Stout
No.
Name of Stout
Brewery
Country of
Origin
1.
Belikin Premium Stout
Belize Brewing Company, Ltd.
Belize
2.
Guinness Extra Stout
Arthur Guinness Son & Co., Ltd.
Ireland
3.
Mackeson Stout
Whitbread & Co., PLC
England
4.
Sierra Nevada Brewing Co.
Stout
Sierra Nevada Brewing Company
U.S.A.
5.
Thos. Cooper & Sons Stout
Cooper & Sons, Ltd.
Australia
Ul
w
54
brewing companies and countries of origin for the 58 brands
of beer sampled.
Assay Microorganism
Ochromonas malhamensis
(ATCC 11532) culture was pur-
chased from the American Type Culture Collection, Rockville, Maryland.
Chemical Reagents and Microbiological Media
All chemical compounds were reagent grade.
Crys-
talline cyanocobalamin (vitamin B-12) was purchased from
ICN Nutritional Biochemicals, Life Sciences Group, Cleveland, Ohio.
The composition and preparation of Ochromonas
malhamensis maintenance medium, vitamin B-12 assay basal
medium, vitamin B-12 standard solutions, volatile preservative for stock solutions and fluids awaiting analysis and
aconitic acid buffer are described in Appendices A through
E.
Glassware Maintenance
Protozoan assays are highly sensitive and specific.
Traces of organic material can promote or inhibit the
growth of the microorganism (Guttman, 1963; Baker and
Frank, 1968) •
Traces of the vitamin to be assayed will
also interfere with the results.
To eliminate these fac-
tors, a meticulous cleaning regimen was developed and ad-
55
hered to for all glassware.
The washing procedure includ-
ed the following sequence of steps:
1.
The glassware was soaked overnight in hot tap water with two percent (20 ml/1 water) Micro detergent (International Products Corporation, Trenton,
N .J •) .
2.
The glassware was scrubbed with a brush if necessary.
3.
The glassware was washed in an ultrasonic cleaning
bath with two percent Micro detergent for 30 minutes.
4.
Each piece of glassware was rinsed six times in
hot tap water.
5.
Each piece of glassware was rinsed six times in
deionized water.
6.
All glassware was air dried in an inverted position.
7.
All glassware was sterilized for 30 minutes at
121°C and 15 psi in the autoclave on the dry goods
cycle with fast exhaust.
Equipment
1. UV-Visible Spectrophotometer-Model 24, Beckman
Instruments, Inc., Fullerton, California
2. pH Meter, Model pH 102, Brinkmann Instruments Inc . .,.
Westbury, New York
56
3.
Gyratory Water Bath Shaker, Model G-76, New Brunswick Scientific Co., New Brunswick,
4.
New Jersey
Mettler Electronic Analytical Balance, Model AC
100, Mettler Instrument Corporation, Princton,
New Jersey
Sartorius Balance, Model 3703, Brinkmann Instruments, Westbury, New York
5.
Autoclave, Model STM-E, Type C, No. 45018,
Market
Forge, Everett, Massachusetts
6.
Ion Exchange Columns, organic and cation removalr
Model D 0803, Barnstead Sybron Corporation, Boston, Massachusetts
7.
Ultrasonic Cleaner, Model B52, Bransonic Co.,
Stamford, Connecticut
8.
Micro-Fernbach Flasks, 25 ml, No. 2522-00010,
Bellco Glass, Inc., Vineland, New Jersey
9.
Screw-capped Media Storage Bottles, 120 ml, borosilicate, No. 5636-00125, Bellco Glass, Inc.,
Vineland, New Jersey
10.
Screw-capped Culture Tubes, 16 x 125 mm, borosilicate, No. 2012-16125, Bellco Glass, Inc.,
Vineland, New Jersey
11.
Pipetman Continuously Adjustable Digital Microliter, Pipette, Model P-5000, Rainin Instrument
Co., Inc., Woburn, Massachusetts
12.
Eppendorf Digital Pipette, 100-lOOOul, No. 22 33
360-7, Brinkmann Instruments, Inc., Westbury, N.Y.
57
13.
Additional equipment:
Fisherbrand Touch Mixer,
Model 12-810, Fisher Scientific Co., Pittsburgh,
Pennsylvania; Corning Hot Plate-Stirrer,
M~del
PC-
351, Corning Glass Works, New Jersey; test tube
racks; sterile disposable pipettes; refrigeratorfreezer; Bausch and Lomb Microscope; and miscellaneous glassware
Methods
The basis for the microbiological assay method used
in the present study was originally described by Butner,
Provasoli and Filfus (1953) •
It was developed by Ford
(1953) and revised by Baker and Frank (1968).
utilizes. the protozoan organism Ochromonas
This method
malh~mensis.
The protozoan method was chosen for this study because it
is the most sensitive and specific way to measure the metabolically and clinically active forms of vitamin B-12 (Baker and Frank, 1968; Voigt and Eitenmiller, 1978).
The
major steps in the protozoan assay method utilizing 0. malhamensis are summarized in Figure 2.
Sample Preparation
First carbon dioxide was removed from the beer sample&
Each beer sample was opened and approximately 50 ml poured
into sterile 250 ml Erlenmeyer flasks.
The flasks were
covered with aluminum foil, placed in a Gyrotory water bath
shaker, and agitated at 75 percent of full shaking power
58
ADDITION OF
ACONITIC ACID BUFFER
+
SAMPLE ASSAY
FLASKS AT 3
CONCENTRATIONS
Bl2 STANDARD CURV
ASSAY FLASKS AT 9
CONCENTRATIONS
5-DAY OLD
CULTURE OF
0. MALHMENSIS
CALCULATE Bl2
CONTENT OF
SAMPLES
Figure 2.
Schematic Outline of Vitamin B-12
Assay Procedure
59
(setting of 7.5 on a scale of 10, maximum of 400 rpm) for
30 minutes.
The samples were then allowed to sit without
agitation for an additional 15 minutes.
This method was
found to be faster and more effective than the Association
of Official Analytical Chemists (1975) method which required shaking the flasks by hand.
The beer samples were prepared for vitamin B-12 analysis employing the methodology described by Baker and Frank
(1968) for urine vitamin analysis. A 1.0 ml aliquot of a
degassed sample was put into a 20 x 125 mm culture tube and
diluted with 1.0 ml of aconitic acid buffer (see Appendix
E) containing 0.1 mg/ml sodium metabisulfite.
The sodium
metabisulfite treatment liberates vitamin B-12 (Butner,
Cury and Baker, 1958).
The tubes containing the samples
were covered with stainless steel caps.
The tubes were
then autoclaved for 20 minutes at 121°C at 15 psi.
After autoclaving, 8 ml of sterile deionized water was
added to each tube to bring the total volume to 10 ml.
each sample bottle of beer, two tubes were prepared.
For
The
beer samples were prepared immediately prior to assay.
Microbiological Assay for Vitamin B-12
Maintenance of Ochromonas malhamensis
(ATCC 11532).
