Download Anti-microbial substances produced by food associated micro

Biochemical Society Transactions
694
12. Okui, S., Uchiyama, M., Mizugaki, M. & Sugawara, A.
(1063) Hiochim. Hiophys. Acta 70, 348-35 1
13. Farbood. M. I., Morris, J. A., Sprecker, M. A,.
Hienkowski, I,. J., Miller, K. l’.? Vock, M. 11. &
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WO 8912-134A
15. Hilton, M. D. & Cain, W. J. (1990) Appl. Environ.
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16. Farbood, M. I., Willis, H. J. & Christenson, 1’. A.
(1086) S. African ZA 8.5 04,306
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U S . Pat. 4,970,163
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Keceived 12 April 1091
Anti-microbial substances produced by food associated micro-organisms
Hans Horn and Christina Msrtvedt
MATFORSK, Norwegian Food Research Institute, Osloveien I , N- I430 As, Norway
Introduction
Fermentative breakdown of carbohydrates results
in a range of small molecular mass organic molecules which exhibit anti-microbial action, including
lactate, propionate, acetate and ethanol. Conditions
favouring the growth of bacteria producing these
metabolic end products were discovered and used
thousands of years ago and are still exploited in the
production of a variety of products. They are
employed in different manners, either as food additives after production in industrial fermentors, or
produced in situ during fermentation of meat, vegetables and milk.
Today there is considerable renewed interest
in the use of naturally produced anti-microbial substances for food preservation and protection owing
to the low energy demand of the fermentation process and the discovery of an abundance of additional anti-microbial activities associated with the
fermentative microbes. Recombinant DNA technology has made it possible to identify and clone
genes encoding these anti-microbials and electrotransformation has enabled the transfer of the genes
to other bacteria, thus opening the era of tailormade starter cultures.
The present paper will survey the antimicrobials produced by food associated microorganisms with the main emphasis on the lactic acid
bacteria and related organisms.
Metabolic end products
Present knowledge suggests that the commonest
form of anti-microbial activity expressed in foods is
that associated with lactic, acetic and propionic
acids.
Lactic acid results from the metabolism of
many different types of bacteria, primarily LactoAbbreviation used: 3-HPA, 3-hydroxypropanal.
Volume 19
bacillus, Lactococcus, Leuconostoc and Pediococcus.
Lactic acid is produced from breakdown of hexoses
via the Embden-Meyerhof-Parnas pathway. Ideally,
in homofermentative breakdown one mole of
hexose gives rise to two moles of lactic acid and two
moles of ATP. The anti-microbial action of lactic
acid is only moderate. The acid produced lowers
the pH and the growth of most spoilage and pathogenic organisms is inhibited to some extent. Lactic
acid causes the leakage of hydrogen ions across the
cell membrane. This results in acidification of the
cell interior and inhibition of nutrient transport. The
energy-yielding metabolism is not influenced [ 11.
Lactic acid is the primary acid produced during fermentation of sausage, sauerkraut, olives, yoghurt,
etc.
In heterofermentative breakdown of sugars,
the dissimilation proceeds via the pentosephosphate shunt. One mole of hexose gives rise to
one mole of lactic acid, ethanol, CO, and ATP,
respectively. However, oxidation of NADH + H +
using alternative hydrogen acceptors (see below)
sometimes leads to the formation of acetic acid and
ATP instead of ethanol. Acetic acid is one of the
most used anti-microbials from micro-organisms.
Industrially it is produced through aerobic oxidation of ethanol by members of the genus Acetobacter. Acetic acid is mainly used as a food additive,
in the form of vinegar. It is added as a preservative
substance and a flavouring agent to many different
foods including mayonnaise, dressings, pickles and
mustard.
Acetic acid has a wide range of inhibitory
activity, inhibiting yeasts and moulds as well as
bacteria. Its action cannot be explained by pH
reduction alone. The undissociated form penetrates
the cell and hereby exerts its inhibitory action,
which is consistent with its anti-microbial activity
increasing with decreasing pH values.
