371
RUSSELL,
C., BHANDARI,
R. R. & WALKER,T. K. (1954). J . gen. Microbiol. 10,
371-376.
Vitamin requirements of thirty -four Lactic Acid
Bacteria associated with Brewery Products
BY C. RUSSELL,* R. R. BHANDARI
AND
T. K. WALKER
College of Technology, University of Munchester
SUMMARY: The vitamin requirements of thirty-four strains of lactic acid bacteria,
representing ten species and isolated from brewery materials, were studied. Individual strains required from one to four vitamins for normal growth on the basal
medium used. Pantothenic acid was required by all the organisms. In a number of
cases growth failed when groups of vitamins, individually non-essential, were
omitted.
As part of a general study of lactic acid bacteria occurring in association with
brewery materials, Bhandari, Russell & Walker (1954) isolated sixty-nine
strains from sour beers and brewer's yeasts. It was felt of interest not only
to identify the species which had been isolated, but also to investigate their
nutritional requirements.
The nutrition of the lactic acid bacteria which occur in milk has been studied
by many workers (reviewed by Dunn, 1949; Knight, 1945), but apart from
a communication by Rainbow (1952) which described the requirements of four
strains of lactobacilli isolated from beer, the literature is devoid of references
to the nutritional requirements of beer lactic acid bacteria.
METHODS
The basal medium used was essentially that of Dunn, Shankman, Camien &
Block (1947), differing only in the concentration of adenine, guanine, xanthine
and uracil which were all used a t a concentration of 12 mg./l. In general,
media were made up from stock solutions of each organic constituent as
desired. Growth of the organisms was measured turbidimetrically against
uninoculated control blanks, using a Hilger ' Spekker ' absorptiometer with
neutral grey filters (H508).Values (expressed as 1000 x drum reading) were
the means of triplicate tubes. The tubes were test tubes 4 in. in length each
containing 3 ml. medium. They were sterilized by autoclaving a t 10 lb./sq.in.
for 2.5 min. (Toennies & Gallant, 1948).
Organisms were maintained in stab cultures and were transferred a t
monthly intervals. Reserve cultures were kept in the presence of CaCO, in
sealed tubes. Though other workers have found yeast-extract glucose broth
to be a suitable inoculum medium for lactic acid bacteria, our organisms grew
slowly or not at all in such a medium. A medium of the following composition
proved satisfactory: 1 yo (w/v) yeast extract (Difco); 0.5 yo (w/v) peptone
(Evans); 1 yo (w/v) glucose; 1.2 yo (w/v) sodium acetate; 0-5 Yo (v/v) each of
* Present address : Department of Biochemistry, Christie Hospital and Holt Radium
Institute, Manchester 20.
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C . Russell, R. R. Bhandari and T . K . Walker
salt solutions A and B (Snell & Wright, 1941). The reaction was adjusted to
pH 5.1-5.3, using Johnson’s pH papers.
The cultures for inoculum were grown in 4 ml. medium in centrifuge tubes.
These tubes were modified by the intrusion of glass prongs into the lumen
about 3cm. from the mouth. A cotton-wool plug was supported by the
platform thus provided and so was not forced down during centrifugation.
The cells were centrifuged, washed twice with 0.9% (w/v) saline and finally
resuspended in 15-20 ml. saline. Pasteur pipettes were used to transfer one
drop (about 0.05 ml.) of this suspension to each culture tube. The tubes were
incubated at 25’ in closed vessels in an atmosphere containing 80-85 % CO,.
This procedure was adopted because the organisms studied normally grow
anaerobically in the presence of a high concentration of CO,.
RESULTS
Individual cultures were incubated for the particular period (5-8 days) which
had been found in preliminary experiments to be optimal for maximum
growth in the complete medium. Some organisms (not dealt with in this
paper) failed to grow after 14 days incubation, thus showing that the complete
medium was not suitable for all lactic acid bacteria.
The vitamin requirements of the bacteria are shown in Table 1, in which the
organisms are arranged in order of their fastidiousness and the vitamins in
order of their relative effect. A vitamin was termed ‘essential’ when its
omission caused growth to decrease to one-quarter of that achieved in the
complete medium; a fall to only one-half the turbidity reached in the control
medium was deemed indicative of a ‘ stimulatory ’ vitamin, in order to facilitate
classification.