0. malhamensis was obtained from the American Type Culture
Collection, Rockville, Maryland.
The culture was trans-
ferred to and maintained in a maintenance medium (see
Appendix A) .
One drop of the old medium containing the
microorganism was transferred to the new medium every five
60
days.
The culture tubes were incubated at 20-22°C on an
isolated counter in the laboratory.
The tubes were exposed
to sunlight through the laboratory windoxs and overhead
fluorescent lighting for approximately 12 hours each day.
Baker and Frank (1968) recommend constant illumination and
an incubation temperature of 28-32°C.
Under the latter
conditions, the microorganism grew too rapidly.
The lower
temperatures and limited illumination that were used in
this study are within the range of variables that will
support the normal growth of
vasoli and Filfus, 1953).
o.
maZhamensis
(Butner, Pro-
Under the stated conditions, the
level of growth in the maintenance culture tubes after five
days was ideal for use in the assay (an absorbance reading
of approximately 0.300 at 525 nm on a UV-visible spectrophotometer) •
Samples from the maintenance culture tubes were regularly checked under a microscope for bacterial contamination.
It was decided to use an inoculum taken directly
from a five day old maintenance culture because of its simplicity.
Assay medium. The vitamin B-12 free assay medium was
prepared according to the formulation of Baker and Frank
(1968)
(see Appendix B).
The medium was prepared monthly
and stored at 4°C with volatile preservative (see Appendix
D).
A 2.5 ml aliquot of this double strength prepared
medium was pipetted into each standard flask or assay flask.
The double strength medium was then diluted to single
61
strength by adding working vitamin B-12 standard solution
(see Appendix C) or prepared beer sample and sterilized
deionized water to the flasks for a total volume of 5 ml.
Inoculum. Baker and Frank (1968) recommend that the
inoculum be prepared by making an aseptic 1:5 suspension of
a 3-4 day-old culture of 0. malhamensis is sterile deionized water.
Under the conditions used to maintain the
microorganism in this study, a minimum of five days was
required to achieve sufficient growth for use in the inoculum.
Preliminary runs of the standard curve were carried
out to compare the inoculum prepared as described above
with an inoculum taken directly from a five-da¥ old maintenance culture.
The latter method was simpler to carry
out and provided less opportunity for bacterial contamination than the Baker and Frank (1968) method.
Using the
latter method it was necessary to take care in maintaining
the organisms uniformly in suspension during the actual
inoculation (Ford, 1953) •
The preliminary runs showed a
slight vitamin B-12 carryover effect from the inoculum
taken directly from a five-day old maintenance culture.
The effect was small, consistent and had no influence on
the shape of the standard curve.
Standard curve preparation.
prepared in duplicate.
The standard curve was
A 2.5 aliquot of basal medium was
added to 25 ml micro-Fernbach flasks.
Working vitamin B-12
standard solution (see Appendix C) and sterile deionized
62
water were then pipetted into the flasks according to the
protocol described in Table 16.
Two control flasks
(Flask
nos •.1 and 2) composed of basal medium and sterile deionized water (representing zero concentration of vitamin
B-12) were included to check the vitamin B-12 carryover
from the inoculum.
Any carryover was later subtracted from
the standard curve values.
Two reference flasks which were
not inoculated were prepared but were not incubated with
the standards· due to restricted space in the bath shaker.
These were refrigerated at 4°C and used as references for
the UV-Visible Spectrophotometer.
Sample flask preparation.
All samples were assayed at
three concentrations in duplicate.
A 2.5 aliquot of basal
medium was added to 25 ml micro-Fernbach flasks.
Prepared
samples and sterilized deionized water were then added to
the flasks according to the protocol described in Table 17.
The protocol resulted in dilutions of 1:50, 1:33.3 and 1.25.
The water bath shaker held a total of 42 flasks.
ard curve required 18 flasks.
The stand-
The remaining 24 flasks al-
lowed for the assay of four beer samples per run (24 flasks:
four samples at three concentration in duplicate) •
Sterilization, inoculation and incubation.
When addi-
tions had been completed, all flasks, both standard curve
and sample assay, were covered with stainless steel caps and
autoclaved for 20 minutes at 121°C and 15 psi with slow exhaust.
This procedure kills all microorganisms including
resistant sporeformers and also drives off the volatile pre-
·'-'
Table 16
Protocol of Vitamin B-12 Standard Curve
Flask
!)O.b
Vitamin B-12
Concentration
J29Lflask
J2QLml
Vitamin B-12
standardc
);!er flask {mll
1. 2e
0
0
3,4
1
5
0.5
5.6
3
15
7,8
6
9,10
a
Basal
Deionized
Mediumd !ml}
water !ml}
0
Total
O.malhamensis
volume per inoculum
flask !mll
!1 drool
2.5
2.5
5.0
+
of Soln.Bc
2.5
2.0
5.0
+
1.5
of Soln.B
2.5
1.0
5.0
+
30
0.3
of Soln.Ac
2.5
2.2
5.0
+
9
45
0.45 of Soln.A
2.5
2.05
5.0
+
11,12
12
60
0.6
of Soln.A
2.5
1.9
5.0
+
13,14
15
75
0.75 of Soln.A
2.5
1. 75
5.0
+
15,16
18
90
0.9
of So1n.A
2.5
1.6
5.0
+
17,18
21
105
1.05 of Soln.A
2.5
1. 45
5.0
+
aAdapted from Baker and Frank (1968).
bEach concentration was assayed in duplicate.
cAppendix C. Solution A (0.1 ng vitamin B12/ml).
Solution B (0.01 ng vitamin B12/ml).
dAppendix B.
eFlask no. 1 and 2 were used as zero concentration.
(jl
w
+$
Table 17
Protocol of Vitamin B-12 Assay Flasks for Beer Samples a
Sample
addition
(ml)
Basal
mediumc
(ml)
Distilled
water
(ml)
Sample
19,20
1
1. od
2.5
1.5
5.0
+
21,22
1
1. 5e
2.5
1.0
5.0
+
23,24
1
2.0f
2.5
0.5
5.0
+
no.
Total volume
per flask
(ml)
0. maZhamensis
inoculum
(1 drop)
Flask
b
no.
25,26
2
1.0
2.5
1.5
5.0
+
27,28
2
1.5
2.5
1.0
5.0
+
29,30
2
2.0
2.5
0.5
5.0
+
31,32
3
1.0
2.5
1.5
5.0
+
33,34
3
1.5
2.5
1.0
5.0
+
35,36
3
2.0
2.5
0.5
5.0
+
37,38
4
1.0
2.5
1.5
5.0
+
39,40
4
1.5
2.5
1.0
5.0
+
41.42
4
2.0
2.5
0.5
5.0
+
aAdapted from Baker and Frank (1968).
bEach concentration was assayed in duplicate.
cAppendix B.
dCorresponds to a sample dilution level of 1:50.
eCorresponds to a sample dilution level of 1:33.3.