Food Biotechnology
Propionibacterium produces propionic acid
from lactic acid. In short, 3 moles of lactic acid give
rise to 2 moles of propionic acid, one mole of acetic
acid, C 0 2 and ATP. In ripe Swiss and Jarlsberg
cheese up to 1% of propionic acid may be detected.
The inhibitory action of propionic acid is partially
caused by inhibition of nutrient transport [2], and
to accumulation in the cell resulting in inhibition of
cell metabolism [3]. Propionates are mainly used as
niould inhibitors, but the organism which causes
rope in bread, Bacillus mesentericus is also inhibited.
Consequently, the main application of propionates
is in the baking industry.
The three organic acids described here have
an optimum anti-microbial function located close to
and below their pK, values. At these pH values a
large fraction of the acids are undissociated, they
solubilize in the cell membrane, block transport of
necessary growth substances, acidify the cell
interior by dissociation and exert other inhibitory
activities on cell growth. The Food and Agriculture
Organization (FAO) and World Health Organization (WHO) have set no limit for the acceptable
daily intake of these acids.
[8] indicate that active transport across the membrane is affected. A more delayed reaction is also
described. After 2 hours exposure to the lactoperoxidase system cell death can be demonstrated.
Cytoplasm is extruded from a polar position, possibly when the last cell division envelope is formed
PI.
When iodine substitutes for thiocyanate,
direct halogenation of bacterial proteins seems to be
the inhibitory mechanism. Hypochlorous acid,
which exhibits multiple effects on bacterial cells,
apparently is formed from chloride [9]. The lactoperoxidase system is not much used as an antimicrobial food additive. In vivo mainly the
H,O,-producing organisms are influenced. The
spoilage psychrotrophs do not activate the system.
By addition of glucose and glucose oxidase or
hydrogen peroxide (at non toxic levels) the lactoperoxidase system is activated and it is possible to
extend the lag period of psychotrophs in refrigerated milk from 2 to 5 days. Residual H20, and the
inhibitory thiocyanate product are both inactivated
by pasteurization. This process has been patented
by Alfa Laval.
The lactoperoxidase system
Reuterin: 3-hydroxypropionaldehyde
In 1924, Hanssen [4] observed that freshly drawn
milk exhibited bactericidal properties. The inhibitory activity was lost after heat treatment (75"C, 15
min) and the activity was mistakenly attributed to
peroxidative enzymes transferred from plants.
Today, it is known that the enzyme responsible,
lactoperoxidase, is present in high concentrations in
milk and other body fluids and that the lactoperoxidase system is dependent on two additional
factors to exhibit its bactericidal effect [ 51. Together
with thiocyanate and/or halides, also present in
milk, and hydrogen peroxide, produced by aerobically growing micro-organisms, an enzyme reaction
product of bactericidal nature is formed.
When thiocyanate is utilized by the lactoperoxidase-HzO, system, it is converted from a
weak to a strong bactericidal agent. The instability
of the actual compound formed has made it difficult
to isolate the active substance and to establish its
exact chemical composition.
According to Bjorck et a.! 1975 [6] the bactericide produced is a low molecular mass compound, easily destroyed by heat, which exerts the
most pronounced activity against Lactococci and
Gram-negative bacteria. The nature of the bactericidal action is not well defined. An immediate reaction has been described [7] where hexokinase or
other glycolytic enzymes are involved. Other results
Recently, research on Lactobacillus reuteri has
revealed the nature of an extremely broad-spectrum
anti-microbial, called reuterin [lo]. When grown on
a mixture of glucose and glycerol, L. reuteri produces a catabolite from glycerol which can act as an
acceptor for the NADH + H + formed from glucose.
The catabolite, 3-hydroxypropanal (3-HPA), is
formed by an enzymatic dehydration of glycerol
and is subsequently reduced to 1,3-propanediol by
an NADH + H+-specific dehydrogenase (Fig. 1).