The maximum number of vitamins required by any organism was four;
LactobaciZZus pamus required pantothenic acid, nicotinic acid, riboflavin and
thiamine. Twelve strains (representing four species) required only pantothenic
acid; all the organisms studied required this vitamin. Nicotinic acid was
essential for twenty-two organisms and stimulated four others. In contrast,
p-aminobenzoic acid (p-AB)was required by only one organism, L. pastorianus
strain W 4 , while choline, inositol, the B6 group, folic acid and biotin were
apparently not required by any of the organisms tested under our conditions.
It was thought of interest to attempt to grow some of the organisms on media
containing only their ‘essential ’ vitamins in addition to amino acids, purines,
uracil, glucose and salts. Vitamin-free casein (Difco 1 yo,w/v), supplemented
with tryptophan and cysteine, was used in place of the mixture of amino
acids; other constituents were present in the same concentration as in the
basal medium.
Eighteen media were prepared according to the vitamin requirements of
eighteen organisms. Only L. pastorianus HH4 grew as well in this ‘essential
vitamin ’ medium as in the complete medium of vitamin-free casein +all
vitamins. Of the remaining organisms the growth of five fell to between
one-quarter and one-half of that in the complete medium, and of twelve
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Nutrition of brewery lactic acid bacteria
373
Table 1. Vitamin requirements
Ca panto- Nicotinic
Organism
thenate
acid
Lactobacillus parvus*
E
E
L . buchneri CC3
E
E
L . leichmannii EE4
E
E
L. rnalefermentansjE
E
L. pastorianus HH4
E
E
L. pastorianus W 4
E
E
L. bifldus DDI
E
E
L. bifidus DD5
E
E
L. bifldus EEl
E
E
L. buchneri a
E
E
L. buchneri p
E
E
L. buchneri 1
E
E
L . frigidus HH5$
E
E
L. pastorianus BB 1
E
E
L . pastorianus BB4
E
E
L . pastorianus DD 4
E
E
L . pastorianus W 5
E
E
L. pastorianus 12
E
E
L . pastorianus 19
E
E
L. pastorianus 23
E
E
Pediococcus damnosus var.
E
E
salicinaceus FF 6
Streptococcus cremoris 15
E
E
Lactobacillus buchneri C
E
N
L . buchneri K
E
S
L . leichmannii A A 1
E
RT
L. leichmannii AA2
E
N
L. leichmannii AA3
E
N
L. leichmannii AA4
E
N
L. pastorianus CC4
E
N
L. pastorianus W1
E
S
L. pastorianus W 10
E
S
L. pastorianus D
E
S
L. pastorianus L
E
N
L . plantarum q5
E
N
Riboflavin
E
E
E
E
N
N
S
N
N
N
N
S
N
N
S
N
N
N
N
N
N
N
N
S
N
N
K
K
N
hT
N
S
N
N
Thiamine
E
N
p-AB
N
N
N
E
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
S
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
B6
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
S
N
N
N
N
N
N
N
N
N
E
N
N
N
N
N
PI;
11'
N
N
N
N
N
N
N
N
N
N
N
S
N
N
N
N
N
N
RT
N
N
N
h
T
E, essential (turbidity below one-quarter of control); S, stimulatory (turbidity onequarter to one-half of control); N, non-essential (turbidity above one-half control): remaining vitamins were all non-essential.
* Russell & Walker (1953b). t Russell & Walker (1953a). $ Bhandari & Walker (1953).
organisms to less than one-quarter of the latter. Thus, the vitamin requirements (shown in Table 1) are not the optimal requirements of seventeen of
the eighteen organisms. The experiment was then repeated with the media
modified by the addition of further vitamins. These were those considered
most likely to improve growth; they were added singly and as a mixture.
Thiamine was the most potent vitamin in that six organisms (L.buchneri p,
L. pastorianus strains 12, 19, 23 and DD4, and Streptococcus cremoris 15)grew
very well in the presence of their essential vitamins+thiamine. It may not
be mere coincidence that in all these cases the essential vitamins were nicotinic
and pantothenic acids. I n eight cases the addition of folk acid (but not p-AB)
resulted in a significant increase in growth. Taken in conjunction with the lack
of requirement for p-AB this would seem t o indicate that the organisms can
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374
C . Russell, R. R. Bhandari and T . K . Walker
synthesize this vitamin at a rate sufficient to satisfy their needs, but that the
synthesis of folic acid is a slower process. Even when all the vitamins found
important when omitted singly were added as a group, three organisms still did
not grow well. These were Lactobacillus buchneri 1, L. pastorianus CC4 and
L. plantarum q5.
DISCUSSION
The finding that all the organisms required pantothenic acid is in accord with
the work of Shankman, Camien, Block, Merrifield & Dunn (1947), and of
Cheldelin, Hoag & Sarett (1945) with other lactic acid bacteria. Other work
in this laboratory has shown that the components of pantothenic acid, supplied
either separately or together, were not used in place of the intact vitamin.