£Corresponds to a sample dilution level of 1:25.
0'1
*"
65
servative (Baker and Frank, 1968).
The flasks were al-
lowed to cool in the autoclave for at least 20 minutes and
then were allowed to cool on a laboratory counter until
they reached room temperature.
The inoculum was taken from a single five-day old
culture of 0. malhamensis.
The microorganisms tends to
clump on the sides and bottom of the tube and it was necessary to vortex the culture tube for a minimum of 30 seconds
to insure a uniform dispersion of cells throughout the tube.
One drop of inoculum was aseptically delivered from a longtipped 1 ml sterile pipette to each standard curve flask
and each assay flask.
When inoculum was taken into the
pipette, it was drawn up and down a number of times to
tain a homogeneous suspension of microorganisms.
mai~
To mini-
mize exposure to contamination, the flasks were inoculated
one at a time, removing the cap only long enough to add the
inoculum.
The Gyratory Water Bath Shaker, Model G-76 (New Brunswick Scientific Co., New Brunswick, New Jersey) has a
stai~
less steel tray which holds 42 25 ml micro-Fernbach flasks.
Each flask was placed in the tray after it was inoculated.
When all 42 flasks had been inoculated, the entire tray was
lowered into the water bath.
The flasks were incubated at 29°c and shaken in subdued light during the day, and darkness during the night,
for 72 hours (Ford, 1953).
Baker and Frank (1968) suggest
66
incubating the flasks under illumination.
Equipment that
could control both temperature and illumination reliably
was not available.
Without a reliable light .source, it was
decided to run the assays in the dark which is fully acceptable for 0. maZhamensis.
The only complication of running
the assays in the dark is the need for additional aeration
(Butner et aZ., 1953), which was provided by shaking the
flasks constantly.
The flasks were shaken at a setting of
0.5 on the Gyrotory Water Bath Shaker (settings range from
0 to 10 with a maximum rpm of 400).
The temperature of the
water bath was checked daily and additional water was added
if necessary.
Absorbance measurement.
At the end of the incubation
period, the standard flasks and the assay flasks were autoclaved at 121°C and 15 psi for 5 minutes to stop further
growth of
o.
maZhamensis.
at room temperature.
The flasks were allowed to cool
Each flask was thoroughly mixed on a
Fisherbrand touch mixer and the turbidity was measured at a
wavelength of 525 nm using a UV-Visible Spectrophotometer
(Beckman, Model 24) equipped with a sipper system.
The
spectrophotometer was set at 525 nm so that the results indicated the amount of cell mass (turbidity) and not green
pigment production which can vary with changes in the assay
method independent of the amount of vitamin B-12 present
(Gurrman, 1963).
The spectrophotometer was first calibrat-
ed with deionized water and then with the uninoculated
67
reference.
The reference was used for reading the ab-
sorbances of the cultures.
Treatment of Data
Standard curve construction.
The means of the ab-
sorbances for each duplicated vitamin B-12 concentration
(ordinate) were plotted on graph paper against the known
concentrations of vitamin B-12 (abscissa) •
A standard
curve was obtained by fitting the data points with a French
curve.
A typical standard curve is shown in linear plot
in Figure 3.
Calculation of vitamin B-12 concentration in beer
samples.
The vitamin B-12 concentration of each sample
assay flask was determined by comparing its absorbance
with the standard curve.
The quantity of vitamin B-12 per
355 ml bottle of beer was then calculated using the calculation outlined in Appendix F.
Since each sample was
assayed at three concentrations in duplicate, the mean and
standard deviation were calculated from the six measurements.
The vitamin B-12 concentrations were expressed in
ng/355 ml bottle of beer.
Statistical Analysis.
An analysis of variance was
used to determine i.f significant differences among the
vitamin B-12 contents in beers within each category existed at the 0.05 level (Linton and Gallo, 1975).
Following
this analysis, Tukey•s Honestly Significant Differences
Test (Linton and Gallo, 1975) was used to determine whether
""'
~
~0.5
1.1'1
N
1..1'\
/8
I
1-
..:; 0.4
LLl
u
z
c::x::
~
~0.3
t:Q
c::x::
I
~
/
0.2
0.1
0
1
2
3
4
5
6
8
9 10 11 12 13 14 15 16 17 18 19 20 21
VIT.I\MIN B-12 CONCENTRATION
Figure 3.
(PGIML)
Typical Vitamin B-12 Standard Curve
~
00
69
specific means
ineach-category~differed
from one another.·
An analysis of variance was used to determine if sigficant differences in vitamin B-12 content existed between
the eight categories of beer at the 0.05 level.
Following
this analysis, Scheffe's Specific-Comparison Test (Mattson,
1981) was used to determine whether specific means differed from one another.
For a number of beer manufacturers, both the lager
beer and the low-calorie beer produced by the same manufacturer were assayed.
The t Test (Linton and Gallo, 1975)
was used to determine if there was a significant difference in vitamin B-12 content between the lager beers and
low-calorie beers at the 0.05 level.
Similar data were also collected from manufacturers
producing_ both a lager beer and a dark beer.
The t Test
was used to determine if there was a significant difference in vitamin B-12 content between the lager beers and
dark beers at the 0.05 level.
Chapter 4
RESULTS AND DISCUSSION
Vitamin B-12 Content of Different Brands of Beer Within
Each Beer Type.
Domestic Lager Beer.
Fourteen brands of domestic
lager beer were assayed for their vitamin B-12 content
(Table 18).
Henry Weinhard's Private Reserve Beer had
the highest vitamin B-12 content with 115 ng per 355 ml
while Schlitz Beer nad the lowest vitamin B-12 content
with 27.7 ng per 355 ml.
The average vitamin B-12 content
for the domestic lagers was 66.7 ng per 355 ml.
When a
one-way between subjects analysis of variance (ANOVA) was
applied to these data, significant differences (p <0.05)
were found.
Tukey's Honestly Significant Differences Test
(Tukey's) was used to determine which brands of beer had
significantly more vitamin B-12 than the others.
The four
lagers with the highest vitamin B-12 content, Henry Weinhard's, Coors, Tuborg and Olympia, were all significantly
different (p<0.05) from the four lagers with the lowest
vitamin B-12 content, L6wenbrau, Lone Star, Michelob and
70
71
Table 18
Vitamin B-12 Content of Domestic Lager Beers*
Brand Name
(Abridged)
Henry Weinhard's
ng/355 ml
115.0 + 24.0
Container
Type**
a
DB
Coors
98.0 +
9.2a,b
DB
Tuborg
97.3 + 31.9a,b
DB
Olympia
92.3 +
DB
Augsburger
86.0 + 33.5a,b,c
DB
Anchor
81.0 + 18.1a~b,c,d
DB
Budweiser
64.0 + 12.3b,c,d,e
DB
Erlanger
63.3 + 16.9b,c,d,e
DB
Miller
57.0 +
6.1b,c,d,e
CB
Hamm's
49.0 +
5.3b,c,d,e
AC
L~enbrau
41.3 +
2.9c,d,e
DB
Lone Star
33.7 +
2.9d,e
AC
Michelob
29.0 +
5.6e
DB
Schlitz
27.7 +
8.5e
DB
Average
66.7 + 28.4
6.4a,b
* Values
are the mean + S.D. of the mean from six replicates. Those values which have no superscripts in
common are significantly different at the 0.05 level.