The N A D H + H + needed for this reaction to proceed, is gained from the main heterofermentative
pathway by the shifi from ethanol to acetic acid production [ 111. The aldehyde has been shown to be
the inhibitory compound. Because it is readily
reduced to the diol, 3-HPA cannot be detected
during the active phase of growth when reducing
power is plentiful, but begins to accumulate in the
growth medium when the cells enter the stationary
phase of growth [12]. Interestingly, 3-HPA is
formed throughout growth when L. reuteri is cocultured with any one of a number of different
Gram positive and Gram negative species, including Eschm'chia coli [13]. This is probably of great
ecological significance, giving L. reuteri an advantage in competition with other members of the gut
bacterial flora. The enzymes involved in glycerol
dissimilation appear to be constitutive in L. reuteri
1991
695
Biochemical Society Transactions
Bacteriocins
Fig. I
The proposed reactions taking place in the utilization
of glycerol as hydrogen acceptor
696
Framed substance is reuterin.
Hz-q-OH
H-?-OH
Hz - C - O H
Glycerol
Glycerol
dehydratase
1
H-T=O
H-q-H
HzC-OH
FNr:I
+3
3-hydroxypropionaldehyde(3-HPA)
Main pathway
reductase
3-HPA
Hz?-OH
H-C-H
I
HzC-OH
I ,3-propanediol
[ 141 in contrast with the situation in Klebsiella pneumoniae, for example, where they are inducible [ 111.
Dissimilation of glycerol is reported to take place by
the same pathway, in Clostridiurn spp. [15], in K.
pneumoniae [ 161 and in other Lactobacillus spp. [ 171.
The actual accumulation and excretion of 3-HPA is
however, a trait confined to L. reuteri alone. The
mechanism underlying lack of accumulation and
excretion in other glycerol dissimilating bacteria is
not yet understood but dismutation and/or isomerization of 3-HPA have been proposed by several
authors [ l l , 12, 171.
Isolation and purification of reuterin led to the
discovery of an equilibrium mixture of 3-HPA consisting of the monomer, hydrated monomer and a
cyclic dimeric form [ 121. Reuterin is produced and
exhibits its bactericidal function at pH 4-9, with an
optimum around pH 6-8. Reuterin has a very
broad activity spectrum. All micro-organisms tested
so far are sensitive, although to different degrees.
Experiments show that it acts as an anti-bacterial,
anti-fungal, anti-protozoal and anti-viral compound
[121.
Even the producer strain itself is influenced.
Lactic acid bacteria are in general more resistant
than other micro-organisms [ 131. These findings
are supported by the demonstration of inhibitory
effect on sulphydryl enzymes [18], essential in
DNA synthesis and other vital cell functions.
Volume 19
Bacteriocins are anti-microbial substances which
are produced and excreted by micro-organisms and
act on the same or closely related species. Bacteriocins contain an essential protein moiety and are
supposed to show a bactericidal mode of action.
A wealth of basic information concerning bacteriocins is available. Most of today's knowledge is
deduced from research on E. coli bacteriocins
(colicins), and the reader is referred to Pugsley and
Oudega, 1987 [ 191 for a detailed review.
Nisin, produced by Lactococcus lactis, is the
best known bacteriocin produced by a food associated micro-organism. Nisin has a well documented activity, a broad inhibitory spectrum and is
affirmed for use in certain foods [201.
Many other bacteriocins have been described,
of which those produced by food associated
bacteria have the greatest potential value. So far
more than 20 different bacteriocins produced by
lactic acid bacteria have been reported [ 19, 201.
Although only a few of these have been purified to
homogeneity, they appear to constitute a heterogenous group of anti-microbials. Estimation of size
based on molecular sieve procedures often indicates
a molecular mass considerably higher than the
actual size determined after purification. During
purification, activity is often reduced severely [2 1,
221.
The genetic determinants involved in bacteriocin production encode at least two different
single traits, i.e. production and immunity. Starter
cultures producing bacteriocins may prove to be
useful in improving the safety of the food fermentation processes. Genes encoding production of
and immunity to bacteriocins may be transferred to
strains used as starter cultures by means of electrotransformation. The genes represent easily selectable genetic markers, which may be used to
enhance the stability of additional genes, when used
in edible vectors and will give the transformed
starter culture an advantage compared with nonbacteriocin-producing sensitive strains.