Shankman et al. (1947) did not find p - A B essential for any of the twentythree lactic acid bacteria they studied, but Isbell (1942) reported it to be
essential for L. arabinosus 17-5, and Pennington (1946), found that it was
required by Leuconostoc rnesenteroides P-60 under certain conditions. It is to
be noted that the requirement for p - A B observed in the present work was
found on a medium which contained folic acid and purines. Evidently Lactobacillus pastorianus W 4 cannot utilize these substances in place of p-AB, unlike
L.pentosus (Snell & Mitchell, 1942).
The lack of requirement for folic acid and biotin makes the brewery lactic
acid bacteria rather different from the milk organisms. Of the latter, Snell
(1948) listed nine which required biotin and three which required folic acid.
Shankman et al. (1947)found two bacteria which were exacting towards biotin
and four to folic acid, out of twenty-three organisms studied. The lack of
a biotin requirement observed with our organisms may be linked to the
presence of asparagine and a high concentration of CO,, although oleate was
absent.
In the absence of an ‘essential’ vitamin, growth generally dropped to much
less than one-quarter of that in the complete medium. Frequently the decrease
was to one-tenth, particularly when pantothenic and nicotinic acids were
involved. Thus, successful assays for these vitamins might be developed with
these organisms; especially is this indicated by the fact that the organisms
were tested on a rich medium over fairly long incubation periods.
There were a number of cases of apparent inhibition of growth by vitamins.
Thus, the omission of p - A B led to increased growth of eight organisms, and
thiamin inhibited two strains. Shankman et al. (1947), stated that p - A B was
inhibitory towards L. casei, while Whiteside-Carlson & Carlson (1949) found
instances of inhibition of Leuconostoc sp. by riboflavin, biotin, folic acid,
p - A B and vitamin B6.
Table 1shows that the vitamin requirements of different strains of any one
species varied greatly. Thus, with respect to Lactobacillus buchneri, strain CC 3
required three vitamins, strains a and p required two and strains C and K each
required only one vitamin. In the case of L. pastorianus, two strains had three
requirements, seven had two requirements, and for five strains only one
vitamin was necessary. This variation in nutritional requirements of strains
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Nutrition of brewery lactic acid bacteria
375
of any particular species is well known (Boivin, 1945; Shankman et aZ. 1947).
However, there appears to be a tendency for strains isolated from the same
source to have the same requirements. Thus, strains AA1, AA2, AA3 and
A A 4 of L. Zeichmannii were all isolated from one yeast and all required only
pantothenic acid; strain EE 4, from a different yeast, required pantothenic
acid, nicotinic acid and riboflavin. Strains GC and @ of L. buchlzeri isolated from
one yeast both required pantothenic and nicotinic acids, whereas strains C
and K, from another yeast, required only pantothenic acid. This trend to
similarity is not shown by strains W1, W 4 and W 5 of L. pastorianus which
were all isolated from one source but which required one, three and two
vitamins, respectively.
Not many attempts appear to have been made to grow lactic acid bacteria
on media which contained only their ‘essential ’ vitamins. However, WhitesideCarlson & Rosano (1951) found that L. dextranicum strain ‘elai’ did not grow
on a basal medium+thiamine, nicotinic acid and pantothenic acid. The
addition of a single vitamin was not effective in restoring growth completely,
although folic acid gave a significant improvement.
Thanks are due to Dr F. Bergel, Roche Products Ltd., for the gift of pyridoxal
acetal hydrochloride and pyridoxamine dihydrochloride. One of us (C.R.) was
a Postgraduate Scholar in Technology of Manchester City Council and one of us
(R. R. B.) held a Scholarship awarded by the Rajasthan Government.
This paper is the second of a serieson lactic acid bacteria associatedwith breweryproducts.
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