**
Container types:
AC=aluminum can.
DB=dark bottle;
CB=clear bottle;
72
Schlitz (Table 18).
Henry Weinhard•s also contained sig-
nificantly more vitamin B-12 than Budweiser, Erlanger and
Miller.
Augsburger contained significantly more vitamin
B-12 than Lone Star, Michelob and Schlitz.
And Anchor
contained significantly more vitamin B-12 than Michelob
and Schlitz.
The fact that significant differences exist between
the various brands of lagers is not unique in this study.
As will be seen in later sections, significant differences in vitamin B-12 content exist among a number of
brands in all eight categories.
The reasons for these
differences are difficult to determine.
The specific
source of the vitamin B-12 in the various beers is not
known.
Yeast do not require vitamin B-12 for their growth
and they do not produce vitamin B-12 (Broquist, 1979;
Peppler, 1979).
Therefore, residual yeast in the various
beers cannot account for the differences in vitamin B-12
content.
The source of vitamin B-12 in beers is microbial
contamination which is true of all foods in which vitamin
B-12 is found.
A number of factors could affect the
levels of contamination and the rate of growth of these
vitamin B-12 producing microorganisms:
the purity of the
original ingredients; the use {or lack of) aseptic techniques in the brewing process; the length of
fermentatio~
and the degree of filtration {Steinkraus, 1979; Darby,
73
1979; Westermann. and Huige, 1979).
This type of informa-
tion is not usually made available for the individual
brands of beer by manufacturers.
The types and quantitiffi
of ingredients and the brewing processes are trade secrets.
The only type of beer that carries nutrient
labe~
ing is low-calorie beer and the information provided is
limited to kilocalories, carbohydrates, protein and fat.
Alcohol content for individual beers is not available on
their labels.
If alcohol content were available or had
been assayed for in this study it would have been at
least a partial indication of the length of fermentation.
Without any of the aforementioned information it is difficult, if not impossible, to determine the reasons for
the differences in vitamin B-12 content among the various
beers.
Paul and Southgate (1978) listed a value of 490 ng of
vitamin B-12 per 355 ml bottle of lager beer (Table 7).
This quantity is more than seven times the amount found
in domestic lagers and almost twelve times the amount
found in imported lagers in this study.
are difficult to account for.
The differences
The most likely explanation
is the difference in assay microorganism.
The value list-
ed by Paul and Southgate (1978) was obtained using Lactobacillus leichmannii.
A number of studies have been carried out comparing
the sensitivity and specificity of L. leichmannii to that
74
of Oehromonas maZhamensis.
the response of L.
Baker et aZ.
(1956) compared
Zeichmannii and 0. maZhamensis to
vit~
L. Zeiehmannii yielded values twice
min B-12 in urine.
as high as 0. maZhamensis and the differences increased
as the vitamin B-12 concentration decreased.
(1960) found that L.
Baker et aZ.
Zeiehmannii yielded values almost
three times as high as O. maZhamensis in human serum and
four times as high in human blood.
Williams et aZ.
(1956)
concluded that 0. maZhamensis be used to check assays using L. Zeichmannii in feed supplements and other natural
products because L.
Zeichmannii will respond to pseudoco-
balamins and other nonspecific factors.
miller and Ware (1979) found that L.
Voigt, Eiten-
Zeichmannii indi-
cated vitamin B-12 activity in enzymatic hydrolysates of
tomato juice, orange juice and spinach while 0. maZhamensis showed no vitamin B-12 activity.
monstrate that
o.
specific than L.
These studies de-
maZhamensis is far more sensitive and
Zeiehmannii.
It is known that L.
Zeichmannii responds to deoxy-
ribosides and a number of clinically inactive vitamin B-12
analogs while 0. maZhamensis does not (see Table 5).
Beers have not been assayed for their deoxyriboside or
vitamin
B~12
analog content.
Westermann and Huige (1979)
reported that four to six percent of the solids in typical lager wort are peptones, peptides, polypeptides and
amino acids.
It is possible that the final beer product
75
contains some materials that are clinically active for
L. Zeichmannii but not for 0. malhamensis or humans.
One way to determine the cause of the differences
between the present study and the results of Paul and
Southgate (1978} is to assay the same samples with both
microorganisms.
The samples could also be assayed for
their deoxyriboside and vitamin B-12 analog content.
Without additional information about the samples or assay,
it is difficult to come to any useful conclusion about
the differences.
Information regarding the type of container the beer
was purchased in is included in the table.
Only six of
the samples carne in containers that were not dark glass
bottles.
There were too few samples of the non-dark glass
type to make any statistical comparisons.
Imported Lager Beer.
Eight brands of imported lager
beer were assayed for their vitamin B-12 content (Table
19}.
Carta Blanca Beer had the highest vitamin B-12 con-
tent with 72 ng per 355 rnl while Asahi Lager Beer had the
lowest with 21.3 ng per 355 rnl.
The average vitamin B-12
content for the imported lagers was 40.5 ng per 355 rnl.
An ANOVA showed that significant differences (p < 0. 05}
existed among the vitamin B-12 contertt of these beers.
Tukey's test indicated that Carta Blanca had significantly
more (p<0.05} vitamin B-12 than Carlsberg, Molson, Kirin,
San Miguel and Asahi (Table 19).
Dos Equis had signifi-
76
Table 19
Vitamin B-12 Content of Imported Lager Beers*
Brand Name
(Abridged)
ng/355 ml
1.7a
Container
Type**
Carta Blanca
72.0 +
Dos Equis
52.3 + 14.2a,b
DB
St. Pauli
47.3 + 19.3a,b,c
DB
Carlsberg
42.7 +
7.3b,c
DB
Molson
38.3 +
4.9b,c
DB
Kirin
26.7 +
8.3b,c
DB
San Miguel
24.0 +
4.4c
DB
As a hi
21.3 +
4.9c
DB
Average
40.5 + 17.0
* Values
DB
are the mean + S.D. of the mean from six replicates. Those values which have no superscripts in
common are significantly different at the 0.05 level.
**
Container type:
DB=dark bottle.
77
cantly more vitamin B-12 than both San Miguel and Asahi.
No other significant differences were found.
Dark Beer. Eight brands of dark beer were assayed for
their vitamin B-12 content (Table 20).
Heineken Special
Dark Beer had the highest vitamin B-12 content with 172.3
ng per 355 ml while Beck's Dark Beer had the lowest with
46 ng per 355 ml.
The average vitamin B-12 content for
the dark beers was 94 ng per 355 ml.