Screening for bacteriocin producing organisms is usually performed with the deferred method
[23] where fresh mature colonies are overlaid with
soft agar containing a sensitive strain. After further
incubation to allow the indicator strain to grow,
bacteriocin producing colonies are seen with clear
inhibition zones (Fig. 2). T o quantify bacteriocin
activity, a microtitre plate assay is used. Twofold
dilutions of sterile bacteriocin extracts are inoculated with a freshly prepared indicator culture. Bac-
Food Biotechnology
teriocin units (BUIml) are usually presented as the
reciprocal of the highest dilution exhibiting growth
inhibition of the indicator organism [24].
The demonstration of bacteriocin production
in synthetic media does not necessarily reflect production and activity in fermented foods. Nutrient
availability, temperature, pH, inoculum size and
other factors will all influence production [ZO]. We
have developed an easy and safe procedure to
demonstrate plasmid encoded bacteriocin activity in
fermented food.
Lactobacillus sake L45, isolated from naturally
fermented dry sausage, produced a bacteriocin
Fig. 2
Result from the deferred assay, demonstrating the
production of the bacteriocin, lactocin S, by L. sake
L45, as clear inhibition zones surrounding the
individual colonies
Fig. 3
Effect of bacteriocin production in fermented sausage
(*) denotes producing strain (Bac', Imm') and (0) the sensitive (Bac-, Irnm-, E$) strain.
*I
* *
108
-
;t(
4..
\
.-n
L
\
Y
n
n
L
0
n
107-
5
z
0
4
8
12
Time (days)
16
20
24
designated Lactocin S [25]. The genes encoding
bacteriocin production and self-resistance are
located on a 50 kb plasmid. A cured strain was
obtained through segregation experiments and
shown to be both bacteriocin negative and sensitive
(Bac-, Imm-) [25]. Electro-transformation was
performed [26] to construct a Bac-, Imm-, Ery'
variant of L. sake L45, with growth characteristics
identical with the parental strain.
Dry sausage was made using a mixture of L.
sake L45 Bat+, Imm' and Bac-, Imm-, Ery'
isolates as the starter culture. After four days the
number of Ery' cells dropped in contrast with the
Bac+, Imm+ count (Fig. 3). This method may prove
useful in demonstrating bacteriocin production in
fermented food, since all additional anti-microbial
activity can be neglected.
1. Busta, F. F. & Foegeding, P. M. (1983) in Disinfection, Sterilization and Preservations (Block, S.S., ed.),
pp. 657-664, Lea & Febinger, Philadelphia, USA.
2. Eklund, T. (1980)J. Appl. Bact. 48,423-432
3. Luck, E. (1980) Antimicrobial Food Additives Characteristics, Uses, Effects, pp. 186-21 1, SpringerVerlag, Berlin, Heidelberg & New York