An ANOVA showed that
significant differences (p<O.OS) existed among the vitamin B-12 content of these beers.
Tukey's test indicated
that Heineken, Tuborg and Carta Blanca contained significantly more (p <0.05) vitamin B-12 content than St. Pauli
Girl, Kronenbourg, L6wenbrau, San Miguel and Beck's (Table
20).
No other significant differences were found.
Low-Calorie (Light) Beer. Ten brands of low-calorie
beer were assayed for their vitamin B-12 content (Table
21).
Amstel Light had the highest vitamin B-12 content
with 74.3 ng per 355 ml while Lite Beer had the lowest
with 20.7 ng per 355 ml.
Lite Beer had the lowest vitamin
B-12 content of all 58 brands tested.
The average vitamin
B-12 content for the low-calorie beers was 38.3 ng per 355
ml.
An ANOVA showed that significant differences (p < 0 •. 05)
existed among the vitamin B-12 content of these beers.
Table 21 shows the statistical relationships between the
beers.
The major relationship of interest indicated that
Amstel had significantly more (p < 0. 05) vitamin B-12 than
all the other low-calorie beers.
There was no apparent
78
Table 20
Vitamin B-12 Content of Dark Beers*
Brand Name
(Abridged)
· ng/355 ml
Container
Type**
Heine ken
172.3 +
8.6a
DB
Tuborg
159.7 + 17.5a
DB
Carta Blanca
144.3 + 18.0a
DB
St. Pauli
71.7 +
4. 2b
DB
Kronen bourg
60.7 +
4.7b
DB
Lowenbrau
49.3 + 11. 9b
DB
San Miguel
47.7 +
2.9b
DB
Beck's
46.0 +
6.5b
DB
Average
94.0 + 54.7
*Values are the mean + S.D. of the mean from six replicates. Those values which have no superscripts in
common are significantly different at the 0.05 level.
**Container type:
DB=dark bottle.
79
Table 21
Vitamin B-12 Content of Low-Calorie (Light) Beers*
Brand Name
(Abridged)
ng/355 ml
Container
Type**
Amstel
74.3 +
6.0a
DB
Schlitz
46.0 +
4.6b
DB
Brisa
44.3 +
5.8b
DB
Hamm's
39.3 +
4.0b,c
AC
Olympia
39.0 +
9.0b,c
DB
Coors
37.0 + 11.3b,c,d
DB
Michelob
29.3 +
6.0b,c,d
DB
Kirin
28.7 +
1.2b,c,d
DB
Budweiser
25.0 +
4.6c,d
DB
Lite
20.7 +
2.3d
DB
Average
38.3 + 15.0
*Values are the mean + S.D. of the mean from six replicates. Those values which have no superscripts in
common are significantly different at the 0.05 level.
**Container types:
DB=dark bottle;
AC=aluminum can.
80
relationship between kilocalorie content of these light
beers and their vitamin
Ale.
B-~2
content.
Five brands of ale were assayed for their vita-
min B-12 content (Table 22).
McEwan•s Scotch Ale had the
highest vitamin B-12 content with 214.3 ng per 355 ml
while Thos. Cooper and Sons Real Ale had the lowest with
35.8 ng per 355 ml.
McEwans Scotch Ale had the highest
vitamin B-12 content of all 58 brands assayed.
The aver-
age vitamin B-12 content for ales was 81.2 ng per 355 ml.
An ANOVA showed that significant differences (p <0.05)
existed among the vitamin B-12 content of these beers.
Tukey•s test indicated that McEwan•s Scotch Ale contained
significantly more (p <0.05) vitamin B-12 than Molson,
Bass, Sierra Nevada and Thos. Cooper (Table 22).
No other
significant differences were found.
Paul and Southgate (1978) list vitamin B-12 values
for ale ranging from 385 ng per 355 ml to 1295 ng per 355
ml (see Table 7) •
It was noted in Chapter 2 that increas-
ing alcohol content of these ales seemed to be related to
increasing vitamin B-12 content.
It would be interesting
to see if McEwan•s Scotch Ale had a higher alcohol content
than the others.
The values reported by Paul and South-
gate (1978) are markedly higher than the values found in
this study.
Since Lactobacillus Zeichmannii was the assay
microorganism, it is again difficult to assume the differences are due to anything other than the choice of assay.
81
Table 22
Vitamin B-12 Content of Ales*
Brand Name
(Abridged)
McEwan's
ng/355 ml
Container
Type**
214.3 + 32.0a
DB
Molson
59.3 +
6.8b
DB
Bass
51.7 +
8.1b
DB
Sierra Nevada
45.3 +
9.7b
DB
Thos. Cooper
35.8 -+
2.0b
DB
Average
81.2 + 74.7
*Values are the mean + S.D. of the mean from six replicates. Those values which have no superscripts in
common are significantly different at the 0.05 level.
**Container type:
DB=dark bottle.
82
Malt Liguor.
Four brands of malt liquor were assayed
for their vitamin B-12 content (Table 23).
Colt 45 Malt
Liquor had the highest vitamin B-12 content with 100.7 ng
per 355 ml while
Carlsber~
Elephant Malt
lowest with 36.7 ng per 355 ml.
L~quor
had the
The average vitamin B-12
content for malt liquor was 69.5 ng per 355 ml.
An ANOVA
showed that significant differences (p <0.05) existed
among the vitamin B-12 content of these beers.
Tukey•s
test indicated that Colt 45 and Mickey's both contained
significantly more (p <0.05) vitamin B-12 content than Old
English or Carlsberg (Table 23) .
No other significant
differences were found.
Porter.
Four brands of porter were assayed for their
vitamin B-12 content (Table 24)·.
Anchor Porter had the
highest vitamin B-12 content with 109.8 ng per 355 ml
while Sierra Nevada Brewing Co. had the lowest with 43.5
ng per 355 ml.
The average vitamin B-12 content for por-
ters was 84.7 ng per 355 ml.
An ANOVA showed that signi-
ficant differences (p<0.05) existed among the vitamin
B-12 content of these beers.
Tukey•s test indicated that
Anchor., Taddy and Yuengling all had signficantly more
(p<0.05) vitamin B-12 tnan Sierra Nevada (Table 24).
Anchor contains significantly more vitamin B-12 than
Yuengling also.
No other significant differences were
observed.
Stout. Five brands of stout were assayed for their
83
Table 23
Vitamin B-12 Content of Malt Liquors*
Brand Name
(Abridged)
Colt 45
ng/355 ml
Container
Type**
100.7 + 13.2a
AC
Mickey's
96.7 +
4.0a
DB
Old English
43.3 +
2.1b
CB
Carlsberg
36.7 +
3.8b
DB
Average
69.5 + 34.2
*Values are the mean + S.D. of the mean from six replicates. Those values which have no superscripts in
common are significantly different at the 0.05 level.
**Container types:
CB=clear bottle.
AC=aluminum cani
DB=dark bottlei
84
Table 24
Vitamin B-12 Content of Porters*
Brand Name
(Abridged)
Anchor
ng/355 ml
109.8 +
Container
Type**
3.8a
DB
Taddy
95.3 +
8.5a,b
CB
Yuengling
91.0 +
4.6b
DB
Sierra Nevada
43.5 +
6.1c
DB
Average
84.7 + 29.0
*Values are the mean + S.D. of the mean from six replicates. Those values which have no superscripts in
common are significantly different at the 0.05 level.
**Container types:
DB=dark bottle;
CB=clear bottle.
85
vitamin B-12 content- (Table 25).
Mackeson Stout had the
highest vitamin B-12 content with 100.3 ng per 355 ml
while Guinness· Extra Stout had the lowest with 48 ng per
355 ml.
The average vitamin B-12 content for stouts was
67.8 ng per 355 ml.
An ANOVA showed that significant
differences (p < 0. 05) existed among the vitamin B-12 content of the beers.
Tukey's test indicated that Mackeson
had significantly more (p <0.05) vitamin B-12 than Belikin, Sierra Nevada, Thos. Cooper and Guinness (Table 25).
Belikin also contained significantly more vitamin B-12
than Guinness.
No other significant differences were
found.
Paul and Southgate (1978) listed values of 385 ng
per 355 ml for stout and
stout (Table 7).
~95
ng per 355 ml for extra
Again the values are much higher than
those found in this study and the possibility that Lactobacillus leiehmannii is measuring more than the
active vitamin B-12 must be considered.
clinical~
It should also
be noted that the current study found that Guinness Extra
Stout had the lowest vitamin B-12 content for stouts,
just the reverse of the high value for extra stout listed
by Paul and Southgate (1978).
Without further informa-
tion about samples in the current study it is difficult
to determine if the two extra stouts are actually similar.
Vitamin B-12 Content of Eight Types of Beer
The previous section listed the average vitamin B-12
86
Table 25
Vitamin B-12 Content of Stouts*
Brand Name
(Abridged)
ng/355 ml
Container
Type**
5.1a
DB
Belikin
73.7 + 15.0b
DB
Sierra Nevada
63.0 + 12.5b,c
DB
Thos. Cooper
54.0 +
1.0b,c
DB
Guinness
48.0 +
2.0c
DB
Average
67.8 + 20.5
Mackeson
100.3 +
*Values are the mean + S.D. of the mean from six replicates. Those values which have no superscripts in
commom are significantly different at the 0.05 level.
**Container type:
DB=dark bottle.
87
values for each type of beer and the values for the highest vitamin B-12 content and lowest vitamin B-12 within
each type.
Table 26 summarizes the average values for
each type of beer.
An ANOVA was applied to the data in
Table 26 and significant differences (p<O.OS) among the
means were found.
Due to the unequal cell sizes and the
small number of samples within certain beer types, the
conservative Scheffe•s Specific-Comparison Test was used
to determine significant differences.
Test, no significant differences (p
Using Scheffe•s
> 0.05) were actually
found between the types of beer with respect to vitamin
B-12 content.
Even though the differences were not found
significant, certain observations about the data can be
noted.
erie
Domestic and imported lagers, as well as low-calbeers, have less than four percent alcohol by weight
while dark beer, ale, malt liquor, porter and stout all
tend to have at least four percent and possibly five to
six percent alcohol by weight.
The three beers with the
lowest alcohol content (four percent or less) appear to
have the least vitamin B-12 while the five beers with the
highest alcohol content (four to five percent or more)
seem to have the most vitamin B-12.
The additional alco-
hol might represent additional fermentation time and
therefore, additional time for vitamin B-12 production.
The quantity of data analyzed at one time in Table 26
may have confounded some significant differences.
Data
88
Table 26
Average Vitamin B-12 Content of Beers
Type of Beer
ng/355 ml
No. of
Samples a
Domestic Lager
66.7 + 28.4
14
Imported Lager
40.5 + 17.0
8
Dark
94.0 + 54.7
8
Low-Calorie (light)
38.3 + 15.0
10
Ale
81.2 + 74.7
5
Malt liquor
69.5 + 34.2
4
Porter
84.7 + 29.0
4
Stout
67.8 + 20.5b
5
aEach sample was replicated six times.
b
Values are the mean + S.D. of the mean. Those values
followed by the same superscript are not significantly
different at the top of the 0.05 level.
89
were available which allowed nine lager beers to be matched with their brand name low-calorie counterparts (Ex.
Olympia and Olympia Gold) •
The lager beers had a mean
value of 57.3 + 26.8 ng vitamin B-12 per 355 ml while the
light beers had a mean of 34.3 + 8.7 ng of vitamin B-12
per 355 ml.
Using a t test it was found that lager beers
had significantly more (p <0.05) vitamin B-12 than their
low-calorie counterparts.
At the present time, low-calorie
beer is literally a watered-down version of the regular
lager beer.
If this is the case, one would expect the
low-calorie beer to have less vitamin B-12 in addition to
less kilocalories, carbohydrates and protein.
Enough data were available to match five lager beers
with their brand name dark beer counterparts (Ex. San Miguel and San Miguel Dark) •
The lager beers had a mean
value of 56.2 + 28.6 ng of vitamin B-12 per 355 ml .. while
the dark beers had a mean value of 94.6 + 53.6 ng of vitamin B-12 per 355 ml bottle.
Using a t Test it was deter-
mined that there was no significant difference (p>0.05)
between lager beers and their brand name dark counterparts.
The major difference between lager and dark beer is the
addition of roasted barley to the latter.
This difference
by itself would not necessarily stimulate the production
of vitamin B-12.
Perhaps a lengthened fermentation to
bring the levels of alcohol up would increase the vitamin
B-12 levels.
In this study no difference was observed.
90
Four other comparisons were made to find out if products produced by the same company had similar vitamin B-12
content per bottle.
An ANOVA was used to analyze the data
for Sierra Nevada Brewing Co. Pale Ale, Porter and Stout.
There was no significant difference (p>O.OS) in vitamin
content of the three Sierra Nevada products.
B~2
A t Test was
used to analyze the data for Carlsberg Lager and Carlsberg
Elephant Malt Liquor.
No significant difference (p>O.OS)
in vitamin B-12 content was found between the two products.
A t Test was used to analyze the data for Anchor Steam Beer
and Anchor Porter.
No significant difference (p>O.OS) in
vitamin B-12 content was detected.
Finally, a t Test was
used to analyze the data for Thos. Cooper and Sons Real Ale
and Stout.
A significant difference (p<O.OS) in vitamin
B-12 content was found between these products.
Many possibilities exist to explain why no significant
differences were discovered in three of the sets of comparisons.
The first possibility is that the sample size was
~
too small to see an effect.
Another possibility is that
the typical brewing company would expose all their products
to a similar microbial environment.
They would also draw
needed brewing ingredients from common storage areas.
Handling techniques on all product lines would be similar.
All these added together could produce a regular contamination of all their products with similar amounts of vitamin
B-12 producing microorganisms.