4. Hanssen, F. W. (1924) B. J. Exp. Pathol. 5,271-280
5. Bjorck, L. P. (1978)J. Dairy Res. 45, 109-1 17
6. Bjorck, L. P., Rosen, C. G., Marshall, V. & Reiter, B.
(1975) Appl. Microbiol. 3, 199-204
7. Reiter, B. & Oram, J. D. (1967) Nature, (London) 216,
328-330
8. Reiter, B. (1976) in Inhibition and Inactivation of
Vegetative Microbes (Skinner, F. A. & Hugo, W. B.,
eds.), pp. 42-48, Academic Press Ltd, London
9. Davidson, P. M., Post, L. S., Branen, A. L. &
McCurdy, A. R. (1983) in Antimicrobials in Foods
(Branen, A. L. & Davidson, P. M., eds.), pp. 371-385,
Marcel Dekker, Inc., New York and Base1
10. Axelsson, L. T., Chung, T. C., Dobrogosz, W. J. &
Lindgren, S. E. (1989) Microb. Ecol. Health Disease
2, 131-136
11. Forage, R. G. & Lin, E. C. C. (1982) J. Bacteriol. 151,
591-599
12. Axelsson, I,. T. (1990) Dissertation Report 44,
Uppsala, Sweden
13. Chung, T. C., Axelsson, L. T., Lindgren, S. E. &
Dobrogosz, W. J. (1989) Microb. Ecol. Health
Disease 2, 137- 144
14. Talarico, T. L. & Dobrogosz, W. J. (1990) Appl.
Environ. Microbiol. 56, 1195-1 197
15. Forsberg, C. V. (1987) Appl. Environ. Microbiol. 53,
639-643
16. Forage, R. G. & Foster, M. A. (1983)J. Bacteriol. 149,
413-419
17. Sobolov, M. & Smiley, K. L. (1960) J. Bacteriol. 79,
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18. Dobrogosz, W. J., Casas, I. A., Pagano, G. A.,
Talarico, T. I,., Sjoberg, B.-M. & Karlsson, M. (1989)
in The Regulatory and Protective Role of the Normal
Microflora (Gruff, R., Midtvedt, T. & Norin, E., eds.),
Wenner-Gren Cent. Int. Symp. Ser., vol. 52, pp.
283-292, Macmillan Press, London
19. Pugsley, A. P. & Oudega, B. (1987) in Plasmids
(Hardy, M. G. ed.), pp. 105-161, IRL Press, Oxford,
Washington DC
20. Daeschel, M. A. (1990) in Biotechnology and Food
Safety (Bills, D. D. & Kung, S-D., eds.), pp. 91-104,
Butterworth-Heinemann,Stoneham,MA, U S A .
21. Schillinger, U. & Lucke, F. K. (1989) Appl. Environ.
Microbiol. 55, 1901-1906
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Received 12 April 1991
DNA probes and the detection of food-borne pathogens using the polymerase
chain reaction
Raymond A. McKee, Christopher M. Gooding, Stephen D. Garrett, Hilary A. Powell, Barbara M. Lund and
Margaret Knox
A.F.R.C., Institute of Food Research, Norwich Research Park, Colney Lane, Norwich NR4 7UA, U.K.
Introduction
In recent years the increased incidence of foodborne microbial pathogens [ 1, 2, 31 has highlighted
the need for more rapid and reliable tests than those
currently available. In the case of short shelf-life
products this would enable manufacturers to
quickly recognize contaminated products.
Standard microbiological tests are available
for the vast majority of microbial contaminants
which may be found in foods. Each identification
procedure will vary with the organism and food
concerned, but would normally involve pre-enrichment, and enrichment incubations. These allow an
increase in cell number, sufficient for detection and
identification using a battery of microbiological and
biochemical tests [4].
These standard microbiological techniques
have been supplemented with recently developed
methods to ease the labour intensive nature of the
process, such as immunological detection [51.
Determination of the presence of living cells within
the sample by ATP monitoring, flow cytometry and
electrical conductance measurement can give an
indication of the need to proceed with microbiological tests.
The use of nucleic acid probes for the detection of microbial pathogens is based on the unique
ability of a molecule of DNA or RNA to hybridize
Abbreviations used: PCR, polymerase chain reaction;
CFU, colony-formingunit,
Volume 19
specifically with a complementary sequence. A
major advantage of nucleic acid probes over other
detection methods is their information content. An
antibody for example may recognize a unique
stretch of six amino-acid residues, but owing to the
redundancy in the genetic code these six amino
acids may be coded for by up to several thousand
different DNA molecules.
There are a number of choices as to which
nucleic acid sequence should be used as the target.
Genomic DNA sequences from known cloned
genes such as those encoding toxins have been
used and offer high specificity. However, even if the
target gene is present on a plasmid it is unlikely to
be present at greater than one hundred copies per
cell. When the number of target organisms per
sample is low [see later, polymerase chain reaction
(PCR) methodologies] this concentration would not
be detected using standard hybridization protocols.
With this in mind a number of commercially available probes target ribosomal RNA (rRNA), as this
can be present at from 1000 to 10000 copies per
cell. One of the drawbacks to this approach is the
high degree of conservation of primary sequence in
rRNA from different species, particularly closelyrelated species.
At the Institute of Food Research we have an
interest in developing DNA probes for the detection
of List& species and in particular List& monocytogenes. W e have used an approach developed by
Ward and his co-workers [6] which allows the