Large sample comparisons of
product lines of breweries would be interesting.
Chapter 5
CONCLUSION AND RECOMMENDATIONS
The results of this study demonstrate that beer
contains vitamin B-12.
Small, measurable amounts of
min B12 were found in all beers assayed.
vit~
The values for
vitamin B-12 content of beers ranged from a low of 38.3
ng per 355 ml in low-calorie beers to a high of 94.0 ng
per 355 ml in dark beers.
The mean value for all beers
assayed was 67.8 ng per 355 ml.
The significance of beer
as a source of vitamin B-12 at these levels could be
challenged.
A bottle of beer containing this quantity of
vitamin B-12 would provide only two percent of the RDA.
However, people may need as little as 100 to 500 ng of
vitamin B-12 per day to avoid vitamin B-12 deficiency
anemia.
A number of individual brands of beer contained
well over 100 ng of vitamin B-12 per 355 ml which is
in the 100 to 500 ng range.
wit~
For people who do not regu-
larly consume animals or animal products, beer could contribute small but significant amounts of vitamin B-12 to
their diets as part of a well-balanced diet.
91
Homemade
92
beers, which are typically exposed to more contaminating
microorganisms and are usually less filtered than commercial beers, might be a better source of vitamin B-12 than
commerical beers.
The values of vitamin B-12 found in beers in this
study were substantially lower than those found by Paul
and Southgate (1978).
The major reason for the large
differences found between the two studies is most likely
the choice of microorganism.
The values reported by Paul
and Southgate (1978) were obtained using Lactobacillus
leichmannii while the present study used Ochromonas malhamensis.
Most studies agree that the protozoan method
using 0. malhamensis is far more specific than the bacterial method using L. leichmannii.
With the differences
in activity of the two microorganisms it is difficult to
compare values obtained through the two different methods.
Future samples should be assayed with both microrganisms
to determine if the two are measuring only the clinically
~
active forms of vitamin B-12.
In studying the vitamin
B-12 content of California red wines using 0. malhamensis
Voigt et al.
(1978) found ranges of vitamin B-12 of from
32 to 71 ng per 355 ml, ranges comparable to those found
in this study.
The data from the present study show that there are
significant differences among the various brands of beer
in each category but there are no significant differences,
·in general, between the categories.
These data indicate
93
that the most important factor in determining the vitamin
B-12 content of a beer in this study is the manufacturer
and not the actual category in which the beer is found.
The range of values found in each category shows that the
basic formulation and methodology for brewing a particular
type of beer are not the major factors involved in the
vitamin B-12 content of the beer.
It seems reasonable
to attribute differences in vitamin B-12 content of beers
in this study to individual brewery manufacturing practices.
Future studies could look closely at these prac-
tices.
A number of directions for future research are indicated by this study.
Alcohol content may be correlated
with vitamin B-12 content in beers and should be assayed
in any study of vitamin B-12 content of alcoholic beverages.
If fermentation time can be obtained, it would be
an important factor to investigate.
tainer type could be determined.
The effect of con-
American homemade beers
could be assayed for their vitamin B-12 content.
The
researcher could make the beer in the laboratory following
recipe books or could advertise for people to volunteer
their home brews.
Homemade alcoholic beverages are sig-
nificant sources of many nutrients around the world and
currently many Americans are returning to "natural" and
"homemade" products.
Finally, the fact that vitamin B-12 has been found
94
in this fermented vegetable product, indicates that it
would be worthwhile to look into the vitamin B-12 content
of other fermented vegetable foods such as sauerkraut
and some of the popular oriental fermented vegetable foods
that are now being produced in the West.
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9th ed.
Rahway~
101
Wokes, F., Badenoch, J. and Sinclair, H.M. 1955. Human
Dietary Deficiency of Vitamin B-12. American Journal
of Clinical Nutrition, 3: 375-382.
APPENDICES
102
APPENDIX A
Maintenance Medium for Ochromonas malhamensis
0. malhamemsis was maintained in 16 x 125 mm screwcapped culture tubes and initially transferred to new
tubes weekly.
The transfer time was reduced to a five
day cycle to provide fresh microorganisms for the assays
which were run every five days.
The medium was based on
the formulation of Baker and Frank (1968) and was composed
of:
Tryticase Peptone
(Baltimore Biological
Laboratory)
200 mg
Difco Yeast Extract
200 mg
Liver Extract Concentrate
(ICN Nutritional Biochemicals, #110337)
10 mg
Sucrose
1000 mg
500 mg
Glycerol (400 pl)
Distilled sterile water
to
100 ml
The medium was prepared in quantities of 500 ml.
tuents were added to a 500 ml volumetric flask.
ConstiThe
constituents were readily soluble in water and heating was
not required to get them into solution.
ed to 6.5 with KOH.
The pH was adjust-
The 10 ml quantities were distributed
into 16 x 125 mm screw-capped culture tubes and autoclaved at 121°C at 15 psi for 30 minutes.
103
The tubes were
104
allowed to cool at room temperature.
After reaching room
temperature, one drop of five day old medium was aseptically delivered from a long-tipped 1 ml sterile pipette
to fresh maintenance medium.
The tubes were inoculated
at room temperature (20-22°C) on an isolated counter in
the lab.
The tubes were exposed to window light and over-
head fluorescent lighting.
Baker and Frank (1968) recom-
mend constant illumination and an incubation temperature
of 28-32°C.
Under these conditions it was found that the
organism grew too rapidly and had to be transferred often.
Under the chosen conditions, the organism grew at a reasonable rate.
Constant illumination and higher tempera-
tures are not necessary requirements for the maintenance
of 0. maZhamensis
(Butner, Provasoli and Filfus, 1953;
Ford, 1953).
The remaining maintenance medium was stored at 4°c
with volatile preservative (see Appendix D) until used.
105
APPENDIX B
Basal Medium for Vitamin B-12 Assay
Using OahPomonas malhamensis
(Double Strength)
The basal medium for assay use must be totally free
of vitamin B-12 while providing all other essential growth
factors for the microorganism.
The medium was based upon
the formulation of Baker and Frank (1968) who recommend
against the use of commercial mixes due to their lack of
reliability from batch to batch.
The medium was composed
of:
(NH ) H citrate
4 2
Caco
150 mg
3
L-Glutamic acid
K Po
3
1000. mg
3000 mg
300 mg
4
(basic)
400 mg
L-Histidine HCl·H o
2
Thiamin HCl*
500 mg
Mgco
3
1 ml
Biotin*
.5 ml
Metals mix*
10 ml
MgS0 ·7H 0
2
4
L-Arginine HCl
Mo Standard (o.1 mg/ml)
((NH ) Mo o
-4H 0 in H 0)
2
2
4 6 7 24
Sucrose
KHC0
3
100 mg
400 mg
1 ml
12,000 mg
300 mg
106
DL-Methionine
400 mg
V Standard (1.0 mg/ml)
(v 2 o 5 in dilute HCl)
Casein Hydrolysate
(Vitamin & salt free)
20
pl
5000 mg
DL-Asparagine
1000 mg
L-Tryptophan
100 mg
p-Aminobenzoic acid*
1 ml
NaCN*
2 ml
Distilled water
to 500 ml
*Added from stock solutions
Fresh stock solutions were prepared monthly as
follows:
1.
Thiamin HCl.
Exactly 100 mg of thiamin HCl were
placed in a 100 ml volumetric flask.
Sterile
deionized water was added to 100 ml.
This dilu-
tion produced a stock solution of 1.0 mg/ml.
4°c in a refrigerator
The solution was stored at
with volatile preservative.
2.
Biotin.
Exactly 10 mg of biotin were placed in
a 1000 ml volumetric flask.
Approximately 10 ml
of sterile deionized water and a few ml of 0.1 M
KOH were added to the flask to dissolve the
vitamin (Stecher, 1968).
Sterile deionized
water was added to the 1000 ml mark.
This dilu-
tion produced a stock solution of 0.01 mg/ml.
107
The solution was stored at 4°C in a refrigerator
with volatile preservative.
3.
Metals mix.
A lOOOX solution that was suffi-
cient for 100 liters of basal medium was prepared with the following constituents:
ZnS0 ·7H o
2
4
17.6 g
Mnso -H 0
4 2
Fe(NH ) (so ) ·6H o
4 2
4 2
2
12.4 g
1.4 g
0.4 g
Cuso -5H o
2
4
Coso ·7H o
4
2
0. 24 g
Citric acid·H o
3.0 g
2
Sterile deionized H o to 1000 ml
2
The constituents were added to a 1000 ml volumetric flask.
The flask was placed on a hot
plate-stirrer with a sterile magnet and heated
to boiling to dissolve the constituents.
The
solution was cooled in a room temperature water
bath and stored in a refrigerator at 4°C with
volatile preservative.
4.
p-Arninobenzoic acid.
Exactly 125 mg of p-Arnino-
benzoic acid were placed in a 100 ml volumetric
flask.
100 ml.
Sterile deionized water was added to
This dilution produced a stock solution
of 1.25 mg/ml.
The solution was stored in a
refrigerator at 4°C with volatile preservative.
5.
NaCN.
All work with NaCN was done under the
108
exhaust hood while wearing gloves.
Exactly 100
mg of NaCN were placed in a 100 ml volumetric
flask.
mark.
Sterile deionized water was added to the
This dilution produced a stock solution
of 1.0 mg/ml.
The solution was stored in a
cabinet at room temperature with volatile
preservative.
One liter of double strength medium was made each
month.
The amount of each constituent was doubled and
added to a 1000 ml volumetric flask.
Approximately 700 ml
of sterile deionized water was added to the flask.
The
flask was placed on a hot plate-stirrer with a sterile
magnet and heated at a moderate heat without boiling.
The
solution was heated just long enough to dissolve the constituents.
water bath.
The flask was then cooled in a room temperature
After cooling, the total volume was brought
up to 1000 ml with sterile deionized water.
The pH of the
basal medium was then adjusted to pH 5.5 with KOH.
The double strength basal medium was stored in 120 ml
screw-capped media storage bottles.
The bottles were
filled to a point that left an absolute minimum of head
space and a drop of volatile preservative was added.
The
bottles were then stored until use at 4°C in a refrigerator.
109
APPENDIX C
Vitamin B-12 Standard Solutions
Vitamin B-12 Stock Solution (0.1 mg/ml).
This solu-
tion was prepared by adding 100 mg of crystalline vitamin
B-12 (ICN Nutritional Biochemicals, Life Sciences Group,
Cleveland, Ohio) to a sterile 1000 ml volumetric flask.
Enough sterile deionized water was then added to bring the ·
volume to 1000 ml.
The vitamin B-12 readily dissolves in
water without the application of heat.
This stock solu-
tion was stored for a maximum of one month at 4°C with
volatile preservative.
Solution A or Vitamin B-12 Working Solution (0.1 ng/m].
First, 1.0 ml of stock solution (0.1 mg/ml) was diluted
precisely to 1000 ml with sterile deionized water to give a
solution of 0.1 pg vitamin B-12/ml.
Second, 1.0 ml of 0.1
pg vitamin B-12/ml solution was diluted precisely to 1000
ml with sterile deionized water to give a working solution
of 0.1 ng vitamin B-12/ml.
This working solution was
stored for a maximum of one month at 4°c with volatile
preservative.
§Qlution B or Vitamin B-12 Working Solution (0.01ng;ffi].
To prepare this solution, a 10.0 aliquot of Solution A
(0.1 ng/ml) was diluted precisely to 100 ml with sterile
deionized water.
This working solution was stored for a
maximum of one month at 4°C with volatile pre~ervative.
110
APPENDIX D
Volatile Preservative
To protect stock solutions and fluids awaiting analysis against microbial action it was recommended that a
volatile preservative be routinely added to the (Baker and
Frank, 1968).
This preservative did not interfere with
assays because it was removed during autoclaving.
The
volatile preservative was composed of (by volume) :
1 part chlorobenzene
1 part 1,2-dichloroethane ("ethylene dichloride")
2 parts n-butyl chloride (1-chlorobutane)
The preservative was dispensed from a dropping bottle and
was added to all solutions which were not intended for
assay use on a particular day.
cient for most solutions.
A few drops were suffi-
For large volumes, 1 ml for
every liter of solution was used.
111
APPENDIX E
Aconitic Acid Buffer
Vitamin B-12 was extracted from the sample using an
aconitic acid buffer in combination with sodium metabisulfite and heat.
The aconitic acid buffer (Baker and
Frank, 1968) was composed of:
trans-aconitic acid
sterile deionized water
5
g
to 1000 ml
The aconitic acid was added to a 1000 ml volumetric flask.
Sterile deionized water was added to precisely 1000 ml.
The pH· was adjusted to 4.5 with KOH pellets.
Sodium meta-
bisulfite was added at a level of 0.1 mg/ml just before
use of the buffer.
The buffer was stored at 4°C in a
refrigerator with volatile preservative.
112
APPENDIX F
Calculation for Obtaining Vitamin B-12 Content
Per Bottle of Beer*
a·c·d
b·e
=
concentration of vitamin B-12 in beer sample
per ml
a
=
vitamin B-12 concentration per ml in assay
flask as derived from standard curve
b
=
ml (volume) of beer used for the assay
c
=
volume of beer sample after dilution with
buffer and water = 10 ml
d
=
total volume in growth flask
e
= ml
=
=
1 ml
5 ml
(volume) of diluted beer sample (c above)
(sample + diluent) added to assay flask =
1 ml, 1.5 ml or 2 ml
To obtain the concentration of vitamin B-12 per bottle
of beer (typical 12-ounce or 355 ml size), multiply the
value from the above calculation by 355 ml/bottle of beer.
*Source:
Baker and Frank, 1968.